EA041083B1 - METHOD FOR INDUCING DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN VITRO - Google Patents

Description

State Licensing Rights

The present invention was made with partial government support under NIH/NINDS grant NS052671 and NIH/NINDS grant P50 NS047085 from the US National Institutes of Health (NIH) and the National Institute of Neurological Disorders and Stroke (NINDS). Accordingly, the Government of the United States has certain rights in the present invention.

The field of technology to which the invention belongs

The present invention relates to the field of stem cell biology, in particular to the field of lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to: human embryonic stem cells (hESC, hESC), in addition to non-embryonic induced human pluripotent stem cells (hiPSC , hPSCs), somatic stem cells, stem cells from sick patients, or any other cell capable of lineage-specific differentiation. In particular, methods are described for ensuring lineage-specific differentiation of hESCs and/or hPSCs into progenitor cells of the ventral plate of the midbrain and then further into large populations of FOXA2+LMX1A+TH+ dopamine (DA) midbrain neurons using novel culture conditions. FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons obtained using the methods of the present invention are further used in a variety of applications including, but not limited to, use in in vitro assays for drug development and as a therapeutic agent for disease reversal , damage or lack of dopamine neurons in the patient. Further, compositions and methods are provided for providing differentiation of FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons from human pluripotent stem cells for use in disease modeling, particularly Parkinson's disease. In addition to natural DA, neurons contain a large number of markers, such as CD142, and neuronal cells of the A9 type.

Prerequisites for the creation of the invention

Cell populations retaining the ability to differentiate into numerous specialized cell types can be used to develop a large number of lineage-specific differentiated cell populations. These cell populations, which retain the ability to further differentiate into specialized cells, contain pluripotent cells. Pluripotent cells can be derived from embryonic and/or non-embryonic stem cells. It is expected that these populations of lineage-specifically differentiated cells will find applications in cell replacement therapy for patients with diseases caused by loss of function of a particular cell population. In addition to direct therapeutic value, lineage-specifically differentiated cells also represent valuable research tools for a variety of purposes, including in vitro screening assays for identification and verification, as well as tests to investigate function or to investigate the delivery of therapeutic molecules to treat diseases associated with certain cell lines.

Previously, embryonic and somatic stem cells have been used as therapeutic agents and model systems for neurodegenerative diseases. In recent years, research and technological developments have been carried out related to the directed differentiation of embryonic and somatic stem cells in the field of diseases of the central nervous system (CNS), such as Huntington's chorea, Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, the results of these studies have shown the low ability of these cells to provide restoration of neuronal functions in the patient when applied in vivo ability of these cells, and that their use often leads to the growth of unwanted tumors in patients.

Accordingly, there is a need for compositions and methods for obtaining cell populations suitable for use both in research and as therapeutic agents for the treatment of diseases caused by the loss of cells with a particular function.

Brief description of the invention

The present invention relates to the field of stem cell biology, in particular the lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to: human embryonic stem cells (hESC, hESC), as well as non-embryonic induced human pluripotent stem cells (hiPSC, iPSC h ), somatic stem cells, stem cells from patients with disease, or any other cell capable of lineage-specific differentiation.

In particular, the present invention provides a method for inducing differentiation of pluripotent stem cells in vitro as described in the following claims 1-32.

1) A method for inducing differentiation of pluripotent stem cells in vitro, including contacting pluripotent stem cells with a signal transduction inhibitor SMAD (Small Mothers Against Decapentaplegic), and contacting said cells with a SHH signal transduction activator (Sonic hedgehog) and a wingless signal transduction activator (Wnt ), and the contact

- 1 041083 of these cells with the Wnt signal transduction activator are carried out in the time interval from the third (3) to the eleventh (11) day after the initial contact of the cells with the signal transduction inhibitor

SMAD to produce a cell population, more than 10% of said cell population expressing both the forkhead box A2 (FOXA2) protein and the LIM homeobox-containing transcription factor 1 alpha (LMX1A).

2) The method of claim 1, wherein said SMAD signaling comprises TGFβ/activin-nodal signaling.

3) The method of claim 1 or 2, wherein said SMAD signaling comprises bone morphogenesis protein (BMP) signaling.

4) The method according to any one of claims 1 to 3, wherein at least 20% of said cell population expresses both the FOXA2 protein and LMX1A.

5) The method according to any one of claims 1 to 4, characterized in that the cells are brought into contact with the signal transduction activator of the SHH protein at least between 24 and 36 hours from the initial contact of the cells with the SMAD inhibitor.

6) A method according to any one of claims 1 to 5, characterized in that the cells are brought into contact with the wingless signal transduction activator (Wnt) at least between 24 and 36 hours from the initial contact of the cells with the SHH protein signal transduction activator.

7) The method according to any one of claims 1 to 6, characterized in that at least 40% of said cell population expresses FOXA2 and LMX1A protein.

8) The method according to any one of claims 1 to 7, characterized in that said pluripotent stem cells are selected from the group consisting of non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent stem cells and modified human pluripotent stem cells, a primate other than human or rodent.

9) The method according to any one of claims 1 to 8, characterized in that said population of cells expressing FOXA2 and LMX1A additionally expresses at least one marker selected from the group consisting of tyrosine hydroxylase (TH), orthodenticle homeobox 2 (OTX2) , nuclear receptor-related protein 1 (NURR1), neuron-specific beta-tubulin class III (Tuj1), trefoil family factor 3 (TTF3), homeobox protein 3 containing a paired domain-like 3 (PITX3), achaete-scute complex (ASCL ), B-cell early factor 1 (EBF-1), B-cell early factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), G-coupled internal rectifying potassium channel (Kir3.2/GIRK2 ), CD142, DCSM1, CD63 and CD99.

10) A method according to any one of claims 1 to 9, wherein said pluripotent stem cells differentiate into said population of cells within 11 days of initial contact of said cells with at least one SMAD signal transduction inhibitor.

11) The method according to any one of claims 1 to 10, characterized in that the pluripotent stem cells differentiate into dopamine neurons no later than 25 days after the initial contact of these cells with the signal transduction inhibitor SMAD.

12) The method of claim 11, wherein at least 20% of said dopamine neurons express FOXA2 and LMX1A and additionally express at least one further human midbrain dopamine neuron marker subtype A9 selected from the group consisting of Girk2, CD142 and DCSM1.

13) The method according to claim 11 or 12, further comprising selecting a population of dopamine neurons expressing CD142 or DCSM1, or both markers.

14) The method according to any one of claims 1 to 13, characterized in that said cell population is characterized by a significantly low level of expression of at least one marker selected from the group consisting of PAX6, EMX2 and LHX2.

15) The method according to any one of claims 1 to 14, further comprising contacting said population of cells with brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cell neurotrophic factor (GDNF), dibutyryl cAMP (dbcAMP), and transforming growth factor type β3 (TGFe3) to produce dopamine neurons.

16) The method of any one of claims 1 to 15, further comprising contacting said population of cells with an FGF signal transduction activator.

17) The method of claim 16 wherein said FGF signal activator is FGF8.

18) The method according to claim 16 or 17, wherein said cells are contacted with said FGF signaling activator for up to 144 hours from the initial contact of said cells with the SMAD signaling inhibitor.

19) A method according to any one of claims 1-18, wherein said SMAD signal inhibitor is selected from the group consisting of LDN-193189, SB431542, Noggin, dorsomorphine, and combinations of the above.

20) The method according to any one of claims 1 to 19, characterized in that said SMAD signal inhibitor

- 2 041083 is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H-imidazol-2-yl] benzamide (SB431542).

21) A method according to any one of claims 1-20, wherein the SHH signal transduction activator is selected from the group consisting of SHHs, Smoothened agonists (SAGs), and combinations of the above.

22) A method according to any one of claims 1 to 21, wherein the SHH signal transduction activator is a Smoothened (SAG) agonist.

23) The method according to claim 21 or 22, characterized in that said SAG is purmorphamine.

24) The method of claim 21, wherein said SHH is a recombinant SHH protein.

25) The method of claim 24, wherein said recombinant SHH protein is at least 95% identical to the N-terminal fragment of mouse SHH.

26) The method according to claim 24 or 25, wherein said recombinant SHH protein is SHH C25II.

27) A method according to any one of claims 1 to 26, wherein said wingless signal transduction activator (Wnt) is a compound capable of reducing glycogen synthase 3beta 3β (GSK3e) signal transduction.

28) A method according to any one of claims 1-26, wherein said Wnt signal transduction activator is selected from the group consisting of CHIR99021, Wnt3A, Wnt1, and combinations of the above.

29) The method according to any one of claims 1 to 28, wherein said Wnt signal transduction activator is CHIR99021.

30) The method according to any one of claims 1 to 29, characterized in that said cells are contacted with two inhibitors of SMAD signaling, which are SB431542 and LDN-193189, and the wingless signal activator (Wnt) is CHIR99021.

31) The method of any one of claims 1 to 30, further comprising transfecting said pluripotent stem cells with a polysialyl transferase (PST) prior to contacting said pluripotent stem cells with an SMAD signaling inhibitor.

32) The method according to any one of claims 1 to 30, further comprising treating said population of cells with exogenous PST.

In particular, methods are described for ensuring lineage-specific differentiation of hESCs and/or hPSCs into progenitor cells of the ventral midbrain plate and then further into large populations of FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons using novel culture conditions. FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons obtained using the methods of the present invention are further used in a variety of applications, including, but not limited to, use in in vitro assays for drug development, neurological research, and as a therapeutic means for reversing a disease, injury or deficiency of dopamine neurons in a patient. Further, compositions and methods are provided for providing differentiation of FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons from human pluripotent stem cells for use in disease modeling, particularly Parkinson's disease. In addition, natural DA neurons contain a large number of markers such as CD142 and A9 type neuronal cells.

A kit containing a first signal transduction inhibitor, a second signal transduction inhibitor and a third signal transduction inhibitor, wherein said first inhibitor is capable of reducing transforming growth factor beta (TGFe, TGF-beta)/Nodal-Activin signal transduction, said second inhibitor is capable of reducing SMAD signal transduction (Small Mothers Against Decapentaplegic), and said third inhibitor is able to reduce glycogen synthase kinase 3-beta (GSK3e) signaling for Wnt(wingless) activation. In one embodiment, said first inhibitor is LDN-193189. In other embodiments, said first inhibitor is selected from the group consisting of LDN-193189, derivatives thereof, and mixtures thereof. In one embodiment, said second inhibitor is SB431542. In other embodiments, said second inhibitor is selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In one embodiment, said third inhibitor is CHIR99021. In other embodiments, said third inhibitor is selected from the group consisting of CHIR99021, derivatives thereof, and mixtures thereof. In one embodiment, said kit further comprises a Sonic hedgehog (SHH) protein signal transduction activator and a fibroblast growth factor receptor (FGF) family signal transduction activator 8. In one embodiment, said kit further comprises brain-derived neurotrophic factor (BDNF), ascorbic acid (AK), glial cell line neurotrophic factor, dibutyryl cAMP and transforming growth factor type β3. In one embodiment, said kit further comprises antibodies selected from the group consisting of antibodies to tyrosine hydroxylase (TH), an antibody to forkhead box A2 protein (FOXA2), and an antibody to LIM and homeobox containing transcription factor 1 alpha (LMX1A). In one embodiment, said set

- 3 041083 additionally contains a cell selected from the group consisting of a stem cell, an embryonic stem cell, an induced pluripotent stem cell and a modified cell. In one embodiment, the implementation of the specified kit further contains instructions to ensure the differentiation of progenitor cells and FOXA2/LMX1A+ dopamine (DA) midbrain neurons. In one embodiment, said kit further comprises instructions for obtaining a cell from a patient with Parkinson's disease (PD).

A composition comprising a population of cells in contact with a first signal transduction inhibitor and a second signal transduction inhibitor, where more than 40% of said cell population is positive for the forkhead box A2 (FOXA2) protein, where said cell population was previously brought into contact with the first signal transduction inhibitor, a third inhibitor of signal transduction and a signal transduction activator of the Sonic hedgehog (SHH) protein, wherein said first inhibitor is capable of reducing transforming growth factor beta (TGFβ)/Activin-nodal signal transduction, said second inhibitor is capable of reducing glycogen synthase kinase 3-beta (GSK3e) signal transduction to activate Wnt(wingless) signaling, and said third inhibitor is capable of reducing SMAD (Small Mothers Against Decapentaplegic) signaling. In one embodiment, said first inhibitor is a small molecule selected from the group consisting of LDN-193189, derivatives thereof, and mixtures thereof. In one embodiment, said second inhibitor is selected from the group consisting of CHIR99021 and derivatives thereof. In one embodiment, said third inhibitor is selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In one embodiment, said Sonic hedgehog (SHH) signal transduction activator is selected from the group consisting of Small Molecular Weight Smoothened Receptor Agonist (SMO) and Sonic hedgehog (SHH) C25II, said agonist being purmorphamine. In some embodiments, said cell population is additionally pre-contacted with fibroblast growth factor 8 (FGF8). In one embodiment, said majority of the cells comprising said population are forkhead box protein A2 (FOXA2) ⁺ , LIM + homeobox-containing transcription factor 1 alpha (LMX1A) ⁺ , NGN2 + and DDC + ventral midbrain progenitor cells. In one embodiment, said population of cells is selected from the group consisting of rodent cells, primate cells, and human cells. In one embodiment, said cells are derived from cells from a patient with Parkinson's disease (PD). In one embodiment, said cell population is at least 50% positive for the forkhead box A2 (FOXA2) protein. In one embodiment, said cell population is at least 60% positive for the forkhead box A2 protein. In one embodiment, said cell population is at least 70% positive for the forkhead box A2 protein. In one embodiment, said cell population is at least 80% positive for the forkhead box A2 protein. In one embodiment, said population of cells is at least 90% positive for the forkhead box A2 protein. In one embodiment, said population of cells is at least 95% to 100% positive for the forkhead box A2 protein.

A composition containing an in vitro population of cells, where the majority of the cells constituting said population of cells are tyrosine hydroxylase (TH) ⁺ , forkhead box protein A2 (FOXA2) ⁺ , LIM + homeobox-containing transcription factor 1, alpha (LMX1A) ⁺ dopamine (DA ) neurons of the ventral plate of the midbrain. In one embodiment, said greater than 40% of said midbrain ventral plate dopamine (DA) neurons are tyrosine hydroxylase (TH+) positive. In one embodiment, said population of cells is at least 50% positive for tyrosine hydroxylase. In one embodiment, said cell population is at least 60% positive for tyrosine hydroxylase. In one embodiment, said population of cells is at least 70% positive for tyrosine hydroxylase. In one embodiment, said cell population is at least 80% positive for tyrosine hydroxylase. In one embodiment, said cell population is at least 90% positive for tyrosine hydroxylase. In one embodiment, said population of cells is at least 95% to 100% positive for tyrosine hydroxylase. In some embodiments, said population of cells contains predominantly dopamine (DA) FOXA2/LMX1A+ midbrain neurons. In one embodiment, said ventral midbrain dopamine (DA) neurons are positive for markers selected from the group consisting of: nuclear receptor NURR1 (NR4A2), class III neuron-specific beta-tubulin (Tuj1), trefoil family factor 3 (TTF3 ), homeobox protein 3 containing a domain similar to the paired and homeobox protein 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3) and transthyretin (TTR). In one embodiment, said FOXA2/LMX1A+ midbrain dopamine (DA) receptor population is positive for a compound selected from the group consisting of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In one embodiment, said marker is selected from the group consisting of a protein and a nucleic acid. In some embodiments, said population of FOXA2/LMX1A+ midbrain dopamine (DA) receptors is capable of engrafting in vivo in a patient selected from the group consisting of a patient with Parkinson's disease (PD). In one embodiment, said dopamine (DA)

FOXA2/LMX1A+ midbrain neurons are able to engraft in vivo and function as dopamine (DA) neurons.

In some embodiments, the present invention provides a composition comprising a population of cells in contact with LDN-193189 and CHIR99021, where more than 40% of said cell population is positive for the forkhead box A2 (FOXA2) protein, and where said cell population has been previously contacted with LDN -193189, SB431542, Sonic hedgehog (SHH) signal transduction activator and CHIR99021. In one embodiment, said Sonic hedgehog (SHH) signal transduction activator is selected from the group consisting of Sonic hedgehog (SHH) C25II and purmorphamine. In one embodiment, said greater than 10% of said cell population is doubly positive for the forkhead box A2 (FOXA2) protein and the LIM homeobox encoded transcription factor 1, alpha (LMX1A). In one embodiment, said majority of said cell population is the ventral midbrain progenitor cell population. In one embodiment, said population is pre-contacted with fibroblast growth factor 8 (FGF8). In one embodiment, said population of cells is selected from the group consisting of rodent cells, primate cells, and human cells. In one embodiment, said human cells are cells from a person with a neurological symptom of Parkinson's disease (PD). In one embodiment, said cell population is derived from induced pluripotent stem cells (iPSCs, iPSCs). In one embodiment, more than 10% of said cell population is selected from the group consisting of doubly positive for forkhead box A2 protein (FOXA2)/LIM-encoded transcription factor 1 alpha (LMX1A) and doubly positive for forkhead box A2 protein (FOXA2) /orthodenticle homeobox 2 (OTX2).

In one embodiment, the present invention provides a method for inducing targeted differentiation of cells into a population of ventral midbrain progenitor cells, comprising a) providing: i) a population of cells, said cell population being selected from the group consisting of a non-embryonic stem cell, an embryonic stem cell, induced non-embryonic pluripotent cell and modified pluripotent cell; and ii) a first signaling inhibitor, a second signaling inhibitor, a Sonic hedgehog (SHH) protein signaling activator, and a third signaling inhibitor, wherein said first inhibitor is capable of reducing transforming growth factor beta (TGFβ)/activinodal signaling, said second inhibitor is capable of reduce SMAD (Small Mothers Against Decapentaplegic) signaling, and said third inhibitor is capable of reducing glycogen synthase kinase 3-beta (GSK3β) signaling to activate Wnt(wingless) signaling; b) contacting said cell population with said first and said second inhibitor, c) after contacting said cell population with said first and said second inhibitor, further contacting said cells with said Sonic hedgehog (SHH) signal transduction activator under conditions for differentiation of the progenitor cell population of the ventral plate of the midbrain; i) after contacting said cell population with said Sonic hedgehog (SHH) signal transduction activator, further contacting said cells with said third inhibitor to differentiate said cell population into a midbrain ventral plate progenitor cell population. In one embodiment, said contact with said first and said second inhibitor is carried out under conditions that allow said differentiated population of ventral midbrain progenitor cells to be obtained. In one embodiment, said contact with said first and said second inhibitor is carried out within 1 hour of planting the cells in vitro. In one embodiment, said contact with said first said second inhibitor is carried out within 48 hours of planting the cells in vitro. In one embodiment, said contact with said first and said second inhibitor is carried out within 62 hours of seeding the cells in vitro. In one embodiment, said cells are contacted with said Sonic hedgehog (SHH) signaling activator under conditions that allow said differentiated population of ventral midbrain progenitor cells to be generated. In one embodiment, said cells are contacted with said Sonic hedgehog signal transduction activator (SHH) at least 24 and up to 36 hours after said cell population is contacted with said first and said second inhibitor. In one embodiment, said contact of said cells with said Sonic hedgehog (SHH) signal transduction activator is up to 144 hours. midbrain midbrain. In one embodiment, said contact of said cells with said third inhibitor is at least 24 and up to 36 hours after said cell population is contacted with said Sonic hedgehog (SHH) signaling activator. In one

- 5 041083 embodiment, said contact of said cells with said third inhibitor is up to 192 hours. and said said second inhibitor. In one embodiment, said first inhibitor is SB431542. In one embodiment, said second inhibitor is LDN-193189. In one embodiment, said third inhibitor is CHIR99021. In one embodiment, said Sonic hedgehog (SHH) signal transduction activator is selected from the group consisting of Sonic hedgehog (SHH) C25II and purmorphamine. In one embodiment, said method further provides fibroblast growth factor 8 (FGF8) and contacts said population of cells with said FGF8 under conditions that allow said differentiated population of ventral midbrain progenitor cells to be obtained. In one embodiment, said contact of said cells in said FGF8 occurs at least 24 hours and up to 36 hours after said cell population is contacted with said first and said second inhibitor. In one embodiment, said contact of said cells with said FGF8 is up to 144 hours. In one embodiment, said population of ventral midbrain progenitor cells contains more than 40 forkhead box A2 (FOXA2) ⁺ cells. In one embodiment, said population of ventral midbrain progenitor cells contains greater than 40% cells positive for forkhead box A2 (FOXA2), LIM homeobox encoded transcription factor 1, alpha (LMX1A). In one embodiment, said method further comprises step e) contacting said midbrain ventral plate progenitor cell population with a neuronal maturation medium, said medium comprising N2 medium, brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial neurotrophic factor cell line, dibutyryl cAMP (dbcAMP) and transforming growth factor type β3, to differentiate progenitor cells of the ventral midbrain plate into dopamine (DA) neurons of the ventral midbrain plate. In one embodiment, said method further comprises step e) contacting said population of ventral midbrain progenitor cells with B27-supplemented neuronal maturation medium to differentiate ventral midbrain progenitor cells into ventral midbrain dopamine (DA) neurons. . In one embodiment, said cells, contacted with B27-supplemented neurobasal medium, are contacted with brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cell-line neurotrophic factor, dibutyryl-cAMP (dbcAMP), and transforming growth factor. type β3 to differentiate ventral midbrain progenitor cells into dopamine (DA) neurons in the ventral midbrain. In one embodiment, said ventral midbrain dopamine (DA) neurons are positive for forkhead box A2 (FOXA2) protein, LIM homeobox gene-encoded transcription factor 1, alpha (LMX1A), nuclear receptor-related protein 1 (NURR1), and tyrosine hydroxylase (TH) . In one embodiment, more than 40% of said midbrain ventral plate dopamine (DA) neurons are positive for tyrosine hydroxylase (TH) ⁺ . In one embodiment, said population of ventral midbrain dopamine (DA) neurons differentiates by at least 25 days after said cell population is contacted with said first and said second inhibitor. In one embodiment, said ventral midbrain dopamine (DA) neurons are positive for molecule-identifying markers. In one embodiment, said markers are selected from the group consisting of tyrosine hydroxylase (TH), forkhead box A2 protein (FOXA2), encoded by transcription factor 1 homeobox gene LIM, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) , alpha, nuclear receptor NURR1 (NR4A2), neuron-specific beta-tubulin class III (Tuj1), TTF3, homeobox protein 3 containing a paired-like domain 3 (PITX3), achaete-scute complex (ASCL), early factor 1 B cells (EBF-1), B cell early factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT) and G protein-coupled internal rectifying potassium channel (Kir3.2/GIRK2) . In one embodiment, said molecule is selected from the group consisting of a protein and a nucleic acid. In one embodiment, said molecule is identified using a marker selected from the group consisting of an antibody, a PCR primer, a nucleic acid sequence, and an enzyme test. In one embodiment, said ventral midbrain dopamine (DA) neurons are capable of engrafting in vivo in a patient with Parkinson's disease (PD) and mediating dopamine (DA) neuron function. In one embodiment, said method further comprises providing a patient in need of dopamine producing neurons, said patient exhibiting at least one neurological symptom, at the step of transplanting dopamine (DA) neurons of the ventral midbrain to said patient to provide dopamine (DA) function. ) neurons. In one

- 6 041083 embodiment, these neurological symptoms are selected from the group consisting of tremor, bradykinesia (severe slowness of movement), hunched posture, postural instability and rigidity. In one embodiment, the implementation of the specified patient is a decrease in the severity of the specified neurological symptom. In one embodiment, said cell is selected from a rodent cell, an omen cell, and a human cell. In one embodiment, said human cells are cells from a person with a neurological symptom of Parkinson's disease (PD). In one embodiment, the present invention provides a method for providing engraftment in vivo for therapeutic treatment, comprising a) providing: i) a population of dopamine (DA) neurons of the ventral plate of the midbrain, where more than 40% of said population express tyrosine hydroxylase (TH); and ii) a subject, wherein said subject exhibits at least one neurological symptom; and b) transplanting said ventral midbrain dopamine (DA) neurons into said subject under conditions allowing engraftment in vivo to provide dopamine (DA) neuron function. In one embodiment, the implementation of these neurological symptoms are selected from the group consisting of tremor, bradykinesia (severe slowness of movement), hunched posture, postural instability and rigidity. In one embodiment, said subject experiences a decrease in the severity of said neurological symptom. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a population of ventral midbrain progenitor cells treated according to the methods of the present invention. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a population of ventral midbrain progenitor cells treated according to a method further comprising the step of contacting said population of ventral midbrain progenitor cells with a neuronal maturation medium. , wherein said medium contains N2 medium, brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cell-line neurotrophic factor, dibutyryl-cAMP, and transforming growth factor type β3 for differentiation of midbrain ventral plate precursor cells into dopamine (DA) neurons in the ventral plate of the midbrain. In one embodiment, said population of ventral midbrain progenitor cells is derived from a population of cells treated in accordance with the method of the present inventions. In one embodiment, said population of ventral midbrain progenitor cells is derived from a population of cells treated according to a method for inducing directed cell differentiation into a population of ventral midbrain progenitor cells comprising a) providing: i) a population of cells, wherein said population the cells are selected from the group consisting of a non-embryonic stem cell, an embryonic stem cell, an induced non-embryonic pluripotent cell, and a modified pluripotent cell; and ii) a first signaling inhibitor, a second signaling inhibitor, a Sonic hedgehog (SHH) protein signaling activator, and a third signaling inhibitor, wherein said first inhibitor is capable of reducing transforming growth factor beta (TGFβ)/activinNodal signaling, said second inhibitor capable of reducing SMAD (Small Mothers Against Decapentaplegic) signaling, and said third inhibitor is capable of reducing glycogen synthase kinase 3-beta (GSK3e) signaling to activate Wnt(wingless) signaling; b) contacting said cell population with said first and said second inhibitor, c) after contacting said cell population with said first and said second inhibitor, further contacting said cells with said Sonic hedgehog (SHH) signal transduction activator under conditions to differentiate a population of progenitor cells of the ventral plate of the midbrain; and d) after contacting said population of cells with said activator of Sonic hedgehog signaling (SHH), further contacting said cells with said third inhibitor to differentiate said population of cells into a population of ventral midbrain progenitor cells. In one embodiment, the implementation of the specified population of dopamine (DA) neurons of the ventral plate of the midbrain is derived from a population of cells selected from the group consisting of animals, primates and humans. In one embodiment, said human cells are from a patient with a symptom of Parkinson's disease (PD).

In one embodiment, the present invention provides a composition comprising a population of cells in contact with LDN-193189 and CHIR99021, where more than 40% of said cell population is positive for forkhead box A2 (FOXA2) protein, and where said cell population has been previously contacted with LDN -193189, SB431542, Sonic hedgehog (SHH) signal transduction activator and CHIR99021, said Sonic hedgehog (SHH) signal transduction activator being selected from the group consisting of Sonic hedgehog (SHH) C25II and purmorphamine. In one embodiment, said cell population is at least 50% positive for the forkhead box A2 (FOXA2) protein. In one embodiment, said cell population is at least 60% positive for the forkhead box A2 protein. In one embodiment, said population of cells is at least 70%

- 7 041083 rich in forkhead box A2 protein. In one embodiment, said cell population is at least 80% positive for the forkhead box A2 protein. In one embodiment, said population of cells is at least 90% positive for the forkhead box A2 protein. In one embodiment, said population of cells is at least 95% to 100% positive for the forkhead box A2 protein. In one embodiment, more than 10% of said cell population is selected from a group consisting of cells that are doubly positive: for forkhead box A2 protein (FOXA2)/LIM-encoded transcription factor 1 alpha (LMX1A) and doubly positive: for forkhead protein box A2 (FOXA2)/LIM homeobox 2 (OTX2). In one embodiment, said cell population is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% positive, at least 95% to 100% positive. In one embodiment, at least 20% of said cell population is positive for a marker selected from the group consisting of Nurr1+, CD142, DCSM1, CD63, and CD99. In some embodiments, said cell population is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% positive, at least 95% to 100% positive. In some embodiments, said cell population is at least 50%, 60%, 70%, 80%, 90% positive, at least 95% to 100% positive. In one embodiment, said cell population is selected from the group consisting of rodent cells, primate cells, human cells, and human cells from a patient with a neurological symptom of Parkinson's disease (PD). In one embodiment, the present invention provides a method for inducing targeted differentiation of cells into a population of ventral midbrain progenitor cells, comprising a) providing: i) a population of cells, said cell population being selected from the group consisting of a non-embryonic stem cell, an embryonic stem cell , induced non-embryonic pluripotent cell and modified pluripotent cell; and ii) a first signal transduction inhibitor, a second signal transduction inhibitor, a Sonic hedgehog (SHH) signal transduction activator, and a third signal transduction inhibitor, said first inhibitor being SB431542, said second inhibitor being LDN193189, said Sonic hedgehog signal transduction activator ( SHH) is selected from the group consisting of Sonic hedgehog (SHH) C25II and purmorphamine, and said third inhibitor is CHIR99021; b) bringing said population of cells into contact with said first and said second inhibitor, wherein said contact with said first and said second inhibitor is carried out under conditions that allow said differentiated population of progenitor cells of the ventral midbrain plate to be obtained, such that said contact with the specified first and the specified second inhibitor is carried out within 48 hours after planting the cells in vitro, c) after bringing the specified population of cells into contact with the specified first and specified second inhibitor, further bringing the specified cells into contact with the specified signal transduction activator Sonic hedgehog (SHH) under conditions for differentiation of the progenitor cell population of the ventral plate of the midbrain; and d) after contacting said population of cells with said activator of Sonic hedgehog signaling (SHH), further contacting said cells with said third inhibitor to differentiate said population of cells into a population of ventral midbrain progenitor cells. In one embodiment, said cells are contacted with said Sonic hedgehog signaling activator (SHH) under conditions that allow said differentiated population of midbrain ventral plate progenitor cells to be obtained such that said cells are contacted with said Sonic hedgehog signaling activator (SHH) is carried out at least 24 and up to 36 hours after contacting said population of cells with said first and said second inhibitor. In one embodiment, said cells are contacted with said third inhibitor under conditions that allow for said differentiated population of midbrain ventral progenitor cells to be obtained such that said cells are contacted with said third inhibitor at least 24 and up to 36 h after contacting said cell population with said Sonic hedgehog (SHH) signal transduction activator. In one embodiment, the ventral midbrain progenitor cell population contains greater than 40% cells. Positive for forkhead box A2 (FOXA2), LIM+homeobox-containing transcription factor 1, alpha (LMX1A). In one embodiment, the method further comprises step e) contacting said population of midbrain ventral plate precursor cells with a neuronal maturation medium, said medium comprising N2 medium, brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), neurotrophic factor glial cell line, dibutyryl cAMP, and transforming growth factor type β3 to differentiate ventral midbrain progenitor cells into dopamine (DA) neurons in the ventral midbrain. In one embodiment, said ventral midbrain dopamine (DA) neurons are positive for a set of markers selected from the group consisting of: forkhead box A2 protein (FOXA2) ^/ LIM homeobox encoded transcription factor 1 alpha (LMX1A) / tyrosine hydroxylase (TH) ; protein forkhead box A2/ encoded by homeobox gene LIM transcription factor 1 alpha/tyrosine hydroxylase/CD142; forkhead box protein A2/encoded

- 8 041083 homeobox gene LIM transcription factor 1 alpha/tyrosine hydroxylase/related nuclear receptor protein 1 (NURR1); forkhead box protein L2/encoded by the LIM homeobox gene transcription factor 1 alpha/tyrosine hydroxylase/CD142/nuclear receptor-related protein 1 and tyrosine hydroxylase/a-synuclein. In one embodiment, said method further comprises step f) sorting said ventral midbrain dopamine (DA) neurons for CD142 expression into a population of cells at least 80% positive for CD142. In one embodiment, said ventral midbrain dopamine (DA) neurons are positive for a marker identifying a molecule selected from the group consisting of tyrosine hydroxylase (TH), forkhead box protein L2 (FOXA2), encoded by the LIM homeobox gene of transcription factor 1, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), alpha, NURR1 nuclear receptor (NR4A2), class III neuron-specific beta-tubulin (Tuj1), TTF3, homeobox protein 3 containing a paired domain-like domain 3 (PITX3), achaete-scute complex (ASCL), early B-cell factor 1 (EBF-1), early B-cell factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT) and conjugated with G-protein of the potassium channel of internal rectification (Kir3.2/GIRK2), CD142, DCSM1, CD63 and CD99. In one embodiment, the method additionally takes a patient in need of dopamine-producing neurons and further performs step e) of treating said patient by transplanting said dopamine (DA) neurons of the ventral midbrain to provide dopamine (DA) neuron function. In one embodiment, the implementation of the specified patient has at least one neurological symptom selected from the group consisting of tremor, bradykinesia (severe slowness of movement), hunched posture, postural instability and rigidity. In one embodiment, said patient is observed to have at least one neurological symptom selected from the group consisting of tremor, bradykinesia (severe slowness of movement), hunched posture, postural instability, and rigidity. In one embodiment, said patient experiences a reduction in at least one of said neurological symptoms.

In one embodiment, the present invention provides an in vivo engraftment method for therapeutic treatment, comprising a) providing: i) a population of dopamine (DA) neurons in the ventral midbrain, wherein greater than 40% of said population expresses tyrosine hydroxylase (TH); and ii) a subject, wherein said subject exhibits at least one neurological symptom selected from the group consisting of tremor, bradykinesia (severe slowness of movement), hunched posture, postural unsteadiness, and rigidity; and b) transplanting said ventral midbrain dopamine (DA) neurons into said patient under conditions suitable for in vivo engraftment to provide dopamine (DA) neuron function. In one embodiment, the implementation of the specified subject is a decrease in the severity of at least one symptom. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a cell population using the additional step of sorting said ventral midbrain dopamine (DA) neurons by CD142 expression into a population at least 80% positive for CD142. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a population of ventral midbrain progenitors after the step of sorting said CD142-expressing dopamine (DA) neurons into a cell population of at least 80 % positive for CD142. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a population of cells selected from the group consisting of animals, primates, humans, and a symptomatic Parkinson's disease (PD) patient. In one embodiment, said population of ventral midbrain dopamine (DA) neurons is derived from a population of cells isolated from a group consisting of animals, primates, humans, and a patient with symptoms of Parkinson's disease (PD).

Definitions.

As used herein, the term disease modeling refers to the process of using an experimental organism or cell cultures in vitro to mimic specific signs or symptoms that a person develops as a result of a disease. In one embodiment, pluripotent stem cells derived from a model animal with a genetic mutation leading to a neurological disorder such as Parkinson's disease (PD) can be grown and differentiated into nerve cells to identify new neuronal characteristics associated with PD. In one embodiment, human pluripotent cells taken from a person with a genetic mutation resulting in a neurological disorder such as Parkinson's disease (PD) can be grown and differentiated into nerve cells having a defect similar to that seen in said person.

As used herein, the term parkinsonism refers to a group of diseases, each of which is associated with a deficiency of dopamine in the basal ganglia, which are part of the brain that controls

- 9 041083 driving movement. Symptoms include tremors, bradykinesia (severe slowness of movement), hunched posture, postural instability, and rigidity. A diagnosis of parkinsonism is made only if at least two of these symptoms are present, one of which must be tremor or bradykinesia. The most common form of parkinsonism is idiopathic, or classic, Parkinson's disease (PD), but in the case of a significant minority of diagnoses, approximately 15 percent of the total, one of the parkinson-plus syndromes (PPS) may be present. These syndromes, also known as atypical parkinsonism, include corticobasal degeneration, Lewy body dementia, multiple system atrophy, and progressive supranuclear palsy. In general, Parkinson's disease involves the dysfunction and death of critical nerve cells in the brain, primarily in an area of the brain called the substantia nigra. Many of these important nerve cells produce dopamine, so if these neurons die, the amount of dopamine that depends on brain differentiation is reduced, resulting in the inability to control movements normally. The gut also contains dopamine cells that are destroyed in patients with Parkinson's disease, which may be an important factor in the GI symptoms that are part of the disease. Complexes of symptoms in different patients are different. The first motor signs of Parkinson's disease include the following: tremors of the hands, arms, legs, jaw, and face, bradykinesia or slowness of movement, stiffness or decreased mobility of the joints and trunk, and postural instability or disturbances in balance and coordination.

In the present text, the term subject refers to a mammal (human and animals, ie, non-human animals) that will be the recipient of a particular treatment, including any type of control. Generally, the terms subject and patient are used interchangeably herein to refer to a human subject.

In the present text, the term non-human animals means all non-human animals, including vertebrates such as rodents, non-human primates, rams, bulls, ruminants, lagomorphs, pigs, goats, equines, canines, felines, birds, etc. etc., but not limited to the above.

As used herein, the term dopamine (dopamine) refers to a chemical compound produced by dopamine neurons that carries signals to the part of the brain that contains neurons that control movement and coordination.

In the present text, the term LSB refers to the combination of the two components LDN-193189 and SB431542, which is capable of reducing or blocking transforming growth factor beta (TGFβ)/activin-Nodal signaling and SMAD (Small Mothers Against Decapentaplegic) signaling in a cell .

In the present text, the term SB431542 refers to a compound capable of reducing or blocking transforming growth factor beta (TGFβ)/activin-Nodal signaling, having CAS number 301836-41-9, molecular formula C ₂₂ H ₁₈ N ₄ O ₃ and name 4-[ 4-(1,3-benzodioxol-5-yl)-5-(2pyridinyl)-1H-imidazol-2-yl]-benzamide. See for example the following structure

In one example, SB431542 is SB431542 Stemolecule™, Stemgent, Inc. Cambridge, Massachusetts, USA. In the present text, the term LDN-193189 refers to the low molecular weight compound DM-3189, IUPAC name 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3yl)quinoline with the chemical formula C ₂₅ H ₂₂ N ₆ . LDN-193189 may act as an inhibitor of SMAD signaling. LDN-193189 is also a highly potent, small molecule inhibitor of ALK2, ALK3, and ALK6, protin tyrosine kinases (PTKs), inhibiting signal transduction of the ALK1 and ALK3 families of TGFe type I receptors, resulting in inhibition of multiple biological signaling, including BMP2, bone morphogenesis protein (BMP) signaling, BMP4, BMP6, BMP7 and activin cytokine and subsequent phosphorylation of SMAD Smad1, Smad5 Smad8 proteins (Yu et al. (2008) Nat Med 14:1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18: 4388-4392, cited source incorporated herein by reference).

- 10 041083

In one embodiment, LDN-193189 is LDN-193189 Stemolecule™, Stemgent,

Inc. Cambridge, Massachusetts, USA.

In the present text, the term glycogen synthase kinase 3β inhibitor or GSK3e inhibitor means a compound that inhibits glycogen synthase kinase 3β, see, for example, Doble, et al., J Cell Sci. 2003;116:1175-1186 (this source is incorporated into the present description by reference). For the purposes of the present invention, a GSK3e inhibitor is capable of activating the WNT signaling cascade, see, for example, Cadigan, et al., J Cell Sci. 2006; 119:395-402; Kikuchi, et al., Cell Signaling. 2007;19:659-671 (this source is incorporated into the present description by reference).

In the present text, the term CHIR99021, or CHIR, or aminopyrimidine, or 3-[3-(2carboxyethyl)-4-methylpyrrole-2-methylidenyl]-2-indoline corresponds to the name 6-(2-(4-(2,4dichlorophenyl)- 5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinnitrile according to IUPAC. CHIR99021 is one example of a small molecular weight glycogen synthase kinase 3β (GSK3e) inhibitor that activates the WNT transmission cascade and is highly selective, showing approximately 1000-card selectivity for a panel of related and unrelated kinases with an IC ₅₀ =6.7 nM for human GSK3e and nanomolar values. IC ₅₀ for homologous GSK3e rodents.

In one embodiment, CHIR99021 is CHIR99021 Stemolecule™, Stemgent, Inc. Cambridge, Massachusetts, USA. In the present text, the term purmorphamine refers to a purine derivative, such as a derivative having a CAS number: 483367-10-8. One example is a compound having the following structure, which activates the Hedgehog cascade, including targeting Smoothened.

In one embodiment, purmorphamine is Stemolecule™ purmorphamine, Stemgent Inc. Cambridge, Massachusetts (USA).

As used herein, the term signaling in relation to a signaling protein refers to proteins that are activated or otherwise affected by the binding of a ligand to a membrane receptor protein or some other stimulus. Examples of signal proteins include SMAD, a WNT complex protein, in another embodiment, a WNT complex protein including beta-catenin, Sonic hedgehog (SHH) proteins, NOTCH, transforming growth factor beta (TGFe), activin, Nodal, glycogen synthase kinase 3β (GSK3e ) and the like. For many cell surface receptors or intrinsic receptor proteins, ligand-receptor interactions are not directly related to cell response. The ligand-activated receptor must first interact with other proteins within the cell to effect the final physiological effect of the ligand on cell behavior. Often, after activation or inhibition of the receptor, there is a change in the behavior of a chain of several interacting cellular proteins. The complete set of changes in cells initiated by receptor activation is called the signal transduction mechanism (signal transmission) or signal transmission cascade.

In the present text, the term LSB/S/F8/CHIR or LSB/SHH/FGF8/CHIR refers to bringing cells into contact with LDN-193189 and SB431542 (i.e. LSB) in addition to S, Sonic Hedgehog protein activator, F8, FGF8 and CHIR in accordance with the present invention. Unlike LSB/S/F8, or SHH/FGF8, or SHH/FGF, which refer to bringing cells into contact with LDN-193189 and SB431542 (i.e. LSB) in addition to S, the Sonic Hedgehog protein activator, F8 , FGF8, but in the absence of CHIR, as previously described. In related abbreviations, LDN/SB refers to bringing cells into contact with LDN, LDN-193189 and SB, SB431542.

As used herein, the term "inhibit" or "block" means a reduction in the level of activity of a particular cell signaling cascade after treatment with a compound (i.e., an inhibitor) compared to the activity of said signaling cascade in a cell that has not been treated with such compound or has been subjected to a control treatment.

As used herein, the term "activate" refers to an increase in the level of activity of a particular cell signaling cascade after treatment with a compound (i.e., an activator) compared to the activity of said signaling cascade in a cell that has not been treated with that compound or has been subjected to a control treatment. Any level of inhibition or activation of a particular signal transduction cascade is considered an embodiment of the present invention if such inhibition or activation results in directed stem cell differentiation.

In the present text, the term SMAD (or Sma Mothers Against Decapentaplegic or Small Mothers Against Decapentaplegic) refers to a signaling molecule.

As used herein, the term WNT or wingless in relation to a ligand refers to a group of secreted proteins (namely, Int1 (integration 1) in humans) that can interact with a WNT receptor, such as the Frizzled family receptor and LRPDerailed/RYK.

In the present text, the term WNT or wingless in relation to the signal transduction cascade refers to a signal transduction pathway consisting of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed/RYK receptors, mediated by or without β-catenin. For the purposes described here, the preferred WNT signaling cascade involves the involvement of β-catenin, i.e. WNT/β-catenin complex.

In the present text, the canonical cascade or classical activation in relation to WNT refers to one of the many underlying Wnt signaling cascades, for example, in the canonical cascade, the main effect of Wnt ligand binding to its receptor is to stabilize the cytoplasmic beta-catenin degradation complex. Other Wnt cascades are non-canonical.

As one example, the small molecule CHIR compound affects the downstream Wnt signaling cascade.

In the present text, the term Sonic hedgehog (SHH or Shh) refers to a protein that is one of at least three proteins in the mammalian signal transduction cascade family called hedgehog, the other is DHH (DHH (desert hedgehog), and the third IHH (Indian hedgehog).Shh interacts with at least two transmembrane proteins by interacting with transmembrane molecules Patched (PTC) and Smoothened (SMO). transducer).In the absence of SHH, PTC normally inhibits SMO, which in turn activates a transcriptional repressor, causing some genes to not be transcribed.If Shh is present and binds to PTC, PTC cannot interfere with SMO function. In the absence of SMO inhibition, some proteins can enter the nucleus and act as transcription factors, allowing certain genes to be activated (cm. Gilbert, 2000 Developmental Biology (Sunderland, Massachusetts: Sinauer Associates, Inc., Publishers).

In the present text, the term activator or activator refers to small molecules, peptides, proteins and compounds for activating molecules leading to directed cell differentiation in accordance with the present inventions. Examples of activators include, but are not limited to: CHIR, Sonic hedgehog (SHH) C25II, Smoothened purmorphamine fibroblast growth factor (FGF), a small molecular weight protein agonist, etc.

As used herein, the term Sonic hedgehog (SHH) signal transduction activator refers to any molecule or compound that activates the SHH signal transduction pathway, including molecules or compounds that bind to PCT or the Smoothened agonist and the like. Examples of such compounds are the Sonic hedgehog (SHH) C25II protein and the small molecular weight agonist of the Smoothened protein purmorphamine.

As used herein, the term Sonic hedgehog (SHH) C25II refers to a recombinant N-terminal fragment of the full-length mouse hedgehog protein capable of binding to the SHH receptor and activating SHH, see for example R&D Systems catalog number: 464-SH-025/CF.

In the present text, the term signals refers to intrinsic and extrinsic factors that control changes in cell structure and function. They can be chemical or physical in nature. In the present text, the term ligand refers to molecules and proteins that bind to a receptor (R), examples include, but are not limited to, transforming growth factor-beta, activins, nodal protein, bone morphogenesis proteins (BMPs), etc.

In the present text, the term signal transduction inhibitor or inhibitor refers to the inhibition of a signaling molecule or signaling molecular cascade, such as in the case of an SMAD signal transduction inhibitor, glycogen synthase kinase 3β (GSK3e) inhibitor means a compound or molecule (e.g., small molecule, peptide, peptidomimetic, a naturally occurring compound, protein, siRNA, antisense nucleic acid, aptamer, or antibody) that disrupts (i.e., reduces, or suppresses, or abolishes, or blocks) the signaling function of a molecule or cascade. In other words, an inhibitor is any compound or molecule that alters any activity of a named protein (signaling molecule, any molecule interacting with a signaling molecule, named related molecule, such as glycogen synthase kinase 3β (GSK3e)) (for example, including the signaling molecule described herein). molecules, but not limited to), as one example, by making direct contact with SMAD signaling, making contact with SMAD mRNA, resulting in conformational changes in SMAD, decreased levels of SMAD protein, or disruption of SMAD interaction with partners in signal transduction (eg, including partners as described here); and downregulation of target SMAD genes (eg, genes as described here). Inhibitors also include molecules that indirectly regulate the biological activity of SMAD by intercepting molecules upstream of the signaling cascade. Accordingly, in one embodiment, an inhibitor of the present invention induces (changes) or changes differentiation from a cell type in the absence of an effect (typical) to another cell type (atypical), one of the methods of the present invention comprising the use of LDN/SB, CHIR and an activator SHH (which can inhibit glycogen synthase kinase 3β) mediated progenitor differentiation into atypical neuronal progenitors. In a preferred embodiment, the inhibitor of the present invention alters or reduces or blocks the original signaling, directing cell differentiation towards an atypical cell type, for example, as described herein for the differentiation of progenitor cells of the ventral midbrain plate and dopamine (DA) FOXA2/LMX1A+ neurons midbrain in accordance with the present inventions. Accordingly, an inhibitor of the present invention is a natural compound or small molecule that alters the activity of a signaling molecule such that it promotes differentiation of the original cell population (day 0) into progenitor cells of the ventral midbrain. If progenitor cells are brought into contact with inhibitors, these small molecules promote further differentiation into dopamine (DA) FOXA2/LMX1A+ midbrain neurons in accordance with the present inventions. Inhibitors are described in terms of competitive inhibition (binds to an active site resulting in exclusion or reduction of binding to another known binding compound) and allosteric inhibition (binds to a protein resulting in a conformational change in the protein which disrupts the binding of the compound to the active site of that protein) in addition to inhibition in addition to inhibition induced by binding to and acting on a molecule upstream of said signal molecule, which in turn causes inhibition of said molecule. In some cases, an inhibitor is referred to as a direct inhibitor, which means inhibition of the signaling target or signal transduction pathway that is the target; for example, a direct gamma-secretase inhibitor is the DAPT molecule, which binds to the gamma-secretase protein.

In the present text, the term derivative refers to a chemical compound with a similar basic structure.

In the present text, the term ventral midbrain progenitor cell, when applied to an in vivo cell located in the midbrain, including the period of embryonic development of midbrain neurons, means a cell that can differentiate into a dopamine-producing cell. In some embodiments, a midbrain ventral progenitor cell refers to a cell in culture that is used to artificially produce an in vitro cell culture that expresses sets of markers that are identical to and overlap with those expressed by cells in vivo, i.e. co-expresses the ventral plate marker FOXA2 and the tegmental markers LMX1A, OTX2, NGN2 and DDC as in cultured cells according to the present inventions around day 11 after initiation of directed differentiation as described here. In a preferred embodiment, the ventral midbrain progenitor cell is FOXA2+LMX1A+ or FOXA2/LMX1A+. In some embodiments, a small number of cells in the differentiated progenitor population are FOXA2/LMX1A/TH+.

In this text, the term DA tegmental neurons or authentic DA midbrain neurons or FOXA2+LMX1A+ dopamine (DA) midbrain lineage neurons or dopamine (DA) ventral plate midbrain neurons or engrafted DA midbrain neurons or mDA neurons or FOXA2+LMX1A+ TH+, or FOXA2/LMX1A/TH, or FOXA2+LMX1A+ NURR1+TH+, or FOXA2/LMX1A/NURR1/TH REFER TO a population of engrafted DA midbrain neurons obtained by the method described here, usually around day 25 or day 25 after initiation directed differentiation. In a preferred embodiment, the authentic midbrain DA neurons are FOXA2+/LMX1A+/NURR1+/TH+. These neurons were designated as engraftable after transplantation experiments in mice and primates, indicating the ability of these neurons to reverse parkinsonism-like neurological conditions while causing fewer problems associated with neuronal overgrowth and teratoma formation. Dopamine (DA) FOXA2/LMX1A+ midbrain neurons in accordance with the present inventions were kept in vitro for several months while maintaining the ability to engraft.

According to the present disclosure, cells used to generate progenitor cells of the ventral plate of the midbrain and dopamine (DA) FOXA2/LMX1A+ midbrain neurons are obtained from various sources, including embryonic and non-embryonic sources, e.g. with a disease

- 13 041083

those. isolated pluripotent cells and modified derived stem cells from patients with Parkinson's disease, cancer stem cells, human or mammalian pluripotent cells, etc.

In the present text, the term stem cell refers to a cell that has the ability to divide for an indefinite period in culture and give rise to specialized cells. The stem cell can be obtained from animals and patients, including humans; for example, a human stem cell is a stem cell that is human. Stem cells can be obtained from a variety of sources, including both embryonic and non-embryonic sources such as cord blood cells, children's cells, and adult cells. For the purposes of the present inventions, adult stem cells generally refer to cells that were not originally derived from a fetus, in other words, cells from infants, from unused umbilical cords, from unused placenta, from children, from adults, etc.

In the present text, the term cord blood stem cells refers to stem cells obtained from the umbilical cord at birth, which have the ability to give rise to at least all blood cells in the body (hematopoietic).

In this text, the term somatic stem cells (adult stem cells) refers to a relatively rare, undifferentiated form of cells found in many organs and differentiated tissues with limited capacity for self-renewal (in the laboratory) and differentiation. The ability of such cells to differentiate varies, but is usually limited by the cell types of the organ from which they originate.

In this text, the term somatic cell refers to any cell in the body, with the exception of gametes (eggs and sperm), sometimes referred to as adult cells.

In the present text, the term neuronal cell line refers to a cell that is involved in the development of the nervous system (both central and peripheral) or neural crest cells during development or in adulthood. Neural roll cell lines include cranial, stem, vagal, and cardiac cells from which mesoderm, cranial cartilage, cranial bones, thymus, teeth, melanocytes, iris pigment cells, cranial ganglia, dorsal root ganglia, sympathetic/parasympathetic ganglia, endocrine cells are derived , cells of the intestinal nervous system and parts of the heart.

As used herein, the term adult stem cell refers to a somatic stem cell, such as a hematopoietic stem cell, which refers to the stem cell in infants, children, and adults that gives rise to all red blood cells and platelets and platelets.

In this text, the term embryonic stem cell refers to a primary (undifferentiated) cell that is derived from one of several sources, including a pre-implantation embryo, an artificially created embryo, i. obtained as a result of in vitro fertilization, etc., but not limited to, capable of dividing without differentiation during long-term cultivation; it is known that these cells have the ability to develop into cells and/or tissues of the three primary germ layers: ectoderm, mesoderm and endoderm.

In the present text, the term endoderm refers to the layer of cells formed from the inner cell mass of the blastocyst; it has the ability to give rise in vivo to the lungs, other respiratory structures and digestive organs, or, in the common gut, as well as a variety of cell types in vitro.

As used herein, the term embryonic stem cell line refers to a population of embryonic stem cells that are cultured under in vitro conditions that proliferate without differentiation for days, months, or years, such as the human WA09 cell line, for example.

In this text, the term human embryonic stem cells or hESCs (hESCs) refers to a type of pluripotent stem cells derived from embryos in early stages up to and including the blastocyst stage, which are capable of dividing without differentiation over an extended culture period and which are known to develop into cells and tissues of the primary germ layers: ectoderm, mesoderm and endoderm.

In the present text, the term induced pluripotent stem cell or iPSC (iPSC) refers to a type of pluripotent stem cell, similar to embryonic stem cells, consisting of somatic (adult) cells reprogrammed in such a way that they go into a state similar to embryonic stem cells, for due to the fact that they are forced to express factors important for maintaining the stemness of embryonic stem cells (ESCs, ESCs). Mouse iPSCs were described in 2006 (Takahashi and Yamanaka) and human iPSCs were described in late 2007 (Takahashi et al. and Yu et al). Mouse iPSCs display important properties of pluripotent stem cells, including expression of stem cell markers, tumor formation from all three germ layers, and participation in the formation of many different tissues when injected into mouse embryos at a very early stage of development. Human hPSCs also express stem cell markers and are able to form cells characteristic of

- 14 041083 all four germ layers. In contrast to the embryonic stem cell, iPSCs are artificially produced by introducing certain embryonic genes (such as the OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi, Yamanaka Cell 126, 663-676 (2006), this source is incorporated herein by refs) into a somatic cell, eg cell lines from induced cells, C14, C72 and the like. Another example of iPSCs are adult human skin cells or fibroblasts transformed with genes (OCT4, SOX2, NANOG, LIN28 and KLF4) cloned into a plasmid, see Yu, et al., Science DOI: 10.1126/science. 1172482 (this source is incorporated into the present description by reference).

As used herein, the term totipotent refers to the ability to give rise to all cell types of an organism plus all cell types that form extraembryonic tissues such as the placenta.

As used herein, the term multipotent refers to the ability to develop into more than one cell type of an organism.

As used herein, the term pluripotent refers to a cell that has the ability to give rise to at least two, but often many, different cell types of an organism. Pluripotent cells often form teratoma after injection into immunosuppressed mice.

In the present text, the term pluripotent stem cell refers to the ability of this cell to develop into at least two different cell types depending on environmental factors, i. growth factors, morphogens, signaling molecules, activators or inhibitors, etc. In some embodiments, a pluripotent stem cell refers to the ability of a cell to develop into any of the three germ layers during an individual's development of an organism, including endoderm, mesoderm, and ectoderm.

In the present text, the term specialized cell refers to a type of cell that performs a specific function in multicellular organisms. For example, groups of specialized cells, such as neurons, work together to form a system, such as the nervous system.

In the present text, the term neuroectoderm refers to a cell or lineage of cell differentiation found in early periods of individual development or during the differentiation of pluripotent stem cells that can give rise to neuronal cell lines.

In the present text, the term cell proliferation markers refers to the expression of molecules associated with rapidly dividing cells that are not normally present in mature slowly dividing or non-dividing cells, i.e. are present in actively dividing cells, as opposed to cells with an extended cell cycle or non-dividing cells. Examples of such markers include the cell proliferation marker Ki67 (Gerdes, et al., Int J Cancer 31:13-20 (1983) cited herein by reference) and G2/M-phase markers of mitosis phosphohistone H3 (Hendzel, et al., Chromosoma 106:348-360 (1997, reference cited herein by reference).

In the present text, the term proliferation refers to an increase in the number of cells.

As used herein, differentiation refers to the process by which a non-specialized embryonic cell acquires the characteristics of a specialized cell, such as a specific type of neuron, brain, heart, liver, or muscle cell. Differentiation control ensures that the cell's genes interact with the physical and chemical conditions around said cell, usually through signaling pathways involving proteins embedded on the cell surface.

As used herein, the term differentiation, as applied to cells of a differentiating cell system, refers to the process by which cells differentiate from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiateable cell) into another cell type, such as a differentiated target cell.

As used herein, the term cell differentiation refers to the pathway by which a less specialized cell (i.e., a stem cell) develops or matures into more advanced form and function (i.e., iPSCs developing into a neural crest progenitor, a neuronal lineage cell, into ventral midbrain progenitor cells to midbrain pathway dopamine (DA) FOXA2/LMX1A+ neurons in accordance with the present inventions).

In the present text, the term undifferentiated refers to a cell that has not yet developed into a specialized cell type.

In the present text, the term default or passive in relation to a cell differentiation pathway refers to a pathway in which a less specialized cell becomes a cell of a certain type of differentiated cell in culture, in the absence of treatment with certain compounds, i.e. under normal cell culture conditions without contact with at least one morphogen. In other words, a default cell is formed when the cell is not brought into contact with a molecule capable of changing the type of differentiated cells (i.e., a morphogen), such as culture treated with LSB alone, but not with SHH activator or Wnt activator to obtain a positive for Forkhead A2 (FOXA2)+ cells in accordance with these

- 15 041083 inventions, results instead in the expression of markers HES5, PAX6, LHX2 and EMX2. Conversely, a non-default cell refers to a differentiated cell type resulting in a cell type other than the default cell, i.e. a non-default cell refers to a type of differentiated cell produced under non-default conditions, for example, a cell according to the present inventions, including Forkhead A2 (FOXA2)+ positive neuronal cell, ventral plate progenitor cell brain and dopamine (DA) FOXA2/LMX1A+ neuron of the midbrain pathway according to the present inventions; etc. The default cell may also be a default cell that, upon contact with the morphogen, becomes a non-default cell, without subsequent contact with the morphogen compound; for example, a non-default ventral midbrain progenitor cell that then becomes a non-default non-dopamine (DA) FOXA2/LMX1A+ midbrain pathway neuron due to lack of contact with a morphogen such as CHIR.

In the present text, the term morphogen refers to a compound that affects cell differentiation, i.e. determines, at least in part, the direction of cell differentiation. A morphogen can also influence a cell in such a way that it differentiates into a non-default cell type.

In the present text, the term directed differentiation refers to the manipulation of stem cell culture conditions to induce differentiation into a particular (e.g., desired) cell type, e.g., ventral midbrain progenitors and dopamine (DA) FOXA2/LMX1A+ neurons of the midbrain lineage in in accordance with the present inventions. In one embodiment, the term directed differentiation, as applied to a cell, refers to the use of small molecules, growth factor proteins, and other growth conditions that promote the transition of a cell from a pluripotent state to a more mature or specialized direction of cell differentiation (e.g., into a central nervous system cell, a nerve cell , a progenitor cell of the ventral plate of the midbrain and a dopamine (DA) FOXA2/LMX1A+ neuron of the midbrain line in accordance with the present inventions, etc.). In one preferred embodiment, the onset of directed differentiation occurs when the cell is brought into contact with LDN/SB on day 0. The passage of cell directed differentiation as described herein results in non-default cell type progenitor cells of the ventral plate of the midbrain and dopamine (DA) FOXA2/LMX1A+ neuron lineage of the midbrain in accordance with the present inventions. In the present text, the term differentiation induction in relation to a cell refers to a change from a default cell type (genotype and/or phenotype) to a non-default cell type (genotype and/or phenotype). Accordingly, the induction of differentiation in a stem cell refers to the induction of the formation, when a cell divides, of daughter cells with characteristics different from said stem cell, such as genotype characteristics (i.e., a change in gene expression as assessed using genetic analysis, such as a microarray) and/ or phenotype (i.e. a change in the expression of a protein, such as the Forkhead A2 (FOXA2) protein, or a set of proteins, for example, cells that are positive (+) for the Forkhead A2 protein (FOXA2) and the LIM homeobox gene-encoded transcription factor 1-alpha ( LMX1A), but negative (-) for PAX6).

In the present text, the term direction (lineage) of differentiation in relation to a cell, for example, the definition of the direction of cell differentiation refers in general to a cell of a genetically defined lineage, the progeny of which is capable of becoming cells of various types or several specific types, depending on in vivo conditions or in vitro cultivation conditions . In other words, the predetermined direction of cell differentiation is determined by the environment, predetermining the specific path of differentiation; thus, a cell becomes one type of cell and not another, for example, progeny of stem cells with a neural lineage of differentiation become nerve cells, not muscle cells or skin cells.

In the present text, the term neurite outgrowth or neuronal outgrowth refers to the observable elongated, membrane-enclosed projections of cytoplasm from cells.

In contrast to neuronal overgrowth, which refers to undesirable unrestricted growth of neurons, i.e. uncontrolled growth of neurons from transplanted cells in the graft engraftment site. In the present text, the term teratoma refers to a noncancerous tumor of any type of tissue derived from transplanted cells.

In the present text, the term teratoma formation refers to the unwanted growth of various types of tissue to form non-cancerous tumors as a result of the growth of transplanted cells.

As used herein, the term dopamine neuron or dopaminergic neuron generally refers to a cell capable of expressing dopamine. Midbrain dopamine neurons, or mDAs, refer to putative dopamine-expressing cells in forebrain structures and dopamine-expressing cells in forebrain structures.

As used herein, the term neural stem cell refers to a stem cell found in adult neural tissue capable of giving rise to neurons and glial (support) cells. Examples of glial cells include astrocytes and oligodendrocytes.

In the present text, the term ventral plate or FP or fp refers to the region of the neural tube in vivo running along the entire ventral midline, also described as the unpaired ventral longitudinal zone of the neural tube or referred to as the signaling center of the neural tube. In other words, the neural tube has been divided into different regions, with the ventral cells closest to the midline making up the ventral plate. In one example of further identifying chick midbrain FP cells can be divided into medial (MFP) and lateral (LFP) regions based on gene expression, mode of induction, and function. Ventral plate cells are found in vivo in several areas of the developing embryo, such as cells in the ventral plate of the midbrain, hindbrain, etc. In vivo, ventral plate cells in the midbrain region are believed to give rise to cells different from cells differentiating from ventral plate cells in other areas. One of the primary markers of the ventral plate in the midbrain region is FOXA2.

In the present text, the term lamina quadrigemina (operculum) refers to the dorsal cells closest to the midline. One of the markers of the quadrigeminal lamina is represented by LMX1A. During embryonic development, the cells of the ventral plate and the plate of the quadrigemina are located in separate positions in the CNS (ventral/dorsal) and differ in diametrically opposed pattern requirements for induction.

As used herein, the term midbrain refers to the region of the developing vertebrate brain between the forebrain (anterial) and hindbrain (posterior). Many areas of the brain originate in areas of the midbrain, including, but not limited to, the reticular formation, which is part of the tire, the region of the brain stem that affects motor functions, the brain stem, which consists of nerve fibers that connect the hemispheres of the brain brain with the cerebellum, and a large pigmented nucleus called substantia nigra. A unique feature of the developing midbrain is the co-expression of the ventral plate marker FOXA2 and the quadrigeminal plate marker LMX1A.

In the present text, the term neuron refers to the nerve cell, the basic functional unit of the nervous system. A neuron consists of a cell body and processes - an axon and one or more dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

In the present text, the term cell culture (cell cultivation) refers to the growth of cells in vitro in an artificial environment, for research or medical treatment.

In the present text, the term culture medium refers to a liquid covering cells in a culture vessel such as a petri dish, multiwell plate, etc., and containing nutrients to nourish and support the cells. The culture medium may also include growth factors that are added to induce the desired changes in the cells.

In the present text, the term neuronal maturation medium or BAGCT medium refers to a culture medium containing N2 medium supplemented with brain-derived neurotrophic factor (BDN), ascorbic acid (AA), glial cell-line neurotrophic factor, dibutyryl-cAMP, and transforming growth factor type β3 for dopamine (DA) FOXA2/LMX1A+ neurons of the midbrain line.

In the present text, the term feeder layer refers to a cell used in co-culture to maintain pluripotent stem cells. In the case of human embryonic stem cell cultures, typical feeder layers include mouse embryonic fibroblasts (MEFs) or human embryonic fibroblasts that have been treated to prevent division in culture.

As used herein, the term cell culture passage refers to the process by which cells are dissociated, washed, and seeded into new culture vessels after a round of cell growth and proliferation. The number of passages cultured cells have undergone is an indication of their age and expected stability.

As used herein, expression in relation to a gene or protein refers to the production of mRNA or protein, which can be observed using assays such as microarray assays, antibody staining assays, and the like.

In the present text, the term pair box 6 gene, or PAX6, refers to a marker other than the default neuroprogenitor cell.

In the present text, the term TUJ1, or class III neuron-specific beta-tubulin in relation to a differentiating cell according to the present inventions, refers to a marker of early neural differentiation of human cells, such as neural progenitor cells, and its expression is found in PNS and CNS neurons.

In the present text, the term homodimer in relation to a SMAD molecule refers to at least two SMAD molecules linked together, for example, by disulfide bonds.

- 17 041083

In the present text, the term bringing cells into contact with a compound in accordance with the present inventions refers to placing said compound in a location that allows contact with a cell to obtain (provide) contacted cells. Bringing into contact can be carried out using any suitable method. For example, in one embodiment, contacting occurs by adding said compound to a tube of cells. Bringing into contact can also be carried out by adding the specified connection in the cell culture.

In the present text, the term attached cell refers to a cell growing in vitro, characterized in that said cell is attached to the bottom or walls of the culture vessel; the attached cell may come into contact with the vessel by means of extracellular matrix molecules and the like, and the use of an enzyme is required to detach said cell from the culture vessel/container, i.e. trypsin, dispase, etc. An attached cell, as opposed to a cell in suspension culture that is not attached and does not require the use of an enzyme to remove the cells from the culture vessel. As used herein, the term marker or cell marker refers to a gene or protein that is indicative of a particular cell or cell type. A marker for a cell may not be limited to a single marker, markers may refer to a pattern of markers, i.e. the designated group of markers may provide identification of a cell or cell type, distinguishing it from other cells or cell types. For example, dopamine (DA) FOXA2/LMX1A+ neurons of the midbrain lineage according to the present inventions express one or more markers that distinguish a ventral midbrain progenitor cell from a less differentiated progenitor cell, i. positive for the Forkhead A2 protein (FOXA2) and positive for the LIM homeobox encoded transcription factor 1 alpha (LMX1A), in contrast to HES5+ and PAX6+ cells, for example, as seen from the expression patterns shown in FIG. 1e and 1f. In the present text, the term positive in relation to a cell, including the combination positive cell, refers to a cell that expresses a marker, as one example, an antibody stains for this marker when using an antibody staining (detection) system or a nucleic acid sequence that hybridizes with the sequence nucleic acid marker, as assessed using a reporter molecule, i.e. a fluorescent molecule that attaches to double-stranded nucleic acid sequences in a detectable amount for quantitative and/or qualitative analysis, greater than the amount for a control or comparable cell. For example, a cell that is positive for such a marker, such as Forkhead A2 (FOXA2) protein, etc., means a cell that expresses FOXA2 mRNA and/or protein as detected during analysis, for example, using a gene chip or antibody , respectively. Such a positive cell can be called FOXA2+. If a cell is positive for more than one marker, for example, if the designation FOXA2/LMX1A+ is used, said cell, or a majority of the population of said cells, is positive for both FOXA2 and LMX1A.

In the present text, the term negative in relation to a cell or population of cells, including a negative cell refers to a cell or population in which there is no detectable marker signal, or a signal is present at the level of control populations. For example, a cell that does not stain using a detection method involving contact with an antibody to Forkhead A2 protein (FOXA2), or a gene chip method that includes detection of FOXA2 mRNA, etc., is FOXA2-, or negative by FOXA2.

In the present text, the terms reporter gene or reporter construct refers to genetic constructs containing a nucleic acid encoding a protein that is easily detected or analyzed, such as a colored protein, a fluorescent protein such as GFP, or an enzyme such as β-galactosidase (gene lacZ).

As used herein, the term GFP refers to any green fluorescent protein DNA sequence, the expression of which in a cell can produce a fluorescent protein commonly used as a marker for the expression of a target gene. Examples of GFPs include GFP sequences isolated from coelenterates such as the Pacific metduza, Aequoria Victoria, and derived synthetic sequences such as eGFP.

The term sample (sample) is used in the broadest sense. In another sense, it includes a sample or culture obtained from any source and includes liquids, solids and tissues. Environmental samples include environmental materials such as surface materials, soil, water, and industrial materials. These examples should not be considered as limiting the types of samples that can be used in the present invention.

The terms purified, purify, purification, isolated, isolate, isolate and their grammatical equivalents in this text refer to the reduction of at least one impurity in a sample. For example, the target cell type is at least 10% pure, preferably at least 30% pure, more preferably at least 50% pure, even more preferably at least 75% pure, and in the most preferred embodiment, at least 90%, with a corresponding reduction in non-target cell types, for example,

- 18 041083 directed differentiation according to the present inventions resulted in the desired increase in purity of differentiated midbrain ventral plate progenitors or dopamine (DA) FOXA2/LMX1A+ midbrain neurons according to the present inventions. In other words, purify and its equivalents refer to the removal of certain cells (eg, unwanted cells) from a sample, either mechanically, such as by sorting in a flow cytometer, or by directed differentiation. For example, to allow differentiation of a purified population of forkhead box A2 (FOXA2) and LIM homeobox gene-encoded transcription factor 1 alpha (LMX1A) positive progenitor cells in accordance with the present inventions, progenitor cells are purified by removing PAX6 neuronal contaminants by sorting a mixed population of cells with obtaining twice positive: protein forkhead box A2 (FOXA2) and encoded by the homeobox gene LIM transcription factor 1, alpha (LMX1A) cells by flow cytometry; dopamine (DA) FOXA2/LMX1A+ midbrain neurons are also purified or selected from nedopamine (DA) (typical cells) using a particular cell culture method, including compositions and methods in accordance with the present inventions. Removal or selection of cells other than FOXA2/LMX1A+ dopamine (DA) midbrain neurons results in an increase in the percentage of target dopamine (DA) FOXA2/LMX1A+ midbrain neurons in the sample. Accordingly, cell type purification leads to enrichment, i.e. increasing the number of target cells, i.e. dopamine (DA) FOXA2/LMX1A+ midbrain neurons, in the sample.

In this text, the term natural when applied to an entity (such as a cell, tissue, etc.) and/or a chemical compound (such as a protein, amino acid sequence, codon, etc.) means that said entity and/or compound occurs / met in nature. For example, a native cell is a cell present in the body that can be isolated from a natural source, such as a fetal cell, and said cell has not been intentionally modified by humans in a laboratory.

In the present text, the term in vitro refers to an artificial environment and processes or reactions that occur in this artificial environment. Examples of In vitro environments are, among others, test tubes and cell cultures.

In the present text, the term in vivo refers to the natural environment (eg, animal or cells) and processes or reactions that occur in the natural environment, such as embryonic cell development, cell differentiation, neural tube formation, etc.

The term derived from, or derived from, or differentiated from, as used in connection with any cell described herein, means a cell that is derived from (e.g., isolated, purified, etc.) a parent cell in a cell line, tissue (such as a disrupted embryo, or liquid) using any manipulation, such as, without limitation, isolation of a single cell, cultivation in vivo, processing and/or mutagenesis. A cell can be obtained from another cell using, for example, chemical treatment, irradiation, induction of expression of new proteins, for example, by infection with a virus, transfection with DNA sequences, contact with (treatment) a morphogen, etc., and selection (for example , by serial cultivation) of any cell type contained in cultured parent cells). The resulting cells can be selected from a mixed population based on response to growth factor, cytokine, selected sequence of cytokine treatment, adherence, non-adhesiveness, sorting procedures, and the like.

In the present text, the term cell refers to a single cell, also to a population of cells (ie, more cells). The population may be a pure population containing a single cell type, such as a population of neurons or a population of undifferentiated embryonic cells. Alternatively, a population may contain more than one cell type, such as a mixed population. It is not intended to limit the number of cells in a population, for example, in one embodiment, a mixed population of cells may contain at least one differentiated cell. In the present inventions, there is no limitation on the number of cell types that can be contained in a population.

In the present text, the term highly enriched population refers to a population of cells, such as a population of cells in a culture dish, expressing a higher amount or percentage of a marker than a reference population, e.g. day 3 with the CHIR compound resulted in a highly enriched population of midbrain ventral plate progenitors relative to LSB treatment alone. In other examples, an enriched population is a population obtained by sorting or separating cells expressing one or more markers from cells not expressing target markers, such as CD142-rich population, A9-rich population.

The term cell biology or cell biology refers to the study of living cells, eg the anatomy and function of cells, eg physiological properties of cells, structure, organelles and interactions with their environment, their life cycle, division and death.

- 19 041083

The term nucleotide sequence of interest refers to any nucleotide sequence (e.g., RNA or DNA) that one of ordinary skill in the art may consider desirable for manipulation for any reason (e.g., to treat a disease, provide improved properties, express a protein of interest in a host cell). , ribozyme expression, etc.). Such nucleotide sequences include, but are not limited to, structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.) and non-coding regulatory sequences that do not code for an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).

In the present text, the term protein of interest refers to the protein encoded by the nucleic acid of interest.

The term gene refers to a nucleic acid sequence (eg DNA or RNA) that contains the coding sequence needed to produce a polypeptide or precursor (eg proinsulin). The polypeptide may be encoded by the full-length coding sequence or by a portion of the coding sequence, as long as the activity or functional properties (eg, enzymatic activity, ligand binding, signal transduction, etc.) of the full-length compound or fragment are retained. The term also encompasses the coding region of a structural gene adjacent to the coding region at both the 5' and 3' ends by about 1 kb or more at either end, i.e. the gene corresponds to the length of the full-length mRNA. Sequences that are located 5' from the coding region and that are present in the mRNA are called 5' non-translated sequences. Sequences that are located 3' or downstream of the coding region and that are present in the mRNA are called 3' non-translated sequences. The term gene encompasses both cDNA and genomic forms of a gene. The genomic form or clone of a gene contains a coding region within which are located non-coding sequences called introns or intermediate regions or intermediate sequences. Introns are gene segments that are transcribed into nuclear RNA (hnRNA, chRNA); introns may contain regulatory elements and enhancers. Introns are removed or excised by splicing from the nuclear or primary transcript; accordingly, introns are absent in messenger RNA (mRNA transcripts). During translation, mRNA determines the sequence or order of amino acids in the resulting polypeptide.

In the present text, the term gene expression refers to the process of translating the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by gene transcription (i.e., as a result of the enzymatic activity of RNA polymerase), and, in in the case of protein-coding genes into protein by translation of mRNA. Gene expression can be regulated at many stages of the process. Up-regulation or activation refers to regulation that increases the production of gene expression products (ie, RNA or protein), and down-regulation or repression refers to regulation that reduces production. Molecules (eg, transcription factors) that are involved in upregulation or downregulation are often referred to as activators and repressors, respectively.

In the present text, the terms nucleic acid molecule encoding, DNA sequence encoding, DNA encoding, RNA sequence encoding, and RNA encoding refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a deoxyribonucleic or ribonucleic acid strand. The order of these deoxyribonucleotides or ribonucleotides determines the order of the amino acids in the chain of the polypeptide (protein). Thus, a DNA or RNA sequence codes for an amino acid sequence.

The term isolated when applied to a nucleic acid, as in the combinations isolated oligonucleotide or isolated polynucleotide, refers to a nucleic acid sequence that has been identified and separated from at least one component or contaminant with which it is commonly found in its natural source. An isolated nucleic acid is a nucleic acid that exists in a form or environment other than those in which it occurs naturally. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA in the state in which they occur in nature. For example, a particular DNA sequence (eg, a gene) exists on the chromosome of a host cell in the vicinity of adjacent genes; RNA sequences, such as a particular mRNA sequence encoding a particular protein, are present in a cell as a mixture with numerous other mRNAs that encode for a variety of proteins. However, an isolated nucleic acid encoding a specific protein includes, by way of example, such a nucleic acid in a cell normally expressing said protein, where the position of the nucleic acid in the chromosome differs from that observed in nature. An isolated nucleic acid, oligonucleotide, or polynucleotide may be in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is used to express a protein, said oligonucleotide or polynucleotide must contain at least a sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single stranded), but may also contain sense and antisense strands (ie, the oligonucleotide or polynucleotide may be double stranded).

As used herein, the term kit refers to any delivery system for delivering materials. In the context of cell differentiation, a kit may refer to a combination of materials for bringing into contact with stem cells, such delivery systems include delivery systems that allow the storage, transport, or delivery of reaction reagents from one location to another in suitable containers (such as test tubes, etc.). and/or with auxiliary materials (e.g. buffers, written instructions for performing cell differentiation, etc.) (e.g. compounds, proteins, detection agents (such as antibodies that bind to tyrosine hydroxylase (TH), forkhead protein box A2 (FOXA2), encoded by the homeobox gene LIM transcription factor 1, alpha (LMX1A), etc.), etc. For example, the kit includes one or more containers (for example, boxes, bags, tubes, eppendorf tubes, capillary tubes, multiwell plates, etc.) containing reaction reagents to inhibit signal transduction cascades, such as an inhibitor to reduce Transforming Growth Factor Beta (TGFβ)/Activin-Nodal such as SB431542 (or a substitute for SB431542) and the like, an inhibitor for reducing signal transduction, SMAD, LDN-193189 (or a substitute for LDN-193189), etc. p., an inhibitor for reducing glycogen synthase kinase 3β (GSK3e), as one example, for activating wingless signaling (Wnt or Wnts), also known as a WNT signaling activator (WNT agonist), such as CHIR99021 (or CHIR99021 substitute) etc.) and the like, a Sonic hedgehog (SHH) signal transduction activator (such as a small molecule receptor agonist (SMO)), for example, Sonic hedgehog (SHH) C25II molecules, purmorphamine and the like, a molecule having an activity fibroblast growth factor 8 (FGF8) such as fibroblast growth factor 8 (FGF8), etc. and molecules associated with neuronal maturation, for example, (BDNF), ascorbic acid (AA), glial cell line neurotrophic factor, dibutyryl cAMP, and transforming growth factor type β3, including molecules capable of replacing these components and/or accessory materials. The reagents in the kit, in one embodiment, may be in the form of a solution, may be frozen, or lyophilized. The reagents in the kit in one embodiment may be contained in separate containers or presented in the form of certain combinations, such as the combination of LSB (LDN-193189 with SB431542), Sonic hedgehog (SHH) C25II molecules with purmorphamine, Sonic hedgehog (SHH) C25II molecules with purmorphamine with CHIR99021 or Purmorphamine with CHIR99021, molecules associated with neuronal maturation, etc.

Brief description of the figures

In FIG. 1 exemplifies the induction and neurogenic conversion of human ES cell-derived midbrain ventral plate progenitors dependent on the addition of CHIR990221. a) Immunocytochemical analysis at differentiation day 11 for the expression of FOXA2 (red), NESTIN (green, upper panels), LMX1A (green, middle panels) and OTX2 (green, lower panels). b, c) Calculation of the data presented in (a). The data of three independent experiments are presented, each of which was carried out in triplicate (mean ± error of the mean (SEM)). Significance levels for individual markers are presented in comparison with data obtained when processing only LSB: ANOVA; Dunnett's test: ***p<0.001; **p<0.01; p<0.05). d) Schematic representation of the culture conditions used for the three processing conditions. e, f) Lists of selected differentially expressed genes at day 11 compared to LSB/S/F8/CHIR conditions with either LSB (e) or LSB/S/F8 (f). g, h) Analysis of temporal gene expression of selected markers characteristic for identification of midbrain (g), forebrain and non-DA (h) ventral progenitors. Segments for scaling correspond to 50 µm.

In FIG. 2 demonstrates, by way of example, immunocytochemical and molecular analyzes of the midbrain DA developmental pathway in LSB/S/F8/CHIR treatment compared to the LSB/S/F8 (ventral/hypothalamic) and LSB (dorsal forebrain) pathways. A) Day 25 immunocytochemical analysis of FOXA2 (blue) expression in combination with Tuj1 (red)/LMX1A (green) (upper panels) and NURR1 (red)/TH (green) (lower panels). B) Quantification of co-expression in cultures treated with LSB/S/F8/CHIR. The data of three independent experiments are presented, each of which was carried out in triplicate (mean ± standard error of the mean (SEM)). C, D) Total gene expression analysis was performed on day 25 (triple samples for all three conditions). Selected lists of the most differentially expressed genes when comparing data at days 13 and 25 under LSB/S/F8/CHIR conditions (C) and when comparing LSB/S/F8/CHIR treatment with LSB (D, left panel) and LSB/S /F8 (D, right panel). E) Analysis of normalized differential gene expression for key DA markers in midbrain neurons. Significance levels for individual markers are presented in comparison with LSB-only processing data: ANOVA; Dunnett's test: ***p<0.001; **p<0.01; p<0.05). Segments for scaling correspond to 50 µm.

- 21 041083

In FIG. 3(1) and 3(2) exemplify in vitro maturation, characterization, and functional evaluation of DA of midbrain neurons derived from ventral lamellae versus DA of midbrain neurons derived from rosette structures. In FIG. 3(1): shows a typical: a) differentiation day 50 immunocytochemistry for TH (red), in combination with LMX1A (green, left panels), FOXA2 (blue, left panels) and NURR1 (green, right panels). b) Counting of TH+, FOXA2+, LMX1+ and NURR1+ cells among the total number of cells in the rosette-derived culture versus the ventral plate-derived culture (LSB/S/F8/CHIR). c) Estimation of neuronal subtypes of serotonin+ (5-HT), and GABA+ at day 50 in the culture of DA neurons obtained from ventral plates and in the culture of DA neurons obtained from rosette structures. d, e) HPLC analysis to measure DA and metabolites, d) representative electrochemical detection chromatogram of DA in a sample of ventral plate-derived cultures, e) comparison of DA, DOPAC and HVA levels among ventral plate- and rosette-derived cultures. f) Immunocytochemical analysis of ventral plate cultures (day 80) for TH (red) and synapsin (green). g-i) Electrophysiological analyzes of ventral plate cultures on day 80 of DA neuron differentiation. Phase contrast image of a chained neuron (g) and corresponding readings (h). i) Power consumption analysis showing the membrane potential oscillation characteristics that are a hallmark of the DA neuron (from 2 to about 5 Hz). Significance levels for individual markers (panels b, c, e) are presented as a comparison of cultures obtained from ventral plates and from rosette structures: Student's t-test: ***p<0.001; **p<0.01; p<0.05). Scaling bars correspond to 50 µm in (a), 20 µm in (f, top panel), 5 µm in (f, bottom panel) and 20 µm in (g) j) In vitro maturation of mouse DA neurons (d65). TH-positive neurons still express FoxA2 and extend the long fibers characteristic of mouse DA neurons, and to: HPLC measurement of DA release: 65 day old TH+ neurons are functional in vitro. In FIG. 3(2) shows a typical overview of cells obtained according to the protocol described in this application DA of midbrain neurons based on ventral plates. a) In contrast to previous strategies (e.g., Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101, 12543-8 (2004)), the new protocol described in this application is based on obtaining LMX1A/FOXA2-positive midbrain ventral plates (left panel) followed by neuronal conversion (middle panel) and maturation of DA neurons (right panel). In mature cells of DA neurons derived from the ventral plates, FOXA2/LMX1A expression is retained.

In FIG. 4 exemplifies the in vivo survival and function of DA of human ventral plate-derived neurons in the brains of mice, rats, and monkeys used as models of Parkinson's disease. ad) Transplantation of DA ventral plate-derived neurons into adult mice with 6-OHDA injury (NOD-SCID strain IL2Rgc null). a) TH expression and graft morphology 4.5 months after transplantation. b) Expression of a specific human marker (hNCAM, blue), TH (green), and FOXA2 (red). c) FOXA2+, TH+ and doubly labeled cells count in ventral plate-derived grafts (mean±SEM, n=4 at 4.5 months post-transplant). d) Amphetamine-induced rotation analysis in ventral plate-derived grafts (blue) versus rosette-derived grafts (green). Scaling bars correspond to 500 µm in (a), 100 µm in (b), and 40 µm in (4c). ep) Transplantation of DA ventral plate-derived neurons into adult rats with 6-OHDA-induced lesions. Immunohistochemical analysis for the simultaneous expression of TH (green) and human specific markers (red) hNA. e) and hNCAM (4f). g) Stereological count of the total number of cells (hNA+), TH+ cells and TH+ cells co-expressing FOXA2. The mean graft volume was 2.6+/-0.6 mm ³ ). hj) High-resolution images showing co-expression of TH (green) with specific midbrain transcription factors FOXA2, PITX3, and NURR1 (red), km) Behavioral analysis of animals treated with DA ventral plate-derived neuron transplants compared to sham-treated animals . k) Rotation asymmetry caused by amphetamine. 1) Walk test: measurement of the akinesia of the forelimbs of the treated side compared to the non-treated side. m) Cylinder test: measurement of preference for the ipsilateral paw versus the contralateral paw when lifting the hind limbs. The transplant animals showed significant improvement in at least three tests (p<0.01 at 4.5-5 months; n=4-6 in each case). np) Immunohistochemical analysis for TH (green) and co-expression (red) with DAT (n), GIRK2 (o) and calbindin (p). Significance levels (panels d, k, 1, m) are as follows: **p<0.01;p<0.05). Scaling bars correspond to 200 µm in (e), 50 µm in (f), 20 µm in (hj), and 40 µm in (np). qt) Transplantation of ventral plate-derived DA neurons into adult rhesus monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesions. q) Overview of a representative area of the graft 1 month after transplantation, marked by the expression of a specific

- 22 041083 human physical cytoplasmic marker SC-121 (green), d) Expression of TH graft surrounded by a halo of TH+ fibers (arrows), s) Examination of the simultaneous expression of SC-121 (red) and TH (green) in the graft nucleus, t) Analysis of the simultaneous expression of FOXA2 (red) in TH+ neurons (green). Scaling bars correspond to 2 mm for (q), 500 µm for (r), 200 µm for (s), and 50 µm for (t). u) outgrowth of fibers from the graft (huNCAM+) into the striatum of the recipient, v) Cells derived from the graft do not differentiate into glial cells. However, the transplants contained several serotonergic fibers in addition to the TH+ cell population.

In FIG. 5 exemplifies the timing of FOXA2/LMX1A midbrain ventral plate progenitor induction mediated by CHIR99021. A) Immunocytochemical analysis of FOXA2 (red)/LMX1A (green) on day 11 of differentiation followed by treatment alone with LSB/S/F8 treatment or in combination with CHIR starting at different marked time intervals. B) Calculation of the percentage of FOXA2+, LMX1A+ and doubly labeled cells on day 11 of differentiation, followed by the start of differentiation under the action of CHIR, as described in (A). Significance levels for individual markers are presented in comparison with conditions without CHIR treatment: ANOVA; Dunnett's test: ***p<0.001; **p<0.01; p<0.05). Segments for scaling correspond to 50 µm.

In FIG. 6 demonstrates by way of example that FGF8 treatment does not play a major role in the induction of FOXA2/LMX1A midbrain ventral plate progenitors. Representative images of FOXA2 (red)/LMX1A (green) expression obtained by immunocytochemistry on day 11 of differentiation. Cells were treated with LSB/CHIR in the presence or absence of SHH (purmorphamine+SHH C25II) and FGF8. Segments for scaling correspond to 50 µm.

In FIG. 7 exemplifies that treatment with a high dose of SHH and/or a small molecule mild agonist (purmorphamine) is required to effectively induce midbrain ventral plates in the presence of CHIR99021. Representative images of FOXA2 (red)/LMX1A (green) immunocytochemistry at day 11 of differentiation. Cells were treated with LSB/F8/CHIR in the presence of various concentrations of SHH (SHH-C25II) and the mild agonist purmorphamine. The segments for determining the scale correspond to 50 µm.

In FIG. 8 shows, by way of example, an analysis of genes differentially expressed in cultures treated with LSB/S/F8/CHIR compared to cultures treated with LSB and LSB/S/F8 at days 11 and 25 of differentiation. a) Hierarchical clustering of total gene expression data obtained for the three conditions on days 0, 1, 3, 5, 7, 11 and 13 (samples were evaluated in triplicate for each condition and on each day). b) Gene enrichment analysis according to GO classes using DAVID; http://david.abcc.ncifcrf.gov. Comparison at day 11 revealed an enrichment for SHH and WNT signaling pathways under LSB/S/F8/CHIR treatment conditions, consistent with activation of the canonical WNT signaling pathway by CHIR99021 treatment. Alternative developmental pathways such as forebrain and diencephalon development and homeobox are other GO elements that are highly enriched at days 11 and 25. c) Real-time validation of data using the Allen Human Gene Expression Database (http://human.brain-map.org) for marker candidates enriched under conditions to generate midbrain DA neurons (LSB/S/F8/ CHIR). TTF3, EBF1, EBF3 and TTR were expressed based on available data on microarrays specific to the human brain region. TTR, the classic transcriptional target of FOXA2, was only weakly expressed in adult substantia nigra, suggesting that it may play a major role in development or that SHH treatment may lead to artificially high levels of TTR expression during in vitro differentiation.

In FIG. 9 exemplifies a differentiation protocol for ventral plate induction and development of midbrain DA neurons in representative independent hESC and hiPSC lines. The data obtained on the hESC H1 line, as well as on the hiPSC 2C6 lines (from a random patient with Parkinson's disease) and SeV6 (based on the Sendai line, not subjected to integration) are presented. The ventral plate protocol described in FIG. 1d and 10 followed by analysis of FOXA2 (red) expression on day 11 and TH (green)/FOXA2 (red) on day 25 of differentiation.

In FIG. 10 is an exemplary schematic summary of the differentiation conditions used for cultures of DA neurons derived from ventral plates and rosette structures. Both protocols used dual SMAD inhibition to accelerate neural pathway selection. In this ventral plate protocol, LDN was used to inhibit BMP, while traditional brain induction was used for rosette structures. Abbreviations are as follows: LDN: LDN-193189, SB: SB431542, SHH (purmorphamine+SHH C25II), FGF8: FGF8, BAGCT: BDNF+ascorbic acid+GDNF+dbcAMP+TGFβ3. As SHH/FGF8, only SHH C25I in the absence of purmorphamine was used in the rosette protocol, following the initial recommendation for structuring cultures of DA neurons derived from rosette structures. Note: Purmorphamine treatment at the rosette stage has shown toxicity at concentrations

- 23 041083 suitable for structuring ventral plate cells. BASF: BDNF+ascorbic acid+SHH/FGF8.

In FIG. 11 demonstrates in vitro maturation of DA neuron cultures derived from ventral laminae as an example. a) Immunocytochemical analysis of TH of ventral plate-derived neurons (green) for DAT expression (red; day 80). b) Extensive areas with Tuj1+ fibers (red) extending >2 mm were observed on day 60 of differentiation. c) At differentiation day 80, GIRK2 co-expression (red) in TH+ neurons (green). Scaling bars correspond to 20 µm in (a), 100 µm in (b), and 20 µm in (c).

In FIG. 12 shows an exemplary immunohistochemical analysis of a short-term (6 weeks) in vivo survival study in the striatum of adult intact (no lesions) mice (NOD-SCID IL2Rgc null strain). Examination of grafts obtained from ventral plates (cells on day 25; 150*103 cells/animal). A) Representative image of the graft nucleus showing TH+ cells surrounded by TH+ fibers of the recipient. An average of 6,200 TH+ cells (n=3) were present in the graft 6 weeks after transplantation. B) FOXA2-expressing cells (red) were found in the graft alone, but not in the surrounding striatum of the recipient, demonstrating the origin of the graft and the distinctive features of midbrain cells. Nearly all TH+ neurons (green) simultaneously expressed FOXA2 (red). However, a significant proportion of FOXA2+ cells simultaneously did not express TH, which suggests that these cells did not acquire the adult DA phenotype or represent another FOXA2+ neuron population that does not contain DA markers of neurons. Segments for scaling correspond to 50 µm.

In FIG. 13 exemplifies a histological long-term (4.5 months) study of 6-OHDA grafted mice (NOD-SCID IL2Rgc null strain) by comparing behavior in mice with ventral plate-derived grafts, grafts derived from rosette structures. Study of grafts obtained from ventral plates. a) An example of one of the largest ventral plate derived grafts. This graft retains the well-described hNCAM+ (red) cytoarchitecture. b) Strong growth of hNCAM+ fibers was observed at the graft periphery. c) Serotonergic (5-HT+) fibers (green) in the graft are largely negative for hNCAM (red), suggesting their origin from the recipient. d) GABAergic neurons and fibers (green) in the graft. The study of grafts obtained from rosette structures. f) Growth of neurons with compression of the brain tissue of the recipient. Most of the cells were positive for both NCAM (red) and DCX (green), suggesting a neuronal origin. f) NCAM+/DCX+ fibers have spread to the untransplanted contralateral region of the brain. g) Multiple DCX+ clusters were found within the graft nucleus. h) Several TH+ cells (green) and fibers were found at the graft periphery, and virtually all rosette-derived TH+ cells were negative for FOXA2 (blue) in vivo. Immunohistochemical study of 4.5-month-old grafts derived from ventral lamellae versus grafts derived from rosette structures. Representative images of the expression of the proliferation marker Ki67 (red) and the neuronal progenitor marker PAX6 (green) (i), the expression of FOXA2/DCX (j), FOXG1/hNCAM (j, inset), and the astrocyte marker GFAP (green) (k) are shown. Scaling bars correspond to 500 µm in (a), 50 µm in (b-d), 500 µm in (e), 50 µm in (g-h), 50 µm in (i), 40 µm in (j), and 20 µm in (k).

In FIG. 14 exemplifies a histological long-term (5 months) study of transplanted SD mice lesioned with 6-OHDA. A) The majority of hNA+ (red) cells expressed Tuj1 (green), indicating characteristic developmental features along the neural pathway. B) GFAP+ fibers (green) within the graft did not simultaneously express hNA (red), suggesting their origin from the recipient. C) Several serotonergic formations of (5-HT+) cells (green) were detected, although most of the serotonergic fibers were obtained from the recipient, as well as in the data on recipient mice (in Fig. 13c). D) Additional example of TH+ neurons (green) within a graft characterized by hNCAM expression (red) to confirm that the cells in the recipient's brain are human cells. Scaling bars correspond to 25 µm in panels.

In FIG. 15 exemplifies histological examination of ventral plate-derived grafts in primate brains. Anterior plate-derived DA neurons derived from H9 hESCs and from H9-GFP hESCs (25 day cultures; 1.25*106 cells/tract, 3 tracts/hemisphere, total 6 tracts) were transplanted into the striatum of adult rhesus monkeys, which were corrupted by MPTP (=2). A) Upper left and right panels: High resolution image reconstruction of representative areas of the graft (1 month post-transplant). The graft cytoarchitecture is illustrated by immunohistochemistry using the human specific marker SC-121 (lack of cross-reactivity with non-human primate tissues). Lower left panel: SC-121+ fibers extending from the graft core. Lower right panel: Higher resolution image of SC-121+ cells showing neuronal progenitor morphologies. B) Examination of GFP expression in the graft nuclei further confirmed the human identity of the cells, complementing the SC-121 data. Note: In each animal, unlabeled H9-derived cells were transplanted into each hemisphere, while cells derived from H9-GFP cells (GFP expression under the control of the EF1a promoter) were transplanted into the other hemisphere. C) High resolution image of a GFP+ graft using DAB detection, showing many GFP+ cells at the graft periphery showing neuroblastic morphology. D) Immunohistochemical studies of areas in the graft core negative for GFP and SC-121. These areas contained a large number of recipient microglia, based on Iba1 expression (red) as well as several ED1+ macrophages (blue), suggesting prolonged inflammation despite ciclosporin immunosuppression. Scaling bars correspond to 500 µm in (A, top panel), 50 µm in (A, bottom left panel), 20 µm in (A, bottom right panel), 2 mm in (b), 500 µm in (C, left panel), 50 µm (C, top right panel), 100 µm (C, bottom right panel), 100 µm (D).

In FIG. 16 exemplifies the generation of TH+ cells from hESCs based on a method using precursor MS5 nurse cells, in which the cells differentiated into DA neuron-like cells via (via) rosette cell intermediates. Top) at the P0 stage, hESCs were contacted with molecules to initiate the induction of neurons from Oct4+ cells into rosette cells using MS5 feeder cells (Perrier et al., 2004). At the P1 stage, rosette cells were developed by contacting the cells with additional molecules to differentiate the cells into P2 stage cells with specific expression patterns, including Pax2+/En1+ DA progenitor cells that further differentiate into TH+/En1+DA neurons. These cells were used for transplantation into rats containing 6OHDA-induced lesions and immunosuppressed by cyclosporine A treatment. These transplantation studies demonstrated very poor in vivo survival of DA-like neurons, including loss of the TH+ phenotype, and revealed problems regarding further growth of undesirable potentially lethal cells in transplanted animals, such as teratomas, as well as the outgrowth of cells of the wrong neural type, which can lead to additional medical problems in patients. A) very few surviving TH+ neurons were observed after 4.5 months post-transplantation (<50 TH+ cells/animal) in grafts from rosette-derived DA progenitor neurons. However, in contrast to TH+ cells, cells labeled with GFP (GFP was under the control of a ubiquitous promoter) survived quite well after transplantation. This suggests that the majority of transplanted surviving cells were non-DA neurons (16B). C) Several transplant-derived cells (hNA+ (green) coexpressed TH (red), suggesting that most transplanted human cells have a non-DA neuron phenotype. Panels 16 D-E demonstrate that D-E despite very poor survival in vivo, there was some (though very limited and very variable) improvement in several behavioral studies such as amphetamine-induced rotations (D), cylinder test, and spontaneous rotations (E).

In FIG. 17 shows an example protocol for obtaining a small number of ventral plate cells. A modified SMAD double inhibition protocol allows the production of ventral plate (fp) cells. High concentrations of SHH were required to induce FoxA2+ ventral plate cells, and this addition of caudal pattern-inducing stimulants such as FGF8, Wnt1, or RA did not reduce FOXA2 expression, but altered regional features. A) Left panel: cells at differentiation day 11 after NSB treatment (Noggin/SB431542); left center panel: NSB+SHH processing (Sonic C25II); center panel: NSB+SHH+RA; center right panel: NSB+SHH+Wnt1; NSB+SHH+FGF8; Note: NSB treatment alone does not induce FoxA2 expression. Only FoxA2+ ventral plate cells were induced in the presence of a high dose of SHH. The addition of RA, Wnt1 and FGF8 did not result in the inhibition of FoxA2 induction. B) Quantitative RT-PCR analysis of FOXA2 and SIX6 gene expression demonstrates the maintenance (or even increase) of FOXA2 expression after treatment with RA or FGF with a concomitant decrease in SIX6 expression to obtain the earliest progenitor ventral plate-like cells. C) Induction of expression of genes - markers of the precursors of the midbrain and ventral plates of the midbrain in the presence of FGF8 and Wnt1.

In FIG. 18 exemplifies an ventral plate cell generation protocol demonstrating high levels of midbrain characteristics compared to low or no levels in cells obtained from the procedures used in FIG. 16 and 17. A) Induction of the ventral midbrain plates in a population of cells exposed to high

- 25 041083 levels of SHH, FGF8 and CHIR resulting in simultaneous expression of FoxA2 and midbrain markers LMX1A and Otx2 at differentiation day 11-1 compared to cells contacted with the molecules described in the other two procedures shown in FIG. 16 and 17 (N/SB and SHH/FGF8, respectively). B) Day 11 total gene expression study comparing three groups of cells exposed to molecules from each of the three treatments, including the third treatment according to the present invention (LDN/SB, SHH/FGF8, LSB/SHH/FGF8/CHIR). Graph B specifically shows genes that are common to all three treatment conditions versus genes that are unique to each individual condition. Plot B is a preliminary analysis used for the microarray results presented in FIG. 1. C) mRNA levels of the midbrain markers FoxA2, LMX1A are highly enriched in the LSB/SHH/FGF8/CHIR treated group compared to the SHH/FGF treated group.

In FIG. 19 exemplifies the in vitro characterization of dopamine neurons derived from the ventral laminae of a midbrain region. A) Simultaneous labeling of FoxA2+ neurons with DA markers of mouse neurons TH, Nurr1 and LMX1A on day 25 of differentiation. B) mRNA expression levels determined by quantitative RT-PCR of DA markers of mouse neurons as well as other midbrain cell types in LDN/SB+SHH/FGF8+SHH/FGF8+CHIR treated groups.

In FIG. 20 exemplifies the potential to compare differentiation along the DA pathway of midbrain neurons in PINK1-mutated PD-iPS cells and wild-type hES (or iPS) cells. The PINK1 Q456X mutant PD-iPSC line was differentiated according to the novel methods for obtaining midbrain DA neurons based on ventral plates according to the present invention, obtaining midbrain differentiation profiles comparable to those obtained in the H9 line. A-C) Immunocytochemical analysis of a PINK1-mutant BP-iPSC line at differentiation day 11 (midbrain progenitor stage) for FOXA2 (red), LMX1A (green) and DAPI (blue) (A), at differentiation day 25 (early post-mitotic stage of DA neurons) for FOXA2 (red) and TH (green) (B) and for NURR1 (red) and TH (green) (C). D-F) Some set of immunocytochemical studies performed using H9 derived cells at differentiation day 11 for FOXA2 (red), LMX1A (green) and DAPI (blue) (D), differentiation day 25 for FOXA2 (red) and TH (green) (E) and for NURR1 (red) and TH (green) (F).

In FIG. 21 exemplifies a PINK1 mutant PD-iPSC line showing a phenotype similar to that of Parkinson's disease with protein aggregation after prolonged differentiation and maturation in vitro. At differentiation day 55, a PINK1 mutant PD-iPSC line showed evidence of expression of α-synuclein (a major component of Lewy body formation in Parkinson's disease patients) in the cytosol of TH+ DA neurons. These cells also showed high expression of ubiquitin (a classic marker of Lewy bodies). In contrast, DA neurons derived from the iPS control line showed expression similar to normal synaptic (as opposed to cytosolic) expression of α-synuclein and very low levels of ubiquitin. A, B) Immunocytochemical analysis of a PINK1 mutant BP-iPSC line at differentiation day 55 for α-synuclein (LB509, red), TH (green) and composite image (A) and for α-synuclein (red) and ubiquitin ( green) (B). C, D) Immunocytochemical analysis of the control iPSC line at differentiation day 55 for α-synuclein (red) and TH (green) (C) and α-synuclein (red) and ubiquitin (green) (D).

In FIG. 22 exemplifies the expression of an aggregated form of α-synuclein. In the brains of patients with Parkinson's disease, dimerized insoluble forms of α-synuclein lead to aggregation into Lewy bodies. The dimerized forms of α-synuclein show phosphorylation of serine-129 in α-synuclein. Cells derived from the PINK1 mutant BP-iPSC line show strong expression of α-synuclein phosphorylated at Ser129 compared to iPSC-derived control cells which show very low levels of expression. A, B) Immunocytochemical analysis of α-synuclein phosphorylated at Ser129 (green) and DAPI (blue) in a cell line derived from PINK1 mutant BP-iPSC at day 55 of differentiation (A) and a control cell line derived from iPSC (B).

In FIG. Figure 23 shows by way of example the differences in α-synuclein expression profiles that are observed depending on the differentiation protocol. The present inventors have demonstrated that authentic midbrain DA neurons have Parkinson's disease-specific vulnerabilities and corresponding specific phenotypes in vitro. DA neurons generated using the classic differentiation protocol based on MS5 stromal feeder cells (Perrier et al., PNAS 2004, incorporated herein by reference) can result in a large number of TH+ neurons. However, based on the data of the present invention, these TH+ cells resulting from differentiation in the classic protocol on MS5 stromal feeders are not authentic midbrain DA neurons. In cultures differentiated by the MS5 protocol, there were a large number of α-synuclein positive cells. However, these cells are

- 26 041083 did not express TH. Moreover, there were no differences in expression profiles between BP-iPSCs and control iPSCs using the MS5 differentiation strategy. These data indicate that α-synuclein was also expressed in other non-DA cell types and that such α-synuclein in non-DA cells does not differ in disease cells compared to iPSC-derived control cells. , in particular, when using the standard differentiation protocol MS5. Finally, the new ventral plate differentiation protocol of the present invention results in a large number of TH+ cells co-expressing α-synuclein. Such TH+ cells express α-synuclein as an expression profile in the cytosol. A, B) Immunocytochemical analysis for α-synuclein (LB509, red), TH (green) PINK1 mutant BP-iPSC line at day 60 of differentiation based on MS5 (A) and control PSC (B). C) Immunocytochemical analysis of the PINK1 mutant BP-iPSC line at day 55-1 of differentiation based on ventral plates for α-synuclein (red), TH (green).

In FIG. 24 demonstrates as an example that DA neurons derived from the PINK1 mutant BPiPSC line are more vulnerable to toxic stimulation. TH+ DA neurons derived from BP-iPSCs using the ventral plate protocol of the present invention were more vulnerable to toxins (valinomycin: mitochondrial ionophore, 5 μM, 48 h) than control cells derived from iPSCs. In contrast, TH+ neurons derived from the classic MS5-based protocol did not show different vulnerabilities between PD-derived cells compared to control cells. AF) Representative immunocytochemical analysis of TH at day 60 of differentiation: normal conditions (no toxin treatment) for both cells derived from Parkinson's disease and cells derived from control iPSC cells obtained using a ventral plate protocol (A, shown cells derived from BP-iPSCs), almost complete degeneration of TH+ DA neurons in BP-iPSCs after toxin treatment (B), partially degenerated TH+ DA neurons from control iPSCs (C), normal conditions for both cultures obtained from Parkinson's disease and and for cells derived from control iPSC cells derived using an MS5-based protocol (D, cells derived from BP-iPSCs), TH+ neurons after exposure to BP-iPSC toxin (E), and cultures derived from control iPSCs ( F) when using the MS5 protocol. G-H) Low-resolution immunocytochemical images of Tuj1 (red) and TH (green) using a ventral plate protocol at differentiation day 60: BP-iPSCs under normal conditions (G) versus toxin-exposure conditions (H) and control iPSCs in normal conditions (I) versus toxin conditions (J). K-N) low-resolution immunocytochemical images for Tuj1 (red) and TH (green) using an MS5-based protocol at differentiation day 60: BP-iPSCs under normal conditions (K) versus toxin exposure conditions (L) and control iPSCs under normal conditions conditions (M) versus toxin conditions (N).

In FIG. 25 exemplifies the scoring of cell survival study data as a function of dose when exposed to a toxin. A cell survival study using alamar-blue dye 48 hours after treatment with valinomycin demonstrated different cell survival in a specific dose range when exposed to the toxin (5 and 10 μM) when comparing BP-iPSCs and control iPSCs (day 60 of differentiation based on the ventral plate according to the present invention). Note: In the present study, overall cell death is assessed, although the most dramatic effects were observed especially in DA neurons (see Fig. 14). Therefore, scoring based on alamar blue data is likely to underestimate the degree of difference in effects observed in DA neuron lines.

In FIG. 26 demonstrates by way of example that human DA transplanted pluripotent stem cell-derived neurons have electrophysiological characteristics typical of those found in substantia nigra pars compacta (SNpc) mice. A) Top - reconstruction of the pacemaker neuron (pacemaker neuron) in the graft site. Below, a typical photomicrograph of a rat-derived brain section in which hES-derived neurons were injected 9 months earlier; the graft is outlined; a high magnification image is shown below as an inset. The section was treated with tyrosine hydroxylase, which is shown in white. B). Above, a typical attached overlay recording from a putative DA neuron in the graft; below - a typical record from the whole cell of the same cell. Recordings were performed in the presence of glutamate and GABA receptor antagonists (50 μM AP5, 10 μM CNQX and 10 μM GABAzine) in order to eliminate synaptic input. These recordings demonstrated that these PS-derived neurons were pacemaker neurons with normal intrasomatic stress trajectories. Other neurons recorded in the transplant sample had similar properties. C) Cell-attached and whole-cell recordings of dopaminergic neurons in adult mouse SNpc are shown for comparison. Abbreviations (CTx=cortex, STr=striatum, SNpc=black body compact fraction, DA=dopaminergic).

- 27 041083

In FIG. 27 exemplifies the A9 surface marker candidate and CD verification in hPSC. a) Venn diagram of transcriptome data from mouse DA neurons derived from mouse ESCs purified by FACS. Among the 107 genes shared between PITX3 and Nurr1, the majority were known DA markers of midbrain neurons, and new markers were also confirmed to be expressed in the ventral midbrain. b) One of these markers was DCSM1, a putative surface marker that appears to be enriched in region A9 based on in situ mRNA expression data (Allen Brain Atlas, Lein, E.S. et al., Genome-wide atlas of gene expression in the adult mouse brain, Nature 445: 168-176 (2007)). c-f) CD test: c) representative 96-well plate (1 of 3x96 plates used to test the entire CD panel). Dark wells show CD markers, which are strongly expressed in DA neurons of hESC on day 25. e) Summary of the results of CD validation in hESC-derived neurons. f) One typical marker was found, CD142, a surface marker enriched selectively in DA neurons at the Nurr1+ stage, which was then isolated by FACS from CD142+ cells versus CD142- cells and analyzed on day 7 post sorting.

In FIG. 28 exemplifies CD142 enriched at the Nurr1+ stage in DA in midbrain neurons and depleted in GABA- and serotonergic neurons. a) Flow cytometry showed representative expression of CD142 at day 25 of differentiation. b) CD142 enriched at the Nurr1+ stage in FOXA2/TH DA midbrain DA neurons in hESCs (e.g., WA09; in Fig. 27f) and validated hiPSC lines (C29 and M3X are two human iPSC lines at day 25 of differentiation). c, d) CD142 depletes GABAergic and serotonergic contaminants after sorting on day 25 and cultured in vitro for 3 weeks. e, f) CD142 depletes GABAergic and serotonergic neurons in vivo 3 months after transplantation. Cells were sorted for CD142 at day 25-1. Note: CD142 cells are also enriched for TH and AADC.

In FIG. 29 shows the intended experimental use of PSA as an example. DA neurons expressing PST and exhibiting PSTnm derived from hESC will be evaluated in vitro for effects on DA phenotype and fiber outgrowth. An in vivo study in a 6OHDA-damaged rat model will test whether fewer PSA-expressing DA neurons can restore the behavior achieved with standard grafts, and whether it is possible that increased PSA expression in DA neurons derived from hESC resulted in recovery as measured in complex motor function studies.

In FIG. 30 shows the use of PST as an example. Overexpression (mouse PST) results in elevated levels of PSA-NCAM in differentiating mouse ES cells. A) Calculation of PST mRNA using quantitative PCR in control cells (Nurr1) and in cells overexpressing PST (Nurr1/PST). Data are presented as fold enrichment of PST levels in Nurr1/PST cells compared to Nurr1 cells. B) PSA immunostaining in cultures of DA neurons on day 14 of differentiation showed elevated PSA levels in Nurr1/PST cells (bar to scale: 100 µm). C) Western blotting of NCAM in differentiated cells. Nurr1/PST cells (lane 2) showed elevated levels of the polysialylated form of NCAM (spots, brackets) compared to controls (lane 1). PSA is removed from NCAM after endoN processing (lane 3). D) PSA spot intensity count, expressed in arbitrary units. E) PSA study using FACS on day 14 of differentiation. Treatment of cells with 20 units of endoN 24 h before the end of differentiation abolished the effect of PST. F) Representative photomicrographs comparing Nurr1 and Nurr1/PST differentiated cells for GFP immunofluorescence and DA markers. Cells sorted for GFP and then seeded back retained the DA phenotype (after sorting). Scale bar: 100 µm.

In FIG. 31 shows an exemplary study of DA neurons derived from ES using FACS. Isolation of GFP+ and SSEA-1- cells using flow cytometry. J1 mouse ES cells were used as a double negative control and a GFP negative control. Approximately 5 to 10% of cells were sorted as positive.

In FIG. 32 demonstrates by way of example that Nurr1/PST transplants were more effective in inducing behavioral recovery in a 6OHDA-damaged mouse model. Nurr1::GFP cells were differentiated and sorted on day 14 to obtain a GFP+/SSEA-1 population. Cells treated with 20 units of endoN were grown for 12 hours before sorting. 55,000 cells were transplanted into 1 ml N2 medium with BDNF and AA. A) Animals were assessed for amphetamine-induced rotation (rotations/min for 20 min) for 3 weeks before transplantation and then 7 weeks after transplantation. Nurr1/PST cells significantly improved performance compared to control Nurr1 cells (2-way ANOVA: p<0.01, Boferroni post-test: *p<0.05, **p<0.01, *** p<0.001; 6 animals in each group). Removal of PSA by endoN abolished the effect of PST (p=0.26). B) At the end of the study, there were more GFP+ cells in the PST transplant than in the control (p<0.05, Student's t-test). C) PSA/GFP immunofluorescence ratio in the nucleus. **p<0.01. Student's t-test (n=5/graft type). D) Immunofluorescence of GFP, TH and PSA. Scale bar: 200 µm. E) Transplanted cells express DA markers. Individual z-levels of confocal micrographs are shown. Scale bar: 20 µm. Values are given as mean +/-SEM.

In FIG. 33 demonstrates PSA augmentation that enhanced striatal innervation of the DA recipient by ES-derived neurons. Increased expression of PSA-NCAM enhanced the outgrowth process. A) Representative photomicrographs of GFP/TH+, GFP/Girk2+ and GFP/synapsin+ processes in controls. The staining of the Nurr1/PST samples was similar. Scale bar: 20 µm. B) Representative z-stack views demonstrating the GFP+ spread process from both transplants, Nurr1 and Nurr1/PST. In the Nurr1/PST graft, there were more GFP+ proliferation processes from the graft (Bar to scale: 50 µm). Inserts demonstrate GFP+/TH+ processes in the same sections. Arrow: growth direction. CE) Calculation of the intensity of GFP+ (C, E) and TH+ (D) processes at different distances from the graft site, normalized by intensity near the injection site (areas were divided into 5 zones at a distance of ~100 µm aside. Normalization was performed with respect to zone I; two-way ANOVA, p<0.01 for both GFP and TH data, n=5/cell type; values are presented as mean +/-SEM). Nurr1/PST grafts sprouted more axons into the recipient's striatum, with growth partially suppressed (E) by endoN treatment. F) Animal recovery correlated with the degree of germination processes. This graft showed a correlation (linear regression analysis) between the intensity of GFP+ axon sprouting in zone IV and recovery in untreated and endoN-treated Nurr1 and Nurr1/PST transplanted animals. Each value represents data from one animal (r ² =0.65, p<0.001, n=17).

In FIG. 34 illustrates the use of PST in methods related to spinal cord injury and motor neuron expression as an example. In the control spinal cord, transplanted GFP Schwann cells (SCs) were separated from the site of injury by recipient scar tissue (A), while PST-modified Schwann cells freely migrated short distances (B). This PSA design resulted in improvements in locomotor activity, intermediate BBB score (shown top line in (C) vs. bottom control line) and hindlimb motor performance (grid walk test; bottom line (D) vs. top line). control). E, H) differentiation of HB9::GFP mouse ESC cells into motor neurons, in which PST administration increases fiber outgrowth and cell migration (arrowheads) in vitro (E, F). Transplantation of these PST cells into the sciatic nerve results in better target innervation as shown in the array of neuromuscular fusions (arrows) in the EDL muscles (G, H).

In FIG. 35 shows the use of the PSTnm enzyme as an example. A) PSA generated by PSTnm inhibits adhesion of Schwann cells in suspension to a monolayer of Schwann cells even more effectively (red line - lowermost line) than PSA generated by overexpression of PST (green line - middle line). B) Immunoblotting of PSA in ESC-derived HB9 motor neurons demonstrated that PSTnm-treated control samples had undetectable levels of PSA. Growth with PSTnm + substrate (CMP - sialic acid) resulted in a large PSA band, which disappeared after treatment with endoN. C, D) Similar to the effects obtained with the PST gene, polysialylation of these cells with PSTnm and substrate during differentiation enhanced axon outgrowth and cell migration (arrowheads). E) PSA immunostaining of DA 30 day old hESC-derived neurons. F) This staining increased significantly after treatment with PSTnm and substrate. G) In vivo injection of PSTnm alone had no effect, while its simultaneous addition with substrate (H) resulted in strong PSA expression in the mouse striatum.

Description of the invention

The present invention relates to the field of stem cell biology, in particular to the field of lineage-specific differentiation of pluripotent or multipotent stem cells, which may include, but are not limited to: human embryonic stem cells (hESC, hESC), in addition to non-embryonic induced human pluripotent stem cells (hiPSC, hPSCs), somatic stem cells, stem cells from sick patients, or any other cell capable of lineage-specific differentiation. In particular, methods are described for ensuring lineage-specific differentiation of hESCs and/or hPSCs into progenitor cells of the ventral plate of the midbrain and then further into large populations of FOXA2+LMX1A+TH+ dopamine (DA) midbrain neurons using novel culture conditions. FOXA2+LMX1A+TH+ midbrain dopamine (DA) neurons obtained using the methods of the present invention are further used in a variety of applications including, but not limited to, use in in vitro assays for drug development and as a therapeutic agent for disease reversal , damage or lack of dopamine neurons in the patient. Further, compositions and methods are provided for providing differentiation

- 29 041083

FOXA2+LMX1A+TH+ dopamine (DA) midbrain neurons from human pluripotent stem cells for use in disease modeling, in particular Parkinson's disease.

The present invention relates to the characteristics of Parkinson's disease (PD), including the selective degeneration of midbrain dopamine neurons (mDA, cmDA) in the patient's brain. Since the main cause of the symptoms of PD is the selective loss of DA neurons in the substantia nigra of the ventral midbrain, PD is considered one of the diseases most suitable for the application of cell replacement therapeutic strategies. Accordingly, numerous attempts have been made to transplant cells into the patient's brain to replace the lost function of midbrain dopamine neurons (mDA). However, these experiments have been neidachian, and the degree of success of modern symptomatic treatments used for patients varies greatly. Accordingly, there is a need for new treatments for patients with PD that would slow the loss of neuronal function.

Human pluripotent stem cells (HPSCs) are a source of cells for use in regenerative medicine. Targeted differentiation of hPSCs into specialized cells such as spinal cord neurons (Li, et al. Nat. Biotechnol. 23, 215-221 (2005), incorporated herein by reference) or dopamine (DA) neurons obtained by methods other than methods according to the present invention. The present inventors have found that, as described above herein, neurons previously referred to as dopamine (DA) neurons (i.e., Perrier, et al Proc Natl Acad Sci USA 101, 12543-8 (2004), which included herein by reference) or DA-like (dopamine-like) midbrain neurons are not authentic midbrain dopamine (DA) neurons (see FIGS. 3, 10, 13 and 16). Accordingly, the authors of the present invention referred to the obtained from the ventral plate dopamine-producing neurons obtained by the methods described here, i. dopamine-producing neurons in accordance with the present inventions as authentic because, unlike the dopamine-producing neurons obtained by the methods described in the publications, when transplanted with authentic dopamine-producing neurons in accordance with the present inventions in rodents and primates, they reverse conditions similar to Perkinsonism and cause less complications, associated with excessive growth of neurons and the formation of teratoma. Also, unlike earlier methods for obtaining dopamine-producing neurons, authentic dopamine neurons in accordance with the present inventions are obtained in a higher percentage relative to the original population, and they also retain the ability to engraft over several months of cultivation.

Accordingly, methods have been developed to obtain authentic midbrain DA neurons using the differentiation methods described here. However, the effective use of hPSCs for cell therapy lags far behind advances in cell culture. Although DA neurons derived from mouse PSCs have been shown to be effective in models of Parkinson's disease (PD) (Tabar, et al. Nature Med. 14, 379-381 (2008); Wernig, et al. Proc. Natl. Acad. Sci. U.S. A 105, 5856-5861 (2008), all sources incorporated herein by reference), DA human PSC-derived neurons generally perform poorly in vivo (Lindvall and Kokaia, J. Clin. Invest 120, 29-40 (2010) , the cited source is incorporated herein by reference). In addition to the lack of compensation for the loss of endogenous neuronal function, there are serious concerns regarding the safety of using hPSC-derived neurons for transplantation, which are related to the fact that they can form teratomas or lead to overgrowth of neurons Roy, et al. nature medical. 12, 1259-1268 (2006); Elkabetz, et al. Gene Dev. 22, 152-165 (2008), source cited herein by reference).

Another possible source of cells for transplantation is DA neurons derived from human ESCs. Previous attempts to use these cells as an initial population of cells to generate differentiated cells, which were considered Da-like human embryonic stem cell-derived midbrain neurons (hESCs) transplanted into a rodent model of PD, were characterized by poor in vivo survival, transplants after transplant. This failure is most likely due to the incomplete differentiation of midbrain DA neurons in vitro, resulting in cells that appeared to be midbrain DA neurons but lacked the ability to engraft and replace the loss of neuronal function. In fact, the authors of the present invention showed that DA-like neurons, which were previously produced in laboratories and described in publications, did not belong to the same cell type as DA neurons of the ventral plate of the midbrain according to the present inventions, and do not have similar functions or ability to engraftment, see, for example, FIG. 16 and 17. Accordingly, the authors also found that in order to achieve directed cell differentiation in the laboratory and obtain cell populations containing a large number of adequately functioning neurons, cells must go through specific developmental stages in order to form a suitable cell population for use in replacement therapy. The authors also found that, at least to obtain engraftable DA neurons in accordance with the present inventions,

- 30 041083 certain stages of development must take place, such as FOX2A/LIM1A+ intermediate stage of the day. The authors found that if the cells do not go through such stages of development, the resulting DA-like neurons do not have the same functional abilities as the midbrain DA neurons in accordance with the present inventions, which are obtained from intermediate

FOX2A/LIM1A+k cells 11 days.

In contrast to what has been previously observed, novel culture techniques are described herein relating to ventral plate cell induction based strategies to obtain human DA neurons that engraft effectively in vivo. Accordingly, the failure to obtain cell populations consisting primarily of committed DA neurons according to the present inventions (i.e., FOXA2+ and LMXIA+ DA neurons capable of effective engraftment) is believed to be the reason for the failure of DA-like transplantation. neurons, i.e. due to incomplete specialization of DA-like cells. Previously, it was assumed that the cause of transplantation failures is the special vulnerability of cells, i.e. DA-like neurons from culture were unable to survive transplant stress. As described here, FOXA2+/LMX1A+ ventral midbrain progenitors were obtained from hPSCs 11 days after treatment with small molecule sonic hedgehog protein activators (SHH) and canonical WNT signaling. These day 11 cells, which are doubly positive for FOXA2+ and LMX1A+, are contacted with additional small molecules to induce further differentiation into engrafted TH+, FOXA2+ and LMX1A+ positive midbrain DA neurons by day 25. These mature DA neurons of the ventral midbrain can be maintained in vitro for several months. A large-scale in vitro molecular study, biochemical and electrophysiological data showed the development and confirmed the identity of midbrain neurons derived from hESC hDA. In vivo survival and function has been demonstrated in animal models of PD in three host species. Long-term survival in mice and rats with 6-OHDA-induced lesions demonstrated sustained survival of midbrain DA neurons, complete recovery of amphetamine-induced rolling behavior, and improvements in forelimb use and akinesia tests. Finally, scalability has been demonstrated by transplantation into parkinsonian monkeys. The excellent survival and function of neurons and the absence of neuronal overgrowth in the three animal models indicate promise for the development of cellular therapeutics for use in PD based on the compositions and methods of the present inventions.

Accordingly, the authors consider the main area of application of their development to be the possibility of obtaining an unlimited source of fully functional midbrain DA neurons from the ventral plate suitable for preclinical and clinical therapeutic applications. In a superficial way, the authors discovered a new protocol for efficient differentiation of smDA neurons from at least populations of pluripotent cells isolated from rodents and humans (human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs, iPSCs)). These studies used mutant iPS cell lines PINK1 derived from a human patient with Parkinson's disease (Seibler, et al., The Journal of Neuroscience, 2011, 31(16):5970-5976, cited source incorporated herein by reference ). Human stem cell populations (hESCs and hPSCs) were differentiated into cells with a midbrain phenotype, which, when brought into contact with molecules for neuronal maturation, gave rise to more authentic engrafting DA neurons. This protocol was used to demonstrate a high yield of hESC progeny by day 11 of directed differentiation of the midbrain DA phenotype (mDA, cmDA), which included the expression of key transcription factors, e.g. TH. Transplantation of these hESC-derived cmDA neurons into immunocompromised rodents and primate hosts, in contrast to the use of previously in vitro derived DA neurons, has shown good in vivo survival of grafted cells and functional recovery of behavioral disorders.

The advantages of using the methods in accordance with the present inventions to obtain DA neurons over other methods will become partially clear from the information below. The use of somatic stem cells and neuronal stem cells in other ways to generate authentic DA midbrain neurons that effectively engraft in vivo has not been successful (for a review, see Kriks & Studer, Protocols for generating ES cell-derived dopamine neurons in Development and engineering of dopamine neurons (Ed. Pasterkamp, et al.) (Landes Biosciences, 2008, reference cited herein by reference.) Pluripotent stem cells such as ES cells were then used as sources of engrafting cells. using murine ES cells have demonstrated the utility of deriving specific lines from in vitro pluripotent cells including neurons (Okabe, et al., Mech. Dev. 59:89-102 (1996); Bain, et al., Dev. Biol. 168v342-357 (1995, all references are incorporated herein by reference.) In fact, DA midbrain neurons were obtained using a strategy directed differentiation genes (Lee, et al., Nat. Biotechnol. 18v675-679 (2000), source cited herein by reference) based on developmental information from early transplant studies (Ye, et al., Cell 93:755-766 (1998), source cited herein text via link). Other directed differentiation strategies have been used to generate other types of neurons, such as somatic motor neurons (Wichterle, et al., Cell 110, 385-397 (2002), cited herein by reference). However, these efforts have not yielded results in the case of cell populations containing a high proportion of DA of midbrain neurons or cells capable of restoring neuronal function in vivo. In fact, the resulting population contained a mixture of cell types in addition to midbrain DA neurons. Further, the inventors developed other methods for obtaining DA of midbrain neurons, described in part below, however, these cell populations were also mixtures of cell types and did not restore neuronal function. Specifically, human ES cells differentiated into early neuroepithelial cells called neural rosettes, followed by the induction of rosette stage cells into cells expressing midbrain neuronal DA progenitor and differentiated markers. These cells continued to show neuronal functional properties in electrophysiology studies, in vitro DA release, and TH-positive production in the synaptic junction immunogold assay (Perrier, et al. From the Cover: Derivation of midbrain dopamine neurons from Human Embryonic Stem Cells. Proc Natl Acad Sci USA 101, 12543-8 (2004) cited herein by reference). However, despite promising results in vitro, transplantation of cells into mice with 6OHDA-induced lesions resulted in very few surviving dopaminergic neurons. This negative result was unexpected given the compelling evidence for functionality of mouse ES-derived DA neurons in vivo (Barberi, et al., Nat. Biotechnol. 21:1200-1207 (2003); Tabar, et al. Nature Med. 14:379 -381 (2008); Bjorklund, et al. Proc. Natl. Acad. Sci. USA 99:2344-2349 (2002); Kim, et al. Nature 418:50-56 (2002), all sources incorporated herein described by reference), robust in vitro functional properties of DA neurons derived from human ES cells (Perrier, et al., From the Cover: Derivation of midbrain dopamine neurons Human Embryonic Stem Cells. Proc Natl Acad Sci USA 101:12543-8 ( 2004), reference cited herein incorporated by reference) and clear evidence that human fetal DA neurons can survive as a xenograft in the striatum (Brundin, et al. Exp. Brain Res. 70:192-208 (1988); Bjorklund, et al., Neuronal replacement by intracerebral neural implants in animal models of neurodegenerative disease. Raven. Press., New. York. 455-492 (1988), all references are incorporated herein by reference). For almost 8 years after the first unsuccessful attempts to transplant DA neurons derived from human ES cells, little progress was observed in this area. Some limited improvement has been observed with primate pluripotent stem cell sources (Sanchez-Pernaute, et al. Long-term survival of dopamine neurons derived from parthenogenetic primate embryonic stem cells (Cynol) in rat and primate striatum. Stem Cells 23:914-922 ( 2005), source cited incorporated herein by reference), cells pretreated with FGF20 or Wnt5A or human ES cells differentiated in the presence of factors secreted by immortalized midbrain astrocytes (Roy, et al., Nature Med. 12:1259- 1268 (2006), source cited herein by reference). However, none of the previous strategies provided an enriched population of DA neurons in accordance with the present inventions for use in engraftment procedures to restore neuronal function in vivo.

I. Cell culture methods for inducing (line) neuronal progenitor cells: Contacting human pluripotent stem cells with SB431542 and LDN-193189 gave a differentiated neuronal cell line.

The following examples describe examples of methods for obtaining neuronal cell lines for use in developments in accordance with the present inventions.

Dual inhibition of SMAD has previously been used as a fast and efficient way to induce a single cell type of neuronal lineage from hPSCs (Chambers, et al., Nat Biotechnol 27, (2009), source cited herein by reference). These neuronal lineages, induced by molecules including Noggin, usually (in the absence of exposure) differentiate along a pathway that provides differentiation into cells of the central nervous system, which corresponds to the fate of nerve cells. In subsequent studies, it was found that the use of low molecular weight dorsomorphin (DM) instead of Noggin, at least in part, similar, but not identical, differentiated cells, with the main differences appearing in cultures (Kim, et al, Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity Stem Cell Rev 6, 270-281, (2010) Zhou, et al., High-Efficiency Induction of Neural Conversion in hESCs and hiPSCs with a Single Chemical Inhibitor of TGF- beta Superfamily Receptors Stem Cells, 504, (2010, references cited herein by reference). The inventors observed that Noggin-derived cells, although at the same developmental stage as LDN-treated cells, expressed essentially the same markers and had similar abilities to develop into different neuronal lineages, also have some differences, such as being in a more anterior position on the anterior-posterior axis (i.e. closer to the forebrain, more cells express

FOXG1, etc.) from LDN-Induced Nerve Cells Accordingly, although LDN has been used instead of Noggin to inhibit BMP, among other signaling pathways, Noggin and LDN may have other types of activity besides BMP inhibition.

Partly due to the high cost of using Noggin, the inventors investigated the possibility of using a BMP inhibitor instead of Noggin to differentiate cells along the neuronal pathway. Accordingly, a small molecule BMP inhibitor, LDN-193189, was used (Yu, et al., Nat Med 14, 1363-1369, (2008) cited herein by reference) and found that it can replace Noggin during development in accordance with the present inventions in combination with SB431542, providing the production of primitive neuroectoderm from PSCh, neuronal fate cells (lines), i.e. CNS cells (Fig. 2A). This combined treatment was designated LSB, according to the combination of the two inhibitors LDN-193189 and SB431542.

Typically, cell differentiation was initiated by treating a highly confluent monolayer with hES or iPSh by doubly inhibiting SMAD signaling. In preferred embodiments, the percent confluence is 50-100%, and 70-80% confluence is most preferred. It will be apparent to one skilled in the art that the initial cell density required to achieve the preferred confluence of the present invention will depend on cell type and size, seeding efficiency, survival, adhesion, and other parameters that can be empirically determined by one of skill in the art without too laborious experimentation. Dual inhibition of SMAD can be achieved using a variety of compounds including Noggin, SB431542, LDN-193189, dorsomorphine, or other molecules that block TGFe, BMP, and Activin/Nodal signaling. In a preferred embodiment, a composition is used containing SB431542 and LDN-193189 (together, LSB) at a concentration of 0.1-250 μM, or more preferably 1-25 μM, or most preferably 10 μM of compound SB431542 and 105,000 nM or , most preferably 100-500 nM of LDN-193189 compound.

II. Formation of DA neurons from hESCs via intermediate rosette cells and results of transplant studies using these DA-like neurons.

Previously, the inventors have used several other methods of directed differentiation, which gave rise to cell populations containing DA-like neurons. These DA-like neurons have been used in transplantation studies, the results of which have raised doubts about the possibility of further use of these cells in therapeutic applications. As examples, the procedures described in Perrier et al., 2004 and Fasano et al., 2010, including induction of neurons based on MS5, resulted in rosette cells and were used to generate day 11 progenitors, (see Figs. 2, 16 and 17 as examples), which were further used to obtain DA-like neurons. These neurons were derived from a small proportion of progenitor cells in the cell populations formed on day 11. Transplantation studies using these neurons have demonstrated poor survival after transplantation and the loss of a DA-like neuronal phenotype in addition to the development of unacceptable nerve cell types observed after transplantation and loss of control of cell growth leading to the formation of teratomas. See fig. 16 and 17.

In particular, at the P0 stage, ESCs were brought into contact with molecules to start the transformation of Oct4+ cells into rosette cells using MS5 feeder cells (Perrier et al., 2004). At the P1 stage, rosette cells were expanded by contacting the cells with additional compounds to allow the cells to differentiate into P2 stage cells with a specific expression pattern, including Pax2+/En1+ DA progenitor cells, which then differentiated into TH+/En1+ DA neurons. These cells were used for transplantation into rats with 6OHDA-induced lesions immunosuppressed with cyclosporin A. Transplantation experiments described have shown poor in vivo survival, loss of the TH+ phenotype, problems with further growth into unwanted, possibly lethal, cells such as teratomas, and development into unacceptable types of neurons that may cause additional medical problems for the patient.

There was a very small number of surviving TH+ neurons 4.5 months after transplantation (<50 TH+ cells/animal) in rosette-derived DA progenitor grafts, FIG. 16A. However, in contrast to TH+ cells, cells labeled with GFP (GFP was under the control of a ubiquitous promoter) showed fairly good survival after transplantation. This suggested that the cells with the best survival after transplantation were non-DA neurons (16B). Several transplant-derived cells (hNA+, green) co-expressed TH (red), which also indicated that the majority of transplanted human cells are non-DA neuron phenotypes, FIG. 16C. Sections of Fig. 16 D-E show that, despite very poor in vivo survival, there was some improvement (variable in low and high degree) in several behavioral tests, such as the amphetamine-induced rotation test (D), the cylinder test, and spontaneous rotations ( e).

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Induction of neurons without feeder cells was performed as previously described (Chambers et al., 2009), but with modifications to obtain ventral plate cells (Fasano et al., 2010, cited source incorporated herein by reference).

Using a modified SMAD dual inhibition method to differentiate pluripotent cells into ventral plate cells, the authors of the present study previously found that high concentrations of SHH were required to induce FP by day 11. For example, in some embodiments, Sonic C25II was added at a concentration of 200 ng/mL. DKK-1 (R&D; 100 ng/ml), FGF8 (R&D; 50 ng/ml), Wnt-1 (Peprotech; 50 ng/ml) and retinoic acid (R&D; 1 mM) were added in some experiments, see Fig. . 17. However, none of the cell populations obtained using previously known methods contained a high proportion of progenitors of FOXA2+/LMX1A+ ventral midbrain cells obtained using the method according to the present invention at day 11.

III. Compounds for Use in Directed Differentiation: Small molecule screening using neuronal cell lines according to the present inventions yielded compounds that allowed cells to differentiate into FOX2A+ and LIMX1A+ neuronal cells by day 11 of culture.

The following examples describe the use of cell examples from Section I to screen for small molecule compounds and determine whether their use will lead to targeted differentiation of a population of cells containing a high proportion of ventral midbrain neurons by day 11 after primary exposure to dual SMAD inhibitors. The results of this screen initially indicated that the SHH-activating compound, together with FGF8 and Wnt activation, efficiently generated FOXA2+/LMX1A+ positive midbrain ventral plastic cells by day 11 of differentiation. Here are the results of exemplary experiments that established which molecules were needed and determined the optimal contact time to obtain FOXA2+/LMX1A+ positive cell populations at day 11.

Recent genetic studies in mice have demonstrated an important role for the FOXA2 transcription factor in the development and survival of midbrain DA neurons. A unique feature of the developing midbrain is the simultaneous expression of the ventral plate marker FOXA2 and the midbrain tegmental marker LMX1A. Typically, the cells of the ventral plate and the roof are located in different areas of the CNS (some ventrally and others dorsally) and require sharply opposite patterns of induction. Recently, the production of region-specific ventral plate precursors from hESCs using a modified dual inhibition protocol has been described. Canonical Wnt signaling was important both for the functioning of the tegmentum and for the development of DA in midbrain neurons.

A. CHIR99021 (CHIR) induced high levels of DA precursors in midbrain neurons by day 11 of culture. As described herein, treatment with CHIR99021 (CHIR), a potent inhibitor of GSK3e known to significantly upregulate WNT signaling, induced LMX1A in FOXA2+ ventral plate progenitors (FIG. 1a). CHIR was more efficient than recombinant Wnt3A or Wnt1 in inducing LMX1A expression. The efficiency of LMX1A induction depended on the time of CHIR treatment, with the maximum effect being achieved on days 3-11 (FIG. 5). CHIR induced FOXA2/LMX1A co-expression and other factors such as FGF8 had only minor effects (FIG. 6). Induction of FOXA2/LMX1A co-expression required significant activation of SHH signaling using purmorphamine, a small molecular weight agonist alone or in combination with recombinant SHH (FIG. 7).

Treatment with SHH (purmorphamine+SHH) and FGF8 (S/F8) agonists in the absence of CHIR99021 showed much lower FOXA2 expression by day 11 and no LMX1A expression at all (Fig. 1a,b). Dual SMAD inhibition (LDN-193189+SB431542=LSB treatment) did not result in FOXA2 expressing cells but did not result in a subset of LMX1A+ cells (Fig. 1a, b). The anterior region marker OTX2 was stably induced in cultures treated with LSB and LSB/S/F8/CHIR, but not under LSB/S/F8 conditions (Fig. 1a, c). Systematic comparisons were made between these three culture conditions (FIG. 1d) using global temporal gene expression profiling. Hierarchical allocation of clusters of differentially expressed genes made it possible to distinguish between these three sets of treatment conditions on day 11 of differentiation (Fig. 8a). FOXA1, FOXA2, and several other downstream SHH targets, including PTCH1, were among the most differentially regulated transcripts in LSB/S/F8/CHIR compared to LSB processing sets (Fig. 1e). Expression of LMX1A, NGN2, and DDC indicated a predetermined developmental pathway into a DA progenitor midbrain neuron as early as day 11 (Fig. 1e, f). In contrast, day 11 LSB cultures were enriched in dorsal hindbrain progenitor markers such as HES5, PAX6, LHX2, and EMX2. Direct comparison of LSB/S/F8/CHIR with LSB/S/F8 treatment (Fig. 1f) confirmed the selective enrichment of DA progenitor markers in midbrain neurons in the LSB/S/F8/CHIR group and indicated the identity of hypothalamic progenitors in cultures treated with 34 041083 LSB/S/F8 based on differential expression of RAX1, SIX3, and SIX6 (see also POMC, OTP expression in Figure 2D below). The complete list of differentially expressed transcripts is in Table. 1, 2 and ontological analysis of the genes in FIG. 8b (DAVID; http://david.abcc.ncifcrf.gov), confirmed enrichment of the canonical WNT signaling pathway by CHIR processing Primary data not yet available at GEO worldwideweb.ncbi.nlm.nih.gov/geo/accession#: [TBD] ). Comparative temporal analysis of gene expression of midbrain DA progenitor markers (Fig. 1g) with non-DA anterior and ventral neuron developmental pathway markers (Fig. 1h) revealed three types of induction: i) LSB: dorsal hindbrain; ii) LSB/S/F8: ventral/hypothalamic; and iii) LSB/S/F8/CHIR: corresponding to midbrain DA progenitor.

Cells obtained using the protocol described here, to ensure development in accordance with the present inventions, were compared with cells obtained by previous methods. In FIG. 2 and 18 show visual comparison examples with CHIR-treated SHH/FGF8+ cells according to the present inventions. In FIG. 19A shows examples of cells doubly labeled with FOXA2/Tuj1 after treatment with LSB/S/F8/CHIR (upper panels) and co-tagged FOXA2 with TH, Nurr1 and LMX1A (lower panels). These combinations of markers make it possible to identify precursors of DA neurons at early stages. In FIG. 19B shows gene expression data (for comparison with FIG. 2E) for key dopamine progenitor markers.

Materials and methods are generally described herein: ESC (H9, H1) and iPSC (2C6 and SeV6) human ESC lines were processed according to a modified ventral plate induction protocol (Fasano, et al., Cell Stem Cell 6:336-347 (2010), cited source incorporated herein by reference) based on dual SMAD inhibition (Dual SMADinhibition) (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009), cited source incorporated herein by reference ). The exposure time of SHH C25II reagents, purmorphamine, FGF8, and CHIR99021 to midbrain ventral plates was optimized to generate new populations of DA neurons (see Fig. 1d). After ventral plate induction, subsequent maturation (11-25 days or longer than 25 days in culture to at least 100 days in culture) was performed in Neurobasal/B27-based differentiation media in the presence of DA neuronal survival and maturation factors (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004, reference cited herein by reference), such as AA, BDNF, GDNF, TGFe3, and dbcAMP (see full method description for details). The phenotype of the obtained population of DA neurons was carefully studied by immunocytochemical methods, quantitative RT-PCR, determination of the global gene expression profile, HPLC analysis for the determination of dopamine, and registration of electrophysiological characteristics in vitro. In vivo studies were carried out in rodents with hemiparkinsonism (mice or rats injected with 6OHDA toxin in one hemisphere of the brain). These studies were performed in non-adult NOD-SCID IL2Rgc mice (Jackson labs) and adult Sprague Dawley rats from Taconic Farms, which were induced with 6-hydroxydopamne damage by stereotactic injections of said toxin as previously described, as well as in two adult rhesus monkeys treated with unilateral injection of MPTP into the carotid artery.

DA neurons were administered by stereotactic injection into the striatum of animals (150x103 cells in mice, 250x103 cells in rats) and a total of 7.5x106 cells (distributed into 6 sites; 3 on each side of the brain) of macaques. Behavioral tests were performed monthly after transplantation, including an amphetamine-induced rotation test, as well as a focal akinesia test (stepping test) and a limb use test (top hat test). Rats and mice were sacrificed at 18-20 weeks, and primates - 1 month after transplantation. The study of the characteristics of the grafts was carried out by stereological analysis of the number of cells and the volume of the graft, as well as through a thorough examination of the phenotype using immunohistochemical methods.

Culture of undifferentiated human ES cells. hESC cell lines H9 (WA-09, XX, 27-55 passages after 10/2009), H1 (WA-01, XY, 30-40 passages after 6/2010) and iPS 2C6 cell lines (Kim, et al. Cell Stem Cell 8:695-706 (2011) cited herein by reference) (XY, 20-30 passages) and SeV6 (XY, 20-30 passages; derived from MRC-5 embryonic fibroblasts using non-integrating 4- Sendai x Factor Vector System (Ban, et al. Proc. Natl. Acad. Sci. USA (2011) 108(34):14234-14239:10.1073/pnas.1103509108, source cited herein incorporated by reference) supported on mouse embryonic fibroblasts at calculated seeding concentrations ranging from 0.5x103/ ^cm2 to 100x103/ ^cm2 for ES on human cells at which cells tend to form clusters (MEF, Global Stem, Rockville, MD) in an optimal environment for ES human cells containing 20% Knockout serum replacement (KSR, Invitrogen, Carlsbad, California) (as previously described (Kim, et al. Cell Stem Cell 8:695-706 (2011) cited herein by reference). The amount of Knockout Whey Replacement used can range from 0 to 40%.

neuron induction. For the induction of dopamine neurons in the ventral plate of the midbrain

- 35 041083 used a modified version of dual SMAD inhibition (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009) cited herein by reference) and ventral plate induction protocol (Fasano, et al. Cell Stem Cell 6:336-347 (2010, cited herein incorporated by reference) based on time exposure to LDN-193189 reagents (100 nM (in concentration range 0.5-50 μM, Stemgent, Cambridge, Massachusetts ), SB431542 (10 µM (range 0.5-50 µM, Tocris, Ellisville, MI), SHH C25II (100 ng/ml (range 10-2000 ng/ml, R&D, Minneapolis, MN), purmorphinamine (2 μM (range 10-500 ng/mL, Stemgent), FGF8 (100 ng/mL (range 10-500 ng/mL, R&D) and CHIR99021 (CHIR; 3 μM (range 0.1 -10 µM, Stemgent) Note: In the ventral plate induction protocol, SHH treatment refers to treatment, i.e. cells in contact with a combination of SHH C25II 100 ng/ml + purmorphamine (2 μM). Cells were plated (35-40x103 cells/cm ² ) and grown for 11 days on matrigel or geltrex gel (used as purchased) (BD, Franklin Lakes, New Jersey, USA) in Knockout Serum Replacement Medium ( KSR) containing DMEM, 15% KSR, 2 mM L-glutamine and 10 μM (in the concentration range 1-25 μM) β-mercaptoethanol. KSR medium was successively replaced with N2 medium starting from day 5 of differentiation, mixing them in ratios of 75%(KSR):25%(N2) on days 5-6, 50% (KSR):50%(N2) on days 7-8 and 25% (KSR): 75% (N2) on days 9-10 as previously described (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009) cited herein by reference). On day 11, medium was changed to Neurobasal/B27 (1:50 dilution)/L-Glut-containing (effective range 0.2-2 mM) medium (NB/B27; Invitrogen) supplemented with CHIR (up to day 13) and BDNF (brain-derived neurotrophic factor, 20 ng/ml in the range of 5 to 100; R&D), ascorbic acid (AA; 0.2 mM (in the concentration range of 0.01-1 mM), Sigma, St. Louis, M1), GDNF (glial cell neutrophic factor, 20 ng/ml (in the concentration range 1-200 ng/ml); R&D), TGFe3 (transforming growth factor type β3, 1 ng/ml (in the concentration range 0.1-25 ng/ml ); R&D), dibutyryl cAMP (0.5 mM (range 0.05-2 mM); Sigma), and DAPT (10 mM (range 0.5-50 nM); Tocris) for 9 days. On day 20, cells were dissociated using Accutase® (Innovative Cell Technology, San Diego, Calif.) and plated at elevated cell density (eg, 300-400,000 cells/cm ² ) on polyornithine (PO) pre-coated dishes; 15 µg/ml (in the concentration range 1-50 µg/ml)/laminin (1 µg/ml) (in the concentration range 0.1-10 µg/ml)/fibronectin (2 µg/ml (in the concentration range 0.1 -20 μg/ml) in differentiation medium (NB/B27+BDNF, AA, GDNF, dbcAMP (in the range of concentrations indicated in this description), TGFe3 and DAPT (in the concentration range indicated in this description) until the desired stage is reached maturation for a particular experiment.

Previously described protocols (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004) cited herein by reference) were used in part to induce DA neurons based on rosette structures, with at least one exception. when dual SMAD inhibition was used to accelerate the initial stage of neuronal induction. Briefly, hESCs were induced for the neural developmental pathway by co-culturing with irradiated MS5 cells in KSR medium supplemented with SB431542 and Noggin (250 ng/ml (in the concentration range 10-1000 ng/ml); R&D), from 2-8 days and SHH+ FGF8 from 6-11 days of differentiation. After 11 days in KSR medium, neuronal rosettes were harvested by hand and grown (P1 step) in N2 medium supplemented with SHH, FGF8, BDNF and AA as described previously (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004) , the cited source is incorporated into the present description by reference). After 5-7 days in the P1 stage, the rosettes were harvested again by hand and ground to powder, followed by incubation in Ca ² /Mg ² free Hank's Balanced Salt Solution (HBSS) for 1 hour and plated onto polyornithine (PO)/laminin coated plates. /fibronectin. Patterning with SHH/FGF8 was continued for 7 days at the P2 stage followed by final differentiation in the presence of BDNF, AA, GDNF, TGFb3 and dbcAMP as described above until the desired stage of maturation for a particular experiment was reached (usually 5-7 days for transplantation experiments). or 32 days for in vitro functional experiments).

Analysis of gene expression. Total RNA was isolated during differentiation on the following days: 0, 1, 3, 5, 7, 9, 11, 13 and 25 for each experiment with conditions: LSB control, LSB/SHH/FGF8 and LSB/SHH/FGF8/CHIR with using the RNeasy kit (Qiagen, Valencia, CA). For biochip analysis, total RNA was processed on an MSKCC Genomic core and hybridized on Illumina Human ref-12 bead biochips according to the manufacturer's instructions. Comparisons were made every day and for every set of conditions using the LIMMA package from Bioconductor (worldwideweb.bioconductor.org). Genes with an adjusted P value less than 0.05 and a fold change in expression greater than two were considered statistically significant. Some of the descriptive bioarray data analyzes and presentation were performed using a commercially available software package (Partek Genomics Suite (version 6.10,0915)). For quantitative RT-PCR analysis, total RNA on day 25 for each set of conditions was subjected to reverse transcription (Quan-36 041083 titech, Qiagen) and the amplified material was analyzed using commercially available Taqman gene expression assays (Applied Biosystems, Carlsbad, CA) with normalization HPRT data. Each point represents 9 technical replicates from 3 independent biological samples. Primary data from biochip experiments are not yet available at GEO worldwideweb.ncbi.nlm.nih.gov/geo).

Surgical intervention in animals. Experiments on rodents and monkeys were carried out in accordance with the instructions of the National Institutes of Health (NIH), and they were approved by the local Institutional Animal Care and Use Committee (IAUCC), the Institutional Biosafety Committee (IBC), and the Embryonic Stem Cell Research Committee (ESCRO).

Mice. NOD-SCID IL2Rgc knockout mice (weighing 20-35 g; Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized with ketamine (90 mg/kg; Akorn, Decatur, IL) and xylazine (4 mg/kg; Fort Dodge , IA). 6-hydroxydopamine (10 µg (in the concentration range 0.1-20 µg), 6-OHDA (Sigma-Aldrich) was injected stereotactically into the striatum with the following coordinates (in millimeters): AP, 0.5 (from bregma ; skull suture was used as a reference point for stereotactic surgery); ML, -2.0; DV, -3.0 (from the dura mater, the membrane that covers the brain, used as a reference point). Mice with successful lesions ( averaging >6 rotations per minute) were selected for transplantation A total of 150 x 10 ³ cells were injected in a volume of 1.5 μl into the striatum at the following coordinates (in mm): AP, 0.5; ML, -1.8; DV, 3.2 Mice were sacrificed 18 weeks after transplantation.

Rats. Adult female Sprague-Dawley rats (Taconic, Hudson, NY, USA) weighing 180-230 g were anesthetized with ketamine (90 mg/kg) and xylazine (4 mg/kg) during surgical procedures. On one side, the medial forebrain bundle of the nigrostriatal tract was injured with stereotactic injections of 6-OHDA (3.6 mg/ml in 0.2% ascorbic acid and 0.9% saline, Sigma in two sites (Studer, et al. Nature Neurosci 1:290-295 (1998, cited herein incorporated by reference.) Rats were selected for transplantation if amphetamine-induced rotation exceeded 6 rotations per minute 6-8 weeks after injection.250 x 103 cells were transplanted into the striatum each animal (Coordinates: AP +1.0 mm, ML -2.5 mm and V -4.7 mm; comb set to -2.5) Control animals received PBS instead Surgical procedures described previously (Studer, and Nature Neurosci 1:290-295 (1998, cited herein by reference) Daily intraperitoneal injections of cyclosporine 15 mg/kg (Bedford Labs, Bedford, OH) were started 24 hours before cell transplantation and continued before sacrifice, after 20 weeks after cell transplantation.

Primates. Two adult (17-18 yr; 10-12 kg; female) rhesus monkeys were rendered hemiparkinsonian by injecting MPTP into the carotid artery followed by weekly intravenous MPTP to create bilateral parkinsonian syndrome (Kordower, et al. Science 290:767- 773 (2000), cited herein by reference). Both animals exhibited Parkinsonian symptoms consistent with moderate to severe injury based on behavioral analysis, including hunched posture, drooping leg, and symptoms of muscle rigidity (non-plasticity of movement), ignorance (motor perception of a lateral stimulus), and bradykinesia (delayed initiation of movement). These parameters can be assessed in monkeys using the Modified Clinical Parkinsonism Rating Scale (CRS). On the day of transplant surgery, animals were administered the tranquilizer ketamine (3.0 mg/kg IM) and dexdomitor (0.02-0.04 mg/kg IM), intubated to maintain stable airway ventilation, and anesthetized with isoflurane. Then they were placed in a stereotactic frame for surgical intervention. Both rhesus monkeys were operated on once, with three intracranial injections of DA cultures derived from human ventral plates based on stereotaxic coordinates (Paxinos, et al. The Rhesus Monkey Brain in Stereotaxic Coordinates (Academic Press, 2000), source cited incorporated herein description by reference). Bilateral cell injections (10 μl per injection; 125,000 cells/μl) were performed in three positions (1 - posterior caudal appendage, 2 - pre-commissural putamen and overlying white matter) with a total volume of 30 μl per hemisphere. An infusion pump attached to a stereotaxic micromanipulator was used to deliver cells at a rate of 1 μl/min using a 50 μl Hamilton syringe with a 28 G needle. After injections were completed, the needle was left in place for 2-5 min to allow the infusate to diffuse out of the syringe tip. and then slowly withdraw the syringe. Immediately after surgery, the animals received an analgesic (buprenex, 0.01 mg/kg IM, twice a day for 72 hours after surgery; meloxicam, 0.1 mg/kg SC, once a day for 72 hours after surgery ), as well as an antibiotic (cefazolin, 25 mg/kg intramuscularly, twice a day) within 72 hours after surgery. Animals received cyclosporine A (Neoral, Sandimmune) orally (30 mg/kg reduced to 15 mg/kg)

- 37 041083 daily, starting 48 hours before surgery until the moment of killing, within one month after transplantation.

behavioral tests. The amphetamine-induced rotation test (mice and rats) and the walking test (rats) were performed before transplantation and 4, 8, 12, 18 weeks after transplantation. Rotational behavior in mice was recorded 10 min after intravenous administration of damphetamine (10 mg/kg, Sigma) and recorded for 30 min. Rotational behavior in rats was recorded 40 min after intravenous administration of d-amphetamine (5 mg/kg) and automatically assessed using the VideoMot2 system (TSE, Germany). Data are presented as mean rotations per minute. Used a modified step test from Blume, et al. Exp. Neurol. 219:208211 (2009) and Crawley, et al. What's Wrong With My Mouse: Behavioral Phenotyping of Transgenic and Knockout Mice (Wiley-Liss, 2000), all sources incorporated herein by reference. Briefly, each rat was placed on a flat surface, the hind legs were raised, gently holding the tail so that only the front paws touched the table. The experimenter pulled the rat back 1 meter at a uniform speed. He counted the number of compensatory steps with the contralateral and ipsilateral paws. Data were presented as percentage of contralateral foot steps/number of compensating contralateral foot+ipsilateral foot steps. The cylinder test was performed by placing a rat on a glass cylinder and counting the number of touches with the ipsilateral paw compared to the contralateral (out of 20 touches) wall of the cylinder, as previously described (Tabar, et al. Nature Med. 14:379-381 (2008), cit. the source is incorporated herein by reference). The processing of tissues for rodents and primates is described below.

Mice and rats: Animals (mice and rats) were injected with elevated doses of phenobarbital intraperitoneally (50 mg/kg) for deep anesthesia and perfused in 4% paraformaldehyde (PFA). The brain was removed, fixed in 4% PFA, and then immersed in 30% sucrose solution for 2-5 days. Sections were prepared on a cryostat after fixation in the O.C.T. (Sakura-Finetek, Torrance, California, USA).

Signs: animals were sacrificed under deep anesthesia with ketamine (10 mg/kg, intramuscularly (IM)) and phenobarbital (25 mg/kg, intravenously (IV)) by heart perfusion with heparinized 0.9% saline, and then with fresh cold 4 % fixing PFA (pH7.4). Immediately after primary fixation, the brain was removed from the skull and fixed in 4% PFA, where it floated freely for 24-36 h. The brains were then washed and resuspended in 10% sucrose with slow shaking at 4°C and left to soak. This process was then repeated in 20% sucrose and then in 30% sucrose. Whole brains were cut along the frontal plane (coronal suture) into serial sections 40 μm thick on a cooled sledge microtome and stored floating in a cryoprotective medium at -20°C.

Immunohistochemistry: Cells were fixed in 4% PFA and blocked with 1% bovine serum albumin (BSA) with 0.3% Triton buffer. The brain tissue section was washed in cold PBS and treated in a similar manner. Primary antibodies were diluted in 1-5% BSA or normal goat serum and incubated according to the manufacturer's instructions, a detailed list of antibodies and sources is given in table. 6. Corresponding Alexa488, Alexa555 and Alexa647-conjugated secondary antibodies (Molecular Probes, Carlsbad, California, USA) with 4',6diamidino-2-phenylindole (DAPI) nuclear counterstain (Thermo Fisher, Rockford, Illinois, USA). For some assays, biotinylated secondary antibodies were used followed by imaging with the DAB (3,3'-diaminobenzidine) chromogen.

HPLC analysis. Reverse-phase HPLC with electrochemical detection to measure the levels of dopamine, homovanillic acid (HVA) and DOPAC (3,4-dihydroxyphenylacetic acid) was performed as previously described (Roy, et al. Nature Med. 12:1259-1268 (2006); Studer, et al Brain Res Bull 41:143-150 (1996, all sources incorporated herein by reference), cultures were sampled in perchloric acid at day 65 of differentiation. For some experiments, DA was measured directly in the medium using the same detection system, but after albumin extraction of dopamine and its metabolites using a commercially available kit, as described previously (Studer, et al. Brain Res. Bull. 41:143-150 (1996 ), the cited source is incorporated herein by reference).

Registration of electrophysiological parameters: Cultures were transferred to a recording chamber on an upright microscope equipped with a 40X water immersion objective (Eclipse E600FN; Nikon); cultures were perfused with saline containing, in mM: 125 NaCl, 2.5 KCl, 25 NaHCO ₃ , 1.25 NaH ₂ PO ₄ , 2 CaCl, 1 MgCl ₂ and 25 glucose (34°C; saturated with 95% O ₂ -5 % CO ₂ , pH 7.4, 298 mOsm/L). The saline flow rate was 2-3 ml/min when passed through an inline heater (SH27B with TC-324B controller; Warner Instruments). Neurons were visualized by video microscopy with a cooled digital CCD camera (CoolSNAP ES ² , Photometries, Roper Scientific, Tucson, Arizona, USA). For registration of electrophysiological parameters, cells similar in shape to neurons with good branching of dendrites were selected. The local patchclamp method on whole somatic cells in the current patching configuration was performed using a MultiClamp 700B amplifier (Molecular Devices). The signals were filtered in the 1-4 kHz range and digitized at 5-20 kHz using a Digidata 1440A (Molecular Devices). The recording contact electrodes were made of borosilicate fiber (Sutter Instruments) drawn on a Flaming-Brown puller (P-97, Sutter Instruments) and had a resistance of 4-6 MΩ in the bath. The electrodes were filled with an internal solution containing in mM: 135 K-MeSO ₄ , 5 KCl, 5 HEPES, 0.25 EGTA, 10 phosphocreatine-di(tris), 2 ATP-Mg and 0.5 GTP-Na (pH 7.3 , the osmolarity was adjusted to 290-300 mosm/l). The bridge amplifier was tuned in such a way as to compensate for the resistance of the electrodes and monitored. The capacitance of the electrodes was compensated. If the series connection increased >20% during clamping, the data was discarded because an increase in resistance indicates a partial technical failure during the measurement.

Cell counts and stereological analyses. The percentage of marker-positive cells on the ventral plate (day 11), FIG. 1, midbrain dopamine progenitors (day 25), FIG. 2, and at the stage of mature DA neurons (day 50 or later), FIG. 3 and 11 were determined in samples obtained in at least 3 independent experiments for each. Images for counting were chosen randomly (uniform distribution), each image was evaluated first by the number of DAPI-positive nuclei, after which the number of cells expressing the marker of interest was counted. Data are presented as mean±SEM (standard error of the mean). The number of human cells (identified by anti-hNA) and TH+ neurons in the graft were embodied and counted at every tenth section if the graft could be identified. Cell count and graft volume were determined using the optical probe of the fractional probe apparatus and the Cavalieri scoring device using the Stereo Investigator software (MBF bioscience, Vermont, UWB) as described in Tabar, et al. Nat. Biotechnol. 23:601-606 (2005); the cited source is incorporated into this text by reference. Data are presented as estimated total cells and total graft volume +/- standard error of the mean (SEM). The following formulations characterize examples of cell culture media for carrying out the present inventions.

hESC Maintenance Medium (1 L): 800 ml DMEM/F12, 200 ml Knockout Serum Replacement, 5 ml 200 mM L-glutamine, 5 ml Pen/Strep, 10 ml 10 mM 15 essential amino acids in MEM minimal medium, 55 μM 13-mercaptoethanol, and FGF-beta (bFGF, final concentration 4 ng/ml).

KSR medium for hESC differentiation (1 L): 820 ml Knock out DMEM, 150 ml Knock out Serum Replacement, 10 ml 200 mM L-glutamine, 10 ml Pen/Strep, 10 ml 10 mM MEM and 55 μM 13- mercaptoethanol.

N2 medium for hESC differentiation (1 L): 985 ml distilled H ₂ O with DMEM/F12 powder, 1.55 g glucose (Sigma, cat. no. G7021), 2.00 g sodium bicarbonate (Sigma, cat. no. S5761), putrescine (aliquot volume of 100 µl from a solution of 1.61 grams in 100 ml of distilled water; Sigma, cat. no. P5780), progesterone (20 µl aliquot of a solution of 0.032 grams in 100 ml of 100% ethanol; Sigma, cat. no. P8783), sodium selenite (aliquots of 60 0.5 mM solution in distilled water; Bioshop Canada, cat. no. SEL888), and 100 mg transferrin (Celliance/Millipore, cat. no. 4452-01), and 25 mg insulin (Sigma, cat. no. 16634) in 10 ml 5 mM NaOH.

Dulbecco's Modified Eagle's Medium (DMEM) with 10% PBS for the preparation of PMEF ((primary mouse embryonic fibroblasts (PMEF)) - feeder cells) (1 L): 885 ml DMEM, 100 ml PBS, 10 ml Pen/Strep and 5 ml of L-glutamine.

Alpha Minimal Maintenance Medium (MEM) with 10% PBS to prepare for MS-5 feeder cell media (1 L): 890 ml Alpha MEM, 100 ml PBS, 10 ml gelatin solution with Pen/Strep (500 ml): dissolve 0 .5 gelatin in 500 ml warm (50-60°C) Milli-Q water. Cool to room temperature.

In summary, below is a brief overview of examples of methods for monitoring the production of mature DA neurons for use in transplantation (see also the conditions shown in Table 7. Day 13 is the ventral plate stage characterized by FOXA2/LMX1A co-expression.

In summary, the following is a brief overview of examples of methods for monitoring the production of mature DA neurons for use in transplantation (see also the conditions shown in Table 7. Day 13 is a midbrain ventral plate stage characterized by co-expression of FOXA2/LMX1A. In addition to FOXA2 and LMX1A expression loss of OCT4 expression and no induction of forebrain markers PAX6 and FOXG1 are observed.Day 25 is the midbrain DA progenitor stage characterized by continued expression of FOXA2/LMX1A and expression of neuronal (TUJ1) and DA markers (NURR1, TH). During the proliferation of Ki67+ cells, the number of progenitors of midbrain neurons PAX6 and FOXG1 are observed, where these markers are undesirable.An Operetta High Content Microscope (Perkin Elmer) was used for unbiased and fast quantitative immunofluorescence data.Real-time qPCR analysis was also performed for each marker to confirm the result of immunofluorescent analyses. In some embodiments, cell lines (cultures) that have passed these preliminary in vitro tests are used for transplantation, see table. 7. In some implementations

- 39 041083 Mature DA neurons at day 25 were cryopreserved without serum (range 20 to 25 days) in culture medium+7% DMSO (range 3-12%) until thawed for use in transplantation. In some embodiments, cell samples are stored in liquid nitrogen. In some embodiments, cells are stored in low temperature refrigerators. Other embodiments contemplate the use of cryoprotectants such as myoinositol, polyvinyl alcohol, serum replacement, caspase inhibitors, in addition to DMSO. After thawing, the cells were examined for viability, marker expression, etc. before transplantation. In some embodiments, thawed cells are tested for retention of function in long-term in vitro and in vivo tests to monitor freezing and storage conditions.

B. Research aimed at identifying additional factors for obtaining functional DA neurons. Additional exclusion and inclusion experiments are contemplated for tissue culture components for use in cell production in accordance with the present inventions. For example, it was shown that although the use of FGF8 led to the production of cells in accordance with the present inventions, it was not a necessary component for obtaining these cells. These experiments will be continued with additional reagents, such as those listed in Table. 8, which can serve as additives to cell cultures in addition to the four main molecules that provided the production of DA neurons in accordance with the present inventions, i.e. i) Alk4/5/7 inhibitor (TGFe-) (SB431542), ii) Alk2/3 inhibitor (BMP) (LDN193189), iii) Smoothened (SHH) agonist (Purmorphamine), and iv) GSK3e- inhibitor (CHIR99021).

As described here, the use of SB431542 and LDN193189 demonstrated efficient conversion of pluripotent stem cells, and the addition of purmorphamine and CHIR99021 to these cells demonstrated induction of the midbrain ventral plate. Other chemicals and proteins have been or may be used to provide long term trophic support and/or accelerated differentiation. Some of these compounds will be used in further tests to determine their role in the cell differentiation described here.

For these experiments, the behavior of other compounds will be compared against the typical limitations for differentiation of DA neurons (Table 7) using 4 main factors (Table 8).

These types of experiments are designed to determine the minimum number of factors required to obtain authentic DA nerve cells in accordance with the present inventions.

C. Implementation to obtain dose-response curves for potentially proliferating impurities (pluripotent hESCs, rosette-like structures of neurons). During development in accordance with the present inventions, the formation of teratomas or excessive overgrowth in the graft was absent for at least 5 months of survival in vivo. To monitor safety in studies of longer duration, aimed at establishing the putative longevity of a graft in humans, a cut-off cell number is studied to determine a clinically significant limit for contaminating problematic cell types, such as undifferentiated hESCs that can develop into teratomas or primitive neuroectodermal progenitors. capable of significant proliferation. Accordingly, some embodiments are designed to enrich cells for use in transplantation with dopaminergic neurons, i.e. reducing the content of impurity cell types before transplantation, see, for example, table. 7 for examples of constraints.

Below is an example of standardized selection in tests to improve validation strategies. In the case of ESCs, a predetermined mixture of undifferentiated (Oct4+/Nanog+) cells with ESP-DA-derived neurons will be used to monitor clinical symptoms indicative of mass effect and/or animal death in animal experiments. Dose response measurements will be made for one human ES cell per 10,000 hESC-DA-derived neurons, 1/5000, 1/1000 and 1/100. The cells will be injected into the striatum and the animals will be subjected to immunosuppressive treatment. Rats will be carefully monitored and euthanized when neurological symptoms appear or no later than 6 months. Brains will be examined for the volume and composition of the graft as described in this text. Cell ratios will be adjusted until a clear threshold for in vivo teratoma formation is established. A similar strategy will be used to determine interfering levels of primitive neuroectodertal progenitors. The presence of early neuronal progenitors is associated with significant potential for proliferation and a wide range of differentiation along the central nervous system pathway as well as the peripheral nervous system (PNS) pathway. Graft study will consist of IHC analysis of cells of rosette-like structures (PLZF expression), their progeny in the CNS (neuronal progenitors expressing Nestin/Sox2 or forebrain progenitors expressing FoxG1), as well as graft volumes and proliferation ratios (% Ki67+ of total surviving cells ).

IV. Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease; it is estimated to affect 4.1-4.6 million patients worldwide and is predicted to

- 40 041083 the figure will double by 2030. This disease is the second most common after Alzheimer's disease, it affects approximately 1 million patients in the US, and another 60,000 patients are diagnosed with this disease each year. The disease carries a significant socioeconomic burden and is associated with high mortality and risk to patients, and the combined direct and indirect costs associated with PD, including healthcare costs and loss of income, are estimated to be approximately $25 billion per year in the US alone. Currently, there is no cure for Parkinson's disease (PD), which is an age-related, progressive disease that leads to disability. The pathogenesis of Parkinson's disease is characterized by a selective loss of DA neurons in the substantia nigra. Accordingly, the main characteristic of PD is the progressive, severe and irreversible loss of dopamine (DA) neurons, resulting in motor dysfunction, ultimately leading to disability. Although pharmacological, exercise-based, and surgical treatments for PD have been developed, none of these approaches provide adequate restoration of neuronal DA function. Long-term management of motor symptoms in patients often remains suboptimal, and despite understanding of the importance of progressive dopamine-non-responsive motor and non-motor symptoms, the fundamental problem of long-term management of dopamine-responsive symptoms continues to be an area of urgent therapeutic need. In PD, numerous pathologies are found that affect both the central and peripheral nervous systems, the main signs of PD (bradykinesia, rigidity and partly tremor) are deeply associated with the loss of DA of neuronal cells and respond to dopamine. Accordingly, the treatment of PD with the use of neuronal cell replacement is envisaged due to the rather selective loss of midbrain neuron DA, which is responsible for most of the motor symptoms of this disease. The brain of a healthy person contains approximately 1 million DA neurons. Accordingly, in one embodiment, it is contemplated that a relatively small number of surviving cells are required to replace DA neurons compared to most other disorders in the CNS.

One of the challenges in developing cell therapy for PD has been identifying a suitable cell source for use in neuronal replacement. This search has been ongoing for more than 30 years, and many potential sources for replacing DA neurons have been proposed (Kriks, Protocols for generating ES cell-derived dopamine neurons in Development and engineering of dopamine neurons (eds. Pasterkamp, R.J, Smidt, & Burbach) (Landes Biosciences, 2008; Fitzpatrick, et al., Antioxid. Redox. Signal. 11:2189-2208 (2009)).In the past, some of these sources have been advanced to early stages of clinical trials, including catecholaminergic adrenal medulla cells, Madrazo , et al., N. Engl. J. Med. 316, 831-834 (1987), carotid body transplants (Arjona, et al., Neurosurgery 53: 321-328 (2003)) or encapsulated retinal pigment epithelial cells (study sferamina, Bakay, et al., Front Biosci 9, 592-602 (2004) However, these trials have largely failed to show clinical efficacy and have resulted in poor long-term survival and low levels of DA release from twisted cells. Another approach has been transplantation of fetal midbrain DA neurons performed in more than 300 patients worldwide (Brundin, et al., Prog. Brain Res. 184, 265-294 (2010); Lindvall, & Kokaia, J. Clin. Invest 120:29-40 (2010) Treatment with human fetal tissue in these patients showed evidence of DA neuron survival and in vivo DA release up to 10 or 20 years after transplantation in some patients. fetal tissue transplantation did not replace the function of DA neurons.In addition, fetal tissue transplantation poses a number of problems, including a small amount and poor quality of donor tissue, ethical and practical difficulties associated with obtaining tissue, and the poorly characterized heterogeneous nature of transplanted cells; these problems are among the factors that contribute to the variability of clinical outcomes.Examples of fetal transplantation are described in the reference kah: Mendez, et al. Nature Med. (2008)); Kordower, et al. N. Engl. J. Med. 332:1118-1124 (1995); Piccini, et al. Nature Neuroscience 2:1137-1140 (1999). However, these clinical results were supplemented by some positive results from early open studies (Lindvall, et al. Science 247:574-577 (1990); Widner, et al. N. Engl. J. Med. 327:1556-1563 (1992); Brundin, et al. Brain 123:1380-1390 (2000); Freed, et al. N. Engl. J. Med. 327:1549-1555 (1992); Freeman, et al. Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson's disease Ann Neurol 38:379-388 (1995) However, two larger NIH (National Institutes of Health) sponsored placebo-controlled clinical trials in the United States produced modest results (Freed, et al. N. Engl. J Med 344, 710-719 (2001) Olanow, et al Ann Neurol 54:403-414 (2003) There are many hypotheses regarding the limited efficacy observed in human fetal transplant trials, including the hypothesis that fetal transplantation may not provide enough cells at the proper her developmental stages for optimal beneficial therapeutic effect. In addition, fetal tissue is rather poorly characterized in terms of cell types and differs in staging and quality variability for each of the tissue samples Bjorklund, et al. Lancet Neurol. 2, 437-445 (2003). Another contributing factor may be the host's indolent inflammatory response to the graft (Bjorklund, et al. Lancet Neurol. 2, 437-445 (2003)).

Conversely, it is expected that the use of a cell source derived from stem cells or another type of compatible cells to obtain cells for transplantation will overcome many of the problems associated with fetal tissue transplantation and provide an unlimited source of DA neurons at the optimal stage for transplantation. After nearly 20 years of attempting to use various potential sources of stem cells, the present inventors have succeeded in obtaining authentic human midbrain DA neurons from pluripotent stem cells capable of reversing neurological defects in mice and primates. This novel differentiation strategy was highly efficient and resulted in robust cell graft engraftment in vivo, induction of functional recovery in PD in disease models, and no adverse events such as inappropriate cell proliferation as evidenced by preclinical data. It is hypothesized that a human ES cell-based treatment strategy for PD may receive FDA approval, as the FDA has approved testing of other human ES cell derivatives in spinal cord injury (Strauss, Nat. Biotechnol. 28:989-990 (2010) and macular degeneration (Schwartz , et al Lancet 379:713-720 (2012)).

Additional implementations are contemplated, including improvement strategies to control cell purity, promote axonal fiber outgrowth, and include new safety/regulatory features of grafting strategies. For example, in some embodiments, a method of cell purification is demonstrated, ranging from simple screening for a surface marker of a cell type of interest (such as CD142) to a targeted enrichment strategy for a specific neuron type. In some embodiments, the use of said method is contemplated in the production of cells for use in humans. In addition, in other embodiments, engineered expression of PSA-NCAM is provided to enhance axonal outgrowth for use in in vivo neural repair. Such use includes stimulation of distant axonal growth in the treatment of motor neuron disease, Huntington's disease, or other disorders primarily affecting projection neurons. Additional implementations of a Good Manufacturing Practice (GMP) compliant source of pluripotent cells are contemplated, and the like. Because transplantation using the methods described herein requires a small number of DA neurons and is based on relatively simple, cost-effective, small molecule techniques designed to induce DA neurons, it is implied that the cost of replacement therapy with DA neurons in terms of the patient will be reasonable. One of the potential biological limitations of the transplant approach in PD is the fact that neuronal degeneration in PD extends to many cell types beyond midbrain dopamine neurons, particularly in the later stages of the disease. In fact, in a long-term study in the late stages of PD, non-responsive to DA symptoms predominate, leading to dysphagia, falls, dementia, and other significant pathological manifestations. However, with the restoration of dopaminergic function, some non-motor symptoms can presumably improve. In addition, it is suggested that the use of hESC-derived DA neurons in the early stages of this disease may prevent some of the secondary symptoms of PD, including the degeneration of dopamine-responsive striatal populations. However, even in the absence of the impact of non-DA-responsive symptoms of this disease, long-term functional dopaminergic recovery of the striatum may represent a significant advance in the treatment of this currently incurable disorder. In the case of Parkinson's disease, there are several alternative therapy options available, including drug-based strategies and surgical approaches such as deep brain stimulation. It is expected that in some implementations, the recovery efficiency will be comparable to the levels achieved with the use of alternative therapy options, or will be higher than the specified levels. It is contemplated that, in other embodiments, the use of therapy with transplantation of said mature DA neuronal cells will be particularly beneficial for a particular subgroup of patients. In other embodiments, the use of said therapy with transplantation of mature DA neuronal cells is contemplated, in addition to existing drug and surgical approaches. One significant advantage of using mature DA neuronal cell transplantation therapy according to the present inventions is the unique non-repairable nature of the post-transplantation period, namely the long-term restoration of neuronal function, which is expected to be exploited in patients with gradual withdrawal of drug therapy. Cell transplantation presumably affects the spectrum of DA-associated symptoms distinct from those responding to drugs or other therapies. Accordingly, in one embodiment, the use of mature DA neuron transplantation with deep brain stimulation (DBS) is contemplated. According to another implementation variant, the use of transplantation of mature DA neurons with therapy is envisaged.

- 42 041083

A) Parkinson's disease and modern therapy options. Significant progress has been made in identifying rare genetic changes that contribute to familial forms of PD. However, in most cases of PD, the contribution of any potential genetic predisposition remains unclear. Traditional therapeutic strategies for PD are limited by the fact that by the onset of clinical symptoms, 30-70% of all DA neurons in the substantia nigra are irreversibly degraded. One of the therapeutic options is the pharmacological replacement of the DA neuronal deficiency using the DA precursor L-DOPA. However, despite a pronounced initial response to L-DOPA therapy in some PD patients, the long-term clinical outcome remains unsatisfactory, and serious side effects of LDOPA therapy, including motor fluctuations and dyskinesias, are often observed in the later stages of the disease. The pulsatile delivery of L-DOPA plays a major role in the development of these motor complications in the later stages, respectively, smoother and more physiological delivery of dopamine, i.e., for example, from transplanted cells in accordance with the present inventions, respectively, would be very desirable. In addition to pharmacological strategies, there are several surgical treatment options. These options include ablation or functional inactivation of cells in the basal ganglia by pallidotomy, or deep brain stimulation by targeting the subthalamic nucleus or the interior of the globus pallidus. Although these surgical options are an alternative for some patients, they provide symptomatic relief but do not restore normal DA function. In addition, side effects of surgical and non-surgical treatments, including malfunction, infection, stroke, bleeding, and the like, have been reported. Other treatment options include delivery of growth factors such as GNTF or neuroturin using direct intraparenchymal infusion or viral expression by gene therapy. Although initial open-label studies in PD showed promising results for GTTP, subsequent controlled trials in a larger group of patients did not confirm any benefit and pointed to a potential safety hazard due to the production of antibodies against GTTP in a subset of patients. When delivered, neuroturin, an AAV (adeno-associated virus)-based HTTP-related molecule, also failed to show any significant clinical benefit in a large, placebo-controlled, multicenter study. Preliminary results from a phase 2 study of injections of AAV-derived glutamic acid decarboxylase (GAD) into the subthalamic nucleus have recently emerged (Lancet Neurology, April 2011), however, the clinical benefits in this study were moderate at best. Although attempts to use strategies based on neurotrophic factors or alternative neuroprotective strategies may bring temporary relief to patients, none of these strategies is able to return / replace DA neurons already lost as a result of the disease, which is the main goal of cell replacement therapy.

B. Cell therapy in PD. Clinical symptoms in PD become evident after the loss of 70-80% of striatal dopamine neurons and approximately 50% of substantia nigra dopamine neurons. However, midbrain dopamine neurons develop by 8.5 weeks after conception, with no conclusive data on lifetime dopamine replacement. Accordingly, the age of dopamine neurons at the time of the onset of the disease is several decades, and there is no natural mechanism for replacing these cells; accordingly, cell transplantation may be necessary to replace these cells in the brain of PD patients. Replacement of DA neurons in PD was carried out in the 1980s using chromaffin cells derived from the adrenal medulla. These hormone-producing cells have been shown to switch the neurotransmitter phenotype from adrenaline to DA when ectopically injected into the CNS. Transplantation using this approach has been performed in several hundred PD patients around the world, but over time it turned out that the survival of the transplanted cells was extremely unsatisfactory, and they provided, at best, a temporary effect. Accordingly, the clinical application of this approach was soon abandoned. In contrast, the use of fetal midbrain transplantation was based on more intensive preclinical studies in rodent models demonstrating sustained long-term engraftment and functional improvement in a panel of DA-related behavioral assays. These preclinical data motivated the continuation of fetal transplantation at several centers in the late 1980s and early 1990s. follow-up histological studies for some patients who died from independent causes. However, the use of fetal transplants raises two potential problems associated with cell therapy methods. The first unexpected problem with the use of fetal transplants was the induction of graft-induced dyskinesias (GIDs) in approximately 15% of patients. Although the mechanism of GID remains controversial, recent evidence suggests that serotonergic neurons are capable of inappropriate storage and release of DA. Another potential

- 43 041083 The mechanism proposed to explain the GID lies in the uneven distribution of DA neurons, leading, respectively, to the formation of hot spots of DA release. However, during the development of the present inventions, methods have been discovered for detecting and reducing the number of serotonergic neurons, which can provide a reduction in the frequency of GID. In addition, injection of mature DA neurons can provide a uniform distribution of mature neurons, including the distribution of dopaminergic fiber endings in the host striatum. Another problem with fetal transplant treatment has been (and is) the limited availability of fetal midbrain tissue at an appropriate developmental stage. An alternative strategy was tested in the clinic to address resource constraints by using porcine fetal DA neurons. However, the survival of DA neurons in these xenografts was unsatisfactory and this approach was abandoned completely. A recent study using retinal pigment epithelium also did not show any benefit. In contrast, the use of human ES cells as sources of transplantable cells is expected to provide an unlimited source of cells to produce dopamine neurons for use in transplantation.

V. Compounds and culture methods have been discovered for the targeted differentiation of FOXA2+ and LMX1A+ positive progenitor neurons into midbrain DA neurons (mesencephalon neurons, mDA) in accordance with the present inventions.

In developing the present inventions, the authors found that the timing of exposure to CHIR99021 determines the induction of FOXA2/LMX1A-n precursors of the ventral midbrain plate. Accordingly, the present inventors tested by FOXA2/LMX1A immunocytochemical assay at differentiation day 11 after LSB/S/F8 (i.e. LSB, S, i.e. SHH cell treatment and FGF8 (F8) treatment alone or in combinations with CHIR starting at different time points: d0-d11 (days 0-11), d1-d11, d3-d11, d5-d11, d7-d11 compared to cell cultures without CHIR treatment in duplicate. quantification of the percentage of FOXA2+, LMX1A+ and dual-labeled cells at differentiation day 11 after differential onset of CHIR exposure as described by the immunocytochemical assay.

A. CHIR99021 (C) is an induction factor for FOXA2+/LMX1A+ cells at day 11 from LSB cultured cells contacted with Hedgehog activator and purmorphamine. The example below describes the use of exemplary methods to test the efficacy of each compound in inducing directed neuronal differentiation of mDA neurons.

This example describes the detection of small molecules and contact timing to enable directed differentiation of FOXA2+LMX1A+ DA neurons in accordance with the present inventions. The following is a summary of some of the experimental findings described herein: When cells with dual SMAD inhibition were treated with SHH (purmorphamine + SHH) and FGF8 (S/F8) antagonists in the absence of CHIR99021, significantly lower FOXA2 expression was observed at day 11 and no expression of LMX1A (Fig. 1a,b). Stable induction of the anterior marker OTX2 occurred in LSB and LSB/S/F8/CHIR treated cultures, but not under LSB/S/F8 conditions (Fig. 1a,c). The present inventors chose a cell population containing pluripotent cells as a starting population and seeded on day 0. Cells were cultured until they reached pre-differentiation preconfluence (60-100% confluence). These cells were contacted with dual SMAD inhibitors (ie exposed to LDN-193189+SB431542=LSB) on day 0. The present inventors continued to monitor the cell population at regular feeding containing fresh LSB until day 11 and found that some of the remaining cells were LMX1A+ cells, however, did not express FOXA2 (Fig. 1a, b). The present inventors seeded in duplicate starting cell populations, then tested for cell types (i.e., gene/protein expression patterns) after contact with mixtures containing any of the following SHH agonists (purmorphamine+SHH) and brought into contact with FGF8 (S/F8), using different exposure schemes, i.e. bringing cells into contact on day 0, or day 1, or day 2, etc., for a certain time, i.e. 24h, 48h, etc. Three examples of primary culture conditions tested are shown below: 1) cells were contacted with LDN/SB (LSB) on day 0, then contacted with fresh LSB until day 5, on day 5 cells were contacted with fresh LDN without SB until day 11, 2) cells were brought into contact with LDN/SB (LSB) on day 0, then brought into contact with fresh LSB until day 5, on day 5 cells were brought into contact with fresh LDN without SB until day 11, while during this period, cells were additionally contacted with fresh purmorphamine, SHH and FGF8 until day 7 and 3) cells were contacted with LDN/SB (LSB) on day 0, then contacted with fresh LSB until day 5, at 5 day, cells were contacted with fresh LDN without SB until day 11, while during this period, cells were additionally contacted with fresh purmorphamine, SHH and FGF8 until day 7, and additionally contacted with fresh CHIR, starting from day 3 of culturing and up to 11 days, with slightly varying indicated p Primary conditions for determining the optimal yield of cell types. B. Comparative in vitro characterization of FOXA2+/LMX1A+ cells derived from the midbrain ventral plate region and DA progenitor cells derived from

- 44 041083 using other techniques. A systematic comparison of the three culture conditions (FIG. 1d) was performed using global gene expression temporal profiling. In hierarchical clustering of differentially expressed genes, the three treatments separated by day 11 of differentiation (Fig. 8a). FOXA1, FOXA2, and a number of other downstream SHH targets, including PTCH1, were among the majority of differentially regulated transcripts in the LSB/S/F8/CHIR or LSB datasets (Fig. 1e). The expression of LMX1A, NGN2, and DDC indicated the determination of the direction of differentiation of the precursors of DA neurons in the midbrain already by day 11 (Fig. 1e, f). In contrast, LSB cultures were enriched in dorsal forebrain progenitor markers such as HES5, PAX6, LHX2, and EMX2. Direct comparison of LSB/S/F8/CHIR treatment with LSB/S/F8 treatment (Fig. 1f) confirms selective enrichment for DA progenitor markers in midbrain neurons in the LSB/S/F8/CHIR group and indicates the presence of hypothalamic progenitors in treated LSBs. /S/F8 cultures based on differential expression of RAX1, SIX3 and SIX6 (see also FIG. 2D, below: POMC, OTP expression). An illustrative list of differentially expressed transcripts is provided, see table. 12; ontological analysis of genes for day 11, fig. 8b (DAVID; http://david.abcc.ncifcrf.gov) confirmed the enrichment in canonical WNT signaling components upon CHIR processing. Raw data not yet available (GEO, worldwideweb.ncbi.nlm.nih.gov/geo/, access no. tbc). Comparative temporal analysis of the gene expression of markers of precursors of DA neurons in the midbrain (Fig. 1g) and markers of the direction of differentiation into antherial and ventral cells that are not DA neurons (Fig. 1h) makes it possible to separate three variants of induction conditions as follows: i) LSB: characteristics dorsal part of the forebrain; ii) LSB/S/F8: ventral/hypothalamic characteristics; and iii) LSB/S/F8/CHIR: characteristics of midbrain DA progenitor neurons.

VI. Further differentiation of FOXA2+/LMX1A+ cells on day 11 into midbrain DA neurons by day 25 and maintenance up to day 65.

For further differentiation of FOXA2+/LMX1A+ progenitors were maintained in neuronal maturation stimulating medium (BAGCT, see Example I). For comparison, two other methods for obtaining progenitor cells of DA neurons were used. The following types of comparisons were made between populations of differentiated cells obtained using previously developed methods and methods in accordance with the present inventions: A) immunohistochemical analysis on day 50 of differentiation in TH in combination with LMX1A, FOXA2 and NURR1, B) Determination of the amount of TH+, FOXA2+ , LMX1+, and NURR1+ cells of total cells for rosette-derived cultures versus ventral plate-derived cultures (LSB/S/F8/CHIR). C) determination of the percentage of serotonin (5-HT+) and GABA+ positive neuronal subtypes (impurities other than DA neurons) at day 50 in rosette-derived DA neuron cultures. And D) HPLC analysis to measure the amount of dopamine and metabolites. Comparison of DA, DOPAC (Dihydroxyphenylacetic Acid) and HVA (Homovanillic Acid) levels between ventral plate and rosette cultures. By day 25, Tuj1+ neurons (Fig. 2A) and cells expressing TH, the rate-limiting enzyme in DA synthesis, were formed in three progenitor cell populations. However, treatment with LSB/S/F8/CHIR gave TH+ cells co-expressing LMX1A and FOXA2 and showing pronounced induction of the nuclear receptor NURR1 (NR4A2) (Fig. 2A, B). Comparison of gene expression in cultures on day 13 and day 25 confirmed the robust induction of other postmitotic markers of DA neurons (Fig. 2C). Characterization of DA neurons at day 25 comparing LSB and LSB/S/F8 treated cultures confirmed enrichment in known midbrain neuron DA transcripts and allowed the identification of several new candidate markers (Fig. 2D, Tables 3-5, Fig. 8b). For example, LSB/S/F8/CHIR cultures (a group of midbrain DA neurons) showed the greatest enrichment in the TTF3 transcript, a gene not previously associated with the development of midbrain DA neurons, but highly expressed in the substantia nigra in humans (Fig. 8c; Allen Brain Atlas: http://human.brain-map.org).

Similar data were obtained for EBF-1, EBF-3 (Fig. 8c), as well as TTR, a known transcriptional target of FOXA2 in the liver. The data obtained during the development of the present inventions indicate enrichment for several PITX genes in DA progenitor cells of midbrain neurons. PITX3, a classic DA marker for midbrain neurons, was also stably expressed at differentiation day 25 (Fig. 2E). Finally, induction of both the ventral midbrain plate and DA neurons can be easily reproduced in independent hESC and hPSC lines (Fig. 9). The data presented here showed that the LSB/S/F8/CHIR protocol, unlike other protocols tested, provided cells with a marker expression profile consistent with the direction of differentiation in midbrain DA neurons.

In vitro and in vivo properties of ventral plate-derived DA neurons were compared with those of DA-like neurons derived from neuronal rosette intermediate cells (FIGS. 10 and 16). Formation from neuronal rosettes is the most widely used strategy for obtaining DA neurons from hPSCs at present. Both ventral plate and rosette-based protocols were effective in generating TH+ neurons capable of long-term in vitro survival (50 days of differentiation; Fig. 3(1)a). However, the percentage of TH+ cells was significantly higher in ventral plate-derived cultures (FIG. 3(1)b). Although NURR1 co-expression was observed in TH+ cells using both protocols, ventral plate-derived DA neurons co-expressed FOXA2 and LMX1A (FIGS. 3(1)a, b and 3(2)).

Few GABA and serotonin (5-HT) positive neurons were observed (Fig. 3(1)c). DA and its metabolites DOPAC and HVA were present in cultures obtained using either protocol, however, DA levels were approximately 8-fold higher in cultures from the ventral plate (Fig. 3(1)d, e). DA midbrain neurons exhibited extensive fiber outgrowth and robust expression of mature neuronal markers, including synapsin, the dopamine transporter (DAT), and G protein-coupled internal rectifier potassium channel (Kir3.2, also referred to as GIRK2, is expressed in DA neurons of the compact parts of the black substance (SNpc)) (Fig. 3(1)f, 11). DA SNpc neurons in vivo exhibit an electrophysiological phenotype that distinguishes them from most other neurons in the brain. In particular, they spontaneously generate spikes at a low frequency (1-3 Hz). In addition, these slow spikes are accompanied by a slow subthreshold oscillating potential. After 2-3 weeks in vitro, the same indicated physiological signs are demonstrated by cultured DA neurons of SNpc mice in the early postnatal period. DA neurons differentiated from hESCs stably (4/4 tests) exhibited this distinct physiological phenotype (Fig. 3(1)g-i).

Maintaining mDA neurons in vitro at day 65 (d65), TH positive neurons were observed still expressing FoxA2 and forming long fibers typical of mDA neurons (Fig. 3(1)j). Measurement of DA release by HPLC showed that TH+ neurons aged d65 are functional in vitro (FIG. 3(1)k).

In summary, the neurogenic conversion of ventral midbrain lamina progenitors and the development of an optimized protocol for differentiation of DA mesencephalon neurons from the ventral lamina are described in this document. Ventral plate-derived DA neurons were generated from human ES cells using small molecule-based SHH activation and canonical WNT signaling early in differentiation (FIG. 3(2)). These hES cells progressed from the dual FOXA2/LMX1A-positive midbrain ventral plate stage to immature Tuj1+ neurons co-expressing FOXA2/LMX1A, and then to mature DA neurons (Fig. 3(2)a,b) characterized by sustained DA release and electrophysiological properties characteristic of DA neurons in the compact substantia nigra (SNpc; A9-type) of the midbrain, including autonomous pacemaker activity (Fig. 3(2)c). Surprisingly, this method was highly efficient, allowing more than half of the cells in the culture dishes to acquire the mature midbrain marker profile (see FIG. 3(2)b).

VII. Characterization of ventral plate-derived midbrain dopamine neurons in vivo as grafted neurons.

The example below describes the use of illustrative methods in accordance with the present inventions in therapeutic cell replacement. One of the major challenges in the art is to be able to obtain hPSC-derived DA midbrain neurons capable of functional engraftment in vivo without the risk of neuronal overgrowth or inadequate differentiation into non-neuronal midbrain cells or development of teratomas. Based on studies with fetal tissue transplantation, the inventors of the present invention suggested that the time of cell cycle exit, which is characterized by the expression of NURR1, may represent a suitable stage for transplantation (approximately 25 days of differentiation, Fig. 2). Initial studies using day 25 cells in adult mice without lesions demonstrated sustained survival of hPSC-derived FOXA2+/TH+ neurons 6 weeks after transplantation (FIG. 12). The long-term survival of FOXA2+/TH+ cells in parkinsonian hosts was tested without leading to neuronal overgrowth. To this end, 6-hydroxydopamine (6-OHDA)-induced lesions (Tabar, et al. Nature Med. 14:379-381 (2008) cited herein by reference) were prepared in NOD-SCID mice with silent IL2Rgc , a lineage that effectively supports xenograft survival, with specific susceptibility to rare tumorigenic cells (Quintana, et al. Efficient tumor formation by single human melanoma cells. Nature 456:593-598 (2008), source cited herein by reference) . Cultures of both ventral plate-derived and rosette-derived DA neurons were engrafted (150x103 cells/animal) without prior purification to detect potential contaminating cells with high proliferative potential. At 4.5 months after transplantation of ventral plate-derived DA neurons, a well-defined central part of the graft was observed, consisting of TH+ cells co-expressing FOXA2 and the human specific marker hNCAM (Fig. 4a-c). Functional analysis showed complete elimination of amphetamine-induced rotational behavior. In contrast, rosette-derived neuronal grafts showed few TH+ neurons, did not significantly reduce rotational behavior (Fig. 4d), and also showed massive excess

- 46 041083 growth of neurons (graft volume>20 mm ³ ; Fig. 13). The intense overgrowth of rosette-derived neuronal cells used for transplantation as described herein was comparable to previous work by the inventors' group with rosette-derived DA grafts (Kim, et al. miR-371-3 Expression Predicts Neural Differentiation Propensity in Human Pluripotent Stem Cells Cell Stem Cell 8:695-706 (2011) cited herein by reference) and others (Hargus, et al. Proceedings of the National Academy of Sciences of the United States of America 107: 15921-15926 (2010), source cited herein by reference). This overgrowth was presumably due to longer survival times (4.5 months versus 6 weeks), lack of FACS purification before transplantation, and host selection of IL2Rgc-silent NOD-SCID mice. The number of proliferating Ki67+ cells in ventral plate-derived grafts was minimal (<1% of the total number of cells), while rosette-derived grafts retained inclusions of proliferating neuronal progenitors. The neuronal overgrowth is thought to be caused by the presence of primitive anterior neuroectodermal cells in the graft (Elkabetz, et al. Genes Dev. 22:152-165 (2008); Aubry, et al. Proc. Natl. Acad. Sci. USA 105: 1670716712 (2008), source cited herein by reference). This hypothesis is supported by the expression of the forebrain marker FOXG1 in rosette-derived but not ventral plate-derived grafts. A small percentage of astroglial cells were present in both ventral plate-derived and rosette-derived grafts, although most GFAP+ cells were negative for human markers, indicating host origin (Fig. 13).

The results for NOD-SCID mice with silent IL2Rgc described herein demonstrated sustained long-term survival of FOXA2+/TH+ neurons, complete reversal of amphetamine-induced rotational behavior, and no evidence of neuronal overgrowth. However, some of these outcomes may be explained by the specific use of NOD-SCID mice with silent IL2Rgc. To test this hypothesis, cultures of ventral plate-derived DA neurons (250x103 cells) were transplanted into adult rats with focal 6-OHDA lesions and pharmacologically induced cyclosporin A immunodeficiency. Five months after transplantation, graft survival was robust (Fig. 4e-h), with an average of over 15,000 TH+ cells co-expressing FOXA2 (Fig. 4g) and human nuclear antigen (hNA) (Fig. 4e); TH+/hNCAM+ fibers extended from the central part of the graft into the surrounding striatum of the host (Fig. 4f). In addition to FOXA2, TH+ cells expressed midbrain DA markers PITX3 and NURR1 (Fig. 4h-j). Behavioral analyzes showed complete elimination of the amphetamine-induced rotational asymmetry, in contrast to sham-transplanted animals that did not improve (FIG. 4k). Transplanted animals also improved in the walking test (Fig. 4l) to measure forelimb akinesia and in the cylinder test (Fig. 4m) in assays independent of pharmacological stimulation of the DA system. For human DA neurons, a late onset of recovery (approximately 3-4 months after transplantation) is expected and depends on the rate of in vivo maturation, eg DAT expression levels (Fig. 4n). The presence of TH+ cells expressing Kir3.2 (GIRK2) or calbindin channels indicates that both SNpc (A9) and ventral tegmental (A10) DA neurons are present in the graft (Fig. 4o, p). As in mice (Fig. 13), serotonergic and GABAergic cells were rare (<1% of total cells) among rat cells, as were host-derived GFAP+ glial cells (7% of total cells; ( Fig. 14. While a small number of serotonin-positive neurons were detected in the graft, hNCAM-negative cells were observed, which were probably host-derived serotonergic fibers (Fig. 14). of neurons in mice, rats, and monkeys, an unexpected recovery of neuronal function was observed in rodent and primate species.Short-term (6 weeks) survival assays were extended to investigate unexpected long-term survival up to 5 months after transplantation into the striatum of host mice with focal 6OHDA lesions and immune deficiency. In fact, a direct comparison of the traditional method of obtaining DA neurons based on e rosettes (Perrier, et al. Proc Natl Acad Sci USA 101, 12543-8 (2004)) and the novel ventral plate-based DA neuron differentiation protocol described herein showed that ventral plate-derived DA neurons were able to mediate long-term engraftment of DA neuron grafts, in contrast to from rosette-based neurons (Fig. 4a-c).

In particular, sustained induction of behavioral recovery in mice with amphetamine-induced rotational behavior in mice with compound 6-OH damage after application of ventral plate-derived neurons (fp, blue line) and rosette-derived (red line) DA neurons (Fig. 4d) showed that ventral plate-derived neurons provided a higher degree of recovery. Mice with transplanted DA neurons,

- 47 041083 obtained from the ventral plate, showed almost complete recovery on the amphetamine scale. Animals with transplants of DA rosette-derived neurons showed a lower improvement in behavioral symptoms, in some cases recovering the original high number of turns over time.

In the 6OHDA-damaged mouse model, sustained graft function was also found. Rats, unlike mice with PD, allow for more sophisticated behavioral tests and to investigate the survival of DA neurons in xenograft models after pharmacological immunosuppression (a treatment that more closely mimics transplantation protocols in humans). Excellent graft survival, evidence of DA fiber outgrowth, and retention of expression of specific transcription factors supported the long-term survival of ventral plate-derived neurons expressing markers of authentic midbrain DA neurons (Fig. 4e-j). A number of functional assays have demonstrated significant improvement in both drug-induced behavior tests (amphetamine-induced rolling) and spontaneous behavior tests (top hat and walking tests).

The results reported here demonstrate excellent graft survival and behavioral outcomes in two independent mouse models. However, the number of DA neurons needed in a mouse or rat is a small fraction of the number needed for transplantation in primates and humans. To investigate the scalability of this protocol, transplant pilot studies were conducted in two adult rhesus monkeys with MPTP-induced lesions. In addition, it was not initially known whether the methods and cells described here would retain their function in primates; this information could help realize the use of these methods and cells in humans. Accordingly, initial studies were conducted in at least two monkeys to investigate the short-term (4-6 weeks) in vivo survival and maintenance of the DA phenotype of midbrain neurons in the primate brain. The studies described here demonstrated sustained survival of TH/FOXA2-positive DA neurons and indicated striatal reinnervation of recipients (Fig. 4q-t). Transplantation of more cells, close to the range needed for future human studies, demonstrated sustained survival of midbrain DA neurons. In addition to these results from short-term studies, a number of longer-term studies were conducted in rhesus monkeys that evaluated cell survival for 3 months and an optimized immunosuppression regimen with cellcept, prograf, and daily prednisone (used as triple therapy in combination) . Surprisingly, sustained survival of DA-derived ES-derived midbrain neurons in primate brains was found, as well as a significantly reduced recipient inflammatory response to transplants compared to the recipient's pronounced microglial response observed in the original transplant study (see Fig. 15).

Specifically, methods associated with studies in monkeys included batches containing 50x106 transplantable DA progenitor neurons obtained on day 25 of differentiation using a ventral plate protocol. The classical dose was 3 mg MPTP-HCL injected into the carotid artery (range 0.5-5 mg). This was followed by a systemic injection of 0.2 mg/kg MPTP IV. Cells were injected into three sites (posterior caudate nucleus and precommissural part of the putamen) on each side of the brain (total of 6 tracts, 1.25x106 cells/tract) and the animals were immunosuppressed with cyclosporin-A. On one side of the brain, DA progenitors from the GFP-expressing H9 subelement were injected, while on the other side, cells derived from unlabeled H9 cells were grafted. Results demonstrating neuronal graft engraftment in rhesus monkeys with continued FOX2A expression and TH production are shown in FIG. 4q-t. One month after transplantation, sustained survival of midbrain DA neurons was observed as assessed based on GFP expression (FIG. 15) and human specific cytoplasmic marker (SC-121) (FIG. 4q). The central part of each graft was surrounded by a halo of TH+ fibers extending up to 3 mm into the host (Fig. 4r). The central part of the grafts consisted of TH+ neurons co-expressing SC-121 (Fig. 4s) and FOXA2 (Fig. 4t). The SC-121 and GFP negative regions within the graft contained host Iba1+ microglia (FIG. 15), indicating incomplete immune suppression.

Thus, engraftment of a new population of DA neuronal cells in primates has been shown; in adult rhesus monkeys with patchy MPTP lesions (3 mg MPTP-HCL (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; concentration range 0.5-5 mg MPTP-HCl) with severe, greater than 95 % loss of endogenous midbrain DA neurons Exposure to MPTP produced observable changes and symptoms similar to those of human Parkinson's disease.

Thus, a new protocol for hPSC differentiation based on the ventral plate was discovered, which accurately reproduces the development of DA in midbrain neurons. The availability of cells with the main features of DA in midbrain neurons will allow them to be used for a wide range of biomedical applications, such as basic development studies, high-performance drug discovery, and disease modeling based on

BP-iPSK. It is important to note that this study has finally developed a method for obtaining a scalable source of FOXA2+/TH+ neurons for neuronal transplantation - a significant step towards cell therapy in PD.

In addition, the production of authentic midbrain DA neurons from hESCs demonstrated excellent in vivo performance (see Fig. 4) and the production of DA neurons at the time of transplantation (approximately 40% of mature neurons in the population at 25-30 days of differentiation, which was higher than the percentage content in the dissection of human ventral midbrain fetal tissue (generally about 10%) (Sauer, et al., Restor. Neurol. Neurosci. 2:123135 (1991)).

Below is a brief description of the materials and methods to be used in assessing the two main parameters after graft healing: survival time and behavioral scores. For short-term studies, for example, to confirm cell survival or phenotypic composition, behavioral assessments are provided; survival testing in animals is carried out approximately 4-8 weeks after transplantation. Long-term studies include behavioral evaluation after transplantation and animal survival for at least 5 months after transplantation. When reviewing improvement strategies, such as modifications to PSANCAM, it is envisaged to include more complex parameters in the behavioral assessment, such as a ladder test to assess forelimb dexterity.

Example protocols for evaluating in vivo performance have at least four major components and include the following: i, induction of lesions, ii, basic behavioral analysis, iii, grafting, and iv, tissue analysis. While these procedures have been used in rats, in some embodiments, these procedures may be used in other species.

i) Induction of focal lesions. Unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) is one of the standard methods for inducing parkinson-like symptoms in rats. 6-OHDA is a neurotoxin that, through retrograde transport through the nigrostriatal pathway to MFB, enters back into the substantia nigra, leading to neuronal cell death as a result of damage to mitochondrial respiratory enzymes. Target neurons include A9 dopaminergic neurons in the substantia nigra compactus (SNC) as well as A10 neurons in the ventral tegmental area. This focal lesion model has been widely studied and is considered an excellent preclinical model for investigating the neurochemical and behavioral consequences of late stages of PD, as it leads to extensive unilateral depletion of dopamine in the caudate nucleus/putamen (CPU) complex. Behavioral consequences are also well documented and include spontaneous and drug-induced rotation, as well as impaired limb function (see below). Bilateral models of parkinsonism may better mimic human disease, however, leading to adipsia and aphagia in rats.

The procedure was performed in anesthetized animals (ketamine/xylazine) by stereotaxic injection at 2 sites along the medial forebrain bundle. The efficiency of induction of a complete focal lesion is largely dependent on the experience of the operator and in the development of the present inventions varied from 60 to 80%. Animals were allowed to recover and then subjected to a series of behavioral tests starting 2 weeks after surgical procedures.

ii) Basic behavioral analysis. Behavioral analysis begins 2 weeks after surgical procedures and continues after transplantation and until the sacrifice of the animal. The purpose of this analysis is 1) to establish that the focal lesion is stable and complete and that the animal did not experience spontaneous reversal of symptoms: a phenomenon that has been observed in animals with partial lesions, 2) to demonstrate the effect of transplantation of dopamine neurons on established behavioral parameters.

(a) Rotational behavior. Rats are observed to establish spontaneous rotations and D-amphetamine-induced (ipsilateral) rotations (10 mg/kg). An indicator of a significant focal lesion is a threshold value exceeded >6 rotations per minute. Apomorphine-induced rotations can also be tested, but positive results are thought to require >80-90% depletion of dopamine innervation in the caudate/putamen region, and are less stable in focal MFB lesions compared to focal CPU lesions. Three data sets are obtained at 2 week intervals and averaged. Rats with rotation scores <6 were excluded from the study.

(b) Walking test. This test is designed to determine the akinesia of the forelimbs. The experimenter holds the rat with one hand, fixing the hind limbs (slightly raising the trunk), and with the other hand fixes the forelimb, which is not monitored. Thus, all the weight is transferred to the other front paw. The rat is slowly moved laterally both in front and behind. Count the number of corrective steps in both directions for both paws.

- 49 041083 (c) Cylinder test. This test is designed to determine the asymmetry of the use of the forelimbs. The rat is placed in a transparent cylinder. During a period of 5 minutes, the behavior of the rat when rising on its hind legs is evaluated. The behavior is analyzed during lifting and lowering. The percentage of simultaneous and asymmetric use of the paws during the indicated movements is determined. This can be done using a computerized video monitoring system. Negative results in these tests are known to correlate with dopamine depletion, and results have been shown to improve after regenerative transplant as described herein.

iii) Transplant. Animals received stereotaxic injections of dopamine neurons into the striatum 3 weeks after the introduction of parkinsonism-inducing lesions if behavioral tests confirmed adequate lesions. The coordinates for injections are well known. After transplantation, the same group of behavioral tests are performed twice a month for varying periods of time (on average, it takes 5 months to achieve a stable behavioral recovery).

iv) Tissue analysis. Animals are sacrificed and perfused. The brain is dissected and prepared for immunohistochemical examination and stereological analysis. Antibodies include TH, FoxA2, Pitx3, Nurr1, Lmx1a, Girk2, DAT to identify and determine the function of dopamine neurons; 5-HT for identification of serotonergic neurons; human NCAM or human nuclear antigen for human identification; nestin, Sox2 to determine the precursors of neurons; Ki-67 to determine proliferation; Oct4, Nanog as pluripotency markers; alpha-fetoprotein; myosin; cytokeratin as markers of multilinearity to exclude the formation of teratomas. Quantifications include graft volume (Cavalieri assessment), total cell number, and dopaminergic cell number (using TH/FoxA2 dual labeling of neurons); and proliferative index (% Ki67+). The listed antibodies are commercially available and have been used in the development of the present inventions.

In some embodiments, the use of Sprague-Dawley (SD) rats is contemplated to better simulate the human situation. SD rats receive daily intraperitoneal injections of cyclosporine (15 mg/kg) starting the day before transplantation until sacrifice. With long-term injection of aqueous forms of cyclosporine (neoral, an oral solution used in humans), a negligible complication rate has been observed.

In some embodiments, examples of methods for scaling cultures of mDA neurons are provided, see Table 1. 9. In particular embodiments, such methods are contemplated for use in obtaining GMP grade cultures for clinical use.

Estimated parameters. In vivo testing is contemplated for use in methods for short and long term evaluation of graft efficacy. The results obtained in the development of the present inventions showed identical tissue characteristics in both cases, with an expected increase in the proportion of TH+ cells with long-term survival due to the differentiation of grafted progenitors.

On average, when transplanting 15,000 TH+/FoxA2+ neurons per 250,000 cells, animals survived for 5 months after transplantation. In short-term survival, significantly fewer double-labeled cells survived and were counted. Accordingly, the expected results in vivo, are given in table. 9 correspond to a conservative estimate. Examples of indicators used to assess the status of success/failure for the composition of transplants are given in table. 10.

For long-term transplants, behavioral evaluation is an essential component of evaluating the performance of hES line products. Guiding principles for defining loss of function and recovery have been best described for amphetamine-induced rotations, where a stable graft should normalize behavior and sometimes lead to contralateral movements due to DA imbalance. The standards given in table. 11 are examples of guidelines identified in the development of the present inventions.

In some embodiments, cell sources for use in differentiation methods of the present inventions include, but are not limited to, WA09, ACT (M09), Bio-Time, and Roslin cell lines. In some embodiments, sources of cells for use in the differentiation methods of the present inventions include, but are not limited to, GMP compliant lines. In some embodiments, cell sources for use in the differentiation methods of the present inventions include, but are not limited to, generating mature DA neurons for use in established graft short-term survival assays. In some embodiments, cell sources for use in differentiation methods according to the present inventions include, but are not limited to, generating mature DA neurons for use in graft engraftment experiments that include behavioral assessments. Control groups receive WA09 category for research and simulated saline. For statistical analysis, ANOVA with Dunnett's post hoc test is used.

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A. Comprehensive behavioral analyzes to assess the degree of reinnervation of the striatum. Standard behavioral tests (as described herein) showed a direct correlation with the number of surviving dopamine neurons in the graft. Rats treated with mature DA neuronal cells in accordance with the present inventions exhibit maximum recovery in behavioral tests with an estimated recovery rate of approximately 30% of neuronal DA, which is necessary to reduce the level of amphetamine-induced rotations. Accordingly, once the survival threshold of DA neurons has been reached in the host (estimate: 800-1200 DA neurons in a rat model), it may be difficult to discern behavioral differences reflecting improvement strategies. A method using micrografting has been described in which several small size grafts are placed within the striatum rather than large size grafts. Controlling the total number of transplanted DA cells, differences in behavioral outcomes were observed when comparing the 2 groups: rats with multiple small grafts had earlier and more extensive behavioral recovery with respect to drug-induced rotations and in walking tests compared to animals with one large graft. size, with the same number of DA neurons and the same level of dopamine release. Surprisingly, there was a clear difference in forelimb dexterity: Animals with widely distributed small size grafts showed improvement, in contrast to rats carrying standard grafts. Forelimb dexterity is thought to be a task that requires spatial and temporal control of DA release and, accordingly, proper communication between transplanted neurons and the host's striatum. Accordingly, in some embodiments, the use of mature DA neurons in accordance with the present inventions for transplantation procedures resulted in the restoration of forelimb use. Accordingly, in some embodiments, mature DA neurons in accordance with the present inventions are introduced into a single site of the striatum. In other embodiments, mature DA neurons according to the present inventions are introduced into at least 2 or more sites within the striatum.

B. Forelimb dexterity test (or ladder test).

The forelimb use test to analyze the stretching and grasping movements performed by the forelimbs was performed before and after the focal lesion, as well as after transplantation. Animals were fasted for 48 hours before the test, and tested daily for 5 days prior to the lesion, and then twice after the lesion with a 3-week interval. After transplantation, the test was repeated 2 more times, at 3 and 5 months. Animals were placed in a plexiglass chamber equipped with a double ladder. Food pellets are placed on 5 steps on both sides when tested before transplantation, and on one side (on the side of the affected limb, contralateral to rotation) after transplantation. Animals are tested for a predetermined time period (eg, 10 min), the number and proportion of pellets eaten (successful retrieval) and pellets that are captured are counted and compared to performance in the individual animal prior to lesion. Several variations of this test were used, differing in the number of repetitions and time characteristics of the test.

VIII. Differentiated using the methods described herein, cells showed electrophysiological responses similar to those of DA cells in situ.

The example below describes the use of illustrative methods in accordance with the present inventions to determine the functional capacity of DA midbrain neurons obtained as a result of differentiation using the methods described herein. DA substantia nigra compact (SNpc) neurons in vivo exhibit an electrophysiological phenotype that distinguishes them from most other neurons in the brain. In particular, they spontaneously generate spikes at low frequency (1-3 Hz). In addition, these slow spikes are accompanied by a slow subthreshold oscillating potential. After 2-3 weeks in vitro, the same indicated physiological features are demonstrated by cultured DA neurons of SNpc mice in the early postnatal period. To determine whether the mDA neurons of the present inventions exhibit a comparable electrophysiological phenotype, the unique electrical response characteristics of the mDA neurons of the present inventions were tested. DA midbrain neurons according to the present inventions were tested on days 80-100 of cultivation by single cell registration. These hESC mDA-differentiated neurons stably (4/4 tests) exhibited the indicated distinct physiological phenotype with specific autonomic spikes and oscillating changes in membrane potential (Fig. 3g-i). This behavior is known as autonomic pacemaker activity, is a specific property of DA of midbrain neurons and, in particular, of the subtype of dopamine midbrain neurons most relevant in Parkinson's disease (DA type of midbrain substantia nigra neurons).

The use of electrophysiological measurements on preparations of live sections is envisaged, i.e. biopsies from grafted areas. In one implementation of DA, type A9 or A10 neurons occur

- 51 041083 grafts are identified in vivo based on testing for autonomic pacemaker activity specific to L9-type dopamine neurons, which are most affected by PD. In other words, L10-type neurons do not have pacemaker activity. Conditions were set for in vivo registration of DA neurons derived from human pluripotent stem cells in live section preparations, see FIG. 26. In particular, the electrophysiological features of transplanted human DA neurons derived from pluripotent stem cells were evaluated and found to have electrophysiological features typical of mouse substantia nigra compact (SNpc) neurons; in fig. 26L top shows a reconstruction of the pacemaker neuron in the graft area. Below is an example micrograph of a section of a rat brain that had been injected with hES-derived neurons 9 months earlier; graft circled; a high magnification image is shown in the inset below. The section was processed to detect tyrosine hydroxylase, which was stained white, Fig. 26b. In addition, above is an example of patch registration in the attached cell configuration from a putative DA neuron in a graft; Below is an example of registering in a whole cell configuration from the same cell. Registration was performed in the presence of glutamate and GABA receptor antagonists (50 μM LR5, 10 μM CNQX, and 10 μM gabasin) to eliminate the synaptic input. The results of this registration showed that the neurons originating from PS cells were autonomous pacemakers with normal intrasomatic tension dynamics. Another registered neuron in the graft sample had similar properties, Fig. 26C. For comparison, the methods of registration in the attached cell configuration and registration in the whole cell configuration from the dopaminergic neuron SNpc of an adult mouse are presented. Abbreviations (CTx=cortex, STr=striatum, SNpc=substance substantia nigra compact, DA=dopaminergic). These data reflect in vivo functional studies in the striatum of transplanted rats months after transplantation. Accordingly, in some embodiments, in vivo functional studies on grafted tissue demonstrate recovery of substantia nigra compact (SNpc).

IX. Directed differentiation of mutant genetically determined PD-iPS PINK1 cells (PINK1 mutation) into DA neurons revealed Parkinson-like anomalies in mature DA neurons.

This example describes the discovery of obtaining large populations of differentiated midbrain DA neurons with characteristics of PD patient neurons using a PD patient cell line, i.e. mutant BP-iPSC PINK1 cells obtained by a method that does not lead to the destruction of the embryo, as a cell population for obtaining FOXA2/LIM1XA/TH+ DA neurons in accordance with the present inventions.

In one embodiment, the present inventors propose isolating a starting population of cells from a patient for use in methods for producing authentic DA neurons in vitro if said patient has a symptom of Parkinson's disease (PD), providing a potential benefit when using the treated cells in in vitro tests for 1) observing differentiation or functional abnormalities compared to authentic DA neurons from humans without a neurological symptom, and then 2) using the observed abnormality to develop a therapeutic treatment to reverse said abnormality; and 3) treating said patient with a therapeutic treatment to reduce, i. reversal of a symptom of Parkinson's disease.

In one embodiment, the authors of the present invention proposed the isolation of a starting population of cells from the body of the same patient to obtain authentic DA neurons for use in transplantation treatment, while the specified patient has a symptom of Parkinson's disease (PD), providing the potential benefit of reducing immunological rejection, those. transplant rejection. In other embodiments, graft rejection is reduced by using a source of original cells isolated from an individual with matching major histocompatibility complex (MHC) antigens (i.e., a twin) or a person with a transplantable level of MHC tissue matching (i.e., a relative). a patient or non-relative who expresses overlapping MHC molecules.

K. Directional differentiation showed that genetically determined PD-iPS PINK1 cells had the ability to develop into DA neurons, similar to midbrain DA neurons. See fig. 20-25. Differentiation of the mutant PD-iPSC line PINK1 Q456X into DA midbrain neurons was performed using a new protocol (method) based on the ventral plate described herein, which provided the production of DA midbrain neurons that showed differentiation profiles comparable to those of differentiated DA neurons. obtained using a new protocol based on the ventral plate of the midbrain from the H9 line. (Fig. 20). This example describes the discovery of obtaining large populations of differentiated midbrain DA neurons with characteristics of neurons in a PD patient using a cell line from a PD patient, i.e. mutant BP-iPSC PINK1 cells obtained by a method that does not lead to the destruction of the embryo, as a population of cells to obtain

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FOXA2/LIM1XA/TH+ YES neurons according to the present inventions.

The PINK1 Q456X mutant PD-iPSC line cells were differentiated into midbrain DA neurons using a new protocol (method) based on the ventral plate in accordance with the present invention, which provides differentiation profiles into midbrain neurons comparable to those for the H9 iPSC line. A-C) Immunocytochemical analysis of mutant PINK1 BPiPSCs at differentiation day 11 (midbrain progenitor stage), for FOXA2 (red), LMX1A (green) and DAPI (blue) (A), differentiation day 25 (early post-mitotic stage of DA neurons), at FOXA2 (red) and TH (green) (B) and on NURR1 (red) and TH (green) (C). D-F) The same group of immunocytochemical assays performed using H9-derived cells, at differentiation day 11, for FOXA2 (red), LMX1A (green) and DAPI (blue) (D), at differentiation day 25, for FOXA2 (red) and TH (green) (E); and on NURR1 (red) and TH (green) (F).

B. Genetically determined PD-iPSCs exhibited a PD-like phenotype with protein aggregation. Fig. 21-24. The present inventors found that PINK1 mutant PD-iPSCs exhibited expression of α-synuclein (a major component of Lewy bodies in PD patients) in the cytosol of TH+ DA neurons at differentiation day 55 using a new ventral plate-based midbrain DA induction protocol ( Fig. 21A-B). A, B). Immunocytochemical analysis of mutant BP-iPSCs of the PINK1 line at day 55 of differentiation to α-synuclein (LB509, red), TH (green); and combined image (A) for α-synuclein (red) and ubiquitin (green) (B). These α-synuclein positive cells also showed high expression levels of ubiquitin (a classic marker of Lewy bodies). In contrast, DA neurons derived from the control iPS cell line displayed normal synaptic (as opposed to cytosolic) expression of α-synuclein and very low levels of ubiquitin (Fig. 21C-D). C, D) Immunocytochemical analysis of a control iPSC line at day 55 of differentiation for α-synuclein (red) and TH (green) (C); and a-synuclein (red) with ubiquitin (green) (D).

C. Expression of the aggregated form of α-synuclein. In the brain of a PD patient, the dimerized insoluble form of α-synuclein leads to aggregation in Lewy bodies. In this dimerized form of α-synuclein, phosphorylation of serine 129 on α-synuclein is observed. On the same day of differentiation, the mutant BP-iPSC-derived PINK1 cells showed strong expression of Ser129-phosphorylated α-synuclein, in contrast to control iPSC-derived cells that showed very low levels of expression (Fig. 22).

In mutant BP-iPSC-derived PINK1 cells, pronounced expression of Ser129-phosphorylated α-synuclein was observed, in contrast to control iPSC-derived cells, which showed very low levels of expression. A, B) Immunocytochemical analysis for Ser129-phosphorylated α-synuclein (green) and DAPI (blue) in BPSC-derived mutant PINK1 cells at differentiation day 55 (A) and corresponding control iPSC-derived cells (B).

D. Differences in α-synuclein expression patterns are observed depending on the differentiation protocol. The present inventors hypothesized that authentic midbrain DA neurons derived from the ventral plate exhibited PD-specific vulnerability and associated specific phenotypes in vitro. DA neurons generated using the classic MS5 stromal feeder cell differentiation protocol (Perrier et al., PNAS 2004, cited herein incorporated by reference) produced significant numbers of TH+ neurons. However, based on the data obtained during the development of the present inventions, the authors of the present invention showed that MS5-based TH+ cells were not authentic midbrain-derived DA ventral plate neurons. Many α-synuclein positive cells were present in cultures differentiated using the MS5 protocol. However, these cells did not co-express TH. In addition, there were no differences in expression patterns between BP-iPSCs and control iPSCs when the MS5 differentiation strategy was applied (Fig. 23AB). These data show that α-synuclein is also expressed in other cell types that are not DA cells, and that such non-DA α-synuclein is not altered in diseased cells relative to iPSC-derived control cells, particularly when standard differentiation protocols are used. with MS5. These cells are represented by DA-like rosette-derived neurons described in published sources (eg, Perrier PNAS 2004). These MS5-based TH+ cells (=DA-like cells) are used for comparison in FIG. 3, 10, 13, and 16. These data show that α-synuclein is also expressed in other non-DA cell types, and that such non-DA α-synuclein is not altered in diseased cells relative to iPSC-derived cells, in in particular, when using standard MS5 differentiation protocols. Finally, the new ventral plate-based differentiation protocol described herein yields a significant number of TH+ cells co-expressing α-synuclein.

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These TH+ cells express α-synuclein according to a cytosolic expression pattern. Fig.

24A, B) Immunocytochemical analysis for α-synuclein (LB509, red), TH (green) of mutant PINK1 BPiPSCs at differentiation day 60 based on MS5 (A) and control iPSCs (B); C) Immunocytochemical analysis of mutant BP-iPSCs of the PINK1 line at day 55 of differentiation based on the ventral plate for α-synuclein (red), TH (green).

E. DA neurons derived from genetically determined PD-iPS cells are more vulnerable to toxic stimulation. Fig. 24-25. BP-iPSC-derived TH+ DA neurons derived from the ventral plate protocol were more vulnerable to toxin stimulation (valinomycin: mitochondrial ionophore, 5 μM (concentration range 1-10 μM), 48 h) than those derived from control iPSCs cells. In contrast, TH+ neurons generated using the classic MS5-based protocol did not show differential vulnerability of BP-iPSC-derived and control iPSC-derived cells (FIG. 24). Alamar blue overall cell viability analysis after 48 h of valinomycin treatment also demonstrated differential cell survival upon toxin stimulation in a specific dose range (5 and 10 μM) when comparing BPiPSCs and control iPSCs (Fig. 25). Reference conditions: BP-iPSC-derived and control iPSC cultures obtained using an MS5-based protocol (D, showing BP-iPSC-derived cells); TH+ neurons after toxin stimulation in BP-iPSC-derived (E) and control iPSC-derived cultures (F) obtained using the MS5 protocol. G-H) Low power immunocytochemical images for Tuj 1 (red) and TH (green) at differentiation day 60 using a ventral plate protocol: BP-iPSCs under normal conditions (G) and under toxin challenge (H); control iPSCs under normal conditions (I) and under toxin stimulation (J). K-N) Low power immunocytochemical images for Tuj 1 (red) and TH (green) at differentiation day 60 using an MS5-based protocol: BP-iPSCs under normal conditions (K) and under toxin challenge (L); and control iPSCs under normal conditions (M) and under toxin stimulation (N).

F. Example of cell viability quantification - dose-response analysis of toxin stimulation. Cell viability assay with alamar blue after 48 h of valinomycin treatment demonstrated differential cell survival when stimulated with toxin in a specific dose range (5 and 10 μM) when comparing BP-iPSCs and control iPSCs (day 60 of differentiation based on the ventral plate). Note that these assays are designed to measure overall rates of cell death, while most of the pronounced effects were observed specifically in DA neurons (see FIG. 14). Accordingly, Alamar blue-based quantification is likely to underestimate the degree of differential effect observed in DA neuronal lines.

X. The proposed large-scale cultivation using the compositions and methods in accordance with the present inventions to obtain exemplary MDA neurons.

The descriptions provided herein provide exemplary methods and applications for large-scale differentiation production of MDA neuronal cells using compositions and methods as described herein. Scalable production (i.e., methods believed to be suitable for successful production of mDA neurons from cultures containing relatively low numbers of cells) has been shown to produce cell populations capable of providing suitable mDA neurons for transplantation at day 25. In particular, for iPSC PINK. See table. 9).

XI. Ways of DA enrichment with midbrain neurons.

Several methods have been developed and tested, before and during the development of the present inventions, to enrich cell populations with DA progenitors of midbrain neurons, by solving problems, for example, by depleting contaminating cell populations, including, but not limited to, contaminating pluripotent stem cells. . Initial studies were performed using primary mouse embryonic neurons, rat embryonic neurons, and mouse ESC-derived populations. In addition to the goal of increasing neuronal populations for use in neuronal enrichment with DA, studies in mouse ESCs have included the development of procedures to prevent teratoma formation, which has been a problem with previous procedures. These strategies included negative selection for cell surface markers expressed on pluripotent cells (such as SSEA1) along with positive selection for cells expressing a neuronal marker (NCAM). A number of genetic reporter strategies involving mouse cells have been proposed for use in identifying neuronal enrichment in neuronal DA transplantation paradigms (eg, identifying cells expressing SOX1, Corin/Lmx1a, Ngn2, TH, Pitx, or DAT). Functional testing was performed on primary cells from Ngn2 reporter mice (Thomposon et al., Exp Neurol. 198(1): 183-98 (2006) and Lmx1A reporter mice, also sorted by Corin (Jonsson, Exp Neurol. 219(1 ):34154 (2009) In the case of populations derived from mouse ESCs, studies were performed using porter mouse ESC lines with SOX1 markers (Barraud et al., Eur J Neurosci. 2005 22(7):1555-69 ), tyrosine hydroxylase TH (TH) (Kelly et al., Minerva Endocrinol. 1991 16(4):203-6), Pitx3 (Hedlund et al., Stem Cells. 2008 26(6):1526-36) and the dopamine transporter DAT (Zhou et al., Stem Cells. 2009 27(12):2952-61) In addition, in the development of the present inventions, the inventors conducted a comprehensive transplantation study with a direct comparison of in vivo performance of three purified mouse ESC-derived populations representing sequential developmental stages of DA neurons: midbrain progenitors (Hes5::GFP), early postmitotic cells ( Nurr1::GFP), and mature DA neurons (Pitx3::YFP). These studies have revealed that Nurr1-expressing developing DA cells are particularly suitable for transplantation and have shown that DA-purified neurons are able to engraft effectively when transplanted in vivo. In addition, these results were used to select cells at differentiation day 25 (Nurr1 expression onset) for use in transplantation of hESC-DA neurons in mice, rats, and rhesus monkey models of PD.

During the development of the present inventions, excellent in vivo performance was observed when obtaining authentic DA midbrain neurons from hESCs (see Fig. 4); the use of the protocols as described herein resulted in approximately 40% DA neuronal recovery at the time of transplantation. This percentage is higher than the percentage of DA neurons obtained from the dissection of human ventral midbrain fetal tissue (generally about 10%). Accordingly, it is expected that the use of the hESC-based source of DA neurons as described herein will provide a further increase in purity when applying cell purification strategies. In addition, genetic reporter lines for use with hESCs similar to those used in mice have been developed, including a human cell line as the Nurr1::GFP line. However, the use of genetic reporters can be problematic in human translation, as GFR is immunogenic in humans and therefore not suitable for use in humans. In addition, FACS sorting to establish a master bank of clinical-grade DA neurons (i.e. obtaining frozen cultures of human DA neurons for use in transplantation) can be difficult given the length of time required to sort batches of approximately 109 cells required for each transplant. in addition to the potential high cost and lower cell recovery from storage. Instead of using genetic reporter systems and cell isolation based on the FACS method, the inventors proposed the isolation of DA neurons based on the expression of surface markers using alternative strategies using cell separation techniques proposed for use in patients with PD. Thus, sorting using magnetic particles (eg, the CliniMACS® system) has been widely used in FDA-approved cell-based methods, and provides fast, cost-effective isolation of up to 10 ¹⁰ cells under Good Medical Practice (GMP) conditions, i.e. conditions approved for the isolation of cells intended for use in humans. Accordingly, in some embodiments, sorting using magnetic particles to enrich for mature DA neurons, such as using CD142 attached to magnetic particles to enrich Nurr1+ neurons for use in transplants, is proposed.

XI I. Identification of cell surface markers for use in methods for obtaining mature DA neurons.

The cell surface marker expression data collected during the development of the present inventions allowed the identification of several new cell surface markers expressed on midbrain DA neurons. In particular, markers have been found to further identify cells, such as specific cells that differentiate into mature DA neurons, mature DA neurons in accordance with the present inventions, and A9 cells. Two main strategies have been employed to identify such surface markers: first, an unbiased screen for gene expression in genetic reporter lines (Fig. 27a) showed the presence of several candidate markers, including CD142 and a marker called DCSM1, which was selectively expressed in midbrain DA neurons and was found to be, in particular, the DA marker of A9-type neurons (Fig. 27b). The second strategy was to use screening for cell surface CD markers in hESC-derived DA neurons, which were tested with 242 commercially available antibodies in a 96-well format (Fig. 27c, d). The results of this type screening (Fig. 27e) identified at least 5 confirmed markers that midbrain DA neurons are rich in, including CD142, a selective marker specific to the Nurr1+ stage of DA neurons (Fig. 27f), in addition to CD63, CD99, and DCSM1.

In particular, as seen from FIG. 27, CD surface marker screening of WA09-derived DA neurons at differentiation day 25 tested with up to 242 individual antibodies. GFP+ motoneurons from hESC-derived cortical neurons, hESC-derived Nkx2.1::GFP+ ventral forebrain progenitors, and several other hESC-derived neuron types.

The new database of surface marker expression profiles was then used to select candidate CD markers selectively enriching any given subtype, eg midbrain DA neurons (FIG. 27). One of the identified markers associated with the differentiation of DA neurons from hESCs was CD142. Selection of cells for CD142 that are specifically enriched, in particular, for hESC-derived DA neurons at the Nurr1+ stage in the depletion of other neuron subtypes. In some embodiments, CD142 is expressed prior to Nurr1+. In some embodiments, the CD142 sorted population of midbrain DA neurons contains Nurr1+ and Nurr1- cells. In some embodiments, the CD142-sorted population of midbrain DA neurons contains Nurr1- cells. In some embodiments, a population of cultured CD142+ sorted Nurr1-DA midbrain neurons begins to express Nurr1 (ie, becomes Nurr1+) within a period of up to two days after sorting.

In addition to CD142, hESC-derived DA neurons were enriched in CD63 and CD99 markers. Accordingly, in some embodiments, DA nerve cell cultures enrich DA neurons by sorting or selecting for markers including, but not limited to, CD142, CD63, CD99, DCSM1, Nurr1+, etc. The CD142 marker is typically present in approximately 30% of the total cells in the population at day 25 of differentiation (FIG. 28a). The selectivity of CD142 for the stage of Nurr1+ DA neurons was confirmed for several independent hESC and hPSC lines (Fig. 28b). Importantly, in addition to enriching DA neurons, CD142 selectively depletes other neuron subtypes such as GABA and serotonergic neurons. (Fig. 28c-f). In vivo studies have been conducted demonstrating the ability of CD142 to lead to the formation of highly pure DA neurotransplants without detectable impurities of GABA and serotonergic neurons. Serotonergic neurons are a cell type that has been implicated in human fetal tissue transplantation as a potential source of graft-induced dyskinesia. Although the use of transplantation methods as described herein using crude cells has already resulted in a paucity of serotonergic neurons, the use of CD142 should reduce this risk to an even greater extent.

A. Markers for the identification of mature A9-type mDA neurons. A9-derived/A10-derived DA neurons have been found to have, in vitro and in vivo, distinct functional characteristics and specific innervation patterns consistent with their role in the functioning of the mesostriatum/mesolimbic system. During the development of the present inventions, the authors found that authentic mDA neurons obtained by methods in accordance with the present inventions (Fig. 4), lead to the appearance of neurons with properties that are more characteristic of A9 than A10. In particular, at least a portion of authentic mDA TH positive (TH+) neurons expressed Girk2, a marker used to detect DA in A9-type neurons. In addition, many mature DA neurons exhibited autonomous pacemaker activity, which is a functional characteristic of A9-type, but not A10-type, DA neurons. However, some TH+ cells obtained in vitro did not belong to the A9 type. Accordingly, the inventors propose enrichment procedures, such as those described herein, to obtain purified populations of authentic A9-type (rather than A10-type) human mDA neurons. As described herein, the inventors have found at least two markers unique to A9 type neurons and at least two markers unique to at least A10 type neurons. Accordingly, in some embodiments, A9 type neurons are identified by markers (Girk2, Aldh1) other than A10 markers (calbindin, Otx2).

B. Determination of a set of markers that increases the output of DA neurons of the midbrain A9 subtype. At least two strategies have been proposed to detect A9-specific surface markers: candidate markers generated using gene expression screening, e.g., as described herein, and candidate CD antibodies using surface marker screening, as described herein. . Populations in another method perform global transcriptome analysis in purified populations of mouse ESC-derived mDA neurons at different stages of differentiation (using BAC transgenic technology; see Fig. 27a,b). The presence of surface markers was detected in the profile of surface markers on DA neurons derived from RCB WA09 using the following exemplary methods. RCB WA09 DA-derived neurons at differentiation day 25 were dissociated and replated in 96-well plates and then treated with 242 CD antibodies and analyzed using the Operetta (Operetta High Content) multivariate analysis scanner. The amount of DA enrichment was tested for at least 5 additional antibodies bound to CD markers identified in these screens (eg CD142, CD63 and CD99). Candidate CD-positive/CD-negative cells were evaluated using a DA QC conformity assay, including FOXA2/TH and TH/Nurr1 expression (see Table 7). In some embodiments, the use of global gene expression profiles to compare unsorted CD142 (CD142+) cells is proposed. In some embodiments, expression of the desired marker in sorted/separated cells has been used for short and long term in vivo studies with

- 56 041083 as described in this document. Among the neuronal DA-specific markers identified in these studies was a surface marker gene named DCSM1 (DA Cell Surface Marker 1). Based on in situ expression data, expression corresponds to the ventral midbrain, however, more surprisingly, expression is at least partially selective for A9 in both developing and adult mouse brains (Fig. 27) and adult brains. human brain. In hESC-DA-derived neurons, many cells expressing DCSM1 were observed. In vitro assays for A9/A10 membership included long-term differentiation of marker-positive cells (day 50 and day 75 of differentiation) and analysis of i) expression of A9, (Girk2, Aldh1) or A10 (calbindin, Otx2) markers in mature neurons, (ii ) differential axonal guidance in response to Netrin-1 and Sema3, and (iii) analysis of A9-enriched neurons with electrophysiological tests. DA A9 neurons exhibited specific functional characteristics as described herein for hESC-derived A9 neurons. Cells expressing DCSM1 and other markers were examined in vivo to confirm i) expression of A9 markers (Girk2, Aldh1) or A10 markers (calbindin, Otx2), ii) DA graft fiber outgrowth, and iii) electrophysiological properties of A9 transplanted cell sections (see Fig. 26).

XII I. Use of polysialic acid (PSA) and polysialyltransferase (PST) enzyme.

Graft integration and extent of DA fiber outgrowth are problematic issues in transplantation techniques, including studies of fetal tissue transplantation in patients with PD. One of the problems encountered in transplanted tissues and cells is the limited growth of fibers from these transplants in the treatment of patients. This problem is especially critical for humans, since recovery in the patient requires extensive reinnervation of the striatum. In previous methods, to achieve adequate reinnervation after tissue transplantation, it was necessary to repeatedly inject portions of cells into different areas of the striatum. Each of the injections can lead to striatal injury and inflammation, and is associated with other surgical risks. Such risks include damage to the blood vessel during cell injection, potentially causing a stroke or seizure in the patient. PSA is a naturally occurring sialic acid homopolymer (namely, alpha-2,8-linked sialic acid) on the cell surface that has been found to be a post-translational modifier (through the action of the polysialyltransferase (PST) enzyme) of other cell surface molecules such as like (polysialylated) nerve cell adhesion molecule (NCAM), NCAM (CD56) and the like. It has been found that PSC is involved in the regulation of the plasticity of certain types of cell behavior that require changes in intercellular interactions, including cell migration and axon outgrowth. Being intensely expressed in the embryonic organism, PSC is downregulated in the tissues of the adult organism, with the exception of local areas of the CNS that maintain structural and physiological plasticity (such as the hippocampus, suprachiasmatic nucleus, SVZ). Accordingly, in some embodiments, polysialic acid (PSA) is provided for use in promoting fiber outgrowth of successfully transplanted cells. Examples of the use of PSC in other cell types are given in WO 2006/042105, incorporated herein by reference in its entirety. In some embodiments, the authors of the present invention contemplate the use of authentic DA neurons in combination with PSCs as described herein.

A. Increasing the number of PSCs in DA neurons for use in patients with PD. Many problems remain in developing methods and generating cells based on previous results in small animal models, including limited survival of transplanted cells and insufficient fiber innervation of the host tissue. The impact of these limitations is expected to be more severe in the larger human striatum and, accordingly, improved survival and innervation is required for effective clinical applications of DA-ESC-derived neurons. Improvement in fiber outgrowth and graft integration in animal models involving at least one and up to multiple injections is expected to reduce the risks associated with repeated injections or insufficient expansion of DA neurons in vivo.

Regulation of cellular interactions by polysialic acid (PSC) is one of the factors contributing to cell proliferation, axonal outgrowth, and directed innervation in vertebrate development, see, for example, Rutishauser, Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat Rev Neurosci 9, 26-35 (2008). PSC was a carbohydrate polymer attached to a neural cell adhesion molecule (NCAM) that attenuated intercellular interactions and thus promoted tissue plasticity. In the glial scar, increased PSC expression in the adult GM promoted the migration of neuronal progenitors from the subventricular zone to the cortex and improved axonal growth (El et al. Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci USA 103, 16989-16994 ( 2006) As described herein, the artificial increase in PSC expression on purified mouse ESC-derived DA neurons resulted in an increase in the number of graft cells, extensive proliferation

- 57 041083 DA fibers of neurons in the host striatum and, unexpectedly, improved recovery of behavioral functions in mice with Parkinson's disease. Further, the use of genetic engineering to increase PSC levels on the surface of DA ESC-derived neurons simultaneously increased in vivo survival and outgrowth of fibers in the host striatum. One exemplary embodiment using the mammalian PST gene, i. mouse or human is shown in FIG. 29. In other exemplary embodiments illustrated in FIG. 29 shows the use of bacterial PST, ie. PSTnm. In particular, as described herein, the increase in the number of PSCs on cells in cell therapy based on mature DA neurons is proposed for use in the treatment of PD.

B. Use of mouse PST to increase PSC expression. In vivo results indicate improved neurite outgrowth and a significant regression of amphetamine-induced rotation in 6-OHDA mice transplanted with PST-modified mouse cells, whereas equal numbers of non-PST-modified cells did not provide this result (Fig. 33). In addition, a correlation was observed between improvement in DA fiber innervation and improvement in behavioral outcomes in a mouse model of PD, for example, when small (injection of approximately 50,000 cells) PSC-positive cell grafts were observed, the latter provided graft integration and fiber outgrowth, accounting for approximately 70% of the captured areas. relative to comparable larger grafts (100,000 or more cells). Comparison of cells derived from DA ESC neurotransplants after PSC stimulation and after control treatment showed the restoration of behavioral functions in the PSC group (each transplant consisted of 55,000 transplanted cells), but was not observed for control cells. When 100,000 DA ESC-derived neurons were transplanted into the brain of mice, behavioral recovery was observed due to DA ESC-derived neurons treated with either PSC or control treatment, which presumably means that the graft volume of 100,000 cells is sufficient for reinnervation. mouse brain in the absence of PSC stimulation. Accordingly, in another embodiment, the artificial expression of PSCs on the surface of authentic DA neurons is proposed for use in procedures for the treatment of patients with PD. When PSC was induced on nerve cells in accordance with the present inventions, said PSC was identical to the PSC polymer naturally occurring in brain cells, thus, in contrast to the use of other cell surface molecules to construct therapeutic cell types, cells engineered to express PSC are presumably have low antigenicity in vivo when used for transplantation procedures in humans. In addition, high levels of PSCs on successfully transplanted cells or endogenous neuronal progenitors (Battista et al., J. Neurosci. 30(11):3995-4003 (2010)), Schwann cells (Ghosh et al., Glia. 60( 6):979-92(2012)), or DA ESC-derived neurons according to the present invention, did not cause detectable side effects when used in various adult rodent models. Artificial expression of effective levels of PSC involves one enzyme, polysialyltransferase (PST), whose only product is this unique glycopolymer. Surprisingly, the amount of protein expressed and the nature of the product of this enzyme remained largely constant and close to those of PSC found naturally in fetal tissues.

As described herein, artificial expression of PSCs on nerve cells is provided for use in transplantation procedures. In particular, the use of PSCs in the preparation of cells for therapeutic use has been proposed to solve problems that arise when using the inducible expression of other types of cell surface markers. In addition, the use of PSC expression provided reproducibility in interspecies protocols for vertebrates (for example, when using mouse PST genes for expression in human cells), since its acceptors also differ in compatible structure in different species and are found on the surface of most cells. Insertion of PST genes into hESCs to increase the number of PSCs on DA neurons. The gene encoding human polysialyltransferase (4PST) was introduced into the hESC cell line (WA01) using a lentiviral vector (pLenty, Invitrogen) and as described herein. Twenty selected clones were expanded and analyzed for PST expression. PST-expressing hESC clones were differentiated to confirm that the PST gene in DA neurons was not silenced. Quantitative determination of PSC-NCAM at different stages of differentiation (days 0, 11, 25, and 50) was performed using FACS analysis and immunofluorescence (Operetta). The indicators of positive clones were compared with a set of qualitative control indicators of DA neurons, shown in Table. 7. At least 3 clones that maintained high, constant levels of PSC-NCAM throughout differentiation and showed good quality controls (Table 7) were selected to assess neurite outgrowth in PST-overexpressing hESC-DA-derived neurons. Sorted control and PST-overexpressing hESC clones were differentiated into DA neurons using a standard protocol as described herein, after which cells were fixed and analyzed at days 25 and 50. The number and length of TH-positive fibers in such cultures were quantified using a microscope.

- 58 041083

Operetta High Content. Neurite number and length, with or without PST, were quantified using the Harmony 3.0 neurite analysis module, and the data were statistically analyzed using two-way ANOVA. PST overexpressing and control hESC clones, selected from the in vitro studies described above, were again differentiated into DA neurons and transplanted into PD model rats. Conducted short-term transplant studies (4-6 weeks) to determine the survival, expression of PSC-NCAM and outgrowth of neurites. All clones that passed short-term in vivo trials with satisfactory performance were used for long-term transplantation studies. In these studies, animals were injected with half or a quarter of the standard (200x103) dose of cells. These studies were aimed at elucidating whether an increase in the number of PSCs leads to an increase in long-term survival after transplantation (5 months), and whether smaller numbers of DA neurons are able to provide the same or better functionality compared to non-PST grafts containing standard cell doses ( Fig. 27).

Additionally, complex behavioral tests were performed, sensitive to the degree of striatal reinnervation, for additional monitoring of differences in the functional potential of PST transplants and control DA of neuronal transplants. After completion of the behavioral tests, the animals were sacrificed and fiber outgrowth was quantified using human specific antibodies NCAM and SC121 and anti-TG antibodies (see also FIG. 29). We measured the intensity and distribution of hNCAM+, SC121+, and TH+ in the graft, as well as the percentage of human cells co-expressing neuronal DA markers (TH, FOXA2) and PSC. The NCAM/TH+ halo density of neurites propagating from the graft was quantified at different distances. Data were compared between groups using two-way ANOVA with Bonferroni correction. Sections were additionally examined to detect qualitative changes (eg branching, thickness, spread and shape of the graft). In addition, some grafts are sectioned for electrophysiological studies (see FIG. 26) for A9 phenotype, synapse formation with the host striatum, and endogenous afferent innervation.

The following example describes the stimulation of polysialic acid expression that improved the functioning of grafts from ESC-derived dopamine neurons in mice with Parkinson's disease.

ESC cells expressing GFP under the control of the Nurr1 promoter (Nurr1::GFP ESC) were stably transfected with a lentiviral vector ubiquitously expressing polysialyltransferase (PST). Transfected cells showed a dramatic increase in PST mRNA content compared to controls (FIG. 30A). PST expression was observed to be sufficient for PSC synthesis on NCAM. Accordingly, PSC-NCAM expression was significantly increased in PST-modified cells on day 14 of DA neuronal differentiation (Fig. 30B-E). Both endogenous and induced PSC on the cell surface of ESC-derived DA neurons can be removed (Fig. 30E) by phage endoneuraminidase (endoN), which cleaves, in particular, the unique alpha-2,8-linked sialic acid polymers in PSC. Surprisingly, PST transfection was not observed to affect the expression of neuronal or midbrain markers in GFB-purified DA neurons (FIG. 30F).

Other studies in hemiparkinsonian mice with 6OHDA-induced damage have shown that transplantation of approximately 100,000 ESC-derived DA progenitor neurons is required to ensure reliable functional recovery, as estimated using the amphetamine-stimulated rotation test. In the course of the present studies, the authors sought to transplant a suboptimal number of cells in order to be able to evaluate improvements due to stimulated expression of PSCs. For transplantation of highly enriched DA neuron populations in FACS-purified cultures depleted of contaminating pluripotent cells, testing for Nurr1-driven GFB expression and lack of SSEA-1 expression was performed on day 14 of differentiation (Fig. 31). In the absence of PST overexpression, when the minimum effective graft size was halved (55,000 Nurr1+DA cells), there was no detectable recovery of behavioral functions. On the other hand, with increased PSC expression, the same number of Nurr1/PST DA neurons provided significant correction of PD-related behavioral disorders (p<0.01; two-way ANOVA), with complete recovery approximately 5 weeks after surgery (Fig. 32A) . Removal of PSCs prior to transplantation by incubation with endoN showed the specificity of stimulation of PSCs, as endoN treatment partially reversed the recovery of functions due to the application of Nurr1/PST (Fig. 32A).

To study the characteristics of transplanted cells, an immunohistochemical study of animals was performed two months after transplantation. There was a difference in the number of living Nurr1+ neurons; those animals transplanted with PST-transfected cells had, on average, twice as many GFR+ positive cells as those transplanted with control cells (9300+/-1400 versus 4230+/-1010 GFR+ cells per

- 59 041083 transplant, for PST or control samples, respectively; fig. 32B, p<0.05, Student's t-test). In addition, Nurr1/PST transplants also showed higher levels of PSC expression in vivo (Fig. 32C, D). At the same time, the proportions of cells expressing the DA markers of midbrain neurons TG and FoxA2 in the center of the transplant were comparable for Nurr1 and Nurr1/PST cells (TG: 62.0%+/-8.0 vs. 51.3%+/-7, 0, p=0.33, FoxA2: 63.2%+/-8.6 vs 55.4%+/-2.0, p=0.3, respectively, Figure 32E).

Neuronal processes mediated by Nurr1 and Nurr1/PST cells indicate comparable levels of TG, Girk2 (G protein-coupled potassium channel of internal rectification) and synapsin (Fig. 33A). Unlike other studies with Schwann cell transplantation (Ghosh, et al. Glia 60, 979-992 (2012)), increased PSC expression has little effect on migration of DA cells from the transplant area. However, distinct changes in neurite outgrowth were observed. As seen in FIG. 33B, Nurr1/PST cells mediated more DA neuronal processes compared to control Nurr1+ cells. When quantifying the intensity of GFB and TG immunofluorescence in five successive zones of 100 µm at different distances from the graft, Nurr1/PST grafts showed a significantly higher relative process density (Fig. 33C, D; p<0.01 for both GFB and for TG, two-way ANOVA). In the quantitative analysis of this effect, the relative density of the processes was normalized by the density observed in the zone closest directly to the center of the graft. This normalization was necessary to compensate for the higher number of viable cells in Nurr1/PST transplants and to confirm the specific effect of PSCs on neurite outgrowth. Specificity has also been demonstrated when PSCs are removed from the cell surface prior to transplantation by treatment with endoN. Accordingly, endoN pretreatment reduced peripheral fiber outgrowth to control levels (FIG. 33E).

These findings have shown that at least some of the effects of PSCs on graft function are the result of increased fiber innervation of the striatum. Accordingly, there was a high correlation between graft performance and the relative level of outgrowth of GFB-positive fibers, eg, into zone IV (Fig. 33F; p<0.001, r2=0.65, n=17). Surprisingly, the association of fiber outgrowth with behavioral function in the experimental groups (control, CPS-stimulated, and endoN-treated) was stable, indicating that graft-host innervation is a predictor of recovery of behavioral function in a mouse model of PD. A number of factors mechanically contributed to increased fiber outgrowth, such as increased permeability of the reactive glia encapsulating the center of the graft, increased sprouting ability, improved outgrowth into surrounding host tissue (eg, easier movement of the growth cone), and prevention of premature formation of bonds with host tissue in the immediate vicinity. proximity to the center of the graft. Typical mechanisms are consistent with the role of PSC, which contributes to the process of growth during normal development and in the nervous system of an adult organism.

The experiments described herein demonstrate the use of artificially stimulated PSC in transplantation of DA neurons, providing superior results compared to transplants from other cell types. The data clearly show that PSC stimulation provides a significant increase in the ability of transplanted DA neurons to innervate the host striatum and attenuate functional impairment in PD. Accordingly, a transition to clinical use involving DA neurons in accordance with the present inventions is proposed to obtain cells prior to transplantation. In some embodiments, cells are genetically modified to express PSCs. In some embodiments, PST can be delivered directly to cells by in vitro treatment with purified enzyme and substrate prior to transplantation. In some embodiments, a CPS-involving strategy is provided to transition to human use in PD transplant to minimize the need for repeat injections and thus reduce the surgical risks associated with said repeat injections.

In other embodiments, this technology is proposed for use with other types and types of cells, for example, to increase the migration of transplanted Schwann cells when creating a bridge (for example, intercellular communication) to resume growth of axons at the site of damage to the spinal cord.

Below are typical materials and methods used in this example.

Animals: 6 weeks old 129S3/SvImJ mice (Jackson Laboratory) were kept under controlled temperature conditions with free access to food and water. Experimental procedures were carried out in accordance with the National Institutes of Health (NIH) and Institutional Animal Care and Use regulations, and were approved by the local Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

6OHDA injection and amphetamine-induced rotation test: Animals were anesthetized with sodium pentobarbital (10 mg/kg) and injected into the striatum on the right side with 2 µl of 6OHDA (4

- 60 041083 µg/µl in saline, 0.5% ascorbic acid). Injection was carried out using a Hamilton syringe in the area with coordinates: 0.5 mm in the posterior and 1.8 mm in the lateral direction from the bregma and 2.5 mm ventral to the GM surface. Prior to surgery, animals received one intraperitoneal injection of desipramine (25 mg/kg, Sigma). Two weeks after surgery, the animals were evaluated in the amphetamine-induced rotation test. Animals were placed on transparent plastic cylinders with a diameter of 30 cm for half an hour, after which they received one intraperitoneal injection of amphetamine (10 mg/kg, Sigma). After 20 minutes and for the next 20 minutes, the number of ipsilateral/contralateral rotations is counted. Animals were tested once a week for seven weeks, then, under deep anesthesia, they were perfused through the heart with PBS and 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed and additionally fixed overnight at 4° C. in 4% paraformaldehyde, and then 40 μm thick sagittal sections were obtained using a vibratome (Pelco-101, Ted Pella). Cell differentiation and transplantation: Nurr1::GFP BAC transgenic reporter line of BAC mouse ESCs (i.e., GFB expression is regulated by the Nurr1 promoter) 5 cells were transfected with a lentivirus (pLenti, Invitrogen) containing the mouse PST gene under the control of the CMV promoter. ESCs were propagated on mitomycin C-treated EFM cells (StemCell Technologies) in DMEM (Invitrogen), 10% FBS (HyClone) supplemented with 1400 units/ml LIF (ESGRO; Invitrogen), 2 mM L-glutamine, 1 mM β-mercaptoethanol, 100 U /ml penicillin and 100 µg/ml streptomycin (Invitrogen). DA cell differentiation was induced according to Barberi et al., NatBiotechnol 21, 1200-1207 (2003), with some modifications. Briefly, cells were differentiated on MS5 feeder cells on gelatin-coated dishes (10,000 cells per 10 cm diameter dish) and cultured for 4 days on serum replacement medium (SRM). On day 4, Sonic hedgehog proteins (SHH, 200 ng/ml) and FGF-8 (100 ng/ml) were added. On the 7th day of differentiation, the medium was replaced with N2 supplemented with SHH, FGF-8, and OFGF (10 ng/ml). On day 11, terminal differentiation was induced by removing SHH, FGF-8 and OFGF and adding ascorbic acid (AA, 200 μM) and BDN (brain-derived neurotrophic factor, 20 ng/ml).

Cells were harvested on days 14-15 with a 45 min Accutase treatment, washed once with N2, and incubated with AlexaFluor-647 conjugated anti-SSEA-1 antibody (BD Pharmingen) for 25 min. Cells were washed once with N2, resuspended in HEPES buffer with 0.1% BSA. DAPI was added to assess viability. FACS sorting was performed on a MoFlo cell sorter and a population of interest was selected for fluorescence of GFB (Nurr1). The population positive for AlexaFluor-647 (SSEA-1) was sorted negatively. For negative control of GFB, unprimed ESCs from J1 mice at the same stage of differentiation were used.

Nurr1::GFP sorted cells were analyzed for viability and resuspended in N2 with BDN and AA to give a final concentration of 55,000 cells/µl. 1 µl was injected into the injured striatum of mice using a 50 µm pointed thin glass capillary at the coordinates: 0.3 mm posterior, 1.5 mm lateral to the bregma, and 2.2 mm ventral to the GM surface. An aliquot of the cell suspension was replated onto matrigel-coated 6 mm dishes for further examination.

For immunofluorescent analysis, cells were fixed with paraformaldehyde for 10 min at 40°C, washed twice with PBS, blocked with 5% BSA (0.1% Triton X-100 in PBS), and incubated with primary antibodies for 2 h at room temperature: rabbit antibody anti-GFP (1:1000, Invitrogen), mouse anti-PSK IgM (1:2000, 5A5), mouse anti-NeuN (1:800, Chemicon), mouse anti-TH (1:1000, Sigma), goat anti-FoxA2 (1:800, Santa Cruz), Goat vs. Engrailed (1:800, Santa Cruz). The cells were then incubated with Cy-conjugated secondary antibodies (1:1000, Jackson).

EndoN Treatment: To remove PSCs from NCAM, the evening before cell collection, cells were treated with 20 units of endoN, a phage enzyme that specifically eliminates PSCs 7-9. Cells were then harvested and injected as above, but resuspended in N2 with BDN and AA and 5 units of endoN. Previously, the authors found that injection of the same amount of endoN alone into injured mice did not improve behavioral function in the animals. In vitro analysis of PST mRNA and PSC-NCAM: For Western blotting, cells were treated with Western Blot Buffer (WB) (PBS with 1% NP40, 150 mM NaCl, 1 mM EDTA, and 1x protease/phosphatase inhibitors added just prior to extraction, at pH 7.4), sonicated twice for 5 seconds, centrifuged and resuspended in Laemmli's buffer (LB). Aliquots that did not contain LB were kept for protein determination. Equal amounts of protein were loaded onto a 6% sodium dodecyl sulfate polyacrylamide gel for gel electrophoresis (BioRad). Proteins were transferred by electrophoresis to polyvinylidene membranes (Millipore). The membranes were blocked for 1-6 h in 0.1% Triton X-100 TBS (TBS-T) with 5% skimmed milk powder and incubated overnight with anti-NCAM antibody (1:10000, Santa Cruz) in TBS-T with 5% milk. Blots were incubated with peroxidase-conjugated secondary antibody (1:10,000, Jackson) and detected using enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech). Protein levels were quantified using pro

- 61 041083 ImageJ software. For RT-PCR analysis, total RNA was extracted with trizol (Sigma), reverse transcription was performed (Qiagen), and 10 μl of the reaction mixture of 2x SYBR and 0.2 μM forward and reverse primers were amplified to a final volume of 20 μl. For FACS analysis, PSC-NCAM cells were harvested with accutase treatment for 45 min, washed once and incubated with mouse anti-PSC IgM (1:250, 5A5) for 25 min on ice, washed once with N2 medium and incubated with Cy3-conjugated anti-mouse IgM antibody (1:250, Jackson) for an additional 25 min on ice. Cells were washed once with N2 and resuspended in 0.1% BSA with 7AAD and analyzed on a FACS Calibur cell sorter. B. In the control, no primary antibody was added.

Immunohistological and stereological procedures: Free-floating coronal sections were blocked in 0.1% Triton X-100, 5% donkey serum in PBS for 30 min at room temperature and incubated for 48 h at 4°C with different antibodies: rabbit against GFP ( 1:300), chicken vs GFB (1:200, Chemicon), mouse vs TH (1:200), mouse IgM vs PSC (1:1000), mouse vs NeuN (1:400), goat vs FoxA2 (1:200) 300), rabbit vs. Girk2 (1:300, Alomone Labs), mouse vs. synapsin (1:200, BD Transduction Laboratories). Sections were then washed and incubated with secondary antibodies: Cy2, Cy3 and Cy5-conjugated donkey antibodies (1:400, Jackson). Cy5-conjugated donkey anti-IgM (1:500 Jackson) was used for PSC. Incubation was carried out for 2 h at room temperature. Sections were washed twice in PBS and mounted in Mowiol (Calbiochem). One in three of the coronal brain sections were analyzed for each immunostaining.

Digital images were obtained using a Zeiss LSM 510 laser scanning confocal microscope with three lasers (argon 488, HeNe 543 and HeNe 633) with a c-Apochromat 40x objective (water immersion). The number of GFR+ and TH+ cells were counted on every third of the sections covering the entire brain using a 40x objective, and the total number of cells per graft was estimated. Cells with double labels were analyzed in separate optical planes along the entire Z axis.

To analyze the percentage of cells labeled with GFR/TH+ and GFR/FoxA2+, 100 GFR+ cells were analyzed for each marker. To analyze the outgrowth process, confocal Z-scans were obtained at 0.8 µm intervals along the entire Z axis (20-40 µm) with a 1 µm aperture using a 40x objective. Sections were scanned from the injection site in the lateral direction until any processes ceased to be observed. Three-dimensional projections covering the entire scanned area were successively combined. To analyze the intensity of GFB and TG, the entire scanned area was divided into five successive 100 µm zones at different distances from the graft, and the intensity was measured using ImageJ software. Data were normalized for intensity in the zone closest to the graft (zone I) to control for any potential differences in graft size.

Statistical analysis: data are presented as mean ± standard error of the mean (SEM). Comparison was performed using a Student's t-test or two-way analysis of variance (ANOVA) followed by a Bonferroni correction. Linear regression analysis and quantification were performed using Pearson's correlation coefficient.

C. Overexpression of the human PST (polysialyltransferase) gene. Groups of injured animals received one of 3 doses: wild-type cells, or cells expressing PST, or cells pre-treated with the PST enzyme. They were subjected to behavioral tests according to the paradigms presented in Table. 11 (amphetamine-induced rotation, cylinder test, step test). Selected doses (eg, 200k, 100k, 50k cells) have been used successfully in mice such that the resulting neurons exhibit results as described herein for mouse PST genes.

D. Direct injection of purified PST enzyme into cells. In other words, a non-transgenic way to induce PST. Pretreatment of cells with the PST enzyme, which results in polysialylation and increased expression of surface PST. Although mammalian PSTs are rare membrane proteins that function in the Golgi apparatus, the purified Neisseria meningitides α2,8-polysialyltransferase (PST-NM) functioned in the extracellular environment using a commercially available non-toxic substrate (namely, CMP-sialic acid) and synthesized a polymer chemically identical to mammalian PST. In one example, an active fragment of said enzyme effectively added PST to therapeutic proteins in vitro, improving in vivo pharmacokinetics. In the presence of CMP-sialic acid, PST was synthesized directly on the surface of a variety of cell types in vitro, including mouse and human ESCs (FIGS. 35A-E). Direct injection of PST-NM and substrate in vivo triggers increased accumulation of PST on the cell surface in areas of the mature brain, including the cerebral cortex, striatum, and spinal cord (Fig. 35G, H). PST synthesized by PST-NM was degraded by endoneuraminidase (endo-H) (Fig. 35B) which removed the induced PST, demonstrating that the synthesized

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PST-NM PST had functional properties comparable to those of endogenous PST (Fig. 35A, C, D). Expression of PST by PST-NM occurred in less than an hour, surpassing the rate of slow transgenic induction of PST. Expression continued for several weeks in vivo, after which the level of PST markers decreased, thus avoiding the side effects of prolonged induction of the PST transgene. Accordingly, the use of in vitro treatment of cells with PST-NM+ substrate has been proposed as a simple alternative strategy for stimulating elevated levels of PST in hESC-DA-derived neurons and other cell types for use in transplantation procedures. In addition, this alternative approach in translation, ie, the transition to use for human transplantation procedures, has the advantages of non-invasiveness, ie, eliminates the use of transgenic methods; these procedures use GMP-compliant reagents to comply with clinical protocols, and provide transient biochemically stimulated PST expression corresponding to the expected time frame for DA fibers exiting the graft area and entering the host brain, which is necessary to prevent some dangerous side effects of using cells designed for transplantation.

The following examples illustrate the enzymatically engineered production of PST on hESC-derived DA neurons using purified bacterial polysialyltransferase, PSTNM, to improve graft efficiency.

Although effective, transfection with the PST genome required genetic modification of hESC cells with limited control during polysialylation. This example describes the discovery that the use of external PST-NM as a gene delivery alternative induced PST formation (see FIG. 35). In FIG. 35A, the adhesion time of PST-treated Schwann cells (SCs) (green line-midline) is increased, while PST-NM-synthesized PST inhibited adhesion. Specifically, (A) PST-synthesized with PST-NM suppresses adhesion of Schwann cells in suspension to a Schwann cell monolayer even more effectively (red line - lower line) than PST obtained using forced PST expression (green line - middle line) . (B) PST immunoblotting of HB9 ESC-derived motor neurons shows that control samples treated with PST-NM alone contained undetectable levels of PST. Incubation with PST-NM and CMP-sialic acid substrate yielded an extensive PST band which disappeared upon treatment with endo-H. (C, D) Similar to the effects obtained with the use of the PST gene, polysialylation of these cells with the PST-NM enzyme and substrate during differentiation enhances neurite outgrowth and cell migration (arrows). (E) PST immunostaining of 30-day-old hESC-DA-derived neurons. (F) Specified staining is significantly enhanced after treatment with PST-NM and substrate. (G) In vivo injection of PST-NM alone had no effect, while co-administration with substrate (H) produced significant levels of PST expression in the mouse striatum.

Accordingly, mature DA neurons externally treated with PST-NM are contemplated for use in obtaining cells for transplantation. Both mammalian PST and PST-NM produced chemically identical PST chains. Elevated levels of PST on hESC-derived DA neurons (FIG. 35F) should persist for several weeks long enough for the DA fibers to exit the graft area. Since PST-NM is removed prior to transplantation, there is no need to take into account the immunogenicity of said enzyme contaminating transplanted cells.

PST-NM was derived from an artificial fragment with enhanced solubility and activity properties (Willis et al., Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology 18, 177-186 (2008)). In hESC cultures, differentiation into DA neurons was induced prior to treatment with PST-NM, substrate, or both. Cultures were analyzed at various time points (from 10 min to 6 h) under treatment using quantitative immunofluorescence (Operetta) and Western blotting to determine the rate and levels of polysialylation. Thus, 25-day differentiated hESC-DA-derived neurons are incubated with optimal concentrations of PST-NM and substrate under the conditions described herein. PST+ mDA neurons are transplanted in a short and long term assay as described here and in FIG. 29.

E. Use in spinal cord injury. Several studies aimed at end-uses in human patients, and using the strategies described herein, have shown that these procedures, including the delivery of PST genes by introducing PST-expressing lentiviral vectors directly into the CNS, to promote axonal regeneration and migration of endogenous progenitors, have great potential. One such strategy for use in humans is for use in spinal cord injury. One type of procedure for spinal cord regeneration used Schwann cell transplants as part of a therapy to repair cell bridges in axonal regeneration in vivo. However, no therapeutic intervention provided recovery of motor function.

- 63 041083 in a patient, including the control of fine muscle movements. As described herein, increased expression of PST on Schwann cells used for transplantation resulted in increased Schwann cell migration and axonal growth, which subsequently resulted in a strong stimulatory effect on motor functions (FIGS. 30A-D). Accordingly, in some embodiments, increased expression of PST on Schwann cells in vitro is contemplated for use in human transplantation procedures with spinal cord injury. Another strategy for using artificial PST expression by overexpressing PST genes in human procedures involved HB9 ESC-derived motor neurons, where introduction of the PST gene into these neurons via lentiviral vector-based gene expression resulted in a dramatic increase in axon outgrowth as in culture. , and after transplantation to mice to restore mechanically induced damage to the sciatic nerve. The latter resulted in improved muscle specific targeting (FIGS. 30E-H). Accordingly, in another embodiment, artificial expression of PST on the surface of ESC-derived motor neurons is used to restore sciatic nerve function.

IVX. Increased safety of transplanted DA neurons.

Described below are exemplary embodiments to further reduce the risk to patient health for use in cell production methods of the present inventions.

Although cells obtained using the methods described herein have exhibited characteristics such as reduced risk to patients when used for transplantation, additional implementation options are being considered to further reduce the potential health risk for patients receiving cell transplants. One of a number of problems in hESC-based cell therapy procedures is the possibility of introducing a contaminant of undifferentiated, undifferentiated cells that, under post-transplantation conditions, develop into cells that cause harm to the patient. In the case of pluripotent cells, one possible detrimental outcome is the formation of a teratoma, which endangers the life of the patient. Teratoma formation resulting from spontaneous cell differentiation using hESC-derived cells following the use of short-term neuronal differentiation protocols has been described. However, the present inventors' use of human ES-derived nerve cells, in contrast to mouse ES-derived counterparts, rarely resulted in teratomas following the application of the appropriate neural differentiation strategies described in the present invention (i.e., monolayer culture). , protocol for dual inhibition of SMAD signaling and culture with cytokines that do not promote proliferation). In fact, no teratoma formation has been observed in the last 10 years in the analysis of several hundred animals with human cell transplants against the background of a number of neural differentiation strategies. Moreover, teratomas have not been observed in PD transplant procedures according to the present inventions using human cells for transplants. The difference in the use of human cells versus the use of mouse cells for transplantation procedures for use in humans is presumably related to the different stages of pluripotency that human/mouse ESCs are at, so the properties of human cells are thought to correspond to the stage of pluripotency described as Epi-SC , in contrast to mouse ESCs, which may be at a different stage of development. At the same time, at least one of the problems with the use of cells in previous transplantation studies, consisting in the constant risk of excessive neuronal growth, which is significantly expressed in the protocol of Perrier et al., PNAS 2004 and similar protocols, is unexpectedly absent when using mature DA neurons and transplantation methods. in accordance with the present inventions. Further, another problem found in transplantation experiments in previous studies has been the formation of hESC-derived neuroepithelial structures (ie, neuronal rosettes) that continue to proliferate in vivo. This expansion of transplanted neuroepithelial cells in vivo has been observed in various non-ironal transplantation paradigms, including transplantation studies using hDA-derived neurons in rodent models of Parkinson's disease and Huntington's chorea. These neuronal rosette-proliferating cells were non-transformed primary cells with a high intrinsic growth potential, which resulted in the formation in transplanted animals of significant size grafts consisting of ectopic, mostly cortical-like tissue. In accordance with the present description, a number of strategies have been used to eliminate contaminants of pluripotent or neuroepithelial cells at the time of transplantation (for example, selection for SSEA-4 (pluripotency marker) or Forse-1 (neuroepithelial marker). These strategies have been somewhat successful when applying differentiation strategies to rosette-based, with overgrowth still observed in the subgroup of transplants selected for Forse1 or negative selection for SSEA-4. non-rosette-forming cells, the problem of neuroepithelial outgrowth was solved.Graft-derived proliferating cells in functional DA-derived hESC-derived neuronal grafts were rarely observed.

Judging by the undesirable results of other transplantation procedures, another safety concern with the use of DA neuron transplantation in humans is the side effects of this therapy, such as the occurrence of graft-induced dyskinesia (GID), which occurs in approximately 15% of patients receiving fetal tissue during participation in transplant research. However, as described herein, the almost complete absence of transplant-derived serotonergic cells, along with the use of a more suitable cell source with the possibility of subsequent purification from unwanted cell types (see text related to Fig. 28), and the ability to control the spread of DA - fibers in vivo (see Fig. 29; namely, the prevention of the formation of foci of neuronal clusters secreting L-DOPA and other compounds) are the main advantages of using the methods in accordance with the present inventions compared with other methods of obtaining cells for use in procedures transplants. Thus, the use of these methods in accordance with the present inventions will presumably minimize the risk to patients.

VX. The use of hESCs in humans to transition to human clinical trials.

In preferred embodiments, human ESCs are provided for use in methods of obtaining and using cells for human transplantation procedures, in other words, cell therapy for the treatment of PD. In particular, the use of human ESCs has numerous advantages over the use of human iSCs in the methods of the present invention, for example, by way of example, in the production of suitable midbrain DA transplantable neurons for use as PD cell therapy. In particular, the use of induced pluripotent stem cells (iSCs) as a source of cells for the formation of DA neurons has a number of advantages, such as providing a genetically compatible source of cells for each patient. However, several recent studies have raised doubts about the safety and complete genetic compatibility of ISCs. Reprogrammed cells have been shown to carry potentially dangerous genetic and epigenetic disorders that are undesirable in clinical use. Further, in studies on mouse iSCs, it was shown that iSC-derived cells are not fully immunocompatible, which is the main objection to their use for transplantation in humans. Further, there are no FDA-approved ISC procedures for transplantation. Finally, the potential creation of GMP-compliant, fully quality-controlled (QC) cell banks for each individual patient would be impractical and too costly. In addition, the genetic stability of hHSCs compared to hESCs has been extensively studied. Although cultured hESCs have been observed to mutate over time, the timing and rate of occurrence of such mutations appear to be different from hESCs, with hESCs generally found to be more genetically stable than iSCs, see Hussein for examples. , et al., Nature 471, 58-62 (2011); Mayshar, et al. Cell Stem Cell 7, 521-531 (2010); Lister, et al. Nature 471, 68-73 (2011); Laurent, et al. Cell Stem Cell 8, 106-118 (2011). In addition, standard operating procedures have been developed for several lines of hESCs that meet the safety requirements under the rigorous testing required by the FDA for products used in cell and gene therapy. Two groups in the US received FDA clearance to transition to the clinical use of hESC-based cell therapy. For example, Geron Corporation has begun a phase I study of hESC-derived oligodendrocyte progenitor cells (GRNOPC1), a Advanced Cell Technology (ACT), Inc. is currently conducting two Phase I/II studies using hESC-derived retinal pigment epithelial cells for the treatment of Stargardt's macular degeneration (study NCT01345006) and progressive dry age-related macular degeneration (study NCT01344993). Finally, the fact that both FDA-approved clinical trials using hESCs are directed to the treatment of disorders of the nervous system demonstrates the advantages of using hESC-based methods to obtain transplant material for the treatment of diseases and injuries of the nervous system. The nervous system is considered to be an immune-privileged area, since in HC foreign tissue (allografts) elicits a weak immune response compared to the same graft placed in the periphery. So, 25 years after transplantation of fetal cells into the human brain, it was found that some allogeneic neurons remained viable in the human brain up to 16 years with transient immunosuppression. Thus, as it turned out, full antigenic compatibility of the cell source and the transplant recipient is not mandatory. Accordingly, hESCs have been proposed as a universal allogeneic source of DA neurons for the treatment of PD, among other diseases, disorders, and injuries of the nervous system. It is expected that patients receiving cells in accordance with the present inventions are offered a clinical diagnosis of PD. Authentic DA neural grafts have been proposed for use in early interventions and in moderate to severe PD, including patients in whom

- 65 041083 there is insufficient control of symptoms with the use of available medications such as levodopa, ancillary drugs, etc. In some embodiments, the administration of neural grafts is proposed in patients who have mild symptoms of early PD (for example, when using neuroimaging to detect dopaminergic deficiency, FDG-PET, symptoms of dyskinesia, etc. It is proposed to assess dyskinesia when monitoring patients before and after transplantation according to the Unified Dyskinesia Rating scale (UDysRS) (Goetz, et al., Mov Disord. 23, 2398-2403 (2008)).Also, patients may have tomograms without signs of dopaminergic deficiency (SWEDDS), in some of These patients may present with dystonia or essential tremor Brain MRI is required to identify patients who have other (non-DOPA) predisposing factors for parkinsonism In some embodiments, patients respond positively to levodopa. and post-transplant parameters and endpoints for monitoring subjects such as assessment of motor function, non-motor function, quality of life, and the use of neuroimaging and other biomarkers. Motor function: The UPDRS and the recently validated MDS-UPDRS are widely used to assess the motor symptoms of PD (Goetz, et al., Mov Disord. 23, 2129-2170 (2008)). Along with this, other tests have been proposed, including tests with a 10-minute or 6-minute walk, TUG test (Timed Up and Go Test), assessment of functional gait, functional reach, etc. for more patient-centered approach to the evaluation of results. Patients are tested during the off as well as on period; rating scales for off periods should be included (e.g. as recommended by the Movement Disorder Society based on the clinimetric properties of validated wasting scales (Antonini, et al. Mov Disord. 26, 2169-2175 (2011)) and dyskinesia scores (e.g., UdysRS (Goetz, et al., Mov Disord. 23, 2398-2403 (2008)). Video recording of standardized examinations of patients in both on and off states is expected. Other functions besides motor: Evaluate primarily cognitive, psychiatric outcomes, and dysautonomy, in addition to cognition, depression, anxiety, apathy, sleep, fatigue, psychosis, and other non-motor symptoms before and after transplantation Quality of life: PD-specific questionnaires are proposed to monitor patient outcomes (PD-QUALIF) and/or well-validated quality of life scales such as SF-36.

Neuroimaging and other biomarkers: Functional imaging has been widely used in surgical-assisted studies of PD. While analysis of graft status involves dopamine-based imaging applications (eg, FDOPA-PET), neuroimaging techniques using other ligands have been proposed, including marker-based imaging, such as those targeting inflammation, as well as non-imaging systemic markers, such as experimental data collection. . Imaging can be used for preoperative planning, such as determining the extent and localization of DA depletion in the basal ganglia, and incorporating PET data into individual surgical planning for each patient. Localization of the graft and the number of injected cells. In some embodiments, the sites for transplantation (introduction) of cells is the shell (Freed, et al. N. Engl. J. Med. 344, 710-719 (2001); Lindvall, et al. Prog. Brain Res. 82, 729-734 (1990); ClearPoint system providing real-time trajectory building and visualization and targeting accuracy This system is placed at the entry site of deep brain stimulation electrodes in patients with PD Cell number and graft composition Fetal studies were performed using a substantially unknown number of DA neurons , as fetal graft material containing multiple cell types was used.Based on the data presented in this document mente, to restore the function of DA neurons, a calculated number equal to 100,000-200,000 viable TH+ neurons was proposed.

Immunosuppression. In some embodiments, immunosuppression of transplant patients is contemplated for at least 6 months and up to continuing throughout the life of the patient. In some embodiments, patients are not immunosuppressed for tissue transplantation.

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Table 1. Gene expression analysis results for genes that are significantly up or down on day 11 of differentiation in the ventral plate-based population treated with SHH/FGF8/Chir compared to the population treated with LSB

column number and W Speakers Multiple change e R- meaning e 7304 FOXA1 22.5512 5.03Е-16 7305 FOXA2 17.3328 5.70E-17 31270 SPON1 16.4393 2.14E-14 31684 SYT4 13.2693 9.61Е-12 4334 COL22A1 8.54042 2.05E-17 6616 FBLN1 8.24162 4.97E-18 32035 TFF3 8.01805 1.08E-13 5324 DKK1 7.95664 1.19E-09 3244 CAPN6 7.58928 1.77E-08 2544 C20ORF56 7.58575 6.71E-15 27591 PKDCC 7.42271 2.02E-11 23584 LOC91461 5.43556 9.86Е-09 5037 DDC 5.19301 2.09Е-08 13598 LDB2 4.76303 2.92E-09 723 AMOT 4.58838 5.68E-08 4978 DBX1 4.40946 2.36E-09 31138 SOX8 4.32227 9.29E-08 30193 SILV 4.3198 2.52E-07 30148 SHISA2 4.31109 1.60E-07 31509 STK39 3.65624 2.90E-06 27661 PLCL2 3.63607 6.81E-11 24425 MGST1 3.59223 2.48E-11 32945 TSPAN7 3.57361 7.36Е-07 29082 RHOU 3.51768 5.89E-06 9283 HS.19193 3.49663 6.24E-13 5478 DOCK10 3.43476 8.18E-07 25094 MMP11 3.40705 2.13E-08

29975 SERPINF1 3.36169 1.97E-10 29042 RGS4 3.33544 7.01Е-06 926 APCDD1 3.33059 3.47E-10 column number and W speakers multiple change e R- meaning e 12640 IRX3 3.32454 1.65E-05 26837 OSBPL10 3.3051 9.55Е-07 32086 THBS4 3.29135 9.77Е-07 14581 LOC100130506 3.1293 5.81E-09 4931 DAB2 3.02851 4.80E-09 12642 IRX5 2.95938 2.73E-08 13774 LMX1A 2.81057 1.42E-06 31516 STOM 2.76317 2.01Е-07 26374 ODZ4 2.75644 3.06Е-06 8210 GSC 2.75053 3.47E-07 26170 NRCAM 2.71264 1.82E-08 28414 RTCH1 2.63379 2.99E-08 31578 SULF2 2.59351 1.42E-07 7366 FREM1 2.5697 2.25E-07 13743 LITAF 2.55681 3.65E-08 26236 NTNG1 2.55383 1.78E-07 25869 NEUROG2 2.55012 5.05Е-06 26132 NPY 2.54575 4.48E-06 25967 NKX2-1 2.52195 0.00023 27101 PCDH18 2.46481 1.69E-05 32557 TNFRSF19 2.45297 4.06Е-08 5759 EFEMP1 2.44308 7.78Е-09 31148 SP5 2.43459 5.34Е-06 28069 PQLC3 2.42685 2.59Е-05 30313 SLC20A2 2.40457 1.24E-06

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30743 SNHG4 2.35277 8.69E-08 27607 PKP2 2.34734 5.34E-05 25520 MYL4 2.3327 3.93E-08 26128 NPTX2 2.29568 1.08E-05 26006 NMD3 2.29039 3.18E-05 16924 LOC391019 2.28656 1.25E-05 19975 LOC646966 2.26444 6.23E-05 29894 SEMA6A 2.25036 0.000844 6161 EYA2 2.23175 0.00024 26105 NPFFR2 2.23095 7.31E-05 4818 CXXC4 2.23047 1.66E-05 2764 C4ORF14 2.22128 5.73E-06 18269 LOC642989 2.22053 0.000735 13059 KIAA1324L 2.21906 4.75E-05 23663 LRIG3 2.21159 4.38E-07 6196 FABP5L2 2.19875 3.09E-05 29347 RPL13A 2.19362 0.000122 12446 IL1RAPL1 2.19134 1.75E-05 28455 PTN 2.18577 9.36E-05 30746 SNHG7 2.18224 4.35E-07 28788 RASL12 2.16856 1.39E-05 17150 LOC401074 2.16782 1.07E-06 30283 SLC16A3 2.16541 6.06E-05 15378 LOC100133008 2.16461 3.15E-05 32180 TLE6 2.16433 6.35E-07 12887 KCNQ1OT1 2.1641 2.97E-06 31210 SPATS2L 2.16393 0.000597 4241 CMTM8 2.16297 0.000392 30933 SNORD25 2.15953 0.000363 2803 C5ORF13 2.15734 3.94E-05 32131 TIGA1 2.15335 1.80E-05 1667 BMP7 2.14281 0.000497 20837 LOC649946 2.14098 8.39E-05 25756 NCRNA00219 2.12543 6.42E-07 8003 GPM6B 2.08967 3.45E-05 5650 DYM 2.08942 0.000179

29606 S1PR3 2.08344 0.000479 27681 PLEKHA5 2.08285 0.000217 11495 HS.570308 2.08044 1.67E-06 26232 NTN1 2.06755 2.49E-08 12635 IRS1 2.06435 0.000109 20404 LOC648343 2.05497 0.000791 29352 RPL15 2.05205 2.82E-06 33800 WNT5A 2.03874 1.08E-06 30481 SLC38A4 2.03653 1.13E-06 12059 HS.7093 2.01993 2.66E-06 22500 LOC728126 2.0183 1.16E-06 16095 LOC136143 2.01775 5.71E-05 12718 ITPR3 2.01384 1.67E-05 4004 CHN2 2.00649 7.18E-06 32162 TIP ARP 2.00538 0.000633 482 ADSS 2.00282 0.000136 2196 C17ORF45 2.00241 6.15E-05 3447 CCDC51 -2.00054 0.000177 5945 ENCI -2.0038 0.000468 27915 POU3F2 -2.00877 2.04E-07 23143 LOC729779 -2.01527 0.000972 23972 MAP1LC3A -2.01892 3.37E-06 7611 GAS6 -2.02463 9.63E-08 17062 LOC399959 -2.02793 2.39E-05 29519 RSPO3 -2.03762 1.61E-05 29038 RGS20 -2.04076 3.60E-09 19157 LOC644936 -2.04097 0.000128 30502 SLC3A2 -2.04467 5.58E-05 33508 VCAM1 -2.05421 2.51E-06 490 AFAP1L2 -2.05673 8.23E-07 23275 LOC730167 -2.06009 0.000652 29215 RNF175 -2.06406 6.81E-05 5923 EMILIN2 -2.06713 2.81E-06 34367 ZNF462 -2.07292 1.63E-05 26244 NUAK1 -2.0732 0.000172 25818 NEBL -2.07351 1.55E-06

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29624 SALL3 -2.0787 2.15E-06 33491 VANGL2 -2.08497 0.000402 30581 SLC7A8 -2.08722 1.40E-08 875 ANXA3 -2.08727 0.000227 34075 ZFP36L1 -2.08803 5.08E-05 15637 LOC100133760 -2.09319 8.14E-05 1227 ASNS -2.10169 8.03E-05 33804 WNT7B -2.1052 3.00E-08 24064 MARS -2.107 0.000146 33725 WDR72 -2.11201 0.000617 31295 SPRY1 -2.12339 2.43E-05 7495 GABARAPL1 -2.12367 1.70E-05 4331 COL1A2 -2.12521 1.97E-05 6821 FILIP 1 -2.1288 0.00026 24246 METRN -2.13118 1.99E-05 12056 HS.66187 -2.13789 2.97E-05 28438 PTGIS -2.14246 7.36E-06 12980 KIAA0367 -2.15304 1.92E-05 1575 BCMO1 -2.16946 7.40E-05 25473 MXRA5 -2.17542 3.63E-06 32537 TNFAIP1 -2.17984 7.66E-06 34064 ZFHX4 -2.23036 0.00046 28787 RASL11B -2.23056 0.000115 5632 DUSP6 -2.23366 0.000732 3149 CACHD1 -2.23514 5.39E-05 3221 CAMKV -2.25983 5.01E-08 7248 FLNC -2.26856 1.13E-05 28307 PRSS8 -2.28456 1.35E-05 12662 ISYNA1 -2.29255 1.40E-05 7524 GADD45A -2.2962 7.46E-07 5921 EMSH2 -2.29995 1.55E-05 941 APLP1 -2.31453 2.03E-07 30576 SLC7A5 -2.32091 0.000757 7474 FZD3 -2.32644 6.13E-06 5940 EMX2OS -2.3303 2.65E-07 1259 ATF3 -2.34034 2.59E-06

33806 WNT8B -2.34155 3.96E-08 31388 SST -2.34663 1.83E-06 3188 CALCB -2.34833 3.49E-06 1075 ARHGEF6 -2.35421 1.54E-08 26885 OTX1 -2.35607 0.00023 26393 OLFM3 -2.35759 2.32E-07 33087 TUBB2A -2.36013 0.000777 31747 TAGLN -2.36809 2.16E-05 5372 DMRTA1 -2.36884 5.28E-08 3614 CD200R1 -2.37345 L25E-14 17147 LOC401056 -2.37854 8.57E-10 33801 WNT5B -2.3794 3.01E-08 28590 RAB11FIP1 -2.40833 6.65E-07 23362 LOC730525 -2.41129 1.59E-06 27658 PLCHI -2.4212 3.31E-07 7000 FLJ32310 -2.42179 5.77E-06 3339 CBX4 -2.43314 1.13E-07 6073 ERRFI1 -2.44539 1.44E-05 32521 TMSB15A -2.45505 1.31E-05 13242 KLHL14 -2.51767 2.71E-11 30201 SIPA1L2 -2.52018 3.67E-06 32760 TRIBI -2.52274 1.73E-06 13641 LFNG -2.53204 5.33E-07 12285 IER3 -2.54594 1.65E-07 28506 PTPRZ1 -2.55447 1.62E-06 1608 BEX2 -2.56331 0.000109 32179 TLE4 -2.56513 0.000135 10888 HS.551307 -2.56708 0.000405 683 ALPK2 -2.57443 2.83E-06 32551 TNFRSF12A -2.59274 4.02E-06 32610 TNRC9 -2.59782 3.23E-07 777 ANKRD1 -2.61554 3.15E-05 13622 LEMD1 -2.62351 2.97E-05 26777 OR7E156P -2.64884 3.51E-07 24377 MGC39900 -2.66479 1.46E-05 12267 Ш1 -2.66562 6.68E-07

- 69 041083

27726 PLP1 -2.69279 9.13E-07 4350 COL4A6 -2.69704 1.17E-07 4320 COL11A1 -2.71039 1.79E-09 3127 CA2 -2.71276 2.35E-08 26151 NR2E1 -2.72059 4.25E-14 31267 SPOCK 1 -2.7283 3.38E-05 13744 LIX1 -2.73818 9.34E-06 32038 TFPI -2.77685 6.23E-05 32776 TRIM24 -2.79151 8.73E-08 32756 TRH -2.79963 3.98E-07 32652 TOX3 -2.81075 9.35E-06 2405 C1ORF21 -2.81457 9.49E-09 5371 DMRT3 -2.82614 4.28E-09 32381 TMEM2 -2.83489 1.83E-05 2437 C1ORF61 -2.85324 3.53E-09 2868 C6ORF141 -2.8569 6.05E-06 1178 ARX -2.87861 0.000122 26245 NUAK2 -2.95666 4.14E-08 33796 WNT2B -2.96258 1.15E-10 28507 PTRF -2.97439 2.07E-07 7662 GCNT1 -2.98108 1.49E-06 5930 EMP1 -2.9918 1.11E-06 31844 TBC1D9 -3.00324 2.53E-09 4352 COL5A2 -3.02084 1.26E-07 4699 CTNNA2 -3.04046 6.91E-09 2516 C20ORF177 -3.04277 4.86E-08 3518 CCL2 -3.04332 9.50E-11 34315 ZNF385B -3.05084 5.56E-07 12058 HS.7023 -3.0642 3.72E-08 23661 LRIG1 -3.07681 3.30E-07 7813 GLI3 -3.07936 1.22E-08 31513 STMN2 -3.09098 4.93E-06 30023 SFRP1 -3.09686 1.13E-06 1128 ARMCX2 -3.09746 2.91E-06 4689 CTGF -3.11416 4.47E-08 6365 FAM181A -3.1422 2.56E-09 31151 SP8 -8.88965 4.42E-20 30024 SFRP2 -9.73482 4.24E-14 13677 LHX2 -10.9076 6.16E-11 5939 EMX2 -11.5085 1.34E-19 21068 LOC650757 -12.1496 7.71E-20 27068 PAX6 -15.7636 2.24E-22 5554 DRD4 -17.3929 3.17E-16

4121 CLDN1 -3.15492 1.93E-06 627 ALDH1A1 -3.15745 1.18E-12 629 ALDH1A3 -3.17887 4.46E-13 33501 VAT IL -3.18253 1.21E-06 4912 CYR61 -3.18856 3.58E-07 12781 KANK4 -3.20054 7.99E-12 3067 C9ORF171 -3.24146 3.73E-11 12363 IGFBP3 -3.34999 1.02E-06 4533 CRIP1 -3.45501 6.62E-08 3740 CDH11 -3.48609 2.23E-08 816 ANKRD38 -3.48622 6.93E-12 12730 JAG1 -3.58463 3.83E-08 12271 Ш4 -3.72402 1.78E-12 810 ANKRD34B -3.76606 1.15E-11 12850 KCNJ13 -3.81092 2.16E-09 6112 ETV5 -3.81591 4.37E-10 9265 HS.181245 -3.86611 1.24E-09 633 ALDH2 -3.87108 1.97E-09 26180 NRIP3 -3.92066 6.08E-08 30413 SLC2A1 -4.11899 1.18E-05 5479 DOCK 11 -4.35251 5.67E-09 25658 NAVI -4.35259 1.02E-10 5349 DLL1 -4.47474 1.58E-07 34418 ZNF533 -4.49025 1.16E-09 327 ACTC1 -4.63509 7.37E-09 1037 ARHGAP15 -4.67561 9.89E-10 31132 SOX3 -4.72958 7.45E-09 26153 NR2F1 -4.73849 1.53E-09 3129 CA4 -4.7904 3.00E-09 33071 TTYH1 -4.79045 8.94E-14 5766 EFHD1 -4.86615 1.16E-09 867 ANXA1 -4.95401 1.41E-08 8500 HES4 -5.24189 1.63E-11 4299 CNTNAP2 -6.14001 2.94E-08 23947 MAMDC2 -6.36522 2.23E-10 7605 GAS1 -6.38074 2.63E-11 26065 NOS2A -20.0139 2.83E-16 1501 BARHL1 -20.6077 2.59E-14 26064 NOS2 -25.1751 7.45E-17 8501 HES5 -38.4549 8.90E-22

- 70 041083

No. column And Set ID data multiple change p-value 31575 SYT4 14.8775 3.24E-12 7195 FOXA1 11.8929 2.65E-13 4225 COL22A1 8.26361 3.21E-17 5215 DKK1 6.39507 1.32E-08 7956 GPR177 5.98201 5.65E-12 13508 LEF1 5.07514 2.45E-11 2435 C20ORF56 5.02955 2.71E-12 4928 DDC 5.0147 3.23E-08 11952 HS.71947 4.81098 5.13E-09 13665 LMX1A 4.7485 3.59Е-10 12427 INHBE 4.72434 3.41E-07 32562 TPBG 4.63162 1.44Е-09 1152 ATF5 4.48583 0.000153048 5369 DOCK10 4.23427 4.29E-08 31414 STOX1 4.20695 3.94Е-10 4869 DBX1 4.17519 5.31E-09 31977 THBS4 4.13195 3.66E-08 2694 C5ORF13 4.0095 7.84Е-10 31469 SULF2 3.83814 8.51E-11 31468 SULF1 3.81974 3.96E-07 34285 ZNF503 3.81092 2.76E-08

Table 2 Gene expression analysis results for genes that are significantly up or down on day 11 of differentiation in the ventral plate-based population treated with SHH/FGF8/Chir compared to the population treated with SHH/FGF8 alone

12531 IRX3 3.73598 3.93E-06 4935 DDIT4 3.73152 3.78E-05 31369 STC2 3.70632 8.48Е-08 No. column And Set ID data multiple change p-value 27482 PKDCC 3.69823 2.20E-07 4257 COLEC12 3.6503 5.20E-06 31926 TFF3 3.62059 1.12E-08 25244 MSX1 3.60243 0.000146141 23475 LOC91461 3.58231 2.08E-06 26777 OTX2 3.47814 3.97E-09 13489 LDB2 3.44124 3.22E-07 13657 LMO3 3.29197 2.61E-12 817 APCDD1 3.21277 6.95Е-10 4822 DAB2 3.18324 1.84Е-09 25760 NEUROG2 3.16851 1.30E-07 31101 SPATS2L 3.15744 2.80E-06 1311 AXIN2 3.13274 1.80E-06 32448 TNFRSF19 3.11104 2.48E-10 4934 DDIT3 3.08248 4.83E-05 26776 OTX1 3.05293 6.14Е-06 28360 PTPN13 3.03567 5.18E-06 994 ARL4A 2.98803 1.45E-07 26728 OSBPL10 2.94508 5.19E-06 32836 TSPAN7 2.86094 1.65E-05

- 71 041083

5665 EFNB2 2.78085 0.000222563 8101 GSC 2.75066 3.46E-07 6426 FAM84B 2.74213 7.89E-07 29866 SERPINF1 2.73192 1.26E-08 924 ARHGAP10 2.72522 3.36E-07 31039 SP5 2.68162 9.57E-07 28210 PSAT1 2.64735 9.82E-06 28933 RGS4 2.6312 0.000148375 13547 LGI1 2.61567 1.30E-06 7369 FZD7 2.61404 2.46E-06 25351 MUSTN1 2.55841 4.85E-07 25411 MYL4 2.55345 4.77E-09 28424 PVRL3 2.55083 1.04E-07 299 ADAMTS9 2.53131 2.88E-08 1555 BMP4 2.52708 9.40E-05 31161 SP0N1 2.5133 0.000145864 26265 ODZ4 2.50312 1.46E-05 33730 XBP1 2.50164 1.72E-06 28766 RBP1 2.49237 6.12E-05 12533 IRX5 2.48929 7.43E-07 1118 ASNS 2.47416 4.50E-06 2770 C6ORF160 2.40219 3.58E-05 12337 IL1RAPL1 2.40145 3.15E-06 12526 IRS1 2.3994 7.70E-06 7221 F0XJ1 2.39492 8.89E-05 28647 RARB 2.37013 9.11E-09 14472 LOC100130506 2.36331 1.36E-06 3979 CITED2 2.36201 2.92E-05 31430 STT3B 2.34069 1.29E-05 26023 NPY 2.3346 1.98E-05 6052 EYA2 2.3192 0.000136915 26991 PCDH17 2.31776 8.71E-05 26127 NTNG1 2.31615 1.30E-06 373 ADSS 2.29099 1.20E-05 6120 FAM107A 2.26256 0.000677009 28387 PTPRM 2.25934 1.41E-05 31572 SYT17 2.257 7.10E-05 7143 FLRT2 2.24042 1.35E-07 32372 TMEM88 2.2394 4.55E-05 27552 PLCL2 2.23488 1.24E-06

5681 EGLN3 2.23059 0.000841858 3837 CHAC1 2.22744 2.61E-05 26045 NR2F2 2.21836 1.71E-05 7196 FOXA2 2.21691 8.01E-05 12950 KIAA1324L 2.18683 6.09E-05 3667 CDK6 2.1717 0.00076066 9540 HS.36053 2.15618 5.44E-07 19012 LOC644860 2.15508 7.00E-06 4366 CPNE8 2.15418 9.80E-06 4936 DDIT4L 2.14147 0.000195501 29691 SDCBP 2.13721 3.10E-05 7144 FLRT3 2.11969 0.00033768 5851 ENPP2 2.10048 0.000485738 1558 BMP7 2.0876 0.000718573 23514 LPAR4 2.08062 1.96E-06 23554 LRIG3 2.07935 1.81E-06 5964 ERRFI1 2.07249 0.000220066 3623 CDCA7 2.07177 0.000280326 30635 SNHG5 2.0594 4.95E-06 32620 TRAM2 2.04784 0.000111169 12809 KCTD6 2.04602 0.000114945 28901 RGL1 2.04043 0.000167278 30628 SNHG1 2.03706 0.000252445 27947 PPPDE1 2.02017 2.69E-05 5434 DPYSL3 -2.00257 0.000139976 23863 MAP1LC3A -2.00377 4.01E-06 24226 MGC18216 -2.01184 0.000204988 4818 D4S234E -2.01244 8.77E-05 4825 DACH2 -2.01277 8.71E-07 4386 CPXM2 -2.02106 2.02E-05 32224 TMEM169 -2.02651 0.000222019 151 ACBD7 -2.03074 6.94E-05 8643 HOOK1 -2.03984 1.85E-05 4278 COPG2IT1 -2.04004 8.62E-07 32981 TUBB3 -2.04044 0.000989413 5887 EPHA1 -2.05082 1.41E-06 28929 RGS20 -2.06283 2.60E-09 28068 PRKCH -2.06869 4.13E-06 33539 WBP2 -2.0692 0.000157614 23711 LY6E -2.07026 8.93E-05

- 72 041083

29803 SEPPI -2.08034 2.48E-05 12713 KCND2 -2.08768 1.63E-08 34309 ZNF533 -2.08871 0.000231468 14009 LOC100129034 -2.09106 4.54E-07 25915 NMU -2.09244 0.000184692 24132 MEST -2.09269 2.92E-06 2715 C5ORF41 -2.09563 1.42E-05 27778 PON2 -2.09758 1.79E-05 24105 MEG3 -2.10214 5.69E-07 31812 TCF7L2 -2.10261 0.000153698 33783 YBX2 -2.1118 6.90E-06 28329 PTGIS -2.11465 9.61E-06 6694 FHDC1 -2.11553 8.86E-06 24175 MFNG -2.11579 2.95E-05 6640 FEZ1 -2.11631 2.58E-06 2407 C20ORF177 -2.12232 3.62E-05 26783 OVOL2 -2.12627 3.53E-07 28883 RFTN2 -2.13498 7.25E-08 19038 LOC644919 -2.13675 2.95E-06 30004 SH3BP4 -2.13785 0.000314453 31020 SOX2 -2.14642 0.000107169 16268 LOC284422 -2.16056 1.07E-05 4819 DAAM1 -2.16701 3.85E-05 32404 TMPRSS2 -2.1693 6.26E-07 26668 OR7E156P -2.17193 1.53E-05 12609 IIPR3 -2.18226 3.09E-06 6372 FAM65B -2.18271 1.37E-06 12635 JARID2 -2.18541 4.46E-05 25024 MOBKL2B -2.19737 5.45E-05 31567 SYT13 -2.19832 2.30E-05 25658 NDRG1 -2.20101 5.25E-07 24318 MGST3 -2.20252 3.24E-07 2147 C18ORF26 -2.2117 2.55E-06 31010 SOSTDC1 -2.21381 2.23E-08 4662 CXCL14 -2.21955 1.81E-08 33161 UCA1 -2.21966 2.19E-05 12176 IER3 -2.21992 2.75E-06 7171 FNBP1 -2.23104 1.98E-05 12199 IFITM3 -2.23167 4.22E-06 25635 NCRNA00153 -2.24173 2.01E-05

27531 PLAC9 -2.24346 9.11E-08 25743 NES -2.2469 3.16E-06 31405 STMN3 -2.25856 0.000796798 7536 GCA -2.27697 1.34E-05 12275 IGSF3 -2.28535 4.08E-06 17038 LOC 401056 -2.2854 2.45E-09 4533 CSRP2 -2.28899 0.000244001 31606 TACSTD1 -2.28933 0.000115538 29372 RRAGD -2.29258 0.000537359 5884 EPCAM -2.29475 0.00099285 28398 PTRF -2.29476 1.92E-05 12672 KANK4 -2.29735 1.73E-08 3505 CD200R1 -2.30795 3.06E-14 33264 UPK2 -2.30847 1.37E-10 24978 mmd -2.317 1.46E-05 6002 ETV4 -2.32842 6.04E-09 27655 PMP22 -2.33585 4.94E-12 5657 EFHD1 -2.33997 7.47E-05 31186 SPRY1 -2.34859 3.56E-06 3631 CDH11 -2.35006 1.77E-05 29555 SAT1 -2.35602 2.21E-05 33268 UPP1 -2.36162 5.38E-08 1968 C14ORF4 -2.36739 2.59E-06 9512 HS.34447 -2.37168 7.49E-08 16243 LOC283953 -2.38022 4.57E-07 3203 CAV2 -2.38208 1.09E-11 26223 NYNRIN -2.40311 1.90E-05 22623 LOC728715 -2.41353 0.000504831 25084 MPPD2 -2.42024 6.42E-10 13403 LAMC2 -2.42245 5.32E-10 25083 MPPD1 -2.42332 3.73E-14 13402 LAMC1 -2.43845 7.40E-05 15722 LOC 100134265 -2.44777 4.60E-06 5836 ENCI -2.45175 1.84E-05 244 ACVR2A -2.46307 1.87E-06 33616 WDR72 -2.46656 6.61E-05 28509 RAB31 -2.47199 1.57E-06

- 73 041083

3629 CDH1 -2.4799 3.05E-06 28905 RGMA -2.50113 4.45E-06 29475 S100A11 -2.50568 3.24E-06 8259 HAPLN1 -2.50809 7.14E-07 3942 CHST7 -2.51075 9.78E-08 12543 ISL1 -2.51716 2.76E-10 28305 PTCHI -2.52418 7.32E-08 32541 TOX -2.5288 5.34E-09 6067 F2RL1 -2.54309 2.36E-08 32334 TMEM54 -2.57701 2.04E-06 3436 CCND1 -2.58061 6.72E-05 707 ANKRD38 -2.58621 4.12E-09 5241 DLL3 -2.5911 8.77E-08 33424 VGF -2.59836 0.000406676 23253 LOC730525 -2.61609 3.32E-07 31368 STC1 -2.63136 1.20E-05 31910 TFAP2C -2.63773 4.72E-08 25012 MN1 -2.6469 8.33E-08 28419 PVALB -2.67012 1.23E-10 6960 FLJ37644 -2.67066 1.78E-12 7417 GADD45G -2.68804 2.14E-06 4709 CXXC4 -2.68958 5.39E-07 8050 GRHL3 -2.73097 4.15E-11 23990 MBIP -2.73995 9.29E-07 5418 DPPA4 -2.75778 5.66E-08 32212 TMEM158 -2.77503 9.30E-06 5896 EPHB1 -2.77518 2.29E-06 31404 STMN2 -2.79651 2.03E-05 12454 INSMI -2.81526 1.15E-09 13142 KLHL24 -2.81638 2.31E-07 9856 HS.475334 -2.82304 1.09E-05 33125 UBL3 -2.85607 2.77E-08 13655 LMO1 -2.90621 4.10E-06 6003 ETV5 -2.90987 5.54E-08 966 ARHGEF6 -2.92751 9.82E-11 31187 SPRY2 -2.94814 6.74E-09 28501 RAB25 -2.97139 5.77E-07 4675 CXCR7 -2.9864 6.13E-07 32652 TRIB2 -2.99455 8.47E-08 28397 PTPRZ1 -3.02099 8.00E-08

31158 SPOCK 1 -3.02198 8.12E-06 25849 NKD2 -3.02555 4.12E-11 25471 MYT1 -3.03712 6.21E-09 6856 FLJ25404 -3.03813 1.95E-08 30304 SLC2A1 -3.05478 0.000275534 12256 IGFBP5 -3.06686 2.00E-06 23779 MAF -3.07491 2.81E-08 28198 PRSS8 -3.08336 6.31E-08 9923 HS.509165 -3.09539 2.57E-07 4243 COL5A2 -3.10066 8.01E-08 3112 CAMKV -3.10668 3.37E-11 29915 SFRP2 -3.13051 3.42E-07 13092 KIT -3.13441 8.27E-12 6684 FGFR3 -3.13726 7.73E-09 8646 HOPX -3.15424 1.07E-14 29497 S1PR3 -3.15587 8.43E-07 33399 VCAM1 -3.15952 2.02E-10 5240 DLL1 -3.18075 1.28E-05 28112 PRODH -3.18595 2.33E-07 23200 LOC730278 -3.27115 1.92E-07 1202 ATP1B2 -3.29446 2.34E-08 6720 FJX1 -3.3039 6.85E-07 3609 CDC42EP4 -3.33348 2.25E-09 12198 IFITM2 -3.35237 3.41E-11 3679 CDKN1C -3.37225 1.15E-09 3202 CAV1 -3.37517 5.73E-13 31171 SPRED1 -3.42954 3.76E-08 30010 SH3GL3 -3.43813 3.73E-11 8335 HDC -3.45787 1.51E-12 8391 HES4 -3.49495 1.00E-08 33335 USP44 -3.50303 6.22E-11 1101 ASCL1 -3.54534 1.26E-11 29945 SFTA3 -3.5611 1.59E-07 13619 LIPA -3.56979 1.34E-08 27178 PDPN -3.72978 5.33E-12 7370 FZD8 -3.74383 7.14E-09 3647 CDH3 -3.80687 3.54E-09 2759 C6ORF141 -3.94476 5.10E-08 13595 LIMCH1 -3.94759 2.52E-11 13513 LEMD1 -3.98954 6.68E-08

- 74 041083

5961 ERP27 -4.02755 8.38E-13 29666 SCRG1 -4.03799 1.96E-17 7286 FRZB -4.04927 2.19E-05 12977 ΚΙΑΛΙ 598 -4.04996 4.12E-09 2380 C20ORF100 -4.16273 2.94E-12 25866 ΝΚΧ6-2 -4.17171 1.46E-10 31030 SOX9 -4.21749 9.69E-10 27805 POU3F1 -4.22404 1.53E-11 7879 GPC4 -4.23918 8.64E-08 17977 LOC642590 -4.36113 1.29E-10 19608 LOC646347 -4.53146 1.77E-08 5238 DLK1 -4.62809 8.12E-05 8401 HEY1 -4.68961 2.52E-12 30592 SMS -4.74656 5.18E-12 29914 SFRP1 -4.84227 1.53E-09 13376 L1TD1 -4.85832 6.96E-08 7984 GPR56 -4.87971 1.88E-13 7367 FZD5 -5.00247 2.43E-11 25858 NKX2-1 -5.06868 2.84E-08 7801 GNG8 -5.39693 2.22E-13 9156 HS.181245 -5.99308 1.43E-12

32962 TTYH1 -6.33607 1.00E-15 5523 DUSP6 -6.34711 8.15E-10 6091 FABP7 -6.56016 2.28E-09 5248 DLX5 -6.56829 8.74E-13 12754 KCNK12 -6.912 1.07E-12 30165 SLC15A3 -7.17663 1.60E-19 6675 FGF8 -7.58196 1.32E-18 31023 SOX3 -7.63996 1.53E-11 8395 HESX1 -8.28818 3.36E-10 28692 RAX -8.87763 6.24E-12 20959 LOC650757 -9.74543 1.18E-18 14470 LOC100130502 -10.333 1.53E-10 13568 LHX2 -14.1638 4.81E-12 30111 SIX3 -15.019 8.24E-13 12197 IFITM1 -16.4704 9.16E-16 25859 NKX2-2 -18.6642 7.93E-20 12254 IGFBP3 -19.1294 8.81E-16 8392 HES5 -33.7356 2.69E-21 30114 SIX6 -39.4411 2.59E-18

No. COLUMN And Column ID multiple changed ie p-value 1209 ASCL1 33.085 1.60E-09 3978 CHGA 28.1727 4.44E-13 31604 STMN2 20.9978 1.47E-08 32159 TH 15.2965 2.12E-08 31606 STMN4 15.2102 3.72E-09 7685 GAP43 12.5729 5.42E-09 4926 D4S234E 10.7914 4.91Е-08 8593 HES6 10.6527 2.94Е-09

Table 3 Gene expression analysis results for genes that are significantly up or down at differentiation day 25 compared to day 13 in the SHH/FGF8/Chir______-treated ventral plate population

4342 COL3A1 10.0606 3.86E-06 12149 HS.7023 9.60731 3.97E-08 5691 EBF3 9.28771 5.13E-11 29820 SCG2 9.22584 1.02E-06 12655 INSM2 9.01409 3.73E-12 27988 POSTN 8.79262 1.09E-10 No. COLUMN And Column ID multiple changed ie p-value 28590 PTPRO 8.70512 3.13E-10 30386 SLC17A6 8.22904 2.02E-06 31767 SYT13 8.1735 3.63E-07

- 75 041083

30390 SLC18A1 8.15448 6.47E-15 5689 EBF1 8.05344 2.30E-06 5029 DCX 8.00552 8.30E-08 2470 C1QL1 7.47022 8.68E-10 12609 INA 6.92121 5.72E-07 26250 NR4A2 6.75723 1.27E-13 29624 RTN1 6.6247 6.52E-07 9374 HS.19193 6.58382 1.95E-08 436 ADCYAP1 6.49154 9.75E-11 1607 BEX2 6.33986 8.93E-09 34098 ZCCHC12 6.19663 9.95E-07 7056 FLJ25404 6.19296 2.14E-06 31605 STMN3 6.17605 8.77E-07 No. column And Column ID multiple changed Not p-value 7997 GNG3 6.1196 1.26E-06 25922 NEFM 6.11347 2.51E-04 12359 ID2 6.07844 1.32E-06 29017 REEP1 6.04393 2.25E-06 30810 SNAP25 6.03677 3.62E-06 25671 MYT1 5.86419 8.19Е-06 33297 UBE2J1 5.76887 6.22E-08 5349 DLL3 5.76451 4.92E-08 29133 RGS4 5.69761 1.87E-06 28123 PPP2R2B 5.56067 2.80E-08 27287 PCSK1N 5.43156 3.22E-09 32126 TFF3 5.26964 3.17E-08 27288 PCSK2 5.22877 9.14Е-10 4533 CRIP2 5.18375 8.40E-08 12506 IL13RA2 5.1246 1.31E-06 32417 TMEM163 5.01765 7.55E-09 30813 SNAP91 4.92061 4.28E-05 13260 KIF5C 4.86241 2.93E-06 15798 LOC100133923 4.83074 3.74Е-07 32412 TMEM158 4.76646 4.81E-06

3770 CDK5R1 4.75524 1.18E-05 5739 EEF1A2 4.75232 9.53E-05 32697 TNRC4 4.75179 1.29E-05 5455 DNER 4.71709 1.52E-05 6700 FAT3 4.68785 1.91E-06 6197 ETS2 4.67343 1.08E-10 25753 NBEA 4.65265 6.99E-07 12944 KCNJ16 4.53077 3.54E-12 3979 CHGB 4.52218 7.76E-05 25562 MXD4 4.445 7.25E-07 3941 CGNL1 4.44195 7.24E-06 26499 ONECUT2 4.37403 2.39E-07 2867 C6ORF141 4.35252 2.58E-10 12362 ID4 4.32183 6.05E-05 26011 NHLH2 4.31928 5.91E-05 2035 C14ORF132 4.31666 9.46E-07 9428 HS.204481 4.29602 1.66E-09 8288 GRM8 4.17846 1.62E-06 7617 GADD45G 4.17663 2.21E-06 4298 CNTNAP2 4.13552 4.10E-05 1408 AUTS2 4.13111 2.79E-05 23822 LRRC4C 4.11703 1.27E-09 31454 SRRM4 4.11574 8.04E-07 4003 CHN2 4.09589 3.30E-07 27665 PITX2 4.08382 1.94E-11 13296 KLC1 4.0528 4.19E-06 27773 PLEKHA6 4.0464 2.57E-07 25618 MYLIP 3.99268 2.96E-06 5036 DDC 3.96553 8.46E-05 33181 TUBB3 3.93922 1.52E-04 24522 MIAT 3.93906 9.87E-05 27411 PEG10 3.9374 3.48E-05 32343 TMEFF2 3.92112 6.49E-07 29821 SCG3 3.92108 6.47E-06 31759 SYP 3.89869 2.51E-04 9425 HS.202577 3.89053 2.85E-07

- 76 041083

33943 XKR4 3.87043 1.49E-06 33178 TUBB2A 3.86265 6.11E-05 12654 INSMI 3.84646 5.59E-05 13236 KIF1A 3.84543 3.27E-05 25721 NAPB 3.81995 3.50E-05 31765 SYT11 3.77827 1.14E-05 5877 ELAVL3 3.70028 9.69E-05 13858 LMO4 3.69359 2.09E-06 4103 CLASP2 3.64468 1.79E-05 2956 C7ORF41 3.63616 5.69E-05 33362 UCHL1 3.61003 5.64E-05 29645 RUNDC3A 3.54448 8.42E-05 24063 MAP1LC3A 3.53736 4.36E-06 5944 ENCI 3.51677 0.000141473 28730 RAB40B 3.48084 4.57E-07 25162 MLLT11 3.4633 3.13E-05 7469 FRMD4A 3.46181 7.40E-06 13070 KIAA0363 3.43032 6.39E-05 13342 KLHL24 3.41191 3.34E-05 7988 GNB3 3.38669 3.31E-09 31840 TAGLN3 3.36355 1.67E-03 12147 HS.66187 3.34592 0.000125172 4549 CRMP1 3.32015 2.42E-04 23453 LOC730525 3.31501 1.64E-03 31514 ST6GALNAC5 3.29449 1.19E-05 27054 PAFAH1B1 3.25816 1.30E-04 32432 TMEM170B 3.25165 2.44E-05 27686 PCIA 3.24255 4.29E-05 1748 BSN 3.2101 0.000169219 6213 EVL 3.17682 3.36E-05 34217 ZMIZ1 3.16249 3.29E-04 16419 LOC283514 3.15057 4.57E-06 32081 TERF2IP 3.13818 3.43E-05 4932 DACH1 3.11457 7.79E-06 5424 DNAJC19 3.09946 1.64E-09 8093 GPM6A 3.07619 1.59E-03

15034 LOC100131718 3.0686 4.01E-05 8706 HIST1H4K 3.06394 1.84E-06 28238 PRICKLE2 3.06341 1.98E-06 31429 SRGAP3 3.0575 7.84E-06 13760 LHFP 3.04812 0.000126565 3181 CADPS 3.03806 6.60E-06 3186 CALCA 3.03372 3.13E-08 1672 BMPR2 3.03171 9.26E-06 25861 NDRG4 3.00851 0.000239868 2265 C18ORF8 2.99462 9.07E-06 4698 CTNNA2 2.962 6.07E-05 1606 BEX1 2.96088 5.37E-05 32769 TPH1 2.96087 2.88E-07 31986 TCEAL7 2.95947 3.43E-05 3322 CBLN2 2.95729 1.12E-04 29639 RUFY3 2.95504 2.59E-05 3851 CELSR3 2.9545 6.27E-04 3177 CADM1 2.95335 1.81E-04 20895 LOC649841 2.94392 2.21E-05 13713 LEMD1 2.93152 9.17E-06 8184 GPR56 2.93095 3.08E-04 31935 TBC1D9 2.92862 6.60E-05 8046 GOLSYN 2.9099 2.80E-06 33004 TSHZ1 2.90158 2.76E-07 5876 ELAVL2 2.90115 0.00136166 12733 IRX5 2.88659 1.21E-04 1254 ATCAY 2.86583 7.41E-04 31763 SYT1 2.86449 1.02E-03 29193 RIMBP2 2.84834 6.29E-06 28741 RAB6B 2.84821 0.000698361 27533 PHF21B 2.8457 6.44E-05 24135 MARCH 2.83633 5.22E-04 10116 HS.505676 2.83499 1.50E-05 8712 HIST2H2BE 2.82687 0.000157254 24061 MAP1B 2.82235 0.000357426 13054 KIAA0182 2.8162 2.66E-04

- 77 041083

34044 ZBTB20 2.81539 8.77E-08 29509 RPRM 2.81168 0.000475144 34007 YPEL5 2.81061 1.16E-04 13609 LANCL2 2.80004 8.07E-06 940 APLP1 2.77078 2.57E-05 29849 SCN3B 2.76654 7.14E-05 29201 RIMS3 2.76205 2.94E-04 27394 PDZD4 2.75594 0.000212095 1364 ATP6V1G2 2.75366 0.0012022 31778 SYT7 2.74946 4.15E-05 32351 TMEM106A 2.73965 0.000217998 7717 GATS 2.72521 3.10E-05 506 AGAP3 2.71696 2.07E-05 24141 MARCH4 2.70659 8.96E-04 5348 DLL1 2.7028 2.05E-04 6212 EVI5L 2.6886 6.79E-05 3196 CALM1 2.68129 8.50E-05 31386 SPRY1 2.67788 1.15E-03 33128 TTR 2.66999 2.76E-05 297 ACPL2 2.66551 1.52E-06 5325 DKK3 2.65119 5.63E-05 23853 LRRN3 2.64615 1.37E-03 32615 TMSL3 2.63733 4.56E-05 29822 SCG5 2.63452 1.42E-04 32000 TCF12 2.63327 1.80E-06 3497 CCDC92 2.6296 2.23E-05 5610 DUSP1 2.60841 1.78E-04 14524 LOC100130053 2.60135 4.53E-04 26279 NRSN1 2.59336 0.00111467 25711 NANOS3 2.58916 4.15E-06 5543 DPYSL4 2.58236 0.000223516 6231 EXOC7 2.58163 4.89E-06 24134 MAPRE3 2.57689 4.85E-05 9382 HS.193784 2.57602 2.18E-06 1608 BEX4 2.57251 9.83E-05

6514 FAM36A 2.56726 7.62E-05 13640 LBH 2.56551 1.34E-05 9920 HS.437111 2.55902 4.44E-05 33904 WSB2 2.55865 5.08E-04 9757 HS.369017 2.55838 4.25E-05 18920 LOC644250 2.55607 0.000475214 27311 PDCD4 2.55148 0.000139052 7562 FZD1 2.54808 2.48E-06 13191 KIAA1688 2.54643 9.31E-05 4292 CNTN2 2.53802 2.29E-04 32474 TMEM200A 2.53495 4.93E-07 6635 FAM89B 2.5341 1.64E-05 23500 LOC730990 2.53197 3.94E-05 1375 ATP9A 2.52973 1.09E-04 8126 GPR137C 2.52944 0.000217768 19248 LOC644936 2.52867 4.31E-04 4916 CYTH2 2.5213 6.31E-06 20654 LOC648921 2.51994 1.76E-03 2445 C1ORF71 2.50491 9.78E-04 21820 LOC652726 2.49851 5.58E-05 16100 LOC100134868 2.49288 0.00076351 27105 PAPSS2 2.49026 0.000148906 32246 TIMP2 2.48501 3.26E-05 27648 PIP5K2B 2.48028 0.000236563 12214 HSBP1 2.47723 1.82E-04 32754 TP53INP2 2.47324 2.42E-05 29037 REM2 2.46751 0.000170127 34742 ZNF84 2.46625 2.39E-04 3872 CENTA1 2.46106 3.74E-04 23918 LY6H 2.45793 0.000317539 29595 RSBN1 2.45785 8.01E-04 1733 BRP44L 2.45488 5.07E-04 2751 C3ORF70 2.45314 1.92E-06 12862 JUN 2.4481 4.37E-04 15357 LOC100132727 2.44481 0.00058635 13224 KIDINS220 2.43944 0.00068445

- 78 041083

33183 TUBB4Q 2.43584 0.00171503 685 ALPP 2.43222 3.60E-05 14002 LOC100128274 2.43042 1.90E-04 20699 LOC649095 2.42655 0.00040167 7123 FLJ35390 2.39837 0.00162187 16740 LOC387856 2.39594 8.21E-04 7033 FLJ22184 2.3897 4.07E-04 20602 LOC648740 2.38616 8.38E-06 10392 HS.538962 2.38583 0.000324449 31615 STOX2 2.38135 4.42E-04 23592 LOC731895 2.3779 0.000838915 32863 TRIM2 2.37536 5.63E-04 30075 SERTAD4 2.36897 0.000363024 26135 NOL4 2.36463 0.000261492 27620 PIK3R1 2.35977 1.01E-07 27073 RAKZ 2.34949 1.62E-04 27855 PMP22 2.34705 7.89E-06 27636 PINK1 2.34687 1.17E-05 3223 CAMSAP 1 2.34021 1.96E-04 9323 HS.168950 2.33738 1.84E-04 17248 LOC401115 2.33367 0.000151535 6270 F3 2.32945 2.00E-08 2256 C18ORF32 2.32914 4.41E-04 777 ANKRD10 2.32837 2.48E-04 5372 DMRTA2 2.32784 5.27E-06 6347 FAM117B 2.32504 9.42E-04 731 AMY1A 2.3232 0.000158787 7610 GABRR1 2.31983 8.23E-08 1520 BAZ2B 2.30948 5.18E-04 7920 GLRA2 2.30858 6.83E-05 10190 HS.522924 2.30739 1.62E-03 7762 GDAP1 2.30342 6.22E-05 31325 SPHK2 2.29983 1.68E-04 31499 ST18 2.2969 9.74E-04 15124 LOC100131989 2.29335 1.57E-04 23665 LOC88523 2.29171 0.000476299

30325 SKP1 2.28675 1.76E-04 2743 C3ORF58 2.28463 0.000119256 27566 PHYHIPL 2.28388 1.72E-03 27332 PDE4D 2.28016 3.70E-05 760 ANK2 2.27803 3.84E-05 1101 ARL3 2.26792 1.O3E-O3 25218 MNX1 2.26631 4.41E-07 7255 FLJ44048 2.26493 6.84E-06 30029 SERINCI 2.26355 3.12E-04 34800 ZSWIM6 2.2634 3.19E-04 13943 LOC100128062 2.26302 0.000498795 18811 LOC644033 2.2611 5.93E-04 20075 LOC646996 2.25787 1.36E-04 7976 GNA01 2.25676 0.00121073 22799 LOC728661 2.25642 0.000599836 979 APPBP2 2.25324 6.41E-04 25716 NAP1L3 2.24879 9.43E-04 14359 LOC100129502 2.24469 1.52E-04 25580 MYCBP2 2.24333 0.000445387 27083 PAM 2.23671 0.00170991 22583 LOC728105 2.2358 0.000601193 29747 SARM1 2.2285 5.83E-07 7602 GABRB3 2.22682 4.00E-04 30261 SIAH3 2.22476 1.89E-05 27664 PITX1 2.22382 0.00102378 19467 LOC645452 2.21703 0.000168591 6533 FAM46A 2.21591 1.24E-03 26308 NT5C2 2.21285 0.00159191 1086 ARID5B 2.21184 0.000139405 494 AFF3 2.21139 1.28E-03 29124 RGS16 2.21012 2.33E-05 10056 HS.475334 2.20669 1.41E-03 3738 CDH10 2.20522 0.000389319 9819 HS.388347 2.2022 2.82E-05 34703 ZNF786 2.20193 1.65E-03 8705 HIST1H4J 2.19897 4.86E-06

- 79 041083

9740 HS.36053 2.19737 3.65E-05 651 ALG13 2.19414 4.86E-05 33588 VASH2 2.19373 0.000586609 28810 RALGPS1 2.19311 0.000999046 5774 EFNB3 2.191 0.000919096 1745 BSCL2 2.18758 8.17E-04 29151 RHBDL3 2.18758 4.86E-05 3740 CDH12 2.18674 0.000112317 24064 MAP1LC3B 2.18347 2.00E-04 32539 TMEM59 2.17758 1.77E-03 33171 TUB AZE 2.17556 2.58E-04 526 AGPAT4 2.17042 1.95E-04 27832 PLXNA1 2.16997 0.000477137 34219 ZMPSTE24 2.16821 0.00130116 34669 ZNF738 2.16494 0.00145642 28749 RAB9B 2.16376 1.41E-03 3350 CCBE1 2.16311 2.69E-04 5875 ELAVL1 2.15865 6.37E-04 13154 KIAA1370 2.15727 5.07E-05 1356 ATP6V1B2 2.15309 0.000839601 6457 FAM181B 2.15303 6.91E-04 25137 MKL2 2.14461 0.000214816 27876 PNMA1 2.14384 0.000290225 23806 LRRC37B2 2.14195 4.81E-04 6851 FGD3 2.139 1.84E-04 1746 BSDC1 2.13842 0.000430355 27997 POTEF 2.13305 0.000655028 2969 C7ORF55 2.12929 5.66E-05 29923 SEC11C 2.12843 8.11E-04 31488 SSX2IP 2.12215 5.19E-06 29277 RNF128 2.12203 5.41E-06 30348 SLC12A2 2.12048 0.000343525 8277 GRK5 2.11347 0.000106085 8573 HEPACAM2 2.11332 2.39E-09 1610 BEXL1 2.10951 2.62E-04

4229 CMIP 2.10246 0.000705627 30224 SHANK2 2.10164 0.000611099 23763 LRP1B 2.09999 4.76E-05 6724 FBXL16 2.09975 8.65E-04 26092 NLRP8 2.09701 0.00161834 17496 LOC440704 2.09699 0.0015232 2543 C20ORF56 2.09698 4.79E-07 32624 TMX4 2.09279 8.41E-04 20006 LOC646821 2.0917 5.47E-05 25809 NCOA6 2.09025 1.61E-03 2811 C5ORF28 2.08938 6.33E-04 3279 CASD1 2.0821 6.10E-05 28256 PRKAR1A 2.08207 0.00119569 6208 EVI1 2.07806 4.85E-05 6920 FJX1 2.07539 1.80E-03 31199 SORBS2 2.07266 7.37E-05 3968 CHD6 2.06939 4.92E-05 34605 ZNF652 2.06441 0.00172675 4838 CYCS 2.06436 1.39E-03 34025 ZADH2 2.05857 3.02E-05 17246 LOC401098 2.05589 1.58E-03 27462 PFN2 2.05533 1.58E-03 27137 PARP6 2.05441 5.65E-04 5651 DYNC1I1 2.04874 0.000834135 7587 GABARAPL2 2.04534 0.00165234 149 AASDHPPT 2.04017 7.58E-04 1970 C12ORF51 2.038 0.000668525 31721 SWOP 2.03603 7.00E-04 1389 ATXN1 2.03408 3.27E-05 20667 LOC648980 2.03345 0.00062575 24523 MIB1 2.03159 3.22E-04 31982 TCEAL3 2.03073 0.00151379 12229 HSD17B7 2.03003 1.50E-03 27402 PDZRN4 2.02983 2.33E-05 6395 FAM13B 2.02944 3.18E-04

- 80 041083

1349 ATP6V0C 2.02572 1.08E-03 12828 JAKMIP2 2.01869 8.10E-04 34227 ZMYND11 2.01687 0.00171037 31603 STMN1 2.00603 0.000940483 29145 RHBDD2 2.00523 6.87E-04 25808 NCOA5 2.00244 0.000102715 30330 SLAIN1 2.00203 1.00E-03 2195 C17ORF45 -2.00166 0.00119767 23469 LOC730746 -2.00168 1.67E-03 30825 SND1 -2.00433 0.00128091 19456 LOC645436 -2.00559 1.69E-03 6242 EXOSC7 -2.00603 0.000798232 17727 LOC442232 -2.00726 7.96E-05 28062 PPIH -2.00996 8.70E-04 23234 LOC729779 -2.01096 4.05E-04 33239 TYK2 -2.01275 0.000787726 27020 P4HA2 -2.01467 1.3OE-O3 4563 CRTAP -2.01592 2.86E-06 29147 RHBDF1 -2.01731 9.21E-04 18367 LOC643007 -2.01867 0.000867101 2331 C1ORF106 -2.02039 2.06E-04 1332 ATP5G2 -2.02135 0.00169599 25354 MRPL45 -2.02158 0.00179523 11023 HS.552799 -2.02363 1.41E-04 17333 LOC402112 -2.02644 0.00178903 4341 COL2A1 -2.03118 0.00112645 14518 LOC100130009 -2.03222 1.54E-05 23928 LYN -2.03236 0.000609191 20489 LOC648294 -2.0327 0.0004778 32244 TIMM9 -2.03469 0.0015035 29412 RPA1 -2.03493 1.70E-03 33756 WDR12 -2.03533 5.96E-04 25781 NCAPD2 -2.03535 0.00132141 19436 LOC645385 -2.03595 0.00151373 2095 C14ORF93 -2.04138 6.93E-06 33243 TYRO3 -2.04437 4.26E-04

13396 KNTC1 -2.04704 1.95E-05 19027 LOC644464 -2.051 0.000744833 1355 ATP6V1B1 -2.0528 0.000263628 32263 TKT -2.05643 0.000380042 1039 ARHGAP19 -2.05734 6.74E-05 4923 CYYR1 -2.05748 1.68E-05 30076 SESN1 -2.05841 2.09E-04 5054 DDX10 -2.06089 0.000505 12782 ITGB3BP -2.06492 6.73E-06 17492 LOC440589 -2.06666 4.64E-04 23246 LOC729816 -2.06873 0.000791801 24128 MAPKAPK3 -2.06933 0.00146997 12221 HSD17B11 -2.07106 2.25E-05 32916 TRIM71 -2.07489 2.28E-05 16186 LOC136143 -2.07533 4.81E-04 31614 ST0X1 -2.07691 0.000191887 28717 RAB38 -2.07726 6.33E-04 21994 LOC653156 -2.07967 1.41E-03 29560 RPSA -2.08096 0.000379606 17241 LOC401074 -2.08141 6.58E-08 8588 HES1 -2.08285 0.000819562 29494 RPLP1 -2.08688 1.3OE-O3 6884 FGFR3 -2.09082 5.89E-06 4977 DBX1 -2.09957 0.000222703 29544 RPS5 -2.10042 0.00138553 29213 RIPK2 -2.10641 0.000218454 14423 LOC100129685 -2.1066 1.19E-03 27816 PLOD3 -2.10673 0.000954258 8805 HNRNP1 -2.10967 1.74E-05 6898 FHL3 -2.1127 5.26E-05 18250 LOC642741 -2.1147 1.02E-03 32523 TMEM45A -2.1169 0.000464867 17691 LOC441876 -2.11785 1.02E-03 1488 BAIAP2L1 -2.11832 3.63E-05 4156 CLEC2D -2.11902 0.000733653 6966 FLJ10986 -2.12606 0.000420546

- 81 041083

14281 LOC100129237 -2.12997 4.96E-04 16802 LOC388556 -2.13532 6.52E-04 25385 MRPS30 -2.13565 3.28E-04 18023 LOC642250 -2.13595 0.00107704 31129 SNRPA -2.13825 6.40E-04 3730 CDCA5 -2.14343 0.0016145 26121 NOB1 -2.14482 3.43E-06 3733 CDCA8 -2.14728 0.000158263 33839 WDYHV1 -2.14889 0.000901318 15213 LOC100132299 -2.14934 3.42E-05 23294 LOC729964 -2.14942 0.000145725 23388 LOC730246 -2.15222 5.52E-04 29474 RPL36 -2.15341 1.12E-03 20693 LOC649076 -2.15494 0.000533848 14136 LOC100128771 -2.15556 6.32E-05 29459 RPL27 -2.15569 8.13E-04 20894 LOC649839 -2.15637 0.000233819 5649 DYM -2.15657 5.43E-04 6676 FANCG -2.15913 2.75E-04 26223 NPY -2.15981 4.99E-04 22700 LOC728428 -2.16464 0.00122727 27540 PHGDH -2.17929 0.000926407 23875 LSM4 -2.18409 0.00036081 15295 LOC100132528 -2.18468 8.34E-04 14997 LOC100131609 -2.187 0.000875169 29442 RPL14L -2.18734 4.55E-04 16653 LOC343184 -2.19197 6.17E-05 5697 EBPL -2.19209 2.05E-05 2763 C4ORF14 -2.19304 0.00128088 4474 CPNE8 -2.19514 2.56E-04 17500 LOC440737 -2.19615 0.000989795 22974 LOC729102 -2.1963 0.000282713 21275 LOC651149 -2.19678 1.31E-O3 17403 LOC440055 -2.19775 1.17E-03 29440 RPL13L -2.19817 2.16E-05 27368 PDK3 -2.20067 3.05E-04

31760 SYPL1 -2.20452 0.000128778 14389 LOC100129585 -2.20627 8.87E-05 29490 RPL8 -2.20725 1.35E-04 15083 LOC100131866 -2.20753 2.67E-05 2915 C6ORF48 -2.20788 0.000428494 19976 LOC646766 -2.21365 0.00033802 28801 RAI14 -2.21753 0.000675636 18557 LOC643433 -2.21779 0.000542767 16862 LOC389141 -2.21829 0.000459763 5715 ECT2 -2.21838 1.41E-03 29558 RPS8 -2.22068 0.00161471 19556 LOC645691 -2.22442 0.00107987 16693 LOC347544 -2.22526 0.000534957 23972 MAD2L1 -2.22823 0.000457464 18286 LOC642817 -2.23024 0.00054939 31588 STK24 -2.23459 3.03E-04 31024 SNORD25 -2.236 5.13E-05 1282 ATIC -2.24113 0.000182776 33110 TTK -2.24337 4.48E-04 23352 LOC730107 -2.24665 0.000445574 8727 HJURP -2.24769 2.63E-05 23237 LOC729789 -2.24864 3.21E-04 29503 RPP40 -2.25604 1.62E-04 17594 LOC441246 -2.25891 4.60E-04 17228 LOC400963 -2.26592 0.000542002 29521 RPS18 -2.26781 0.00151457 32392 TMEM144 -2.27099 5.66E-08 7652 GALK1 -2.27206 9.50E-04 27192 PCDH18 -2.27211 0.000113843 4297 CNTNAP1 -2.2794 2.43E-04 23041 LOC729279 -2.28142 5.88E-05 5840 EIF3H -2.28165 0.000193064 8828 HNRPC -2.28204 0.00128473 30493 SLC27A3 -2.29001 2.85E-04 5742 EEF1D -2.29339 0.000126152 30601 SLC44A1 -2.29419 0.000172089

- 82 041083

3866 CENPN -2.2944 0.000143689 29449 RPL22 -2.29465 0.00105741 20804 LOC649447 -2.29999 0.000262246 28036 PPAT -2.3009 0.000393145 25509 MTP18 -2.30185 3.55E-05 3538 CCNA2 -2.30784 0.000412267 33891 WNT5A -2.3104 3.36E-04 28110 PPP1R3C -2.31108 3.02E-06 1659 BMP2 -2.32278 2.31E-05 18725 LOC643863 -2.32406 1.02E-03 359 AD -2.3269 2.14E-05 13751 LGMN -2.32715 4.54E-05 12250 HSP90AB1 -2.32781 1.49E-03 5850 EIF4B -2.32818 1.13E-O3 13724 LEPREL1 -2.32851 0.000169338 17534 LOC440991 -2.33062 1.54E-04 29559 RPS9 -2.33092 1.06E-04 3724 CDC7 -2.33576 0.000342965 27035 PABPC4 -2.33611 0.000164425 33241 TYMS -2.34068 0.00106577 30229 SHC1 -2.34608 0.00105777 21754 LOC652624 -2.34734 2.21E-04 5846 EIF3M -2.34987 0.00181367 15032 LOC100131713 -2.35114 0.000201749 29432 RPL10A -2.35345 0.00131867 24235 MCM6 -2.35636 0.00108431 19046 LOC644511 -2.36244 0.000514797 5044 DDIT4L -2.36718 0.000107365 545 AHCY -2.3677 0.000431822 18592 LOC643531 -2.37199 1.01E-03 5744 EEF1G -2.37611 0.000630065 5765 EFHD1 -2.38043 1.20E-03 32637 TNFRSF10B -2.38066 1.53E-05 26099 NME1-NME2 -2.38426 4.44E-05 22757 LOC728564 -2.38494 3.69E-04 8826 HNRPA1P4 -2.38609 3.87E-05

17458 LOC440359 -2.38691 5.63E-04 28560 PTPN13 -2.39211 0.000413752 15180 LOC 100132199 -2.39337 2.68E-05 4955 DARS -2.39728 1.56E-03 1882 C11ORF1 -2.39828 2.51E-05 23282 LOC729926 -2.39973 7.65E-04 15041 LOC 100131735 -2.40035 2.35E-05 22829 LOC728732 -2.40089 6.11E-05 3973 CHEK1 -2.40178 1.16E-06 600 AKR1A1 -2.40346 0.000695089 15586 LOC100133372 -2.40481 1.53E-04 31607 STOM -2.40533 3.40E-07 22710 LOC728453 -2.40686 0.0001068 29457 RPL26 -2.4112 0.000285349 29921 SEC11A -2.41182 0.00102718 16776 LOC388275 -2.41288 5.56E-06 29438 RPL13A -2.41592 0.000922424 3795 CDKN3 -2.41856 0.000112421 30404 SLC20A2 -2.41879 6.23E-06 32648 TNFRSF19 -2.42065 6.93E-05 31239 SP5 -2.42085 2.37E-09 23974 MAD2L2 -2.42585 0.000123115 12602 IMPA2 -2.42996 1.90E-06 17525 LOC440927 -2.43391 0.00119949 29414 RPA3 -2.43719 0.00177478 16156 LOC127295 -2.43999 5.06E-04 29565 RPUSD3 -2.44425 1.98E-07 16979 LOC390578 -2.44648 8.18E-08 15104 LOC 100131940 -2.44655 2.19E-05 26103 NME4 -2.44757 0.00114224 8825 HNRPA1L-2 -2.45235 5.46E-04 1537 BCAR3 -2.45546 0.000368573

- 83 041083

14206 LOC100129028 -2.45901 3.51E-04 16672 LOC345041 -2.46013 0.000101781 3125 SAN -2.46417 0.000205741 32089 TET1 -2.46774 3.21E-05 12231 HSD17B8 -2.47022 0.000519001 4479 CPS1 -2.47967 2.21E-06 25464 MT AZ -2.48169 8.09E-05 5758 EFEMP1 -2.48833 1.42E-05 28160 PQLC3 -2.48845 0.000585543 30245 SHMT2 -2.48964 0.00117427 23103 LOC729423 -2.50041 6.49E-06 12741 ISG20L1 -2.50404 9.70E-05 29539 RPS3 -2.50954 0.000103446 22857 LOC 728791 -2.5096 5.69E-06 23391 LOC 730255 -2.50972 1.22E-03 24097 MAP4K2 -2.51767 5.17E-06 7686 GAPDH -2.51826 0.00128279 2433 C1ORF57 -2.51861 1.49E-03 2262 C18ORF56 -2.51885 0.000156088 1792 BUB1 -2.52119 9.50E-06 5831 EIF2S3 -2.52373 3.38E-04 29039 RENBP -2.53102 4.92E-07 29481 RPL39L -2.53153 0.00013954 17141 LOC399804 -2.5389 3.38E-05 34552 ZNF581 -2.54051 9.37E-05 1156 ARRDC4 -2.54054 2.88E-05 8789 HMMR -2.54277 8.17E-05 28953 RBMX -2.5493 5.57E-05 32179 THEM2 -2.55298 5.30E-04 13239 KIF20A -2.55316 1.13E-05 1403 AURKA -2.55681 5.93E-05 27066 PAICS -2.55763 0.00111653 5836 EIF3D -2.56061 6.72E-05 13252 KIF2C -2.56757 1.70E-06

14814 LOC 100130980 -2.57639 4.33E-05 31821 TAF1D -2.57677 6.27E-06 27653 PIR -2.58392 2.26E-06 7564 FZD2 -2.58518 0.000146094 28778 RAD51AP1 -2.59104 5.06E-06 14695 LOC 100130562 -2.59563 3.41E-05 8547 HEATRI -2.59617 5.95E-07 22591 LOC728126 -2.59998 3.37E-05 29483 RPL4 -2.62451 7.56E-05 25783 NCAPG -2.62632 6.41E-05 20366 LOC647856 -2.6267 1.31E-05 29462 RPL29 -2.62809 4.36E-07 23274 LOC729903 -2.63121 4.28E-05 15975 LOC 100134393 -2.6327 2.71E-04 9745 HS.363526 -2.6341 0.000643774 561 AIF1L -2.63663 1.37E-04 8597 HEXB -2.63709 1.51E-03 5993 EPDR1 -2.63912 0.00156376 7966 GMPS -2.64155 1.99E-04 27563 PHYH -2.64322 2.14E-05 4100 CKS1B -2.64512 1.68E-04 7898 GLDC -2.64809 0.00143371 5513 DPH5 -2.6481 2.11E-05 18914 LOC644237 -2.66097 0.0011589 20185 LOC647285 -2.66563 1.82E-04 22536 LOC727984 -2.67684 0.000223687 8625 HIBADH -2.68374 0.00144114 684 ALPL -2.69266 2.39E-06 17636 LOC441506 -2.70546 6.10E-05 30602 SLC44A2 -2.70757 1.24E-03 16741 LOC387867 -2.71424 2.45E-05 23060 LOC729340 -2.7157 0.000999351

- 84 041083

27743 PLCD1 -2.72 1.14E-07 22597 LOC728139 -2.72434 4.77E-05 28530 PTGR1 -2.72675 7.39E-05 5741 EEF1B2 -2.7289 4.84E-05 18360 LOC642989 -2.73236 0.000921883 20058 LOC646942 -2.74335 5.41E-05 16737 LOC387825 -2.74954 5.93E-06 16236 LOC148430 -2.76485 2.32E-05 29441 RPL14 -2.76505 4.49E-06 31176 SNX5 -2.77426 4.41E-08 13592 LAMA1 -2.77535 2.89E-07 16814 LOC388707 -2.77694 1.99E-06 29179 RHPN2 -2.78226 0.00110049 23133 LOC729500 -2.78295 8.63E-06 2524 C20ORF199 -2.79683 8.26E-05 27370 PDLIM1 -2.79852 0.000250318 27030 PABPC1 -2.7998 0.000107149 7486 FRZB -2.81189 5.35E-04 17023 LOC391075 -2.82637 0.00139394 6705 FBL -2.8276 2.59E-05 13708 LEF1 -2.83207 1.15E-05 4101 CKS2 -2.83376 0.000537912 13438 KRT19 -2.83803 2.34E-05 28410 PSAT1 -2.8451 8.29E-07 33979 YAP1 -2.84569 0.00106156 29435 RPL12 -2.85091 4.23E-05 22553 LOC728031 -2.85482 8.14E-05 29524 RPS2 -2.85918 1.32E-05 23861 LRTM1 -2.85972 1.48E-12 26404 NUSAP1 -2.86459 9.00E-05 20473 LOC648249 -2.86802 4.01E-06 23319 LOC730029 -2.87607 3.58E-05 24516 MGST1 -2.87812 2.82E-06 349 ACVR1 -2.88477 0.000447642 30500 SLC29A1 -2.91375 2.94E-06 17086 LOC391833 -2.91531 4.06E-05

20614 LOC648771 -2.91567 7.23E-05 27493 PGM1 -2.92082 0.000287518 30836 SNHG6 -2.92725 2.13E-05 20015 LOC646849 -2.95184 3.75E-05 16783 LOC388339 -2.95309 4.55E-05 3785 CDKN1A -2.95807 0.000310093 29436 RPL12P6 -2.97114 2.40E-06 5258 DIMT1L -2.97803 1.83E-06 15940 LOC100134304 -2.98223 4.08E-08 13733 LGALS1 -2.98473 0.00160731 27902 PODXL -3.00627 2.35E-08 16630 LOC341315 -3.02065 1.10E-04 481 ADSS -3.02244 4.06E-06 29557 RPS7 -3.06985 2.69E-06 1406 AURKB -3.07612 5.67E-07 23373 LOC730187 -3.08276 4.32E-06 25214 MND1 -3.08698 3.39E-07 14412 LOC 100129657 -3.08986 0.00120608 33282 UBE2C -3.09218 4.69E-06 30114 SFRP1 -3.10477 0.000250519 17015 LOC391019 -3.10602 2.05E-05 28602 PTTG1 -3.11309 2.05E-05 17541 LOC441013 -3.11866 6.73E-07 20657 LOC648931 -3.12515 3.24E-09 31661 SUCLG2 -3.13505 5.75E-07 1559 BCL2L12 -3.13599 4.40E-07 32222 TIGA1 -3.13783 7.09E-05 7770 GDF15 -3.14204 1.42E-07 2878 C6ORF160 -3.15022 0.000111744 15382 LOC100132795 -3.21797 1.51E-05 4930 DAB2 -3.2203 2.91E-05 32933 TRIP6 -3.23132 1.32E-06 20091 LOC647030 -3.23731 4.86E-05 26048 NKD1 -3.24181 4.37E-06 6320 FAM107A -3.24905 0.000449795

- 85 041083

16552 LOC286444 -3.25137 6.05E-06 28975 RBPMS2 -3.82727 3.95E-06 26390 NUP37 -3.26236 3.70E-05 23218 LOC729742 -3.84552 7.82E-07 19783 LOC646294 -3.28211 1.06E-06 13597 LAMB1 -3.85107 1.24E-07 2210 C17ORF61 -3.28907 2.68E-05 6291 FABP7 -3.85422 0.000335025 19553 LOC645688 -3.29172 3.95E-06 12606 IMPDH2 -3.85952 4.79E-06 25847 NCRNA00219 -3.29327 7.23E-05 13834 LITAF -3.86 4.66E-05 28663 QPRT -3.29832 2.04E-05 3953 CHCHD10 -3.91295 4.20E-06 31361 SPON1 -3.32269 5.27E-06 2439 C1ORF64 -3.9544 5.48E-10 30835 SNHG5 -3.32459 0.000204237 8473 HAUS4 -3.96227 4.95E-12 4596 CSDA -3.3629 0.000363294 925 APCDD1 -3.97854 6.09E-08 455 ADM -3.40482 4.11E-05 5952 ENO3 -4.00663 6.90E-09 23228 LOC729768 -3.41997 2.20E-05 27019 P4HA1 -4.01147 0.00032695 1753 BST2 -3.42742 1.17E-07 4724 CTSL2 -4.01208 1.47E-06 20685 LOC649049 -3.46913 1.63E-06 19348 LOC645173 -4.02454 2.71E-06 17292 LOC401537 -3.50501 3.79E-07 28605 PTTG3P -4.02805 3.37E-06 6707 FBLN1 -3.53279 3.88E-06 4239 CMTM7 -4.08386 7.09E-09 28241 PRIM1 -3.53892 1.17E-06 8089 GPI -4.08821 1.18E-05 8754 HLA-E -3.54293 2.03E-07 1616 BGN -4.11601 0.000348095 643 ALDOA -3.55096 0.000184641 23196 LOC729679 -4.2525 4.55E-08 14422 LOC100129681 -3.55393 9.42E-07 3694 CDC20 -4.28351 3.38E-06 8773 HMGB2 -3.56419 9.08E-05 32729 TOP2A -4.29945 1.43E-06 32964 TRPM4 -3.5945 2.41E-06 30828 SNHG1 -4.38641 2.77E-06 12398 IFITM2 -3.59918 1.38E-03 3243 CAPN6 -4.44794 4.50E-07 32177 THBS4 -3.6116 6.59E-07 30284 SILV -4.59486 1.80E-07 3540 CCNB1IP1 -3.62989 3.35E-08 15342 LOC 100132673 -4.69254 2.18E-07 5477 DOCK10 -3.64495 7.09E-07 28387 PRSS23 -3.67253 0.000258012 29716 SALL4 -4.95909 2.66E-08 26369 NUDT7 -3.67565 7.62E-05 3541 CCNB2 -5.23718 4.33E-08 26207 NPM3 -3.69114 1.35E-06 30511 SLC2A3 -5.39887 5.64E-07 23887 LTA4H -3.69913 6.82E-05 13691 LDHA -5.76362 0.000569306 12399 IFITM3 -3.70541 0.000779898 7500 FST -5.82949 8.74E-09 8769 HMGA1 -3.72752 3.30E-08 1543 BCAT1 -6.13131 3.52E-06 323 ACTA2 -3.74071 1.75E-04 5323 DKK1 -6.17493 4.94E-08 1530 BBS9 -3.7636 3.82E-07 4343 COL4A1 -6.21986 6.18E-07 7501 FSTL1 -3.80754 0.000136961 8078 GPC3 -6.35513 0.000466041 27805 PLIN2 -3.82123 4.77E-10 4333 COL22A1 -6.371 1.87E-08 32774 TPM2 -6.8139 1.85E-08 2430 C1ORF54 -7.81915 1.70E-07 965 APOE -9.10671 6.03E-07 13804 LIN28 -26.8821 1.20E-13

- 86 041083

SPON1

31361

4.93795

4.69E-08

Table 4. Gene expression analysis results for genes that are significantly up or down on day 25 of differentiation in a ventral plate-based population treated with SHH/FGF8/Chir compared to a control column treated with LSB

column number and Column ID multiple changed Not p-value 13857 LMO3 4.83578 7.25E-08 13865 LMX1A 4.63407 1.51E-06 2543 C20ORF56 4.4431 3.63E-13 32126 TFF3 25.0585 2.47E-14 12733 IRX5 4.2929 1.45E-06 1209 ASCL1 14.9328 1.97E-07 7988 GNB3 4.13634 1.52E-10 7395 FOXA1 12.9897 4.15E-14 32159 TH 4.08743 0.000370318 29133 RGS4 11.0181 6.65E-09 5346 DLK1 4.02203 1.73E-05 27988 POSTN 8.20403 2.16E-10 31775 SYT4 4.00669 0.000372793 7396 FOXA2 7.98132 1.60E-14 9428 HS.204481 3.88697 6.71E-09 4342 COL3A1 7.58625 2.49E-05 6197 ETS2 3.70155 3.07Е-09 12731 IRX3 7.42399 1.64E-06 5036 DDC 3.61254 0.000194062 9374 HS.19193 7.27196 7.36Е-09 26250 NR4A2 3.55879 9.29E-10 3978 CHGA 7.14305 2.77E-08 2867 C6ORF141 3.48146 6.97Е-09 2470 C1QL1 7.1366 1.38E-09 26928 OSBPL10 3.27941 3.81E-07 30390 SLC18A1 7.04751 3.52E-14 5372 DMRTA2 3.26437 1.81E-08 22823 LOC728715 6.5801 8.79Е-09 33930 XVR1 3.2414 0.000921089 column number and Column ID multiple changed ie p-value 27664 PITX1 3.23009 1.10E-05 9819 HS.388347 3.14523 8.26E-08 30239 SHISA2 3.13411 4.23E-05 12456 IGFBP5 6.15962 3.65E-05 2743 C3ORF58 3.10109 1.50E-06 27665 PITX2 6.10679 6.79E-14 623 ALCAM 3.00487 0.000307411 12655 INSM2 5.18438 1.70E-09 6533 FAM46A 2.99243 3.45E-05 12944 KCNJ16 4.98753 8.89E-13 28590 PTPRO 2.94164 4.45Е-05 436 ADCYAP1 4.94597 2.43E-09 18750 LOC643911 2.93448 1.14E-06

- 87 041083

31669 SULF2 2.92397 8.02E-05 19806 LOC646345 -2.02311 0.000183202 5348 DLL1 2.92274 8.36E-05 23846 LRRIQ1 -2.02718 0.000541683 30066 SERPINF1 2.76873 0.000156193 27746 PLCE1 -2.02761 0.000217281 5349 DLL3 2.75425 0.000173675 2727 C3ORF39 -2.03023 0.000131521 3186 CALCA 2.71125 2.10E-07 26072 NLGN3 -2.03154 2.45E-05 30590 SLC39A8 2.67444 2.25E-05 2770 C4ORF22 -2.03489 0.00113778 32000 TCF12 2.66777 1.46E-06 3491 CCDC88C -2.03515 0.00112864 3941 CGNL1 2.63848 0.00120585 25751 NAV3 -2.03716 8.63E-05 32769 TPH1 2.62647 1.95E-06 30413 SLC22A17 -2.03749 0.000703407 25941 NENF 2.55992 0.000599552 13014 KDELC2 -2.04133 0.00147391 29509 RPRM 2.53138 0.00135525 27749 PLCHI -2.0426 0.000234672 32144 TGFBR3 2.46754 8.27E-07 16489 LOC284988 -2.04353 0.000785305 13689 LDB2 2.40983 2.56E-05 9938 HS.440518 -2.04573 0.000285223 2850 C6ORF117 2.4009 0.000508473 28076 PPM1H -2.04637 0.00038276 31806 TACSTD1 2.38661 0.00154495 429 ADCY3 -2.05728 0.000712376 24375 MFNG 2.31488 5.04E-05 31199 SORBS2 -2.05812 8.31E-05 5424 DNAJC19 2.30405 4.42E-07 17624 LOC441453 -2.0676 3.05E-06 12986 KCNS3 2.28802 0.000136062 13793 LIM2 -2.07092 0.00040641 27855 PMP22 2.27371 1.36E-05 33165 TUB -2.07384 0.000187156 4003 CHN2 2.26467 0.000550787 3462 CCDC65 -2.07613 0.00151516 5691 EBF3 2.26453 0.000571924 4305 COBL -2.07819 0.000485834 3187 CALCB 2.11359 2.49E-06 6912 FILIP 1 -2.07924 9.69E-05 28325 PROXI 2.08192 0.000135872 5195 DGCR6 -2.07943 0.000935686 25218 MNX1 2.05528 3.37E-06 33905 WSCD1 -2.08212 0.00033792 6270 F3 2.01261 5.71E-07 12933 KCNIP1 -2.08721 3.72E-05 2245 C18ORF10 -2.00297 0.00129421 4933 DACH2 -2.09051 9.24E-05 28546 PTN -2.003 0.000354472 25960 NEUROG2 -2.09361 0.00111856 5920 EMID2 -2.00456 1.77E-05 25930 NEK2 -2.09683 0.00144313 24405 MGC11082 -2.00498 2.75E-05 3288 CASP3 -2.09925 0.000130992 5949 ENKUR -2.00742 0.000107398 12971 KCNMB4 -2.09934 0.00140267 26398 NUP93 -2.01111 0.00124009 3125 CA14 -2.10277 0.00147559 10837 HS.545615 -2.01378 2.65E-05 17153 LOC399959 -2.10829 0.000816932 12688 IQCC -2.01383 0.00019749 15855 LOC100134073 -2.10866 0.000366572 6196 ETS1 -2.01751 0.000848186 26322 NTM -2.10973 0.000159062 27290 PCSK5 -2.02079 0.00122771 7595 GABRA2 -2.11173 0.00169771 16112 LOC100192378 -2.0216 4.35E-05 5618 DUSP18 -2.11289 8.90E-05

- 88 041083

4289 CNTFR -2.11375 0.00132713 32380 TMEM132D -2.22662 0.000463067 3760 CDH8 -2.11381 7.41E-05 2705 C3ORF15 -2.2299 0.000615847 3196 CALM1 -2.11418 0.00157715 27781 PLEKHG1 -2.23885 1.49E-05 13615 LARGE -2.11685 2.37E-05 29607 RSPH9 -2.24091 0.000109859 32933 TRIP6 -2.11829 0.00047459 23965 MAB21L1 -2.24222 2.00E-05 2242 C17ORF97 -2.12666 0.000851745 25942 ΝΕΟΙ -2.24511 2.27E-05 5478 DOCK 11 -2.12763 0.000306132 34157 ZFP106 -2.24688 0.00129693 7377 FNDC4 -2.13338 0.000482465 33083 TTC29 -2.24927 0.000317648 29672 RYR3 -2.13573 0.000392208 3415 CCDC19 -2.25412 0.000638581 30669 SLC7A6 -2.13865 0.000299041 30209 SH3GL2 -2.2573 0.00175354 29816 SCD5 -2.14266 9.40E-05 839 ANKS1B -2.25784 6.46E-05 12212 HS6ST2 -2.14751 0.00020473 3472 CCDC74A -2.25911 3.66E-05 4213 CLSTN2 -2.15117 2.14E-05 27561 PHTF1 -2.26039 0.00113655 16924 LOC389816 -2.15626 4.60E-09 26505 OPCML -2.26062 2.34E-05 2102 C15ORF26 -2.15663 0.000242005 23859 LRRTM4 -2.26387 0.00050229 1103 ARL4C -2.16184 0.00049625 27040 PACRG -2.26701 8.08E-05 31151 SNX10 -2.16614 0.000623725 24318 MELK -2.27019 0.000423662 16382 LOC255783 -2.17123 0.000592438 10017 HS.452398 -2.27201 0.000477927 30372 SLC16A14 -2.17701 0.000382805 9237 HS.147562 -2.27279 0.000798954 13177 KIAA1598 -2.1779 0.000634174 26976 OTX1 -2.27327 0.000877908 28587 PTPRM -2.17848 0.000334455 8046 GOLSYN -2.27724 0.000100581 8661 HIST1H2AC -2.18157 0.00018782 1074 ARHGEF6 -2.27982 0.000748746 30015 SEPT6 -2.18483 0.00101243 4639 CSRNP3 -2.28361 0.000213299 6572 FAM65B -2.18593 1.46E-05 27547 PHLDA1 -2.28667 2.70E-05 4614 CSMD2 -2.18777 0.000579739 3537 CCNA1 -2.28864 6.92E-05 19737 LOC646168 -2.18864 0.000417767 26071 NLGN2 -2.29131 0.000215422 2429 C1ORF53 -2.19182 0.0017844 5325 DKK3 -2.29195 0.000372444 17855 LOC641785 -2.19315 0.000255891 6326 FAM108C1 -2.29624 0.000567659 5570 DSCR6 -2.20122 0.00137487 28721 RAB3B -2.30577 0.000211905 13835 LIX1 -2.20141 6.32E-06 2372 C1ORF158 -2.3174 0.00184147 23979 MAF -2.20457 6.38E-05 32499 TMEM231 -2.32173 0.000378145 4734 CTXN1 -2.21039 0.0007813 5550 DRD1IP -2.3265 0.00129147 2049 C14ORF159 -2.2127 0.000171696 12785 ITGB5 -2.32837 0.00143999 30388 SLC17A8 -2.21639 0.000916084 30937 SNORA79 -2.32935 0.000394221 26228 NQO1 -2.21974 0.000753734 2538 C20ORF46 -2.34101 0.000827542 23817 LRRC46 -2.22159 0.000756129 5939 EMX2OS -2.34991 1.28E-05

- 89 041083

3153 CACNA1E -2.35127 0.000755994 4965 DBC1 -2.53221 0.000370596 28906 RBKS -2.35486 1.25E-05 32507 TMEM31 -2.53343 2.39E-06 3806 CDRT4 -2.36622 0.000173226 1927 C11ORF75 -2.53649 0.000498243 32129 TFPI -2.36876 0.000644776 6740 FBX015 -2.54184 0.00139562 5763 EFHC1 -2.36977 0.00112503 27370 PDLIM1 -2.54956 0.000681773 3746 CDH18 -2.37821 6.60E-06 728 AMPH -2.55801 6.39E-06 33006 TSHZ3 -2.38415 0.00160322 33718 VWC2 -2.56363 0.000340976 6901 FHOD3 -2.38572 0.00106294 13724 LEPREL1 -2.56798 4.43E-05 13805 LIN28B -2.38688 0.000393414 1057 ARHGDIB -2.573 5.20E-06 1806 C10ORF107 -2.39735 0.000750154 5765 EFHD1 -2.59294 0.000474204 32034 TCTEX1D1 -2.40988 0.00160003 3758 CDH6 -2.6075 0.000303268 31146 SNTN -2.42893 0.000945171 7043 FLJ23152 -2.60944 0.000143522 30057 SERPINB6 -2.43305 1.01E-05 31282 SPATA17 -2.61656 0.00020388 28580 PTPRD -2.43513 6.24E-05 7106 FLJ33590 -2.61873 0.00025779 28210 PRDM8 -2.44594 0.000113613 16264 LOC151162 -2.62005 0.000666466 2614 C22ORF15 -2.45467 0.000112958 32440 TMEM178 -2.62313 0.00011953 12892 KBTBD9 -2.45472 2.16E-06 1987 C12ORF69 -2.62861 5.06E-06 6246 EXT1 -2.4653 0.000117948 2970 C7ORF57 -2.63494 0.000118643 25946 NETO2 -2.46601 0.000568595 23830 LRRC6 -2.63632 0.000197619 493 AFF2 -2.4707 0.0012124 6173 ESMI -2.63913 0.000368518 31610 STOML3 -2.48765 0.000560404 153 ABAT -2.64114 3.81E-06 31577 STIL -2.49103 0.000199457 2793 C4ORF47 -2.64571 0.00134045 4826 CYB5D1 -2.49147 0.000128822 2301 C19ORF51 -2.64617 7.44E-05 2078 C14ORF45 -2.49501 0.000787004 34155 ZFHX4 -2.66127 5.12E-06 33501 USP13 -2.49771 6.06E-05 5762 EFHB -2.66338 0.000148296 34226 ZMYND10 -2.50015 0.000105158 6920 FJX1 -2.66433 8.09E-05 28507 PTCHD1 -2.50016 0.00120417 12872 KANK4 -2.66744 0.000457332 5322 DKFZP781N1041 -2.50483 0.000661286 26104 NME5 -2.66836 0.000118819 3253 CAPSL -2.50626 0.000844682 3148 CACHD1 -2.67846 0.000100474 14289 LOC100129268 -2.508 0.000176359 25628 MYO16 -2.67972 8.67E-05 17551 LOC441054 -2.5088 0.00166136 28838 RAPGEF2 -2.68049 7.06E-05 26395 NUP62CL -2.51081 0.000373281 5553 DRD4 -2.68408 0.000500735 1776 BTG3 -2.51582 0.00131261 4087 CITED2 -2.68818 0.00192224 33005 TSHZ2 -2.51649 3.77E-05 23790 LRRC26 -2.68889 2.44E-06 4705 CTNND2 -2.52798 0.00038507 2631 C22ORF42 -2.69089 0.00039382 29792 SCARNA11 -2.53148 3.35E-06 25564 MXRA5 -2.69302 0.00192861

- 90 041083

12836 JAZF1 -2.70808 5.58E-05 10979 HS.551307 -3.04712 1.13E-05 13346 KLHL29 -2.71506 5.11E-06 29794 SCARNA13 -3.05568 4.35E-05 25749 NAVI -2.71696 0.000248061 32378 TMEM132B -3.06408 1.54E-06 29306 RNF175 -2.71799 3.04E-05 2690 C2ORF77 -3.07752 4.19E-05 13815 LINGO2 -2.71868 2.30E-05 27660 PITPNM1 -3.09871 2.62E-05 25208 MMRN1 -2.73481 0.000116097 32963 TRPM3 -3.10437 0.000937085 3918 CFDP1 -2.73575 0.000150699 3640 CD36 -3.10624 0.000121554 31242 SP8 -2.73862 5.04E-05 26070 NLGN1 -3.1408 9.93E-05 1311 ATP1B3 -2.7498 8.64E-05 5749 EFCAB1 -3.15705 0.000423897 25939 NELLI -2.7509 0.00086776 3121 CAIO -3.15947 0.000537722 32531 TMEM51 -2.77628 6.84E-06 3215 CAMK2N1 -3.16181 0.000561888 15778 LOC100133887 -2.78135 1.17E-05 8016 GNRH1 -3.16554 1.25E-05 815 ANKRD38 -2.79552 0.000168264 13037 KHDRBS3 -3.16572 0.000269437 33758 WDR16 -2.80194 0.000532777 5681 E2F7 -3.17981 2.36E-07 12801 ITM2C -2.81062 7.32E-05 8174 GPR37 -3.18311 2.85E-05 32847 TRH -2.84178 0.00148109 29510 RPRML -3.19177 0.000271322 2881 C6ORF165 -2.84456 6.28E-05 4349 COL4A6 -3.1938 5.88E-05 15644 LOC100133542 -2.84572 9.65E-05 27887 PNOC -3.19863 4.88E-05 29811 SCARNA8 -2.84646 9.99E-06 1543 BCAT1 -3.20615 0.000858798 33964 XPR1 -2.85352 5.49E-06 28813 RALYL -3.21662 0.000509498 25940 NELL2 -2.86423 0.00024831 30531 SLC32A1 -3.24737 0.0002618 5455 DNER -2.87832 0.00127284 25216 MNS1 -3.25707 0.000109366 33807 WDR63 -2.88707 4.43E-05 3364 CCDC109B -3.26787 0.000147649 3038 C9ORF116 -2.89396 0.000215965 4004 CHODL -3.28962 0.00114785 2553 C20ORF85 -2.89405 7.84E-05 965 APOE -3.29237 0.00155578 5531 DPY19L1 -2.90287 0.000183171 5764 EFHC2 -3.29611 0.000143319 31955 TBR1 -2.90815 7.60E-06 29002 RDH10 -3.30034 0.000369857 8655 HIST1H1C -2.91043 9.46E-05 29600 RSHL3 -3.30753 6.17E-05 3401 CCDC146 -2.91057 0.000155147 12317 HTRA1 -3.31522 0.000437969 31935 TBC1D9 -2.91662 6.93E-05 2398 C1ORF194 -3.31829 1.21E-05 7421 FOXJ1 -2.94694 0.000577632 5895 ELMOD1 -3.32218 0.000142072 32783 TPPP3 -2.95648 0.000353912 3066 C9ORF171 -3.33251 4.78E-05 5337 DLG2 -2.96987 1.13E-06 33592 VAT1L -3.3402 0.000247714 27810 PLK4 -2.98102 1.71E-06 12399 IFITM3 -3.3542 0.0016376 24315 MEIS2 -3.03243 0.00109977 28319 PROM1 -3.35504 5.84E-05 31243 SPA17 -3.03709 0.000523067 29034 RELN -3.43956 1.30E-05

- 91 041083

2397 C1ORF192 -3.45738 0.000124675 31257 SPAG6 -3.86214 1.74Е-05 586 AKARI -3.46559 1.94Е-05 7865 GJA1 -3.88524 0.000511853 5032 DDAH1 -3.46896 1.42E-05 3274 CASC1 -3.88963 2.00Е-05 3766 CDK2AP2 -3.4706 3.31E-06 7696 GAS1 -3.95396 2.45E-07 3652 CD47 -3.47334 6.18E-05 7805 GFRA2 -4.00209 0.000116201 4283 CNR1 -3.48521 9.70Е-05 3050 C9ORF135 -4.09611 3.21E-05 12694 IQCG -3.4934 2.59Е-05 23853 LRRN3 -4.2728 1.34Е-05 10300 HS.537002 -3.49808 0.000142247 29603 RSPH1 -4.35627 1.08E-05 12506 IL13RA2 -3.50471 5.79Е-05 6458 FAM183A -4.40302 2.73E-05 28709 RAB31 -3.51372 7.79Е-05 3220 CAMKV -4.47543 4.97Е-05 28536 RTH2 -3.54413 0.000188892 27284 PCP4 -4.64191 1.37E-05 31283 SPATA18 -3.54742 5.71Е-05 29723 SAMD3 -4.7132 2.41E-06 3071 C9ORF24 -3.55395 9.04Е-05 31223 SOX3 -4.74012 8.45Е-06 7432 FOXN4 -3.57299 5.14Е-09 29396 ROPN1L -4.86578 3.17E-06 27171 RVHZ -3.57487 0.000447274 29972 SEMA3C -5.03965 4.16E-05 32270 TLE4 -3.57637 7.16Е-05 3695 CDC20B -5.17405 5.72E-07 28981 RCAN2 -3.61105 0.000608261 4298 CNTNAP2 -5.33902 4.01Е-06 32069 TEKT1 -3.61846 8.36Е-05 2006 C13ORF30 -5.4414 1.12E-06 17491 LOC440585 -3.62588 0.000175217 33600 VCAN -5.47499 0.000917643 5660 DYNLRB2 -3.6297 6.28E-05 1936 C11ORF88 -5.55748 2.96E-06 5938 EMX2 -3.66175 1.17E-06 30115 SFRP2 -6.27713 3.81E-08 24314 MEIS1 -3.68295 0.000402893 27159 PAX6 -6.64236 3.94Е-10 13856 LMO2 -3.74164 7.37E-06 866 ANXA1 -7.3691 4.96Е-06 6004 ERNV1 -3.77917 0.000119962 3561 CCNO -9.3158 2.19E-08 7504 FSTL5 -3.80048 0.000392914 13768 LHX2 -9.53566 3.56Е-05 1119 ARMC3 -3.82142 2.66E-05

No. column And Column ID Multiple changed ie p-value

Table 5. Gene expression analysis results for genes that are significantly up- or down-expressed at day 25 of differentiation in the ventral plate-based population treated with SHH/FGF8/Chir compared to the population treated with SHH/FGF8 alone_________

32126 TFF3 24.0181 3.37E-14 1209 ASCL1 22.1637 1.69E-08 9374 HS.19193 14.7045 1.42E-11 7395 FOXA1 13.8748 2.ZOE-14

- 92 041083

13865 LMX1A 10.953 4.33E-10 12456 IGFBP5 9.56964 1.58E-06 4342 COL3A1 9.49528 5.64Е-06 12361 ID3 9.45077 Z.ZZE-08 27988 POSTN 9.10453 7.82E-11 7396 FOXA2 8.85174 5.07Е-15 3941 CGNL1 8.71553 1.37E-08 30390 SLC18A1 8.37911 4.77E-15 5689 EBF1 8.02385 2.37E-06 2470 C1QL1 7.76325 5.90E-10 22823 LOC728715 7.73246 1.77E-09 27665 PITX2 7.32855 7.16E-15 12655 INSM2 7.04572 4.97E-11 26250 NR4A2 6.75415 1.28E-13 8156 GPR177 6.28979 1.08E-05 13857 LMO3 6.20624 4.64Е-09 33930 XVR1 5.8436 6.74Е-06 12944 KCNJ16 5.71955 1.37E-13 27821 PLS3 5.62251 8.45E-07 5372 DMRTA2 5.61111 8.09Е-12 7988 GNB3 5.55425 2.56E-12 31669 SULF2 5.51821 6.78E-08 33128 TTR 5.48234 2.88E-09 2543 C20ORF56 5.42131 2.00E-14 32762 TPBG 5.30583 7.23E-05 436 ADCYAP1 5.20834 1.29E-09 26977 OTX2 5.10526 6.89E-06 3978 CHGA 5.07311 7.78E-07 column number and Column ID Multiple changed And p-value 32000 TCF12 5.05883 1.27E-10 4003 CHN2 4.53859 9.76Е-08 2867 C6ORF141 4.43629 1.97E-10 32417 TMEM163 4.40645 3.65E-08 26245 NR2F2 4.38488 8.32E-05

5036 DDC 4.37185 3.55Е-05 6700 FAT3 4.28732 4.74Е-06 9428 HS.204481 4.25031 1.93E-09 8288 GRM8 4.16549 1.67E-06 28590 PTPRO 4.13365 8.06Е-07 32159 TH 4.10876 0.000355952 31361 SPON1 4.063 4.56Е-07 34166 ZFP36L1 4.05459 0.000219198 13689 LDB2 3.9497 1.90E-08 32057 TEAD2 3.94608 6.68E-06 12731 IRX3 3.93674 0.000270763 28942 RBM47 3.91109 0.000154758 30066 SERPINF1 3.84276 3.72E-06 1666 BMP7 3.83782 1.66E-05 25444 MSX1 3.74709 3.45E-05 8593 HES6 3.66092 5.43Е-05 12149 HS.7023 3.65165 0.000170814 30239 SHISA2 3.60636 8.64Е-06 9819 HS.388347 3.52316 1.46E-08 7564 FZD2 3.48734 3.69E-06 26928 0SBPL1O 3.44485 1.91E-07 27664 PITX1 3.40016 5.92E-06 2743 C3ORF58 3.31658 5.91E-07 30590 SLC39A8 3.30002 1.28E-06 32144 TGFBR3 3.28727 6.00E-09 18750 LOC643911 3.2583 2.43E-07 17491 LOC440585 3.24609 0.000469294 24375 MFNG 3.24122 3.31E-07 5346 DLK1 3.21349 0.000161289 7562 FZD1 3.15302 8.07Е-08 3186 CALCA 3.14299 1.75E-08 27772 PLEKHA5 3.09727 0.000150905 26335 NUAK1 3.08699 1.10E-05 34155 ZFHX4 3.05463 6.49E-07 27855 PMP22 3.03785 1.12E-07 4491 CPVL 3.02972 0.000317992

- 93 041083

7421 FOXJ1 3.01784 0.000461499 2850 C6ORF117 3.00725 3.47E-05 27105 PAPSS2 2.97981 1.49E-05 12358 ID1 2.94361 0.000160308 33004 TSHZ1 2.93637 2.29E-07 5691 EBF3 2.92465 2.26E-05 1086 ARID5B 2.92012 2.33E-06 32769 TPH1 2.88914 4.24E-07 5424 DNAJC19 2.87234 6.43E-09 6208 EVI1 2.86069 2.04E-07 13858 LM04 2.85216 4.81E-05 12733 IRX5 2.84963 0.000139911 5879 ELF1 2.78573 1.43E-06 23752 LRIG1 2.77491 0.000210665 23822 LRRC4C 2.77041 5.37E-07 4780 CXCR4 2.72468 5.12E-07 27696 PKNOX2 2.7166 1.59E-05 31514 ST6GALNAC5 2.70116 0.00012729 32854 TRIL 2.66182 5.76E-06 7617 GADD45G 2.62149 0.000385727 5349 DLL3 2.62131 0.000304562 8399 GULP1 2.58556 2.34E-07 16419 LOC283514 2.55654 7.18E-05 12986 KCNS3 2.51225 3.59E-05 6270 F3 2.50649 4.09E-09 32177 THBS4 2.48547 9.17E-05 6457 FAM181B 2.48092 0.0001071 12644 INPPL1 2.47363 7.08E-06 28624 PVRL3 2.47112 3.32E-05 2885 C6ORF173 2.45642 9.25E-05 25218 MNX1 2.44999 9.15E-08 3835 CEBPD 2.44764 0.000160544 6197 ETS2 2.43482 2.74E-06 8206 GPR98 2.37781 0.000437636 30664 SLC7A2 2.36837 5.80E-11 7853 GINS2 2.3541 7.75E-05

29887 SDC2 2.34674 3.00E-05 27402 PDZRN4 2.29374 2.31E-06 16112 LOC100192378 2.2867 4.57E-06 21088 LOC650494 2.27924 4.94E-06 8583 HERC5 2.27663 2.89E-07 1775 BTG2 2.27014 6.72E-06 30719 SLK 2.24997 3.57E-05 19806 LOC646345 2.232 3.69E-05 25137 MKL2 2.22229 0.00012672 7610 GABRR1 2.21011 2.33E-07 8573 HEPACAM2 2.17226 1.17E-09 29151 RHBDL3 2.16408 5.80E-05 27016 P2RY5 2.16065 5.91E-06 30261 SIAH3 2.14795 3.46E-05 30517 SLC2A8 2.13975 8.81E-05 7457 FREM1 2.12587 7.87E-08 1102 ARL4A 2.11255 0.000248586 29277 RNE128 2.07512 8.41E-06 29124 RGS16 2.06237 7.60E-05 8211 GPRC5C 2.04839 5.12E-05 26002 NGF 2.01869 9.29E-08 33375 UCP2 2.00568 0.000336807 11039 HS.553187 -2.00024 0.000182185 7972 GNAI1 -2.00345 0.000400577 1241 ASTN1 -2.00503 0.000267753 29957 SEL1L3 -2.00817 9.40E-06 9721 HS.348844 -2.06728 9.46E-05 403 ADAMTS5 -2.09516 8.41E-07 5268 DIRAS2 -2.10442 0.000137286 1042 ARHGAP22 -2.11082 9.20E-05 24440 MGC27121 -2.11477 1.75E-08 28124 PPP2R2C -2.12982 0.000152152 2812 C5ORF30 -2.14976 2.66E-06 8046 GOLSYN -2.15553 0.000225269 7626 GAGE12C -2.16085 1.15E-07 728 AMPH -2.19127 7.33E-05

- 94 041083

28104 PPP1R1C -2.2262 3.10E-07 28251 PRKACB -2.27738 6.28E-05 26252 NR5A1 -2.3262 1.83E-08 153 ABAT -2.33538 2.64E-05 28721 RAB3B -2.33887 0.000174687 31151 SNX10 -2.35266 0.000210353 26936 0SBPL8 -2.36481 0.000356878 30232 SHC4 -2.36756 5.88E-06 28689 RAB15 -2.37701 4.57E-05 31222 S0X20T -2.37929 5.44E-06 10203 HS.525171 -2.37949 0.000112764 27287 PCSK1N -2.39396 0.000122619 30200 SH3BGRL2 -2.39696 0.000352607 23918 LY6H -2.3986 0.000426406 31543 STAMBPL1 -2.40424 0.000210764 12256 HSPA12A -2.4161 5.00E-06 12209 HS3ST5 -2.42056 1.64E-08 1764 BTBD3 -2.43171 9.02E-05 32742 TOX2 -2.47449 6.41E-06 896 AP1S2 -2.4814 0.000411095 13764 LHFPL4 -2.49494 0.000177306 12801 ITM2C -2.50635 0.000289334 1641 BLCAP -2.52288 0.000363648 30231 SHC3 -2.53075 2.70E-06 33964 XPR1 -2.55331 2.67E-05 28266 PRKCE -2.55567 1.75E-07 26521 OPTN -2.57271 0.000282119 5325 DKK3 -2.57389 8.27E-05 33325 UBL3 -2.58024 0.000136606 6859 FGF13 -2.58447 3.31E-05 32210 THY1 -2.60259 0.000107047 30015 SEPT6 -2.63038 0.000101624 30351 SLC12A5 -2.64265 0.000195945 607 AKR1C4 -2.66634 0.000217452 7635 GAGE2B -2.67196 4.87E-10 13177 KIAA1598 -2.67619 4.25E-05

1986 C12ORF68 -2.67913 0.000114615 25516 MTSS1 -2.69402 0.000290278 32535 TMEM55A -2.70095 5.71E-06 25864 NDST3 -2.72344 9.29E-06 33591 VAT1 -2.74928 0.000228555 26498 ONECUT1 -2.77372 1.86E-05 12634 INPP1 -2.79095 5.83E-05 5703 ECEL1 -2.83259 0.000107266 16740 LOC387856 -2.84534 0.000116969 5631 DUSP6 -2.85825 8.35E-05 31568 STC1 -2.8677 0.000365825 227 ABLIM2 -2.8948 2.50E-06 27415 PELI2 -2.9135 2.07E-05 2538 C20ORF46 -2.92994 6.05E-05 4639 CSRNP3 -2.9364 6.80E-06 31628 STS-1 -2.94215 9.63E-07 30210 SH3GL3 -2.94559 0.000343973 10017 HS.452398 -3.01786 1.26E-05 4705 CTNND2 -3.02031 4.91E-05 24516 MGST1 -3.02712 1.36E-06 4283 CNR1 -3.13389 0.000271812 13313 KLF6 -3.16185 3.73E-05 7613 GADI -3.18763 0.000116615 29400 RORB -3.1967 5.32E-08 27566 PHYHIPL -3.21046 3.97E-05 25628 MYO16 -3.21287 9.14E-06 25284 MPPD2 -3.23371 0.000114488 30531 SLC32A1 -3.23601 0.000270573 29034 RELN -3.32027 1.95E-05 26058 NKX2-1 -3.35392 4.84E-06 30114 SFRP1 -3.38198 0.00010787 4965 DBC1 -3.3928 1.25E-05 31763 SYT1 -3.47973 0.00017165 29624 RTN1 -3.48718 0.000208889 3121 CAIO -3.49466 0.000217475 24095 MAP4 -3.54296 0.000118613

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9476 HS.223856 -3.61087 5.18E-05 32343 TMEFF2 -3.6559 1.52E-06 713 AMHR2 -3.65957 2.10E-08 28417 PSD2 -3.70127 0.000123589 7106 FLJ33590 -3.74166 4.19E-06 4386 COPG2IT1 -3.9994 7.98E-05 3207 CALY -4.03219 3.21E-06 30824 SNCG -4.48956 2.76E-06 5455 DNER -4.58982 1.95E-05 5550 DRD1IP -4.64486 5.89E-07 26253 NR5A2 -4.70942 1.65E-11 12954 KCNK12 -4.77483 2.25E-06 5352 DLX1 -4.89802 0.000316228 26332 NTS -4.92922 0.000411631 33565 UTS2 -5.26698 2.91E-07 3220 CAMKV -5.35563 1.08E-05 2631 C22ORF42 -5.4488 2.15E-07 29220 RIT2 -5.49937 9.53E-12 31359 SPOCK2 -5.66406 5.68E-06 12746 ISLR2 -5.6746 0.000247482 32591 TMOD1 -5.689 7.66E-12 3215 CAMK2N1 -5.84508 2.30E-06 33592 VAT1L -6.26024 7.74E-07 27971 POMC -6.74106 6.72E-05 26219 NPTX2 -7.85511 1.15E-05 25939 NELLI -8.58778 1.80E-08 33624 VGF -8.61516 1.37E-05 31223 SOX3 -10.3701 8.71E-09 30314 SIX6 -12.5834 1.04E-12 26965 OTP -14.2469 6.08E-10 12743 ISL1 -21.0507 6.48E-05 31479 SST -21.3179 0.000281588 12746 ISLR2 -5.6746 0.000247482 32591 TMOD1 -5.689 7.66E-12 3215 CAMK2N1 -5.84508 2.30E-06

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Table 6 shows an exemplary list of antibodies used as markers, including the concentration (ie, dilution) of antibodies used as an example source of antibodies.

In some embodiments, bound antibodies are identified using secondary antibodies conjugated to Alexa488, Alexa555, or Alexa647 (Molecular Probes, Carlsbad, CA, USA).

In some embodiments, secondary biotinylated antibodies have been used to identify bound primary antibodies, followed by imaging of ______ using DAB (3,3'-diaminobenzidine) chromogen ______

antibody (primary) dilution source location nuclear antigen human 1:100 Millipore Billerica, MA adhesive molecule human cells 1:100 Santa Cruz Santa Cruz, CA Tyrosine hydroxylase (TH) 1:1000/1:500 Pel-Freez/ImmunoStar Rogers, AR/Hudson, WI β-tubulin III 1:500/1:2000 Covance Littleton, CO/ Princeton, NJ doublecarotene 1:100 Millipore Billerica, MA specific Loya human Nestin 1:300 R&D Minneapolis, MN Nestin #130 1:50 R. McKay NINDS, NIH FoxA2 1:100 Santa Cruz Santa Cruz, CA Pitx3 1:100 Millipore Billerica, MA β-catenin 1:100 BD Franklin Lakes, NJ collagen 1:100 Oncogene LaJolla, CA cytokertin 1:100 DAKO Glostrup DK

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Oct-4 1:200 Santa Cruz Billerica, MA Ki-67 1:200/ 1:400 Zymed / DAKO San Francisco, CA polyclonal GABA 1:2000 Sigma St. Louis, MI serotonin (5-HT) 1:2000 Sigma St. Louis, MI polyclonal GF AR 1:2000 DAKO Glostrup, DC calbidin 1:300 Abeam Cambridge, MA VMAT2 1:200 Millipore Billerica, MA DAT 1:1000/1:2000 Millipore Billerica, MA GFP 1:1000 Molecular Probes Carlsbad, CA Girk2 1:200 Abeam/Alomone Cambridge, MA / Israel FoxGl 1:100 NeuraCell Rensselaer, NY Rahb 1:100 Covance Princeton, NJ Otx2 1:2000 Strategic Diagnostics Newark, D.E. Lmxla 1:2000 Millipore Pittsburgh, PA synapses 1:1000 Sigma St. Louis, MI Iba-1 1:200 Millipore Billerica, MA ED-1 1:200 Millipore Pittsburgh, PA NCAM Human (Eric-1) 1:100 Santa Cruz Santa Cruz, CA specific to human cytoplasmic (SC-121) 1:1000 Stem Cells Inc. Newark, CA Nurr-1 1:1500 Perseus Proteomics Japan

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Table 7. Examples of envisaged differentiation in DA neurons with cell type restriction

cell type marker description analysis assumed limits FP/ DA midbrain FOXA2/L MX1A IHC: _______ co- expression (a), day 13 & day 25; > 50% validation of quantitative PCR-RT precursor of DA neurons FOXA2/T H; TH/NURR1 IHC: _______ co- expression (a), per day > 25% 25; validation quantitative PCR-RT Pluripotent cells OST-4; IHC: @ on day 25; validation quantitative <2% PCR-RT Proliferating cells K167 IGH < 25% predecessor (forebrain) FOXG1; PAX6 IHC: @ on day 25; validation quantitative < 10% PCR-OT output YES neurons TH/FOXA 2 IHC: _______ co- expression (a), on day 25 from hESCs, > IDA p/ESCch sown per day 0 survival In vivo THFOXA 2 (in vivo) Histology in vivo (а), 4 weeks after engraftment > 2,000 / animal 200 thousand cells

Table 8. Examples of experiments to determine the significance of some factors in obtaining DA neurons

I l)N <B H RM CHIR ^HH BDNh GDXl AD I)hc 1)DR1 |||||||jj|||||j|j

IIi111I । ! is!

i : ! J +as ί ί

111111111^^ |||||||||^^ ||||||||||«^ +/- R.D pj'p-Hp 'p j |.\f f-P pB 1h ΡΡ'ΡΡ'ΡΒΡ'Β aa j Iр.т rrj р 'р.jλ|.(Ρί:.Ηρ', ·Μ\:'j рРрр; f1р ?

' rur-,4 ΐ (ΡΗν Η ppp D fr' DchD. RD * [dr

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Table 9. Examples of methods for scaling up the culture of smDA neurons, in particular, in obtaining cultures of the GMP level for use in the clinic

pilot scale n rom hedge th scale ι«ιό ( \ > 1) clinical scale ha b 1\1orazhs1111 :>ic iciiKoniiiiii>i 1 bottle - 1x10 ^b per 6 cm cup 7 vials - 7x10 ⁶ x 15 cm x 1 42 bottles 7x10 ⁶ x 15 cm x 6 Pass US 1 claoers 1:1 - 1:5 on 6 cm cups 1:1 - 1:5 on 15 cm cups 1:1-1:5 on 15 cm cups Passage 2 classrooms 2 x 15cm cups 6 x 15cm cups 36 x 15 cm cups Passage 3 \ccutase (neural 11 H.IX boiling) 1 x 15 cm dish at high density (approximately 3.0x10 ⁷ cells) 3 x 15 cm high density dishes (approximately 9.0x10 ⁷ cells) 18 x 15 cm high density dishes (approximately 5.4 x 10 ⁸ cells) Passage 4 \kk y ia ;a (day 1 5-20) 1:1 1:1 1:1 Krpokonserv approximately 1.2 x approximately 3.6 approximately anna (day 10 ⁸ , assuming 50% loss after cryopreservation = approximately 6 x 10 ⁷ viable cells x 10 ⁸ , assuming a loss of 50% after cryopreservation = approximately 1.8 x 10 ⁸ viable cells 2.16 x 10 ⁹ , with approximately 50% loss after cryopreservation and = approximately 1 x 10 ⁹ viable cells

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Table 10. Example of in vivo evaluation of hESC line products - graft composition

Cell type Marker Assay description Assumed output limits for DA neurons TH FOXA2 ; Theological > 5,000 per i (estimated total 1200,000 inoculated i TH/FoxA2* cells (IHC) per

S graft

Proliferation coefficient Ki67 (MIB-1) GCI on percentage of number of cells K167, general <1% Pluripotent cells OST-4 / Nanog IGH <0.5% Serotonergic neurons 5-HT (serotonin) IGH < 1% predecessors FOXGI; PAX6 IGH < 10% forebrain Derivatives teratoma myosin; cytokeratins; afetoprotein IGH <1%

Table 11. Example of in vivo evaluation of hESC line products - behavioral tests

Test Parameter Analysis description Suggested limits

Lefetamines Total number of twists to the side < 1st twists (*) twists/min damage after twists/min {ipsi-minus, intraperitoneal :

contralateral) amphetamine injections j

Step test % compensation contralateral steps / total compensation Initiation of stepping using the limb on the contralateral side With. 40-50% on ΐ test cylinder With % use and psylateral limb/total number Spontaneous; Min. ipsi study - improvement limb compared compared to the state contralateral limb transplant 20% from BEFORE

(*) In some embodiments, rats make >6 rotations/min, stably transplanted grafts.

Experimental part.

The following examples serve to illustrate specific embodiments and aspects of the present invention and are not intended to limit its scope. In the following descriptions of experiments, the following abbreviations apply: N (H) (normal); M (molar); mM (millimolar); µM (micromolar); mole (mole); mmol (millimole); µmol (micromole); nmol

- 101 041083 (nanomole); pmol (picomole); g (gram); mg (milligram); mcg (micrograms); ng (nanogram); pg (picogram); l (liter); ml (milliliter); µl (microliter); cm (centimeter); mm (millimeter); µm (micrometer);

nm (nanometer); U (unit); min (minute); s and sec (second); deg. (degree); pen (penicillin), strep (streptomycin) and °C (degree Celsius).

Example I

Materials and methods.

Brief description of methods: Human ESC (H9, H1) and iPSC (2C6 and SeV6) lines were processed according to a modified ventral plate induction protocol (Fasano, et al., Cell Stem Cell 6:336-347 (2010), the source cited is incorporated herein by reference) based on dual SMAD inhibition (Dual SMAD-inhibition) (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009), the source cited is incorporated herein by reference). The exposure time of SHH C25II reagents, purmorphamine, FGF8, and CHIR99021 to midbrain ventral plates was optimized to generate new populations of DA neurons (see Fig. 1d). After ventral plate induction, subsequent maturation (11-25 days or longer than 25 days in culture to at least 100 days in culture) was performed in Neurobasal/B27-based differentiation media in the presence of DA neuronal survival and maturation factors (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004, reference cited herein by reference), such as AA, BDNF, GDNF, TGFe3, and dbcAMP (see full method description for details). The phenotype of the obtained population of DA neurons was carefully studied by immunocytochemical methods, quantitative RT-PCR, determination of the global gene expression profile, HPLC analysis for the determination of dopamine, and registration of electrophysiological characteristics in vitro. In vivo studies were carried out in rodents with hemiparkinsonism (mice or rats injected with 6OHDA toxin in one hemisphere of the brain). These studies were performed in non-adult NOD-SCID IL2Rgc mice (Jackson labs) and adult Sprague Dawley rats from Taconic Farms, which were induced with 6-hydroxydopamne damage by stereotactic injections of said toxin as previously described, as well as in two adult rhesus monkeys treated with unilateral injection of MPTP into the carotid artery.

DA neurons were injected stereotactically into the striatum of the indicated animals (150x103 cells in mice, 250x103 cells in rats) and a total of 7.5x106 cells (distributed into 6 sites; 3 on each side of the brain) in macaques. Behavioral tests were performed monthly after transplantation, including an amphetamine-induced rotation test, as well as a focal akinesia test (stepping test) and a limb use test (top hat test). Rats and mice were sacrificed at 18-20 weeks, and primates - 1 month after transplantation. The study of the characteristics of the grafts was carried out by stereological analysis of the number of cells and the volume of the graft, as well as through a thorough examination of the phenotype using immunohistochemical methods.

YES neuron. hESC lines H9 (WA-09, XX, 27-55 passages after 10/2009), H1 (WA-01, XY, 30-40 passages after 6/2010) and iPSC lines 2C6 (Kim, et al. Cell Stem Cell 8:695-706 (2011) cited herein by reference) (XY, 20-30 passages) and SeV6 (XY, 20-30 passages; obtained from MRC-5 embryonic fibroblasts using a non-integrating 4-factor Sendai vector system (Ban, et al. Proc. Natl. Acad. Sci. US A (2011) 108(34):1423414239:10.1073/pnas.1103509108, cited source incorporated herein by reference) were maintained on embryonic fibroblasts mice at calculated inoculation concentrations ranging from 0.5x103/ ^cm2 to 100x103/ ^cm2 per human ES cells at which cells tend to form clusters (MEF, Global Stem, Rockville, MD) in an optimal medium for human ES cells, containing 20% Knockout serum replacement (KSR, Invitrogen, Carlsbad, California) (as previously described (Kim, et al. Cell Stem Cell 8:695-7 06 (2011), the source cited is incorporated herein by reference). The amount of Knockout serum replacement used can range from 0 to 40%.

neuron induction. A modified version of SMAD dual inhibition (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009), cited herein incorporated by reference) and a ventral plate induction protocol (Fasano, et al. Cell Stem Cell 6:336-347 (2010, reference cited herein by reference) based on treatment for a certain time with LDN-193189 reagents (100 nM (in the concentration range of 0.5-50 μM, Stemgent, Cambridge, Massachusetts), SB431542 (10 µM (range 0.5-50 µM, Tocris, Ellisville, MI), SHH C25II (100 ng/ml (range 10-2000 ng/ml, R&D, Minneapolis , MN), purmorphinamine (2 µM (in the concentration range 10-500 ng/mL, Stemgent), FGF8 (100 ng/mL (in the concentration range 10-500 ng/mL, R&D) and CHIR99021 (CHIR; 3 µM (in concentration range 0.1-10 µM, Stemgent) Note: in the ventral plate induction protocol astinok processing SHH refers to processing, i.e. implementation of contact, cells with a combination of SHH C25II 100 ng / ml + purmorphamine (2 μm). Cells were seeded (35-40x103 cells/cm ² ) and grown for 11 days on matrigel or geltrex gel.

- 102 041083 (used as purchased) (BD, Franklin Lakes, New Jersey, USA) in Knockout serum replacement (KSR) medium containing DMEM, 15% KSR, 2 mM L-glutamine, and 10 µM ( in the concentration range of 1-25 μM) β-mercaptoethanol. KSR medium was successively replaced with N2 medium starting from day 5 of differentiation, mixing them in ratios of 75%(KSR):25%(N2) on days 5-6, 50% (KSR):50%(N2) on days 7-8 and 25% (KSR): 75% (N2) on days 9-10 as previously described (Chambers, et al. Nat. Biotechnol. 27:275-280 (2009) cited herein by reference). On day 11, medium was changed to Neurobasal/B27 (1:50 dilution)/L-Glut-containing (effective range 0.2-2 mM) medium (NB/B27; Invitrogen) supplemented with CHIR (up to day 13) and BDNF (brain-derived neurotrophic factor, 20 ng/ml in the range of 5 to 100; R&D), ascorbic acid (AA; 0.2 mM (in the concentration range of 0.01-1 mM), Sigma, St. Louis, MI), GDNF (glial cell neutrophic factor, 20 ng/ml (in the concentration range 1-200 ng/ml); R&D), TGFe3 (transforming growth factor type β3, 1 ng/ml (in the concentration range 0.1-25 ng/ml ); R&D), dibutyryl cAMP (0.5 mM (range 0.05-2 mM); Sigma), and DAPT (10 mM (range 0.5-50 nM); Tocris) for 9 days. On day 20, cells were dissociated using Accutase® (Innovative Cell Technology, San Diego, Calif.) and plated at elevated cell density (eg, 300-400,000 cells/cm ² ) on polyornithine (PO) pre-coated dishes; 15 µg/ml (in the concentration range 1-50 µg/ml)/laminin (1 µg/ml) (in the concentration range 0.1-10 µg/ml)/fibronectin (2 µg/ml (in the concentration range 0.1 -20 μg/ml) in differentiation medium (NB/B27+BDNF, AA, GDNF, dbcAMP (in the range of concentrations indicated in this description), TGFe3 and DAPT (in the concentration range indicated in this description) until the desired stage is reached maturation for a particular experiment.

Previously described protocols (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004), cited herein incorporated by reference) were used in part to induce DA neurons based on rosettes, with at least one exception when the double SMAD inhibition was used to accelerate the initial stage of neuronal induction. Briefly, hESCs were induced for neural pathway development by co-culturing with irradiated MS5 cells in KSR medium supplemented with SB431542 and Noggin (250 ng/mL (in the concentration range 10-1000 ng/mL); R&D), from 28 days and SHH+FGF8 from 6-11 days of differentiation. After 11 days in KSR medium, neuronal rosettes were harvested by hand and grown (P1 step) in N2 medium supplemented with SHH, FGF8, BDNF and AA as described previously (Perrier, et al. Proc Natl Acad Sci USA 101:12543-8 (2004) , the cited source is incorporated into the present description by reference). After 5-7 days in the P1 stage, rosettes were harvested again by hand and ground to powder, followed by incubation in Ca ² /Mg ² free Hank's Balanced Salt Solution (HBSS) for 1 hour and subcultured onto polyornithine (PO)/laminin coated plates. / fibronectin. Patterning with SHH/FGF8 was continued for 7 days at the P2 stage followed by final differentiation in the presence of BDNF, AA, GDNF, TGFb3 and dbcAMP as described above until the desired stage of maturation for a particular experiment was reached (usually 5-7 days for transplantation experiments). or 32 days for in vitro functional experiments).

Analysis of gene expression. Total RNA was isolated during differentiation on the following days: 0, 1, 3, 5, 7, 9, 11, 13 and 25 for each experiment with conditions: LSB control, LSB/SHH/FGF8 and LSB/SHH/FGF8/CHIR with using the RNeasy kit (Qiagen, Valencia, CA). For biochip analysis, total RNA was processed on an MSKCC Genomic core and hybridized on Illumina Human ref-12 bead biochips according to the manufacturer's instructions. Comparisons were made every day and for every set of conditions using the LIMMA package from Bioconductor (worldwideweb.bioconductor.org). Genes with an adjusted P value less than 0.05 and a fold change in expression greater than two were considered statistically significant. Some of the descriptive bioarray data analyzes and presentation were performed using a commercially available software package (Partek Genomics Suite (version 6.10,0915)). For quantitative RT-PCR analysis, total RNA at day 25 for each set of conditions was subjected to reverse transcription (Quantitech, Qiagen) and the amplified material was analyzed using commercially available Taqman gene expression assays (Applied Biosystems, Carlsbad, CA) with HPRT normalization. Each point represents 9 technical replicates from 3 independent biological samples. Primary data from biochip experiments are not yet available at GEO worldwideweb.ncbi.nlm.nih.gov/geo).

Surgical intervention in animals. Experiments on rodents and monkeys were carried out in accordance with the instructions of the National Institutes of Health (NIH), and they were approved by the local Institutional Animal Care and Use Committee (IAUCC), the Institutional Biosafety Committee (IBC), and the Embryonic Stem Cell Research Committee (ESCRO).

Mice. NOD-SCID IL2Rgc knockout mice (weighing 20-35 g; Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized with ketamine (90 mg/kg; Akorn, Decatur, IL) and xylazine (4 mg/kg; Fort

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Dodge, I.A.). 6-Hydroxydopamine (10 μg (in the concentration range 0.1-20 μg), 6-OHDA (Sigma-Aldrich) was injected stereotactically into the striatum with the following coordinates (in millimeters): AP, 0.5 (from bregma; as a point skull suture was used as a reference point for stereotactic surgery) ML, -2.0; DV, -3.0 (from the dura mater, the membrane that covers the brain, used as a reference point) Mice with successful lesions (median > 6 rotations per minute) were selected for transplantation A total of 150x103 cells were injected in a volume of 1.5 µl into the striatum at the following coordinates (in mm): AP, 0.5; ML, -1.8; DV, 3.2. sacrificed 18 weeks after transplantation.

Rats. Adult female Sprague-Dawley rats (Taconic, Hudson, NY) (180-230 g) were anesthetized with ketamine (90 mg/kg) and xylazine (4 mg/kg) during surgical procedures. Unilateral lesions of the medial anterior fasciculus of the nigrostriatal tract have been produced by stereotaxic injection of 6-OHDA (3.6 mg/mL in 0.2% ascorbic acid and 0.9% saline, Sigma) at two sites (Studer, et al. Nature Neurosci. 1:290-295 (1998), the source cited is incorporated herein by reference). Rats were selected for transplantation if the number of rotations induced by amphetamine exceeded 6 rotations per minute 6-8 weeks after injection. 250x103 cells were transplanted into the striatum of each animal (Coordinates: AP+1.0 mm, ml -2.5 mm and V-4.7 mm; comb set to -2.5). Control animals received PBS instead. Surgical procedures have been previously described (Studer, et al. Nature Neurosci. 1:290-295 (1998) cited herein by reference). Daily intraperitoneal injections of cyclosporine 15 mg/kg (Bedford Labs, Bedford, OH) were started 24 hours before cell transplantation and continued until sacrifice, 20 weeks after cell transplantation.

Primates. Two adult (17-18 yr; 10-12 kg; female) rhesus monkeys were rendered hemiparkinsonian by infusing MPTP into the carotid artery followed by weekly intravenous MPTP to create bilateral parkinsonian syndrome (Kordower, et al. Science 290:767-773 (2000), the source cited is incorporated herein by reference). Both animals exhibited Parkinsonian symptoms consistent with moderate to severe injury based on behavioral analysis, including twisted posture, leg drooping, and symptoms of muscle rigidity (non-plasticity of movement), neglect (motor perception of lateral stimulus), and bradykinesia (delayed initiation of movement) . These parameters can be assessed in monkeys using the modified Parkinsonian Clinical Rating Scale (CRS). On the day of transplant surgery, animals were administered the tranquilizer ketamine (3.0 mg/kg IM) and dexdomitor (0.02-0.04 mg/kg IM), intubated to maintain stable airway ventilation, and anesthetized with isoflurane. Then they were placed in a stereotactic frame for surgical intervention. Both rhesus monkeys were operated on once with three intracranial injections of DA cultures derived from human ventral plates based on stereotaxic coordinates (Paxinos, et al. The Rhesus Monkey Brain in Stereotaxic Coordinates (Academic Press, 2000), source cited included herein) via link). Bilateral cell injections (10 μl per injection; 125,000 cells/μl) were performed at three positions (1-posterior caudal appendage, 2-precommissural putamen and overlying white matter) with a total volume of 30 μl per hemisphere. An infusion pump attached to a stereotaxic micromanipulator was used to deliver cells at a rate of 1 μl/min via a 50 μl Hamilton syringe with a 28 G needle. After injections were completed, the needle was left in place for 2-5 min to allow the infusate to diffuse out of the syringe tip, and then slowly withdraw the syringe. Immediately after surgery, animals received an analgesic (buprenex, 0.01 mg/kg IM, twice daily for 72 hours after surgery; meloxicam, 0.1 mg/kg SC, once daily for 72 hours after surgery). intervention) and an antibiotic (cefazolin, 25 mg/kg IM, twice a day) within 72 hours after surgery. Animals received cyclosporin A (Neoral, Sandimmune) orally (30 mg/kg reduced to 15 mg/kg) daily starting 48 hours before surgery until sacrifice, for one month after transplantation.

behavioral tests. Amphetamine-induced rotation test (mice and rats) and focal akinesia test (rats) were performed before transplantation and 4, 8, 12, 18 weeks after transplantation. Rotational behavior in mice was recorded 10 min after intraperitoneal injection of d-amphetamine (10 mg/kg, Sigma) and recorded for 30 min. Rotational behavior in rats was recorded for 40 min after intraperitoneal injection of d-amphetamine (5 mg/kg) and automatically assessed using the TSE VideoMot2 system (Germany). Data were presented as mean rotations per minute. The test for focal akinesia has been modified from that described by Blume et al. Exp. Neurol. 219:208-211 (2009) and Crawley, et al. What's Wrong With My Mouse: Behavioral Phenotyping of Transgenic and Knockout Mice (Wiley-Liss, 2000), each of which is incorporated herein by reference. Briefly, each rat was placed on a flat surface; her hind legs were raised by a gentle lift of the tail so that the front paws touched

- 104 041083 tables. The experimenter pulled the rat back a distance of 1 meter at a constant speed. The number of compensating steps was counted with both the contralateral and ipsilateral paws. Data were presented as the percentage of the number of compensating steps with the contralateral paw/the number of compensating steps with the contralateral paw + the number of steps with the ipsilateral paw. The cylinder test was performed by placing each animal in a glass cylinder and counting the number of touches of the wall of the cylinder with the ipsilateral paw and the number of touches with the contralateral paw (per 20 touches) as previously described (Tabar, et al. Nature Med. 14:379-381 (2008), the cited source is incorporated into the present description by reference).

Fabric processing.

Mice and rats: Animals (mice and rats) received an increased dose of pentobarbital (50 mg/kg) intraperitoneally to induce deep anesthesia, and then they were perfused with 4% paraformaldehyde (PFA). The brain was removed, fixed in 4% PFA, and then soaked in 30% sucrose solution for 2-5 days. Sections were obtained using a cryostat after the introduction of O.C.T. (Sakura-Finetek, Torrance, California).

Primates: Animals were sacrificed after deep anesthesia with ketamine (10 mg/kg, intramuscularly (IM)) and pentobarbital (25 mg/kg, intravenously (IV)) followed by cardiac infusion with 0.9% salin with heparin followed by fresh cold fixative solution 4% PFA (pH7.4). Immediately after initial fixation, the brains were removed from the skull and post-fixed by immersion in 4% PFA for 24-36 hours. They were then washed and kept in 10% sucrose in a shaker with slow agitation at 4°C, allowing them to sink. The process was then repeated in 20% sucrose and 30% sucrose. Whole brains were cut coronally into successive 40 μm sections on a cold sledge microtome and stored free-floating in a cryopreservative medium at -20°C.

Immunohistochemistry: Cells were fixed in 4% PFA and blocked with 1% bovine serum albumin (BSA) with 0.3% Triton. Sections of brain tissue were washed in cold PBS and treated in the same way. Primary antibodies were diluted in 1-5% BSA or normalized goat serum and incubated according to the manufacturer's recommendations. A complete list of antibodies and sources is given in Table. 6. Appropriate secondary antibodies conjugated to Alexa488, Alexa555 and Alexa647 (Molecular Probes, Carlsbad, Calif.) were used with 4',6diamidino-2-phenylindole (DAPI) nuclear contrast stain (Thermo Fisher, Rockford, Illinois). Biotinylated antibodies were used for some assays followed by imaging with the DAB chromogen (3,3'diaminobenzidine).

HPLC analysis. To measure dopamine, homovanillic acid (HVA) and DOPAC (3,4dihydroxyphenylacetic acid) levels, reverse phase HPLC with electrochemical detection was performed as previously described (Roy, et al. Nature Med. 12:1259-1268 (2006); Studer, and Brain Res. Bull 41:143150 (1996), each of which is incorporated herein by reference). Culture samples were taken in perchloric acid on the 65th day of differentiation. In some experiments, DA was measured directly in the medium using the same detection system, but followed by extraction of dopamine and its metabolites using a commercially available kit, as previously described (Studer, et al. Brain Res. Bull. 41:143-150 (1996 ), the cited source is incorporated into the present description by reference).

Recording of electrophysiological parameters: Cultures were transferred to a recording chamber on a straight microscope equipped with a 40X water immersion objective (Eclipse E600FN; Nikon); cultures were perfused with saline containing, in mM: 125 NaCl, 2.5 KCl, 25 NaHCO ₃ , 1.25 NaH ₂ PO ₄ , 2 CaCl, 1 MgCl ₂ and 25 glucose (34°C; saturated 95% O ₂ -5% CO ₂ ; pH 7.4; 298 mOsm/L). The saline flow rate was 2-3 ml/min when passed through an inline heater (SH-27B with TC-324B controller; Warner Instruments). Neurons were visualized by video microscopy with a cooled digital CCD camera (CoolSNAP ES ² , Photometries, Rona Scientific, Tucson, Arizona, USA). For registration of electrophysiological parameters, cells similar in shape to neurons with good branching of dendrites were selected. The method of local clamping of the potential (patch-clamp) on whole somatic cells in the current clamping configuration was carried out using a MultiClamp 700B amplifier (Molecular Devices). The signals were filtered in the 1-4 kHz range and digitized at 5-20 kHz using a Digidata 1440A (Molecular Devices). The recording contact electrodes were made of borosilicate fiber (Sutter Instruments) drawn on a FlamingBrown puller (P-97, Sutter Instruments) and had a resistance of 4-6 MOM in the bath. The electrodes were filled with an internal solution containing in mM: 135 K-MeSO ₄ , 5 KCl, 5 HEPES, 0.25 EGTA, 10 phosphocreatinadi(tris), 2 ATP-Mg and 0.5 GTP-Na (pH 7.3, osmolarity adjusted to 290-300 mosm/l). The bridge amplifier was tuned in such a way as to compensate for the resistance of the electrodes and monitored. The capacitance of the electrodes was compensated. If the series connection increased >20% during clamping, the data was discarded, since an increase in resistance indicates partial technical problems during the measurement.

Cell counts and stereological analyses. Percentage of cells positive for brand

- 105 041083 ru, on the ventral plate (day 11), fig. 1, midbrain dopamine progenitors (day 25), FIG. 2, and at the stage of mature DA neurons (day 50 or later), FIG. 3 and 11 were determined in samples obtained in at least 3 independent experiments for each. Images for counting were chosen randomly (uniform distribution), each image was first evaluated by the number of DAPI-positive nuclei, after which the number of cells expressing the marker of interest was counted. Data are presented as mean ± SEM (standard error of the mean). The number of human cells (identified by anti-hNA) and TH+ neurons in the graft were counted at every tenth section if the graft could be identified. Cell count and graft volume were determined using the optical probe of the fractional probe apparatus and the Cavalieri scoring device using the Stereo Investigator software (MBF bioscience, Vermont, USA) as described in Tabar, et al. Nat. Biotechnol. 23:601-606 (2005); the cited source is incorporated into this text by reference. Data are presented as estimated total cells and total graft volume +/- standard error of the mean (SEM). The following formulations characterize examples of cell culture media for carrying out the present inventions.

The following formulations characterize examples of cell culture media for carrying out the present inventions.

hESC Maintenance Medium (1 L): 800 ml DMEM/F12, 200 ml Knockout Serum Replacement, 5 ml 200 mM L-glutamine, 5 ml Pen/Strep, 10 ml 10 mM 15 essential amino acid solution in MEM minimal medium, 55 μM 13-mercaptoethanol, and FGF-beta (bFGF, final concentration 4 ng/ml).

KSR hESC differentiation medium (1 L): 820 ml Knock out DMEM, 150 ml Knock out Serum Replacement, 10 ml 200 mM L-glutamine, 10 ml Pen/Strep, 10 ml 10 mM MEM and 55 μM 13- mercaptoethanol.

N2 hESC differentiation medium (1 L): 985 ml distilled H ₂ O with DMEM/F12 powder, 1.55 g glucose (Sigma, cat. no. G7021), 2.00 g sodium bicarbonate (Sigma, cat. no. S5761) , putrescine (100 µl aliquot of a 1.61 gram solution in 100 ml distilled water; Sigma, catalog # P5780), progesterone (20 µl aliquot of a 0.032 gram solution in 100 ml 100% ethanol; Sigma, catalog # P8783), sodium selenite (60 aliquot of a 0.5 mM solution in distilled water; Bioshop Canada, cat. no. SEL888), and 100 mg transferrin (Celliance/Millipore, cat. no. 4452-01), and 25 mg insulin (Sigma, cat. no. catalog 16634) in 10 ml of 5 mM NaOH.

Dulbecco's Modified Eagle's Medium (DMEM) with 10% PBS for the preparation of PMEF ((primary mouse embryonic fibroblasts (PMEF)) - feeder cells) (1 L): 885 ml DMEM, 100 ml PBS, 10 ml Pen/Strep and 5 ml of L-glutamine.

Alpha Minimal Maintenance Medium (MEM) with 10% PBS to prepare MS-5 Feeder Cell Medium (1 L): 890 ml Alpha MEM, 100 ml PBS, 10 ml gelatin solution with Pen/Strep (500 ml): Dissolve 0.5 gelatin in 500 ml warm (50-60°C) Milli-Q water. Cool down to room temperature.

Example II.

This example describes the detection of small molecules and the establishment of the processing time to ensure directed differentiation of neurons FOXA2+LMX1A+DA according to the present invention.

The following description is a summary of some of the experimental findings described in the application: Treatment of cells subjected to dual SMAD inhibition with SHH (purmorphamine+SHH) and FGF8 (S/F8) agonists in the absence of CHIR99021 resulted in a significant reduction in FOXA2 expression at day 11 and no expression at all. LMX1A (Fig. 1a, b). The precursor marker OTX2 was strongly induced in cultures treated with LSB and LSB/S/F8/CHIR, but not under LSB/S/F8 conditions (Fig. 1a, c).

The authors of the present invention have previously used several other methods of directed differentiation, which resulted in a population of cells containing DA-like neurons. These DA-like neurons have been used in transplantation experiments, which have revealed problems in the further use of these cells in therapeutic applications. For example, the procedures described in Perrier et al., 2004 and Fasano et al., 2010, including the induction of MS5 neurons, resulted in rosette cell structures and were used to generate progenitors at day 11, see eg, FIG. 2, 16, and 17, which were subsequently used to obtain DA-like neurons. These neurons were derived from a low percentage of progenitor cells from cell populations at day 11. Transplantation experiments using these neurons have demonstrated poor survival after transplantation and loss of the DA-like neuron phenotype, in addition to the generation of unacceptable neurons observed after transplantation, along with loss of growth control, leading to the development of teratomas. See fig. 16 and 17.

In particular, hESC cells in the P0 phase were brought into contact with molecules to initiate neuronal induction of Oct4+ cells into rosette cells using MS5 feeder cells (Perrier and

- 106 041083 others, 2004). At the P1 stage, cells of rosette structures developed as a result of cell contact with additional molecules to differentiate cells into P2 stage cells with a characteristic expression pattern, including Pax2+/En1+DA progenitor cells, and then differentiated into TH+/En1+DA neurons. These cells have been used to engraft rats with 6OHDA-induced lesions and cycosporin A-induced immunosuppression. Transplantation experiments described have shown poor in vivo survival, loss of the TH+ phenotype, problems associated with further proliferation into unwanted, possibly lethal, cells such as teratomas , and growth into unacceptable types of neurons that can cause additional medical problems for the patient.

A very small number of surviving TH+ neurons 4.5 months after transplantation (<50 TH+ cells/animal) was observed in grafts from DA progenitor neurons derived from grove structures, FIG. 16A. However, in contrast to TH+ cells, cells labeled with GFP (GFP was under the control of a ubiquitous promoter) showed fairly good survival after transplantation. This suggested that the cells with the best survival were non-DA neurons (16B). Several transplant-derived cells (hNA+, green) co-expressed TH (red), which also indicated that the majority of transplanted human cells are non-DA neuron phenotypes, FIG. 16C. Sections of Fig. 16D-E show that, despite very poor in vivo survival, there was some improvement (variable in low and high degree) in several behavioral tests such as the amphetamine-induced rotation test (D), cylinder test, and spontaneous rotations. (E).

Induction of neurons without feeder cells was performed as described previously (Chambers et al., 2009), but with modifications to obtain ventral plate cells (Fasano et al., 2010). Using a modified SMAD dual inhibition method to differentiate pluripotent cells into ventral plate cells, the authors of the present study previously found that high concentrations of SHH were required to induce FP at day 11. For example, in some embodiments, Sonic C25P is added at a concentration of 200 ng/mL. DKK-1 (R&D; 100 ng/mL), FGF8 (R&D; 50 ng/mL), Wnt-1 (Peprotech; 50 ng/mL), and retinoic acid (R&D; 1 mM) were added in some experiments, see Fig. . 17. However, none of the cell populations obtained using previously known methods contained a high percentage of progenitors of FOXA2+/LMX1A+ ventral midbrain cells obtained using the method of the present invention at day 11.

As shown in the present description, the cell population containing pluripotent cells was chosen by the present inventors as the initial population and seeded on day 0. Prior to differentiation, the cells were grown to a state close to full population (between 60-100% confluence). These cells were contacted with dual SMAD inhibitors (i.e., treated with LDN-193189+SB431542=LSB) on day 0. The present inventors maintained a cell population with intermittent feeding containing fresh LSB until day 11 and found that some of the remaining cells were LMX1A+ but did not express FOXA2 (Fig. 1a, b). The authors seeded a duplicate of the original cell population and then tested the cell type (i.e., gene/protein expression patterns) after treatment with mixtures containing any of the following SHH agonists (purmorphamine+SHH) and FGF8 (S/F8) contacted with cells in various processing modes, i.e. cells in contact on day 0, or day 1, or day 2, etc. within a certain period of time, i.e. 24h, 48h, etc. Three primary examples of culture conditions were studied, namely: 1) cells were contacted with LDN/SB (LSB) on day 0, then contacted with fresh LSB until day 5, on day 5 cells were contacted with fresh LDN without SB until day 11, 2) cells were contacted with LDN/SB (LSB) on day 0, then contacted with fresh LSB until day 5, on day 5 cells were contacted with fresh LDN without SB until day 11, during which time the cells were additionally contacted with fresh purmorphamine, SHH and FGF8 until day 7, and 3) cells were contacted with LDN/SB (LSB) on day 0, then contacted with fresh LSB until day 5, on day 5 cells were contacted with fresh LDN without SB until day 11, during which time the cells were additionally contacted with fresh purmorphamine, SHH and FGF8 until day 7, and also additionally contacted with fresh CHIR from day 3 of culture to day 11 with several variations of these primary conditions. These experiments were aimed at determining the optimal yield of cell types. Systematic comparisons of these three culture conditions (FIG. 1d) were made by determining the global temporal profile of gene expression. See examples in FIG. 8 and tab. 1-6. Hierarchical allocation of clusters of differentially expressed genes made it possible to distinguish between these three sets of treatment conditions on day 11 of differentiation (Fig. 8a). FOXA1, FOXA2, and several other downstream SHH targets, including PTCH1, were among the most differentially regulated transcripts in LSB/S/F8/CHIR compared to LSB processing sets (Fig. 1e). Expression of LMX1A, NGN2, and DDC indicated a predetermined developmental pathway into a DA progenitor midbrain neuron as early as day 11 (Fig. 1e, f). In contrast, day 11 LSB cultures were enriched

- 107 041083 dorsal hindbrain progenitor markers such as HES5, PAX6, LHX2, and EMX2. Direct comparison of LSB/S/F8/CHIR with LSB/S/F8 treatment (Fig. 1f) confirmed the selective enrichment of DA progenitor markers in midbrain neurons in the LSB/S/F8/CHIR group and indicated the identity of hypothalamic progenitors in LSB-treated cultures. /S/F8, based on the differential expression of RAX1, SIX3, and SIX6 (see also POMC, OTP expression in Fig. 2D). Example lists of differentially expressed transcripts on day 11 are shown in Table. 1, 2 and 25 days in the table. 3-5, and ontological analysis of genes in FIG. 8b (DAVID; http://david.abcc.ncifcrf.gov), confirming the enrichment of the canonical WNT signaling pathway upon CHIR treatment. Primary data is not yet available at GEO worldwideweb.ncbi.nlm.nih.gov/geo/accession#: [TBD]). Comparative temporal analysis of gene expression of midbrain DA progenitor markers (Fig. 1g) with non-DA anterior and ventral neuron developmental pathway markers (Fig. 1h) revealed three types of induction: i) LSB: dorsal hindbrain; ii) LSB/S/F8: ventral/hypothalamic; and iii) LSB/S/F8/CHIR: identical to midbrain DA progenitor.

Example III.

Differentiation of DA neurons. For further differentiation, progenitor cells were maintained in neuronal maturation medium (BAGCT - see Materials and Methods). The following types of comparisons were made between differentiating cell populations obtained by previously known methods and methods according to the present invention: A) Immunocytochemical analysis at differentiation day 50 for TH in combination with LMX1A, FOXA2 and NURR1, B) Cell counts for TH+, FOXA2+, LMX1+, and NURR1+ among total cells, comparing rosette-derived cultures with ventral plate-derived cultures (LSB/S/F8/CHIR), C) Calculation of percent subtypes of neurons serotonin+ (5-HT), and GABA+ (admixture of neurons, non-DA) at day 50 in ventral plate-derived culture and DA culture of rosette-derived neurons, and D) HPLC analysis to measure dopamine and metabolites: comparison of DA, DOPAC and HVA levels between ventral plate culture and culture obtained from rosette structures. By day 25, Tuj1+ neurons (FIG. 2A) and cells expressing TH, the rate-limiting enzyme for DA synthesis, were generated in three cell populations. However, treatment with LSB/S/F8/CHIR resulted in the formation of TH+ cells that co-expressed LMX1A and FOXA2 and showed strong induction of the nuclear receptor NURR1 (NR4A2) (Fig. 2A,B). Comparison of gene expression in cultures at day 13 and day 25 confirmed strong induction of other postmitotic markers of DA neurons (Fig. 2C). Characterization of the identity of DA neurons at day 25 compared to cultures treated with LSB and LSB/S/F8 confirmed enrichment for known transcripts of midbrain DA neurons and identified many new marker candidates (Fig. 2D, Tables 3-5, Figs. 8b). For example, the most enriched transcript in LSB/S/F8/CHIR (midbrain DA group) was TTF3, a gene that was not previously associated with the development of midbrain DA neurons but is highly expressed in human substantia nigra (Fig. 8c; Allen Brain Atlas: http://human.brain-map.org).

Similar data were obtained for EBF-1, EBF-3 (Fig. 8c), as well as for TTR, a known transcriptional target for FOXA2 in the liver. The data obtained during the development of the present invention indicated the enrichment of several PITX genes in midbrain DA progenitor cells. PITX3, a classic DA marker for midbrain neurons, was also highly expressed at differentiation day 25 (Fig. 2E). Finally, induction of both midbrain ventral plates and DA neurons can be easily reproduced in independent hESC and hiPSC lines (Fig. 9). The data presented herein demonstrate that the LSB/S/F8/CHIR protocol, in contrast to other protocols tested, produces cells expressing a set of markers corresponding to the midbrain neuronal DA developmental pathway.

The in vitro and in vivo properties of DA-like neurons derived from the ventral plate were compared with DA-like neurons derived using intermediate rosette-shaped neuronal structures (FIGS. 10 and 16). Obtaining rosette-shaped neuronal structures is currently the most widely used strategy for obtaining neurons DA neurons from hPSCs. Protocols based on both ventral plates and rosette structures were effective in generating TH+ neurons with long-term in vitro viability (50 days of differentiation; Fig. 3a). However, the percentage of TH+ cells was significantly higher in ventral plate cultures (FIG. 3b). Although TH+ cells co-expressed NURR1 in both protocols, ventral plate-derived DA neurons co-expressed FOXA2 and LMX1A (Fig. 3 a, b).

Several GABA- and serotonin (5-HT)-positive DA neurons were found (Fig. 3c), also its metabolites DOPAC and HVA were present in cultures obtained by both one and the second protocol, but DA levels were approximately 8 times higher in ventral plate cultures (Fig. 3d, e). DA midbrain neurons exhibited strong fiber outgrowth and strong expression of neural maturation markers, including synapsin, the dopamine transporter (DAT), and the G protein-coupled upstream potassium channel Kir3.2, also called GIRK2, express

- 108 041083 located in DA neurons of the compact part of the black body (substantia nigra pars compacta, SNpc) (Fig. 3f, 11). DA SNpc neurons in vivo exhibit an electrophysiological phenotype that distinguishes them from most other neurons in the brain. In particular, they generate spontaneous peaks (1-3 Hz) with a low frequency. Moreover, such generating rare peaks is complemented by a slow subthreshold vibrational potential. After 2-3 weeks in vitro, DA SNpc neurons grown from early postnatal mice exhibited the same physiological features. DA neurons differentiated from hESC cells consistently (4/4) exhibited this distinctive physiological phenotype (Fig. 3g-i).

Maintenance of mouse DA neurons in vitro until day 65 showed that TH-positive neurons continue to express FoxA2 and have extended fibers typical of mouse DA neurons, Fig. 3a. Measurement of DA release by HPLC showed that 65 day old TH+ neurons are functional in vitro, FIG. 3b.

Example IV.

Engraftment of new cell populations of DA neurons in rodents, namely mice and rats with damaged neurons.

One of the challenges in this field is the ability to obtain midbrain neurons from hPSC hDA cells that are functionally engrafted in vivo without the risk of neuronal overgrowth or unacceptable differentiation into non-midbrain neurons or formation of teratomas. Based on the study of transplantation of embryonic tissues, the authors of the present invention suggested that the time of the end of the cell cycle, the marker of which is the expression of NURR1, may be an appropriate stage for transplantation (approximately 25 days of differentiation, Fig. 2). Initial experiments using cells at day 25 in undamaged adult mice showed a high survival rate of hPSC-derived FOXA2+/TH+ neurons 6 weeks after transplantation (FIG. 12). We tested the long-term survival of FOXA2+/TH+ cells in parkinsonian recipients without subsequent neuronal outgrowth. For this purpose, 6-hydroxydopamine (6-OHDA) was used to create lesions (Tabar, et al. Nature Med. 14:379-381 (2008) cited herein by reference) in NODSCID IL2Rgc knockout mice, strain , which effectively provides xenograft survival with particular sensitivity to rare oncogenic cells (Quintana, et al. Efficient tumor formation by single human melanoma cells. Nature 456:593-598 (2008), the cited source is incorporated herein by reference). Cultures of DA neurons, both derived from ventral laminae and rosette structures, were transplanted (150 x 10 ³ /animal) without prior purification in order to identify potential contaminating cells with high proliferative potential. Four and a half months after transplantation, the ventral plate-derived DA neuron grafts showed a well-formed graft nucleus composed of TH+ cells co-expressing FOXA2 and the human hNCAM specific marker (Fig. 4a-c). Functional analysis showed elimination of amphetamine-induced rolling behavior. In contrast, DA neuron grafts derived from rosette structures showed only a few TH+ neurons and did not elicit significant facilitation of rotational behavior (Fig. 4d), and massive neuronal outgrowth was also detected (graft volume >20 mm3 ⁾ . ; Fig. 13). Extensive outgrowth of neurons derived from rosette structures used in transplantation as described in this application was compared with previous work with DA grafts derived from rosette structures by the group of authors of the present invention (Kim, et al. miR-371-3 Expression Predicts Neural Differentiation Propensity in Human Pluripotent Stem Cells Cell Stem Cell 8:695-706 (2011) cited herein by reference) and others (Hargus, et al. Proceedings of the National Academy of Sciences of the United States of America 107:15921-15926 (2010), the source cited is incorporated herein by reference). Overgrowth was likely due to longer survival times (4.5 months versus 6 weeks), lack of FACS clearance prior to transplantation, and selection of NOD-SCID IL2Rgc knockout recipients. The number of proliferating Ki67+ cells was minimal in ventral plate-derived grafts (<1% of total cells), while rosette-derived grafts contained foci of proliferating neuronal progenitors. It has been suggested that neuronal outgrowth was caused by primitive neuroectodermal progenitor cells in the graft (Elkabetz, et al. Genes Dev. 22:152-165 (2008); Aubry, et al. Proc. Natl. Acad. Sci. USA 105:16707- 16712 (2008), the source cited is incorporated herein by reference). This hypothesis was supported by the expression of the forebrain marker FOXG1 in grafts derived from rosette structures rather than from ventral laminae. A small percentage of astroglial cells were present in both ventral plate-derived and rosette-derived grafts, although most GFAP+ cells did not contain human markers indicative of the origin of the recipients (Fig. 13).

The results obtained in NOD-SCID IL2Rgc knockout mice described in this application demonstrated a high long-term survival of FOXA2+/TH+ neurons, complete

- 109 041083 an increase in amphetamine-induced rolling behavior and no evidence of neuronal outgrowth. However, some of these results may be specific to the use of NOD-SCID IL2Rgc knockout mice. To test this hypothesis, cultures of ventral plate-derived DA neurons (250x103 cells) were transplanted into adult rats with 6-OHDA lesions subjected to pharmacological immunosuppression using cyclosporine A. Five months after transplantation, graft survival was high (Fig. 4e- h) with an average of over 15,000 TH+ cells simultaneously expressing FOXA2 (FIG. 4g) and human nuclear antigen (hNA) (FIG. 4e); TH+/hNCAM+ fibers extended from the graft nucleus into the recipient's surrounding striatum (Fig. 4f). In addition to FOXA2, TH+ cells expressed the PITX3 and NURR1 DA markers of midbrain neurons (Fig. 4h-j). Behavioral studies showed complete elimination of amphetamine-induced rotational asymmetry, in contrast to sham transplant animals, which did not show any improvement (Fig. 4k). Transplanted animals also showed improvements in the focal akinesia test (Fig. 4l) measuring forelimb akinesia and in the cylinder test (Fig. 4m), studies independent of pharmacological stimulation of the DA system. A late onset of recovery (approximately 3-4 months after transplantation) is expected for human DA neurons, depending on in vivo maturation rates such as DAT expression levels (Fig. 4n). The presence of TH+ cells expressing Kir3.2 channels (GIRK2) or calbindin indicated that DA neurons were present in the graft, both SNpc (A9) and ventral tegmental area (A10) (Fig. 4o, p).

As in mice (Fig. 13), serotonergic and GABAergic cells were rare in rat cells (<1% of total cells), and predominantly recipient-derived GFAP+ glial cells were present (7% of total cells ( Fig. 14. Although a few serotonin-positive neurons were found in the graft, hNCAM-negative cells were found, which were likely serotonergic fibers obtained from the recipient (Fig. 14).

Example V.

Transplantation of a population of new DA neurons to primates with damaged neurons.

The results reported herein demonstrated superior graft survival and behavioral outcomes in two independent mouse models. However, the number of DA neurons required for the brains of mice or rats represent a small fraction of the large number of cells required for transplantation into primates and humans. To test the scalability of this protocol, pilot transplantation studies were conducted in two adult rhesus monkeys with MPTP-induced lesions.

Portions of 50x106 transplantable DA progenitor neurons were obtained on day 25 of differentiation using a protocol based on the use of ventral plates. The classic dose for inducing Parkinsonian-like conditions was 3 mg MPTP-HCL injected into the carotid artery (range 0.5-5 mg). Then systemic injections of MPTP were performed at a concentration of 0.2 mg/kg MPTP intravenously. Cells were injected into three sites (posterior caudate nucleus and precommissural putamen) on each side of the brain (total of 6 nerve bundles, 1.25x106 cells/bundle), animals were subjected to immunosuppression using cyclosporine A. On one side, the brain was injected DA precursors from the H9 subclone expressing GFP, and cells derived from unlabeled H9 cells were transplanted to the second side. Results showing transplantation of rhesus monkey neurons with continued FOX2A expression and TH production are shown in FIG. 4q-t. One month after transplantation, a high survival rate of midbrain DA neurons was observed, as assessed by GFP expression (FIG. 15) and a human-specific cytoplasmic marker (SC-121) (FIG. 4q). Each graft core was surrounded by a halo of TH+ fibers extending up to 3 mm into the recipient (Fig. 4r). The graft nuclei consisted of TH+ neurons coexpressing SC-121 (Fig. 4s) and FOXA2 (Fig. 4t). The SC-121- and GFP-negative regions in the grafts contained Iba1+ recipient microglia (FIG. 15), indicating incomplete immunosuppression.

As a result, a cell population of new DA neurons was transplanted into primates, namely, adult rhesus monkeys with damage caused by MPTP (3 mg MPTP-HCL (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; in the concentration range 0 5-5 mg MPTP-HCl) and characterized by a severe >95% loss of endogenous midbrain DA neurons.MPTP treatment produced observable changes and symptoms similar to Parkinson's disease in humans.

Example VI.

Comparative potential of differentiation along the pathway of DA formation in midbrain neurons in PINK1-mutant PD-iPSC cells and wild-type hESCs (or iPSCs).

The present example describes that large populations of midbrain DA neurons that have developed properties of neurons of Parkinson's disease (PD) patients when a cell line of such patients, namely PD-iPSC cells mutated in PINK1, is obtained in a non-destructive manner. 110 041083 embryo was used as a cell population to obtain FOXA2/LIM1XA/TH+ DA neurons according to the present invention.

The PINK1 mutant BP-iPSC line Q456X was differentiated using the novel protocol for obtaining midbrain DA neurons based on ventral plates (method) according to the present invention, which results in midbrain differentiation profiles comparable to those obtained for the H9 iPSC line. (Fig. 20). A-C) Immunocytochemical analysis of a PINK1 mutant BPiPSC line at differentiation day 11 (midbrain progenitor stage) for FOXA2 (red), LMX1A (green) and DAPI (blue) (A), differentiation day 25 (early post-mitotic stage of DA neurons) for FOXA2 (red) and TH (green) (B), and for NURR1 (red) and TH (green) (C). D-F) The same set of immunocytochemical assays performed using cells derived from H9 at differentiation day 11 for FOXA2 (red), LMX1A (green) and DAPI (blue) (D), at differentiation day 25 for FOXA2 (red) and TH (green) (E), and for NURR1 (red) and TH (green) (F).

In the BP-iPSC line, mutant in PINK1, a BP-like phenotype of protein aggregation was found, followed by prolonged differentiation and maturation in vitro. The authors of the present invention found that BP-iPSC mutant in PINK1 showed evidence of expression of α-synuclein (the main component of Lewy bodies in patients with Parkinson's disease) in the cytosol of TH+ DA neurons on the 55th day of differentiation using a new protocol for DA induction of midbrain neurons based on ventral plates, (Fig. 21A-B). A, B) Immunocytochemical analysis of a PINK1 mutant BP-iPSC line at differentiation day 55 for α-synuclein (LB509, red), TH (green) and composite image (A) and α-synuclein (red) and ubiquitin (green) ) (B). These α-synuclein-positive cells also showed high expression of ubiquitin (a classic marker of Lewy bodies). In contrast, DA neurons derived from the iPS control line showed expression of normal synaptic (as opposed to cytosolic) α-synuclein and very low levels of ubiquitin (Fig. 21C-D). C, D) Immunocytochemical analysis of the control line iPSC line at day 55 of differentiation for α-synuclein (red) and TH (green) (C) and α-synuclein (red) and ubiquitin (green) (D).

Expression of the aggregated form of α-synuclein. In the brains of patients with Parkinson's disease, dimerized insoluble forms of α-synuclein lead to aggregation in the form of Lewy bodies. In the dimerized form of α-synuclein, phosphorylation of serine-129 in α-synuclein is observed.

On the same day of differentiation, cells derived from PINK1-mutant BP-iPSCs showed strong expression of Ser129-phosphorylated α-synuclein, in contrast to cells derived from control iPSCs, which showed very low levels of expression (Fig. 22). Cells derived from BP-iPSCs mutated in PINK1 showed strong expression of Ser129-phosphorylated α-synuclein, in contrast to cells derived from control iPSCs, which showed very low levels of expression. A, B) Immunocytochemical analysis of Ser129-phosphorylated α-synuclein (green) and DAPI (blue) in PINK1-mutant BP-iPSC-derived cells at differentiation day 55 (A) and matched iPSC-derived control cells (B ).

Differences in α-synuclein expression patterns were observed depending on the differentiation protocol. The present inventors observed that authentic ventral plate-derived midbrain DA neurons exhibited Parkinson's disease-specific vulnerability and corresponding specific phenotypes in vitro. The use of DA neurons derived using the classical differentiation protocol based on MS5 stromal feeder cells (Perrier et al., PNAS 2004, cited herein by reference) results in the formation of a large number of TH+ neurons. However, based on the data obtained during the development of the present invention, the authors of the present invention showed that MS5-derived TH+ cells were not authentic DA ventral plate-derived midbrain neurons. In cultures differentiated using the MS5 protocol, there were many α-synuclein-positive cells. However, these cells did not show simultaneous expression of TH. Moreover, there was no difference in expression patterns between BP-iPSCs and control iPSCs using the MS5 differentiation strategy (Fig. 23A-B). These data indicate that α-synuclein is also expressed in other non-DA cell types and that such α-synuclein from non-DA cells is not altered by disease when compared to iPSC-derived cells in particularly when using the standard differentiation protocol MS5. DA-like neurons derived from rosette structures have been described in publications (eg, Perrier PNAS 2004). Such MS5-based TH+ cells (=DA-like) were used for comparison in FIG. 3, 10, 13, and 16. These data indicate that α-synuclein is also expressed in other non-DA cell types, and that such α-synuclein from non-DA cells is not altered in disease compared to cells obtained from control iPSC cells, in particular

- 111 041083 when using the standard MS5 differentiation protocol. Finally, the use of the new ventral plate-based differentiation protocol described in this application results in the formation of a large number of TH+ cells coexpressing α-synuclein. Such TH+ cells express α-synuclein in a cytosolic expression pattern. Fig. 24A, B) Immunocytochemical analysis of α-synuclein (LB509, red), TH (green) of the PINK1 mutant BP-iPSC line at day 60 of differentiation based on MS5 (A) and control iPSC line (B). C) Immunocytochemical analysis of the PINK1 mutant BP-iPSC line at day 55 of differentiation based on the ventral plate for α-synuclein (red), TH (green).

Typical DA neurons derived from PINK1-mutant BP-iPSC cells are sensitive to toxin stimulation. BP-iPSC-derived TH+ DA neurons obtained using the ventral plate protocol were more sensitive to toxic treatment (valinomycin: mitochondrial ionophore, 5 μM (in the concentration range 1-10 μM), 48 h) than control cells obtained based on iPSC. In contrast, TH+ neurons generated using the classic MS5 protocol showed no differential sensitivity for PD cells compared to control cells (Fig. 24). A-F) Representative immunocytochemical analysis of TH at day 60 of differentiation: Normal conditions (no toxic treatment) for both BP cells and control cells derived from iPSCs using a ventral plate protocol (A, cells derived from BP-iPSCs are shown ), almost complete degeneration of TH+ DA neurons during subsequent toxic treatment with BP-iPSCs (B), partially degenerate TH+ DA neurons from control iPSCs (C). The overall cell survival test with alamar-blue after 48 h of valinomycin treatment also showed different cell survival in the specific dose range for toxic treatment (5 and 10 μM) when comparing BP-iPSCs and control iPSCs (Fig. 25).

Normal conditions for both BP cultures and cultures based on control iPSCs obtained using an MS5-based protocol (D, cells derived from BP-iPSCs are shown), TH+ neurons after toxic treatment obtained from a culture of BP-iPSCs (E ) and the control culture iPSC (F) obtained according to the MS5 protocol. G-H) images of low power immunocytochemical studies for Tuj 1 (red) and TH (green) according to the ventral plate protocol on day 60 of differentiation: BP-iPSCs under normal conditions (G) and toxic treatment conditions (H) and control iPSCs in normal conditions (I) and conditions of toxic treatment (J). K-N) images of low power immunocytochemical assays for Tuj1 (red) and TH (green) from MS5-based protocol at differentiation day 60: BP-iPSCs under normal conditions (K) and toxic treatment conditions (L) and control iPSCs under normal conditions ( M) and toxic treatment conditions (N).

An example of scoring in a cell survival test versus dose in a toxic treatment. A cell survival test with alamar-blue after 48 h of valinomycin treatment showed different cell survival in the characteristic dosage range of toxic treatment (5 and 10 µM) when comparing BP-iPSC and control iPSC (day 60 of differentiation based on ventral plates). Note: These tests examine overall cell death, although particularly marked effects were seen on DA neurons (see Fig. 14).

Therefore, alamar blue scoring is likely to underestimate the extent of the various effects observed in DA neuron lines. Documents incorporated herein by reference:

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Li, and dp. Nat. Biotechnol.

23, 215-221 (2005); Perrier, and dp. Proc Natl Acad Sci U S 101, 12543-8 (2004); Perrier, and dp. Proc

Natl Acad Sci U SA 101, 12543-8 (2004); Tabar, and dp. nature medical. 14, 379-381 (2008); Perrier, and dp. Proc Natl Acad Sci U SA 101, 12543-8 (2004); Wernig, and dp. Proc. Natl. Acad. sci. U. S. A 105, 5856-5861 (2008); Lindvall, and dp. J.Clin. Invest 120, 29-40 (2010); Roy, and dp. nature medical. 12, 1259-1268 (2006); Elkabetz, and dp. Gene Dev. 22, 152-165 (2008); Kittappa, and dp. PLOS. Biol. 5, e325 (2007; Ferri, and dp. Development 134, 2761-2769 (2007); Roelink, and dp. Cell 76, 761-775 (1994); Liem, and dp. Cell 82, 969-979 (1995) Fasano, and dp. Cell Stem Cell 6, 336-347 (2010) ;

Chambers, and dp. Nat. Biotechnol. 27, 275-280 (2009) ; Muroyama, and dp. Gene Dev. 16, 548-553 (2002) ; Joksimovic and dp. Nat Neurosci 12, 125-131 (2009) ; Lyashenko, and dp. Nat. Cell biol. 13, 753-761 (2011) ; VanDunk, and dp. J. Neurosci. 31, 6457-6467 (2011) ; Huang, and dp. Nat. Protocol. 4, 44-57 (2009) ; Costa, and dp. Mol. Cell biol. 9, 1415-1425 (1989) ; Elkabetz, and dp. Gene Dev. 22, 152-165 (2008) ; Soldner, and dp. Cell 136, 964-977 (2009) ; Guzman, and dp. J. Neurosci. 29, 1101111019 (2009) ; Nedergaard, and dp. J. Physiol 466, 727-747 (1993) ; Ferrari, and b.p. Eur. J. Neurosci.

24, 1885-1896 (2006) ; Olanow, and b.p. Trends Neurosci. 19, 102-109 (1996) ; Zetterstrom, and bp.

Science 276, 248-250 (1997); Quintana, and b.p. Nature 456, 593-598 (2008) ; Kim, and b.p. diets Neural Differentiation Propensity in Human Pluripotent Stem Cells. Cell Stem Cell 8, 695-706 (2011) ;

Hargu, and b.p. Proceedings of the National Academy of Sciences of the United States of America 107, 15921-15926 (2010) ; Aubry, and b.p. Proc. Natl. Acad. sci. U. SA 105, 16707-16712 (2008); Blume, et al., Exp. Neurol. 219, 208-211 (2009); Ban, et al., Proc. Natl. Acad. sci. U.S.A(2011); Studer, et al., Nature Neurosci. 1, 290-295 (1998); Kordower, et al., Science 290, 767-773 (2000); Paxinos, et al., The Rhesus Monkey Brain in Stereotaxic Coordinate.s (Academic Press, 2000); Crawley, What's Wrong With My Mouse: Behavioral Phenotyping of Transgenic and Knockout Mice (Wiley-Liss, 2000);

Studer, et al., Brain Res. Bull. 41, 143-150 (1996); Tabar, et al., Nat. Biotechnol. 23, 601-606 (2005); and Placantonakis, et al., Stem Cells 27, 521-532 (2009).

Example VII.

Characteristic conditions were established for recording in vivo DA of neurons derived from human pluripotent stem cells in thin-layer preparations; see representative results in FIG. 26.

Electrophysiological measurements are being considered for use in thin layer preparations, namely from biopsies of graft areas. In one embodiment, DA graft-derived A9 neurons, as opposed to A10, can be identified in vivo based on testing for autonomic rhythm generating activity that is characteristic of A9 dopamine neurons that are most harmed in Parkinson's disease. In other words, A10 type neurons do not have activity in creating a rhythm.

Characteristic conditions were established for recording in vivo DA of neurons derived from human pluripotent stem cells in thin-layer preparations; see fig. 26. In particular, measurements have been made on transplanted human DA neurons derived from human pluripotent stem cells and found to have electrophysiological features typical of those found in the substantia nigra pars compacta (SNpc) portion of mice. , fig. 26A, which is a top view of a reconstruction of a pacemaker neuron (pacemaker neuron) at the graft site. The bottom view shows a typical photomicrograph of a rat-derived brain section in which hES-derived neurons were injected 9 months earlier; the graft is outlined; a high magnification image is shown below as an inset. The section was treated with tyrosine hydroxylase, which is shown in white, FIG. 26b. Next, the top view shows a typical attached overlay recording from what is supposed to be in the graft; the view below is a typical recording from an entire cell of the same cell. Recording was performed in the presence of glutamate and GABA receptor antagonists (50 μM AP5, 10 μM CNQX and 10 μM GABAzine) in order to eliminate synaptic input. These recordings demonstrated that these PS-derived neurons were pacemaker neurons with normal intrasomatic stress trajectories. Other neurons recorded in the graft sample had similar properties, Fig. 26C. For comparison, cell attachment and whole cell recordings of dopaminergic neurons in adult mouse SNpc are shown. Abbreviations (CTx=cortex, STr=striatum, SNpc=black body compact fraction, DA=dopaminergic). These data show in vivo functional studies in the striatum of transplanted rat one month after transplantation.

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Thus, in some embodiments of the present invention, in vivo functional studies in transplanted tissues demonstrate recovery of a portion of the black body compacts (substantia nigra pars compacta, SNpc).

Example VIII.

Exemplary methods for identifying cell surface markers for use in the methods of the present invention. In particular, CD142 was identified using these methods.

The two main strategies for identifying surface marker candidates are: Unbiased testing of gene expression in genetic reporter lines (Fig. 27a), which identified several candidate markers, including a marker named DCSM1, which is selectively expressed in midbrain DA neurons and appears in features of DA markers in A9 type neurons (Fig. 27b). The second strategy is to use the check for CD cell surface markers in hESC-derived DA neurons testing 242 commercially available antibodies in a 96-well format (Fig. 27c, d). This validation (FIG. 27e) resulted in the identification of at least 5 confirmed markers enriched in DA midbrain neurons, including CD142, a marker selectively indicative of Nurr1+ DA neuronal stage (FIG. 27f). Using the neuronal DA cellular procedure described herein, CD142 typically labeled approximately 30% of the total cell population at differentiation day 25 (FIG. 28a). The selectivity of CD142 to the Nurr1+ DA neuronal stage was confirmed in multiple independent hESC and hiPSC 1 lines (FIG. 28b). In addition to enrichment in DA neurons, enrichment in CD142-positive cells resulted in selective depletion of unwanted neuron subtypes such as GABA- and serotonergic neurons. (Fig. 28c-f). In vivo studies have confirmed the ability of a population of CD142-positive cells to give rise to transplants with high purity of DA neurons, which allows to overcome the problems of contamination by GABA- and serotonergic neurons. Although the transplantation procedure using crude cells has already yielded a small number of serotonergic neurons, the use of progenitor selection based on CD142 is expected to further reduce the risk of introducing serotonergic neurons, a type of contaminating cell that has resulted in failed transplantation into human embryonic tissues such as potential source of unwanted dyskinesia induced by transplantation into human embryonic tissues.

Example IX.

This Example describes methods for transforming cells with human PST genes to enhance PSA expression on the cell surface. The present Example also shows exemplary methods for using cells having increased PSA expression on the cell surface.

In particular, the present Example shows insertion of PST genes into hESCs to enhance PSA expression in DA neurons. The gene encoding human polysialyltransferase (hPST) was introduced into the hESC line (WA01) using a lentiviral vector (pLenty, Invitrogen). Twenty selected clones were grown and analyzed for PST expression. PST-expressing hESC clones were differentiated to ensure that PST expression was not downregulated in DA neurons. PSA-NCAM counts at different stages of differentiation (days 0, 11, 25 and 50) were made using FACS analysis and immunofluorescence (Operetta). Positive clones had to match the QC parameters of DA neurons given in Table 1. 7. At least 3 clones that retained high, unchanged levels of PSA-NCAM during differentiation and well matched QC parameters (Table 7) will promote the establishment of axonal outgrowth in DA neurons expressing PST and derived from hESC. Matched control and PST-expressing hESC clones were differentiated into DA neurons using the standard protocol described herein followed by cell fixation and analysis at days 25 and 50. The number and length of TH-positive fibers in these cultures were assessed using a high contrast Operetta microscope. The axon analysis module in Harmony 3.0 software counted axon number and length, with or without PST, and the data were statistically analyzed using two-way ANOVA. PST expressing clones and control hESC clones, which were selected based on the in vitro studies described above, were again differentiated into DA neurons and transplanted into Parkinson's disease model rats. Short-term transplants (4-6 weeks) were made to determine survival, PSA-NCAM expression, and axonal outgrowth. Each clone that met short-term in vivo parameters was subjected to long-term graft studies. In these studies, animals received half or a quarter of the standard (200x103) cell dosage. These studies were aimed at elucidating whether elevated PSA resulted in increased long-term survival after transplantation (5 months) and whether fewer DA neurons were able to compete with or outperform non-PST transplants transplanted at a standard cell dosage (Fig. 27).

In addition, a comprehensive behavioral test sensitive to striatal reinnervation was performed in order to further characterize the functional potential of DA neuronal grafts.

- 114 041083 with PST versus controls. Animals were sacrificed after completion of the behavioral tests and fiber outgrowth was scored using human NCAM and SC121 specific antibodies and anti-TH antibodies (see also FIG. 29). Graft intensity and spread of hNCAM+, SC121+ and TH+ were measured, as well as the percentage of human cells co-expressing DA neuron markers (TH, FOXA2) and PSA. The NCAM/TH+ densities of axon halo propagating from the graft were calculated at different distances. Data between groups were compared using a two-way ANOVA in the post-hoc Bonferroni test. In addition, qualitative changes in sections (eg, branching, thickness, graft distribution, and shape) were studied. In addition, some grafts were subjected to thin layer electrophysiological evaluation in terms of A9 phenotype, synapse formation with the recipient's striatum, and endogenous afferent nerve innervation.

X example.

The following example demonstrates an increase in polysialic acid expression that improves the function of ES-derived dopamine neuron grafts in parkinsonian mice.

ES cells expressing GFP under the control of the Nurr1 promoter (Nurr1::GFP ES cells) were stably transduced using a lentiviral vector ubiquitously expressing polysialyltransferase (PST). Transduced cells showed a significant increase in PST mRNA compared to controls (FIG. 30A). PST expression has been found to be essential for PSA synthesis in NCAM. Accordingly, PSA-NCAM expression was significantly upregulated in PST-modified cells on day 14 of DA neuron differentiation (Fig. 30BE). Both endogenous surface PSA and surface PSA in induced DA cells of ES-derived neurons can be removed (Fig. 30E) using phage endoneuraminidase (endoN), which specifically cleaves PSA-unique alpha-2,8-linked sialic polymers. acids. Surprisingly, PST transduction did not affect the expression of neuronal or midbrain markers in GFP-purified DA neurons (FIG. 30F).

Other studies on 6OHDA-damaged hemiparkinsonian mice have shown that transplantation of approximately 100,000 ES-derived DA progenitor neurons is required to obtain strong functional recovery as measured in the amphetamine-induced rotation test. In the present studies, it was sought to transplant a suboptimal number of cells in order to achieve growth by upregulating PSA expression. In order to transplant populations highly enriched in DA neurons that are reduced in contaminating pluripotent cells, FACS-purified cultures on day 14 of differentiation were tested for Nurr-controlled GFP expression and for the absence of SSEA-1 expression (Fig. 31). . Without PST overexpression, a halving of the minimum effective graft size (55,000 Nurr1+ DA cells) did not lead to a detectable recovery of behavior. In contrast, when PSA expression was upregulated, the same number of Nurr1/PST DA neurons resulted in a significant correction of behavioral deterioration in Parkinson's disease (p<0.01; two-way ANOVA) with complete recovery approximately 5 weeks after surgery (Fig. 32A ). Removal of PSA before transplantation by endoN induction showed the specificity of PSA increase, as endoN treatment partially inverted the functional recovery obtained with Nurr1/PST (FIG. 32A).

To test the characteristics of the transplanted cells, the animals were subjected to immunohistochemical studies two months after transplantation. A difference was found in the number of surviving Nurr1+ neurons, as animals transplanted with PST-transduced lines had, on average, twice as many GFP+ cells as animals transplanted with control cells (9,300+/-1,400 versus 4,230+/-1010 GFP+ cells in the transplant). in PST samples versus controls, respectively, Fig. 32B, p<0.05, Student's t-test). Moreover, Nurr1/PST g transplants also showed higher levels of PSA expression in vivo (Fig. 32C, D). However, the proportions of cells expressing the DA markers of midbrain neurons TH and FoxA2 in the graft nucleus were comparable for Nurr1 and Nurr1/PST cells (TH: 62.0%+/-8.0 vs. 51.3%+/-7.0 p= 0.33, FoxA2: 63.2%+/-8.6 vs. 55.4%+/-2.0, p=0.3, respectively; Fig. 32E).

The neuronal processes that originate from Nurr1 and Nurr1/PST cells showed comparable levels of TH, Girk2 (G protein-coupled upstream potassium channel) and synapsin (FIG. 33A). In contrast to other studies with transplanted Schwann cells (Ghosh, M., et al. Extensive cell migration, axon regeneration, and improved cells after spinal cord injury. Glia 60, 979-992 (2012)), increased PSA expression had little effect on the migration of DA cells from the graft site. However, there were clear changes in the outgrowth of axons. As shown in FIG. 33B, there were more DA neuronal processes derived from Nurr1/PST cells compared to Nurr1+ controls. When the intensity of GFP and TH immunofluorescence was counted in five consecutive 100 µm zones away from the graft, the Nurr1/PST grafts showed

- 115 041083 showed a much higher process density (Fig. 33C, D; p<0.01 for GFP and TH, two-way ANOVA). When quantifying this phenomenon, the relative density of processes was normalized to the density observed in the zone closest to the graft core. This normalization was required in order to compensate for the large number of surviving cells in Nurr1/PST transplants and to confirm the specific effect of PSA on axon outgrowth. Specificity was also demonstrated when PSA was removed from the cell surface during endoN treatment prior to transplantation. This endoN pretreatment resulted in a reduction in distal fiber outgrowth back to control levels (FIG. 33E).

These findings demonstrated that at least some of the effects of PSA on graft function are the result of improved striatal fiber innervation. Accordingly, a strong correlation was observed between graft performance and the relative extent of outgrowth of GFP-positive fibers, eg in zone IV (Fig. 33F; p<0.001, r2=0.65, n=17). Surprisingly, the fiber outgrowth/behavior ratio was constant across the experimental groups (control, PSA-boosted, and endoN-treated), indicating that graft-recipient innervation was the parameter determining behavioral recovery in the parkinsonian mouse model. Several factors mechanically contributed to increased fiber outgrowth, such as improved permeability of the reactive glia surrounding the graft core, improved sprouting ability, increased outgrowth into surrounding recipient tissues (eg, easier growth conical translocation), and protection from premature association with recipient tissues. in close proximity to the graft core. The characteristic mechanisms correlate with the role of PSA in promoting growth during normal development and in the mature nervous system.

The experiments described in this application demonstrated the use of genetically engineered PSA in transplantation of graft DA neurons, which provided superior results compared to transplants from other cell types. The data clearly showed that increasing the amount of PSA provided a significant increase in the ability of transplanted DA neurons to innervate the graft striatum and alleviate the functional deficit in Parkinson's disease. Therefore, it is proposed to transfer the method to clinical use, including the use of DA neurons according to the present invention, to provide cells before transplantation. In some embodiments of the present invention, cells will be genetically manipulated to express PSA. In some embodiments of the present invention, PST can be delivered directly to cells by treatment with a purified enzyme and substrate, in vitro, prior to transplantation. In some embodiments, the present invention contemplates the use of a PSA strategy for transplantation in humans in the case of transplantation for Parkinson's disease to minimize the need for multiple injections and therefore reduce the risks from surgery associated with such multiple injections.

In other embodiments, the present invention contemplates the use of this technology for other cell types and species, for example, increasing the migration of transplanted Schwann cells when creating an intercellular bridge (for example, intercellular interaction) to resume growth of axons at the site of injury to the spinal cord.

The following are typical materials and methods used in this Example.

Animals: 129S3/SvImJ mice (Jackson Laboratory), 6 weeks old, were kept at a controlled temperature with free access to food and water. Experiments were performed in accordance with NIH guidelines and the Institute's Animal Use Guidelines and were approved by the local Institutional Animal Care and Use Committee (IAUCC), the Institutional Biosafety Committee (IBC). ).

6OHDA injection and amphetamine injection test: Animals were anesthetized with sodium pentobarbital (10 mg/kg) and 2 μl 6OHDA (4 μg/μl saline, 0.5% ascorbic acid) was injected into the right striatum. Injections were made with a Hamilton syringe at the coordinates: 0.5 mm behind, 1.8 mm laterally in relation to the bregma and 2.5 mm inward in relation to the surface of the brain. Prior to surgery, animals received one intraperitoneal injection of desipramine (25 mg/kg, Sigma). Two weeks after surgery, the animals were evaluated in the amphetamine-induced rotation test. They were placed in transparent plastic cylinders with a diameter of 30 cm for half an hour, after which they received a single intraperitoneal injection of amphetamine (10 mg/kg, Sigma). After 20 min, the number of ipsilateral/contralateral rotations was counted for the next 20 min. Animals were assessed weekly for seven weeks, then subjected to deep anesthesia and perfused through the heart with PBS and 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were removed and additionally fixed overnight at 4° C. in 4% paraformaldehyde, then 40 μm thick sagittal sections were cut using a vibratome (Pelco-101, Ted Pella).

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Cell differentiation and transplantation: A cell line with the Nurr1::GFP BAC reporter ES from BAC transgenic mice (namely, GFP expression was driven by the Nurr1 promoter) was transduced with lentiviruses (pLenti, Invitrogen) containing the mouse PST gene under the control of the CMV promoter. ES cells were expanded on mitomycin C-treated mouse embryonic fibroblasts (MEF) (StemCell Technologies) in DMEM (Invitrogen), 10% FBS (HyClone) supplemented with 1,400 units/ml LIF (ESGRO; Invitrogen), 2 mM L-glutamine, 1 mM β-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). DA differentiation was induced according to Barberi et al., Nat Biotechnol 21, 1200-1207 (2003) with modifications. Briefly, cells were differentiated on MS5 feeder cells in gelatin coated trays (10,000 cells/10 cm tray) and grown for 4 days in Serum Replacement Medium (SRM). On day 4, Sonic hedgehog (SHH, 200 ng/ml) and FGF8 (100 ng/ml) were added. On the 7th day of differentiation, the medium was changed to N2 with the addition of SHH, FGF8 and bFGF (10 ng/ml). On day 11, final differentiation was induced by removing SHH, FGF8 and bFGF, and adding ascorbic acid (AA, 200 μM) and BDNF (20 ng/ml).

Cells were harvested on day 14-15 treated with Accutase® for 45 min, washed once with N2 medium and incubated with anti-SSEA-1 antibody conjugated to AlexaFluor-647 (BD Pharmingen) for 25 min. Cells were washed once with N2 medium, resuspended in HEPES buffer with 0.1% BSA. DAPI was added to assess survival. FACS was performed using a MoFlo cell sorter and the population of interest was sorted for GFP (Nurr1) fluorescence. Populations with a positive signal from AlexaFluor-647 (SSEA-1) were discarded. Untreated J1 mouse ES cells at the same stage of differentiation were used as controls without GFP.

Nurr1::GFP sorted cells were examined for survival and resuspended in N2 with BDN and AA to a final concentration of 55,000 cells/µl. One μl was injected into the injured striatum of the mouse using a 50 μm pointed glass capillary at the coordinates 0.3 mm posterior, 1.5 mm lateral to the bregma, and 2.2 mm inward from the brain surface. An aliquot of the cell suspension was transferred to 6 mm trays coated with matrigel gel for further characterization.

For immunofluorescent analysis, cells were fixed with paraformaldehyde for 10 min at 4°C, washed twice with PBS, blocked with 5% BSA (0.1% Triton X-100 in PBS), and incubated with primary antibodies for 2 h at room temperature: rabbit anti-GFP (1:1000, Invitrogen), mouse anti-PSA IgM (1:2000, 5A5), mouse anti-NeuN (1:800, Chemicon), mouse anti-TH (1:1000, Sigma), goat against FoxA2 (1:800, Santa Cruz), goat antibodies against Engrailed (1:800, Santa Cruz). The cells were then incubated with secondary antibodies conjugated to Cy3 (1:1000, Jackson).

EndoN Treatment: To remove PSA from NCAM, the day before harvest, cells were treated with 20 units of endoN, a phage enzyme that specifically cleaves PSA 7-9. The cells were then harvested and injected as described above, except they were resuspended in N2 with BDNF and AA and 5 units of endoN. The authors of the present invention determined that the injection of the same amount of endoN alone in mice with lesions did not improve the behavior of the animals.

In vitro PST and PSA-NCAM mRNA analysis: For Western blot analysis, cells were treated with WB buffer (PBS with 1% NP40, 150 mM NaCl, 1 mM EDTA, and 1x protease/phosphatase inhibitors added just prior to extraction, at pH 7, 4) and sonicated twice for 5 sec, centrifuged and resuspended in Lamley's buffer (LB). Aliquots without LB were kept for protein determination. Equal amounts of proteins were loaded onto a 6% sodium dodecyl sulfate polyacrylamide electrophoresis gel (BioRad). Proteins were transferred by electrophoresis onto polyvinylidene membranes (Millipore). The membranes were blocked for 1-6 h in 0.1% Triton X-100 TBS (TBS-T) with 5% skimmed milk powder and incubated overnight with anti-NCAM antibodies (1:10,000, Santa Cruz) in TBS-T with 5% milk. The membranes were then incubated with peroxidase conjugated secondary antibodies (1:10,000, Jackson) and detected using the ECL detection method (Amersham Pharmacia Biotech). Protein levels were calculated using ImageJ software.

For quantitative RT-PCR analysis, total RNA was extracted with Trizol (Sigma), reverse transcribed (Qiagen) and amplified with 10 µl of 2x SYBR reaction mix and 0.2 µM forward and reverse primers to a final volume of 20 µl. For PSA-NCAM analysis by FACS, cells were harvested with Accutase® treatment for 45 min, washed once and incubated with mouse anti-PSA IgM (1:250, 5A5) for 25 min on ice, washed once with N2 medium and incubated with Cy3-conjugated anti-mouse IgM (1:250, Jackson) for a further 25 min on ice. Cells were washed once with N2 medium and resuspended in 0.1% BSA with 7AAD and analyzed in a FACS Calibur cell sorter. Primary antibodies were not added as a control.

Immunohistological and stereological procedures: Free floating coronal sections were blocked with 0.1% Triton X-100, 5% donkey serum in PBS for 30 min at room temperature and incubated for 48 h at 4°C with various antibodies: rabbit anti-GFP (1 :300), chicken anti-GFP (1:200, Chemicon), mouse anti-TH (1:200), mouse anti-PSA IgM

- 117 041083 (1:1000), mouse anti-NeuN (1:400), goat anti-FoxA2 (1:300), rabbit anti-Girk2 (1:300, Alomone Labs), mouse anti-synapsin (1:200 , BD Transduction Laboratories). Sections were then washed and incubated with secondary antibodies: donkey antibodies conjugated to Cy2, Cy3 and Cy5 (1:400, Jackson). For PSA, donkey anti-IgM antibodies conjugated to Cy5 (1:500 Jackson) were used. Incubation was carried out for 2 h at room temperature. Media was washed twice with PBS and mounted in Mowiol (Calbiochem). Each of the three coronal brain sections was analyzed for each antibody staining.

Digital images were obtained using a Zeiss LSM 510 laser scanning confocal microscope with three lasers (Argon 488, HeNe 543 and HeNe 633) with a 40x c-apochromatic objective (water immersion). The number of GFP+ and TH+ cells was counted in every third section, covering the entire brain through a 40x objective, and the total number of cells in the graft was estimated. Double-labeled cells were analyzed in one optical plane along the entire z-axis.

To evaluate the percentage of GFP/TH+ and GFP/FoxA2+ labeled cells for each marker, 100 GFP+ cells were analyzed. To analyze outgrowth processes, confocal scans along the z-axis were performed at intervals of 0.8 µm along the entire z-axis (20-40 µm) with a 1 µm aperture through a 40x objective. Sections were scanned from the injection site laterally to the site where no overgrowth was observed. 3-D projections covering the entire scanned area were sequentially composed. For the analysis of GFP and TH intensity, the entire scanned area was divided into five consecutive 100 μm graft-derived zones and the intensities were measured using ImageJ software. Data were normalized for intensity in the zone closest to the graft (zone I) to control for any potential differences in graft size.

Statistical analysis: Data are presented as mean ± standard error of the mean (SEM). Comparisons were made using Student's t-test or two-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni test. Linear regression analysis was performed and calculated using Pearson's correlation.

Example XI.

The following example demonstrates the enzymatic construction of PSA in hESC-derived DA neurons using a purified bacterial polysialyltransferase, PSTnm, to improve transplant efficiency.

Although effective, transfection of the PST gene forced modifications of hESCs with limited control during polysialylation. This example describes the discovery that external PSTnm induced PSA, instead of gene delivery, (see, Fig. 35). In FIG. 35A, PST-treated Schwann cells (SC) (green line-middle line) had increased adhesion time, while PSA derived from PSTnm inhibited adhesion. In particular, A) PSA derived from PSTnm inhibits adhesion of Schwann cells in suspension into a Schwann cell monolayer even more effectively (red line - lowermost line) than PSA derived from overexpression of PST ((green line - middle line) B) PSA immunoblotting in ESC-derived HB9 motor neurons demonstrates that control samples treated with PSTnm alone had undetectable levels of PSA. Incubation with PSTnm + substrate CMP-sialic acid results in a large PSA band which disappears upon treatment with endoN. C, D) Similar to the effects obtained with the PST gene, polysialylation of these cells by PSTnm and substrate during differentiation enhances axonal outgrowth and cell migration (arrowheads). E) PSA immunostaining on day 30 of DA of hESC-derived neurons. F) This staining is significantly enhanced after treatment with PSTnm and substrate. G) In vivo injection of PSTnm alone had no effect, although its simultaneous administration with substrate H) resulted in a large level of PSA expression in the striatum of mice.

Thus, mature DA neurons treated externally with PSTnm are considered for use in obtaining n cells for graft. Both mammalian PST and PSTnm produce chemically identical PSA chains. Elevated PSA levels in hESC-derived DA neurons (FIG. 35F) should persist for several weeks, sufficient for the DA fibers to exit the graft nucleus. Since PSTnm is removed prior to transplantation, the immunogenicity of this enzyme, which contaminates transplanted cells, should not be taken into account.

PSTnm was derived from an engineered fragment with improved solubility and activity characteristics (Willis et al., Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme. Glycobiology 18, 177-186 (2008)). hESC cultures were induced to differentiate into DA neurons before PSTnm treatment, substrate treatment, or both. Cultures were examined at various time points during treatment (from 10 min to 6 h) by quantitative immunofluorescence (Operetta) and Western blotting to determine the rate and levels of polysialylation. So, on day 25, differentiated DA neurons derived from hESC will be incubated with optimal concentrations of PSTnm and substrate under the conditions described in this application. PSA+ YES neurons will be transplanted in short and long term assays as described herein and in FIG. 29.

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All publications and patents mentioned in the present description above are incorporated into the present description by reference. Various modifications and variations of the described methods and systems of the present invention will be obvious to a person skilled in the art, which do not go beyond the scope of the present invention and do not change its essence. While the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention as claimed should not be unduly limited to these specific embodiments. Indeed, various modifications of the described methods for carrying out the present invention, which are obvious to a person skilled in the field of cell biology, neuroscience, cancer cell biology, molecular biology, biochemistry, chemistry, organic synthesis, or related fields, are included in the scope of the following claims.

Claims (32)

CLAIM 1. A method for inducing differentiation of pluripotent stem cells in vitro, which includes contacting pluripotent stem cells with a signal transduction inhibitor SMAD (Small Mothers Against Decapentaplegic), and contacting said cells with a SHH signal transduction activator (Sonic hedgehog) and a wingless signal transduction activator (Wnt ), and the contact of these cells with the Wnt signal transduction activator is carried out in the period from the third (3) to the eleventh (11) day after the primary contact of the cells with the SMAD signal transduction inhibitor to obtain a cell population, more than 10% of the specified cell population expresses as a protein forkhead box A2 (FOXA2) and LIM homeobox containing transcription factor 1 alpha (LMX1A). 2. The method of claim 1, wherein said SMAD signaling comprises TGFβ/activin-nodal signaling. 3. The method of claim 1 or 2, wherein said SMAD signaling comprises bone morphogenesis protein (BMP) signaling. 4. The method of any one of claims 1 to 3, wherein at least 20% of said cell population expresses both the FOXA2 protein and LMX1A. 5. The method according to any one of claims 1 to 4, characterized in that the cells are brought into contact with the signal transduction activator of the SHH protein at least between 24 and 36 hours from the initial contact of the cells with the SMAD inhibitor. 6. A method according to any one of claims 1 to 5, characterized in that the cells are brought into contact with the wingless signal transduction activator (Wnt) at least between 24 and 36 hours from the initial contact of the cells with the SHH protein signal transduction activator. 7. The method according to any one of claims 1 to 6, characterized in that at least 40% of said cell population expresses FOXA2 and LMX1A protein. 8. The method according to any one of claims 1 to 7, characterized in that said pluripotent stem cells are selected from the group consisting of non-embryonic stem cells, embryonic stem cells, induced non-embryonic pluripotent stem cells and modified human pluripotent stem cells, a primate other than human or rodent. 9. Method according to any one of claims 1 to 8, characterized in that said population of cells expressing FOXA2 and LMX1A additionally expresses at least one marker selected from the group consisting of tyrosine hydroxylase (TH), orthodenticle homeobox 2 (OTX2) , nuclear receptor-related protein 1 (NURR1), neuron-specific beta-tubulin class III (Tuj1), trefoil family factor 3 (TTF3), homeobox protein 3 containing a paired domain-like 3 (PITX3), achaete-scute complex (ASCL ), B-cell early factor 1 (EBF-1), B-cell early factor 3 (EBF-3), transthyretin (TTR), synapsin, dopamine transporter (DAT), G-coupled internal rectifying potassium channel (Kir3.2/GIRK2 ), CD142, DCSM1, CD63 and CD99. 10. A method according to any one of claims 1 to 9, wherein said pluripotent stem cells differentiate into said population of cells within 11 days of initial contact of said cells with at least one SMAD signaling inhibitor. 11. The method according to any of claims 1 to 10, characterized in that the pluripotent stem cells differentiate into dopamine neurons no later than 25 days after the initial contact of these cells with the SMAD signaling inhibitor. 12. The method of claim 11, wherein at least 20% of said dopamine neurons express FOXA2 and LMX1A and additionally express at least one further human midbrain dopamine neuron marker subtype A9 selected from the group consisting of Girk2, CD142 and DCSM1. 13. The method according to claim 11 or 12, further comprising selecting a population of dopamine neurons expressing CD142 or DCSM1, or both markers. 14. The method according to any one of claims 1 to 13, characterized in that said cell population is characterized by a significantly low level of expression of at least one marker selected from the group consisting of PAX6, EMX2 and LHX2. - 119 041083 15. The method according to any one of claims 1 to 14, further comprising contacting said population of cells with brain-derived neurotrophic factor (BDNF), ascorbic acid (AA), glial cell neurotrophic factor (GDNF), dibutyryl cAMP (dbcAMP), and transforming growth factor type β3 (TGFe3) to produce dopamine neurons. 16. The method of any one of claims 1-15, further comprising contacting said population of cells with an FGF signaling activator. 17. The method of claim 16 wherein said FGF signal activator is FGF8. 18. The method according to claim 16 or 17, characterized in that said cells are contacted with said FGF signal transduction activator for up to 144 hours from the initial contact of said cells with the SMAD signal transduction inhibitor. 19. The method of any one of claims 1-18, wherein said SMAD signal inhibitor is selected from the group consisting of LDN-193189, SB431542, Noggin, dorsomorphine, and combinations of the foregoing. 20. The method according to any one of claims 1 to 19, characterized in that said SMAD signal inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H- imidazol-2-yl]benzamide (SB431542). 21. The method of any one of claims 1-20, wherein the SHH signal transduction activator is selected from the group consisting of SHHs, Smoothened agonists (SAGs), and combinations of the foregoing. 22. The method of any one of claims 1-21, wherein the SHH signaling activator is a Smoothened (SAG) agonist. 23. The method according to claim 21 or 22, wherein said SAG is purmorphamine. 24. The method of claim 21, wherein said SHH is a recombinant SHH protein. 25. The method of claim 24, wherein said recombinant SHH protein is at least 95% identical to the N-terminal fragment of mouse SHH. 26. The method according to claim 24 or 25, wherein said recombinant SHH protein is SHH C25II. 27. A method according to any one of claims 1 to 26, wherein said wingless signaling activator (Wnt) is a compound capable of reducing glycogen synthase 3beta 3β (GSK3e) signaling. 28. The method of any one of claims 1-26, wherein said Wnt signal transduction activator is selected from the group consisting of CHIR99021, Wnt3A, Wnt1, and combinations of the above. 29. A method according to any of claims 1 to 28, wherein said Wnt signal transduction activator is CHIR99021. 30. A method according to any one of claims 1 to 29, characterized in that said cells are contacted with two inhibitors of SMAD signaling, which are SB431542 and LDN-193189, and the wingless signal activator (Wnt) is CHIR99021. 31. The method of any one of claims 1 to 30, further comprising transfecting said pluripotent stem cells with a polysialyl transferase (PST) prior to contacting said pluripotent stem cells with an inhibitor of SMAD signaling. 32. The method of any one of claims 1-30, further comprising treating said population of cells with exogenous PST.