WO2016019366A1 - Caractérisation et différenciation de lignées de cellules souches humaines - Google Patents

Caractérisation et différenciation de lignées de cellules souches humaines Download PDF

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WO2016019366A1
WO2016019366A1 PCT/US2015/043343 US2015043343W WO2016019366A1 WO 2016019366 A1 WO2016019366 A1 WO 2016019366A1 US 2015043343 W US2015043343 W US 2015043343W WO 2016019366 A1 WO2016019366 A1 WO 2016019366A1
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cells
cell
neural
medium
ectodermal
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Julie V. HARNESS
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Harness Julie V
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present teachings relate to novel methods, for differentiating pluripotent stem cells into ectodermal cells, as well as compositions, kits and assays implementing the novel methods and/or ectodermal cells generated from these novel methods.
  • Stem cells are the building blocks of all cells of the human body, and stem cells can differentiate into many different mature cell types. Stem cells have the capacity to differentiate, or turn into mature, specialized cells. There are many different types of stem cells, including pluripotent stem cells, which are limited to differentiating into the specific cell types of the tissues in which they reside.
  • Human pluripotent stem cells can differentiate into all of the more than 200 types of cells in the human body, can be expanded readily, and have many applications in the field of medicine.
  • Human pluripotent stem cells are either embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Both hESCs and iPSCs have the capacity to be maintained and expanded in an undifferentiated state.
  • hESCs Human embryonic stem cells
  • IVVF in vitro fertilization
  • hESCs Human embryonic stem cells have great potential to differentiate. They undergo tissue-restrictive differentiation. At one of the first diffemtiation points, hESCs differentiate into epiblasts. Epiblasts can then differentiate into the major tissues of the body, including neurons. In the next step, various growth factors and differentiation signals can induce the epiblasts to differentiate into ectodermal cells, such as neuroectodermal cells that are committed to developing into cells of the skin and nervous systems.
  • hPSC lines can also be obtained from individuals without the use of blastocysts. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to behave like hESCs by being forced to express genes necessary for maintaining the iPSCs.
  • iPSCs Induced pluripotent stem cells
  • Huntington ' s Disease is a neurodegenerative genetic disorder that results primarily in the loss of medium spiny projection neurons (MSN) of the striatum.
  • MSN medium spiny projection neurons
  • Current animal models do not sufficiently recapitulate the complex cascade of neurodegenerative events in the human.
  • Current cellular models are complicated by primary extraction and purification methods or are confounded by contaminant progenitor populations.
  • Currently available cell models and therapeutic biologies, such as human fetal ganglionic eminence cells are lacking either in quality or accessibility. In order to facilitate therapeutic relevance, cell models must have high fidelity to the actual human tissue of interest.
  • a method of differentiating pluripotent cells into ectodermal cells comprising the sequential steps of: a. incubating pluripotent cells in striatal medium (SM), wherein the SM medium contains growth factors and proteins associated with cell differentiation and wherein no more than 0-150 ng/ml of sonic hedgehog (SHH) can be added to the medium; and b. monitoring the pluripotent cells for markers associated with differentiated ectodermal cells selected from the group consisting of: transcription factors, RNA, proteins, and cell surface antigens.
  • SHH sonic hedgehog
  • optionally SHH can be added along with an inhibitor of SHH.
  • the differentiated ectodermal cells are selected from the group consisting of cells of the lateral ganglionic eminence (LGE), telencephalon, rhombencephalon, mesencephalon, prosencephalon, cells of the neurogenic placodes, adenohypophyseal placode, lens placode, nasal placode, epipharyngeal placode, trigeminal placode, otic placode, posterior pituitary, hair follicles, teeth, olfactory epithelium, melanocytes, mechanoreceptors, glomeruli, mitral cells, mouth epithelium, cranial sensory ganglia, cranial sensory nerves, spinal dorsal root ganglia, sensory spinal nerve roots, sympathetic ganglia, adrenal medulla, Neural tube, motor spinal nerve roots, dorsal root ganglia, ganglia of the autonomic nervous system, spinal cord and motor neurons in it, optic ve
  • a method of differentiating pluripotent cells into ectodermal cells comprising the sequential steps of: a. incubating pluripotent cells in striatal medium (SM), wherein the SM medium contains growth factors and proteins associated with cell differentiation and wherein no more than 0-150 ng/mi of sonic hedgehog (SHH ) can be added to the medium; and b. monitoring the cells for sequential expression of GSH2, FoxPl and DARPP32, wherein the sequential expression can be confirmation of differentiated ectodermal cells.
  • SHH can be added along with an inhibitor of SHH.
  • a method of differentiating pluripotent cells into ectodermal cells comprising the sequential steps of: a. providing and incubating a plurality of ceils in a first medium, wherein the plurality of cells can be a plurality of substantially undifferentiated pluripotent cells; b. incubating the ceils in a second medium which can be free or essentially free of at least one bone rnorphogenie protein (BMP); c. incubating the ceils in a third medium comprising Noggin in an amount sufficient to promote differentiation in a plurality of the cells; and d.
  • BMP bone rnorphogenie protein
  • BDNF brain-derived neurotrophic factor
  • differentiating pluripotent cells express at least one or more of said markers GSH2, DLX2, FoxPl, FoxP2, DLX2, CTIP2, GAB A, calbindin and DARPP-32 during the course of maturation; and wherein no more than 0-150 ng/mi of sonic hedgehog (SHH) can be added to any one of th e m edia in the method of differentiating pl uripotent cells into ectodermal cel ls; and wherein a plurality of differentiating pluripotent cells differentiate into ectodermal cells.
  • SHH sonic hedgehog
  • BDNF can be gradually increased up to about day 45.
  • no more than 0-75 ng/ ' ml, no more than 0-50ng/ml, no more than 25ng/ ' ml and no SHH can be added to the medium.
  • the plurality of differentiating pluripotent cells yield at least 65% to greater than 92% mature ectodermal ceils.
  • the differentiated ectodermal cells exhibit at least one characteristic selected from the group consisting of: the cells are no longer proliferating, the cells have completed migration to their final location of residence, the cells have stopped dividing, and the cells have a functional connection to cell types that medium spiny neurons could connect to if grown in vivo within an organism.
  • the cells in the second medium are incubated in derivation medium for at least 3 days.
  • the cells in the first medium are cultured in conditioned medium (CM) for at least 3 days.
  • the undifferentiated cells display immunocytochemical staining for markers of pluripotency comprising at least one or more of Oct4, SSEA3, SSEA4 and Tra 1 -81.
  • the derivation media comprises at least one of at least 5ng/ml-150ng/ml Dickkopf-related protein (D K), and 5ng/ml- 150ng/ml Brain-derived neurotrophic factor (BDNF) and the ceils can be cultured from day 10 to upwards of day 130.
  • D K Dickkopf-related protein
  • BDNF Brain-derived neurotrophic factor
  • the differentiated ectodermal cells are differentiated neural cells or epidermal cells. In some embodiments, the differentiated ectodermal cells are differentiated neural cells that can be at least one of LGE, medium spiny neurons (MSN) and striatal-specific differentiated cells. In some embodiments, the
  • differentiated ectodermal cells express GSH2 by day 19.
  • the differentiated ectodermal cells express GSH2 by day 19.
  • differentiated ectodermal cells express at least one or more markers of MSN differentiation selected from the group consisting of: DARPP-32, Tuj-1 , Map2, Ctip2, GABA, calretinin and calbindin at about day 66 and the markers of MSN differentiation identify the ceils as mature MSN cells.
  • the pluripotent cells are selected from the group consisting of a non-diseased cell, a diseased ceil, a non-diseased person or a person who can be at risk for development of a neurological disease and wherein the pluripotent cells are derived from the group consisting of: induced pluripotent stem cells and neural progenitor ceils.
  • the diseased cells have a symptom of disease characterized by polyglutamine repeats that can be at least 30, at least 40, at lest 50, at least 60, at least 70 and at least 80 repeats.
  • the neurological disease is selected from the group consisting of an autosomal dominant, autosomal recessive, X-lmked dominant, X-linked recessive, and spontaneous gene mutations.
  • the diseased cells can be selected from the group consisting of HD (Huntington's disease), PD (Parkinson ' s Disease), LRRK2-associated PD, DRPLA (Dentatorubropallidoluysian atrophy), SBMA (Spinobulbar muscular atrophy or Kennedy disease), SCA2 (Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA17 (Spinocerebellar ataxia Type 17), SCAT (Spinocerebellar ataxia Type 1), and SCAT (Spinocerebellar ataxia Type 7).
  • the person at risk for development of a neurological disease is selected from a person who is: a genetic disease carrier for a neurological disease, exposed to a toxic environmental substance or event, a traumatic brain injury patient, a person with an infection, a person with an autoimmune disease, suffering from an inflammation, and a cancer patient.
  • the person who can be at risk for development of a neurological disease donated said cells and has a predisposition to a disease having a symptom characterized by at least one of delirium, dementia, autonomic dysfunction, tremor, neuropsychiatric problems, Autism, Autism spectrum disorder, Schizophrenia, sensor and sleep difficulties, bradykinesia, and cognitive disturbances.
  • a differentiated ectodermal cell population produced by a method comprising: a. providing and incubating a plurality of cells in a first medium, wherein the plurality of cells comprise pluripotent cells; b. incubating the ceils in a second medium which can be free or essentially free of at least one bone morphogenic protein (BMP); c. incubating the cells in a third medium comprising Noggin in an amount sufficient to promote differentiation in a plurality of the cells; and d.
  • BMP bone morphogenic protein
  • BDNF brain-derived neurotrophic factor
  • Si H i sonic hedgehog
  • the SHH can be added to any of said media on or after day 7 to day 24 during said incubations.
  • SHH can be added in conjunction with an SHH inhibitor to any one of the media.
  • SHH can be added in an amount of at least 0-150 ng/ml to any of said media.
  • an inhibitor of SHH can be added in an amount sufficient to inhibit SHH activity.
  • a method for treating a subject having a neural disease comprising: a. administering to a subject a therapeutically effective amount of the neural cells prepared by the disclosed methods, and b. determining whether the neural cells positively impacts the severity of the neural disease in the subject, wherein impact can be determined by clinical presentation of the subject.
  • the subject can be a mammal.
  • the subject can be a primate.
  • the subject can be a human.
  • a method of screening a compound for an effect on a neural cell comprising: a. contacting a cell culture with a compound, wherein the cell culture comprises neural cells prepared by the disclosed methods, and b. determining any phenotypic, expression, functional or metabolic changes in the neural cells that result from contact with the compound thereby determining the effect of the compound on the neural cell.
  • a method of screening a compound for an effect on a neural cell comprising: a. contacting a cell culture with a compound, wherein the cell culture comprises cells chosen from neural cells prepared by the disclosed methods and express at least one of DARPP-32, Ctip2, calretinin and calbindin, wherein the neuronal cells expressing at least one of DARPP-32, Ctip2, calretinin and cal bindin are progeny of a cell culture comprising cells expressing SSEA3, SSEA4, and Tra 1-81; and b.
  • the cells are MSN cells.
  • a method of selecting or optimizing a method of treating a subject with a neural disease comprising: a. administering to the subject a treatment comprising a therapeutically effective amount of the neural ceils prepared by the disclosed methods and b. assessing an improvement and/or a side effect caused by administering the neural cell treatment for the neural disease in the subject, wherein impact can be determined by clinical presentation of the subject.
  • the subject can be a mammal.
  • the subject can be a primate.
  • the subject can be a human.
  • a method for identifying an agent for treating a neural disease in a subject comprising: a. administering an agent to a cell culture, wherein the cell culture comprises neural cells prepared by the disclosed methods, and b. determining any phenotypic, expression, functional or metabolic changes in the neural cells that result from contact with the agent thereby determining the effect of the agent on a neural cell.
  • the subject can be a mammal.
  • the subject can be a primate.
  • the subject can be a human.
  • an assay for evaluating a therapeutic treatment comprising: a. exposing cells prepared by the disclosed methods to a therapeutic agent; and b. evaluating the cells for an affect selected from the group consisting of cell death, cell toxicity, cell function, cell expression and cell respiration and cell signaling.
  • the evaluation is selected from the group consisting of a survival assay, a functional assay, an expression assay, a metabolic assay and an oxidative stress assay.
  • a method for an in vitro model of LR K2-associated Parkinson ' s disease comprising: a. co-culturing MSNs cells prepared by the disclosed methods and dopaminerginc neurons; and b. measuring functional connectivity with MEAs; or c .measuring cell death; or d. detect expression of at least one marker selected from the group consisting of caspases and lactate dehydrogenase.
  • the dopaminerginc neurons are either mutant or wildtype dopaminerginc neurons.
  • the MSNs are either mutant or wildtype MSNs.
  • the mutant MSNs express mutant LRRK2.
  • a method for high throughput screening of a neurologic factor a. co-culturing MSN cells prepared by the disclosed methods and neuronal cells isolated from a patient with a neurological disease or an addiction disorder; b. exposing the co-culture to a factor; and c. measuring readout.
  • the neurological disease is selected from the group consisting of Huntington ' s Disease, Parkinson ' s Disease, Levodopa- unresponsive Parkinsonism, Multiple System Atrophy with Parkinsonism (LSA-P), Alzheimer's Disease, Dementia with Lewy bodies, Spinocerebellar ataxia ' Type 3, Schizophrenia.
  • the addiction disorder is selected from the group consisting of opiate abuse, cocaine abuse, alcohol abuse and metharnphetamme abuse.
  • the factor is selected from the group consisting of a pharmaceutical, a chemotherapeutic, a therapeutic, a small molecule, a biologic, an antibody, a hybrid antibody, an antibody fragment, a siRNA, an antisense RNA, an aptamer, a protein, or a peptide.
  • readout is selected from one or more assays of the group consisting of: cell death, cell viability, cellular chemistry, cellular function, mitochondrial function, cell aggregation, cell morphology, cellular protein aggregation, gene expression, cellular secretion, metabolism, or cellular uptake.
  • a differentiated ectodermal cell population produced by a method comprising: a. providing and incubating a plurality of cells in a first medium, wherein the plurality of cells can be a plurality of substantially undifferentiated pluripotent cells; b. incubating the cells in a second medium which can be free or essentially free of at least one bone morphogenic protein (BMP); c. incubating the cells in a third medium comprising Noggin in an amount sufficient to promote differentiation in a plurality of the cells; and d.
  • BMP bone morphogenic protein
  • incubating the cells in at least a fourth medium comprising increasing the amount of brain-derived neurotrophic factor (BDNF) gradually up to about day 45, w ierein no more than 0-200 ng/ml of sonic hedgehog (SHH) can be added to any one of the media in the method of differentiating pluripotent cells into differentiated ectodermal cells; and wherein a plurality of the pluripotent cells differentiate into differentiated ectodermal cells.
  • BDNF brain-derived neurotrophic factor
  • SHH sonic hedgehog
  • the media can be a factor selected from the group consisting of forskolin, retinoic acid, retinaldehyde dehydrogenase 3 (RALDH3), or modulation of glutamate and GAB A receptors.
  • the factor can be activin A.
  • the differentiated ectodermal cells are selected from calcineurin positive MSN population, neuroectodermal cells, Deep Short-Axon Cells (dSAC), GL-dSACs, GCL-dSACs, Somatostatin cells, basket cells , chandelier cells, and calbindin cells.
  • kits comprising a first, second, third and fourth containers, a. said first container including therein a derivation medium formulation for incubating induced pluripotent stem cells; b. said second container including therein a differentiation medium formulation which can be free or essentially free of at least one bone morphogenic protein (BMP) for culturing induced pluripotent stem cells without substantial differentiation; c. said third container including therein a differentiation medium formulation comprising Noggin in an amount sufficient to promote differentiation in a plurality of the incubating induced pluripotent stem cells; and d.
  • BMP bone morphogenic protein
  • said fourth container including therein a maturation medium for incubating differentiated induced pluripotent stem cells; wherein no more than 0-150 ng/ml of sonic hedgehog (SHH) can be added to any one of the media during said incubations; and wherein a plurality of the differentiating induced pluripotent stem cells differentiate into differentiated ectodermal cells.
  • the media supplements can include one or more of vasoactive intestinal peptide (VIP), fibroblastic growth factor (FGF), bovine FGF ( ⁇ -FGF), Noggin, sonic hedgehog (SHH), DK , BDNF, collagenase IV, and TrpLE, and optionally, directions can also be included.
  • the media supplement can be activin A.
  • the medium formulation in the first, second, third and fourth containers can be a liquid.
  • the media formulation in the first, second, third and fourth containers can be a freeze-dried particulate that can be
  • the medium formulation in the first, second, third and fourth containers is sterile.
  • the first, second, third and fourth containers are suitable for including therein a volume of media of about 100-250 ml, 250-500 ml, or 500-1000 ml.
  • FIG. 1 depicts microscopic photographs of cells with HD following thawing at various stages of development.
  • FIG. 1 A HD blastocysts and morulae at various stages of development immediately following thaw
  • FIG. IB Expanded SuBi-HDl blastocyst after hatching, day 2 after thaw
  • FIG. 1C Adherent inner cell mass and trophoblast cells on matrigel day 5 after thaw
  • FIG. ID Adherent inner cell mass on day 7 after thaw
  • FIG. IE Exapanded inner cell mass on day 11 after thaw
  • FIG. IF SuBi-HDl at passage 6
  • FIG. 1G FISH testing for trisomy 12 and 17 (arrows)
  • FIG. 1H Karyotype analysis
  • FIG. II PCR assay for CAG repeats in the Huntingtin gene.
  • FIG. 2 depicts microscopic photographs of stage-specific marker expression. MSN derivates of the SuBi-HDl line (FIG. 2D-I) and SuBil line (FIG. 2A, 2C, 2J-2L). Pooled samples (FIG.2B).
  • FIG. 2A Quantification rings overlaid upon a DARPP-32
  • FIG. 2B Histogram of stage-specific marker expression quantification
  • FIG. 2C Western blot for DARPP-32 expression
  • FIG. 2D Merged image corresponding with FIG. 2E and 2F
  • FIG. 2E Hoechst and FIG. 2F: FITC for GSH2 expression at day 19 of differentiation
  • FIG. 2G Merged image corresponding with FIG. 2H and FIG. 21, 2H: Hoechst and 21: FITC for FoxPl expression at day 45 of differentiation
  • FIG. 2J Merged image corresponding with FIG. 2K and 2L
  • Scale bars represent ⁇ (FIG. A) and 50 ⁇ (FIG. 2D-2L).
  • FIG. 3A-C cells in early stage of differentiation express high levels of GSH2 and nestin, low levels of FoxPl and low levels of Map2, day 25.
  • FIG. 3D- 3H cells in middle stage of differentiation express high levels of FoxPl, moderate levels of Map2 and nestin, and virtually no DARPP-32, day 42 and 48, respectively.
  • FIG. 3I-3K cells in final maturation stage of differentiation express low levels of FoxPl and nestin, and high levels of Tuj l, Map2, DARPP-32 and Calbindin, day 66. Scale bars represent ⁇ .
  • FIG. 4 depicts an electrophysiology study of SuBi-HDl derivates at day 138 of differentiation.
  • FIG.4A-C and 4E-G DARPP-32 immunocytochemistry after electrophysiology
  • FIG. 4A is a merged image showing colocalization of the markers in FIG. 4B and 4C.
  • FIG. 4E is a merged image showing colocalization of the markers in FIG. 4F and 4G.
  • FIG. 4B, 4F :
  • FIG. 4C, 4G Biocytin labeled with streptavidin 594.
  • FIG. 4D Quantification of spontaneous post synaptic currents when in ACSF and in NBQX/APV to block glutamatergic inputs.
  • FIG. 4H Trace of spontaneous activity recorded from the cell presented in FIG. 4A-D.
  • Figure 5 depicts cross sections of rat brain after cell transplantation. 5A:
  • Fluorojade C labeling to identify striatal lesion 5B: Ku80 and 5C: DARPP-32 co-labeling to verify survival of the transplanted cells.
  • Figure 6 illustrates media supplements during differentiation of MSN ' s. All panels: nuclei stained with pale methyl green, GSH2 positive nuclei are stained with DAB (dark brown). 6A: Negative Control, purplish-greenish color are nuclei, brown is positive GSH2 staining. If there is brown on top of purplish-greenish, that means there is more GSH2, a marker of differentiation to an MSN cell fate. 6B, 6D: MSN ' s differentiated in the presence of SHH, 6C: Media supplements for cells of Fig. 6B, including SHH, 6E: Media supplements for cells of Fig.
  • FIG. 6D including SSH and Noggin
  • 6F, 6H MSN's differentiated in the absence of SHH, but in presence of Noggin
  • FIG. 6G Media supplements for cells of Fig. 6F, including Noggin but without SHH
  • FIG. 61 Media supplements for cells of Fig. 6H, including Noggin but without SHH.
  • the present teachings can be implemented for the differentiation and maturation of ectodermal and epidermal cell lineages.
  • Examples of where mature ectodermal and epidermal derived cell lineages are useful include the derivation of disease-relevant cell populations enables drug discovery [15,16].
  • the isolation of hepatocytes from cadaveric liver led to the development of hepatotoxicity screening, which in turn became a widely used method of determining drug toxicity and metabolism, enabling clinical trial [17,18].
  • drug screening burgeoned after identification of CHO cells transduced with genes of interest for cancer research, and will do so again after derivation of cardiomyocytes from hESCs [19,20,21].
  • MSNs medium spiny neurons
  • HD Huntington ' s Disease
  • chemotherapeutic agents and research into the effects of neurological disease on surrounding tissues and organs.
  • animal reagent free' can refer to cells not grown on animal cells and not utilizing any animal proteins, e.g., a serum replacement can be an example of a reagent lacking any animal protein compared to fetal bovine serum.
  • the term ' derivation' can refer to creation of a cell population from a different starting population.
  • the starting population is most likely to be the cells of an inner cell mass, a single pluripotent cell (in the case of a clonal line) or a differentiated cell type (e.g. fibroblasts before induction of pluripotency by introduction of reprogramming factors).
  • the starting population is most typically a line of pluripotent cells.
  • ' differentiation and ' differentiated , and ' differentiate , are used interchangeably and can refer to a continuum of the developmental timeline of a cell. Generally this can be starting in the undifferentiated, pluripotent state and continuing forward through the point of pluripotency toward a more committed fate.
  • ectodermal celF can refer to any cell of the central nervous system or the epidermis.
  • tissues comprised of ectodermal cells include but are not limited to: telencephalon, lateral ganglionic eminence (LGE), rhombencephalon, mesencephalon, prosencephalon, cells of the neurogenic placodes, adenohypophyseal placode, lens placode, nasal placode, epipharyngeal placode, trigeminal placode, otic placode, posterior pituitary, hair follicles, teeth, olfactory epithelium, melanocytes, mechanoreceptors, glomeruli, mitral cells, mouth epithelium, cranial sensory ganglia, cranial sensory nerves, spinal dorsal root ganglia, sensory spinal nerve roots, sympathetic ganglia, adrenal medulla, Neural tube, motor spinal nerve roots
  • FoxP 1 ' can refer to forkhead box protein 1.
  • FOXP2' can refer to forkhead box protein 2.
  • CTIP2 ' and ' BCL1 lb ' are used interchangeably and can refer to B-cell CLL/lymphoma 1 IB, a zinc finger protein (ZFP) and regulator of MSN differentiation.
  • ZFP zinc finger protein
  • Map2 can refer to microtubule-associated protein
  • Oct4' and POU5Fr are used interchangeably and can refer to octamer-binding transcription factor 4, also known as POU domain, class 5, transcription factor 1.
  • GABA ' can refer to gamma-aminobutyric acid.
  • ' SSEAS ' As used herein the acronyms ' SSEAS ' , ' SSEA4 , and ' SSEAS ⁇ are used interchangeably and can refer to cell surface glycosphingolipids 3 and 4, which are stage- specific embryonic antigens often complexed together.
  • Tra- 1 -8 ⁇ can refer to teratocarcinoma-related antigen 1-81.
  • incubating ' and ' Gultaring ' are used interchangeably and can refer to maintaining living cells at any temperature for a period of time.
  • the cells can reproduce, grow, reach maturity, remain static, or decline in number as a result of cell death.
  • Incubating includes, but is not limited to, a temperature from about 0°C to 4°C, 1°C to about 10°C, 10°C to about 37°C etc.
  • the term ' induction' can refer to stimulation of a cell population to begin changing to another cell population. For instance, the induction of pluripotent cells into a neural cell fate is brought about by a shift in media and/or growth factors.
  • kit can refer to any system for delivering materials.
  • such delivery systems can include elements allowing the storage, transport, or delivery of reaction components such as cells, tissue culture media, buffering components, additives, nutrients, antigens, antibodies and the like in the appropriate containers from one location to another commonly provided with written instructions for performing the assay.
  • Kits can include one or more enclosures or boxes containing the relevant reaction reagents and supporting materials.
  • the kit can comprise two or more separate containers wherein each of those containers includes a portion of the total kit components. The containers can be delivered to the intended recipient together or separately.
  • Mature cells can refer to cells of the ectodermal lineage that have grown to reach a developmental stage, i.e., terminally differentiated, where they are no longer proliferating. Most of these adult cells in the ectodermal lineage have completed migration to their final location of residence within an organism, they have stopped dividing and instead create a functional connection to other relevant cells. Mature cells can be evaluated based on assays such as marker expression, function, integration and non-division and so on.
  • the phrase ' medium spiny neuron cell' can refer to the predominant species of neuronal cells within the striatum, the subcortical part of the forebrain.
  • the terms ' multipotent cell' and ' progenitor' and ' progenitor cell' are used interchangeably and as used herein, can refer to a type of stem cell that is capable of giving rise to a limited number of cell types within the body of an organism, (e.g. a human body).
  • An example would be a neural progenitor cell that is capable of giving rise to cells of the neural lineage (e.g., neurons and glia), but not blood cells as blood cells are of mesoderm lineage.
  • the phrase ' neural induction ' can refer to the process of inducing undifferentiated ectoderm to form neural tissue in response to signals from the mesoderm during embryonic development or during culturing of pluripotent cells.
  • the term ' pluripotent cell' can be a type of stem cell that can be capable of giving rise to all cell types within an organism.
  • the term ' induced pluripotent stem celf can refer to a type of pluripotent stem cell that can be generated directly from adult cells, (i.e. cells that are differentiated into their mature cell fate).
  • Sonic hedgehog (SHH) ' in the novel methods described herein can be added in an amount no more than 0-150 ng/mf to any of said media during said incubations.
  • ' sonic hedgehog (SHH) ' in any of the methods described herein can be added in an amount no more than 0-100 ng/mf to any of the media during the incubations.
  • ' sonic hedgehog (SHH) ' in any of the methods described herein can be added in an amount no more than 0-75 ng/mf to any of the media during the incubations.
  • ' sonic hedgehog (SHH) ' in any of the methods described herein can be added in an amount no more than 0-50 ng/mf to any of the media during the incubations.
  • ' sonic hedgehog (SHH) ' in any of the methods described herein can be added in an amount no more than 0-35 ng/mf to any of the media during the incubations.
  • ' sonic hedgehog (SHH) ' in any of the methods described herein can be added in an amount no more than 0-10 ng/mf to any of the media during the incubations.
  • SHH sonic hedgehog
  • SHH can be capable of inducing a pluripotent cell to an ectodermal cell fate, from a neural pattern to a differentiated neural cell, e.g., a striatal cell, a medium spiny neuron cell, and so on.
  • the term ' sub-confluence ' can refer to a cell feeder- free system in which there can be about 85% coverage of cell growth within a cell flask.
  • the sub-confluent flask could be split as the next day it would be 100% confluent, cells fully covering the surface of the flask.
  • stem celf can refer to a biological cell that is not differentiated but can differentiate into any cell type specific to any tissue.
  • the stem cells can reproduce by mitosis to produce more stem cells.
  • ' striatum can refer to neural cells within the subcortical part of the forebrain referred to as the striatum.
  • the striatum can refer to neural cells within the subcortical part of the forebrain referred to as the striatum.
  • 90-95% of the striatum comprises spiny projection neurons, commonly referred to as medium spiny neurons (MSN).
  • MSN medium spiny neurons
  • the term ' substantially ' can refer to an amount that is less than 100% but can reflect a predominant amount, characteristic or type within a non-uniform composition.
  • tissue culture one could describe a cell population within a tissue culture flask is comprising >50% undifferientiated cells (udc ' s) and ⁇ 50% stromal cells (differentiate quickly and provide growth factors and support undifferentiated cells.
  • udc ' s undifferientiated cells
  • stromal cells differentiate quickly and provide growth factors and support undifferentiated cells.
  • Substantially can refer to any unit of measurement such as weight amount, a ratio, percentage, or any other unit of measurement that the term substantially is referring to.
  • Substantially can also refer to greater than 60% of an amount that is referred to.
  • Substantially can also refer to greater that 70% of an amount that is referred to. Substantially can also refer to greater than 80% of an amount that is referred to. Substantially can also refer to greater than 90%, of an amount that is referred to.
  • Substantially can also refer to greater than 93% of an amount that is referred to. Substantially can also refer to greater than 95% of an amount that is referred to. Substantially can also refer to greater than 97% of an amount that is referred to. Substantially can also refer to greater than 98% of an amount that is referred to. Substantially can also refer to greater than about 99% of an amount that is referred to. Substantially can also refer to 100% of an amount that is referred to.
  • the phrase ' substantially undifferentiated pluripotent cells in a media ' can refer to the percentage of pluripotent cells in said media and the percentage can be 50%) or more, 60%> or more, 70%> or more, 80%> or more, 90%> or more, 93% or more, 95% or more, 97% or more, 98%> or more, 99% or more, or 100%.
  • the phrase ' totipotent cells ' can refer to a fertilized egg, a zygotic cell or a blastomere comprising up to eight cells.
  • a totipotent stem cell is capable of giving rise to an entire living organism and all of the cell types that comprise the organism.
  • disclosed herein are methods, compositions and kits that provide a cellular model of HD that can be used for drug discovery, study of disease etiology and progression, predictive toxicology and transplantation.
  • Currently available cell models and therapeutic biologies such as human fetal ganglionic eminence cells, are lacking either in quality or accessibility [1 1 ,12, 13].
  • Direct blastocyst derivation of model lines for many genetic disorders, including HD is feasible due to the reliability and ethically justified process of pre- implantation genetic diagnosis.
  • the resulting hESC lines have high proliferative capability, enabling the large number of cells required for screening and exploratory studies.
  • Non- blastocyst, induced pluripotent cell populations are comparably amenable for use in all of the disclosed methods.
  • blastocysts were obtained, with consent, from a fertility clinic that used pre-implantation genetic diagnosis to identify HD-positive blastocysts.
  • the HD cell line used for deriving differentiated ectodermal cells carried 44 polyglutamine repeats, and did not show dramatic signs of pathology at early developmental stages. It is likely that pathology would increase with higher repeat numbers; this could be confirmed by investigating a number of such hESC lines, carrying a variety of CAG repeat lengths [14]. While others have found transcriptional differences between neural cells derived from diseased (between 40 and 51 CAG repeats) and normal lines [14], examination of what differences were most pronounced in MSNs themselves was evaluated.
  • the most prominent roadblock to utilizing differentiated ectodermal cells, is the inability to lead uncommitted neuronal cells to assume an MSN fate.
  • the differentiation protocol disclosed herein produces a high- purity MSN cell population with clinical and research utility.
  • non-blastocyst pluripotent stem cells or induced pluripotent stem cells can be obtained form a vendor such as Cellular Dynamics (Madison, WI), NINDS Human Genetics Repository at Coriell, or Science Exchange
  • a sample of cells e.g., fibroblasts, B-lymphocytes, or another cell type
  • a stem cell bank e.g., Coriell Institute for Medical Research, Camden, NJ, NINDS Human Genetics Repository at Coriell, or ATCC, Manassas, VA
  • a tissue or fluid sample can be obtained from a patient and then reprogram the cells isolated from the tissue or fluid sample into an iPS line.
  • the methods described herein further include inducing differentiation of a pluripotent cell line with or without a genetic variation of interest.
  • the genetic variation can be a trinucleotide repeat.
  • the repeat can be a CAG (poly glutamine) repeat.
  • CAG poly glutamine
  • HD Huntington ' s Disease
  • transcription factors, receptors and so on factors known to differentiate a pluripotent cell to a specific cell lineage
  • factors known to differentiate a pluripotent cell to a specific cell lineage direct cell differentiation to a desired lineage and/or cell population of interest.
  • the now terminally differentiated cells are characterized by the loss of indefinite growth when cultured in vitro, slowing growth and exhibition of properties related to the specialization of the cell fate the cell has assumed.
  • the pluripotent cells as described herein can be differentiated into any cell type including cells arising from the endoderm, mesoderm or ectoderm. Cells of the ectoderm lead to a differentiated cell fate which forms the nervous system and epidermis (skin). Mesoderm originating cells are capable of forming bone, connective tissue, dermis, muscle and blood. The endoderm provides cells capable of differentiating into the internal organs, including the digestive tract and glands, gut, intestines, respiratory system and bladder.
  • the cell population is differentiated into a
  • neuroectodermal neuronal, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic neuronal, parasympathetic neuronal, sympathetic peripheral neuronal, or glial cell population, such as a microglial
  • central nervous system astrocytes, oligodendrocytes, ependymal cells, radial glia
  • peripheral nervous system PNS
  • Schwann cells, satellite cells cell population, or the precursors of any of the preceding, including neural stem and progenitor cells.
  • pluripotent cells are ' induced ' (induced pluripotent stem cells, (iPSC)) from differentiated cells using viral vectors and techniques to genetically reprogram adult cells (post-mitotic) to have the capacity to develop into any cell type.
  • Sources of cells successfully induced to pluripotentcy include skin and foreskin cells.
  • medium spiny neurons derivate from pluripotent cells including, human embryonic stem cells (hESC), iPSC and neural progenitors.
  • iPSC ' s can be made for most any known cell type.
  • the use of multipotent cells, e.g. from cord blood
  • an antagonist of SHH in an amount sufficient to inhibit some or all SHH activity is included.
  • the disclosed methods provide more mature MSN cells for drug discovery, screening of neurological factors, including but not limited to a pharmaceutical, a chemotherapeutic, a therapeutic, a small molecule, a biologic, an antibody, a hybrid antibody, an antibody fragment, a siRNA, an antisense RNA, an aptamer, a protein, or a peptide, treating a subject having a neural disease, predictive toxicology, therapeutic screening, screening potential pharmaceuticals, evaluation of toxicity of chemotherapeutic agents, cell therapy via transplantation, study of disease etiology and progression, and research into the effects of neurological disease on surrounding tissues and organs
  • cryopreservation straws For thawing and subsequent culture, straws were individually removed from liquid nitrogen, held in the air for 30 seconds, then immersed in a 37°C water bath for 45 seconds. Straw ends were cut and contents expelled into an empty dish. Biological materials were immediately transferred by mouth pipet to balanced 37°C 0.5M sucrose in 75% Quinn ' s modified Human Tubal Fluid medium (mHTF) (Irvine Scientific, Irvine, CA) and 25% Serum Substitute Supplement (SSS) (Irvine Scientific). After 10 minutes, materials were transferred to balanced 37°C 0.2M sucrose in 75% mHTF, 25% SSS.
  • mHTF Human Tubal Fluid medium
  • SSS Serum Substitute Supplement
  • Derivation medium consisted of KO-DMEM F12, 1% B27, lng/ml T3, 25 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin and lOng/ml hyaluronic acid (HLA) supplemented with lOng/ml vasoactive intestinal peptide (VIP) and 50ng/ml FGF (FIG. 1 A). After 24 hours, cells were transferred to derivation medium and FGF was decreased to 20ng/ml.
  • CM consisted of KO-DMEM-F12 (Invitrogen) with 20% KOSR, 1% non-essential amino acids, 0.5% GlutaMAX-I (all from Life Sciences Inc.) and 70 ⁇ 1/1 ⁇ -mercaptoethanol (Sigma-Aldrich).
  • This medium was supplemented with 4ng/ml ⁇ -FGF (GF003-AF, Millipore) and conditioned on irradiated mouse embryonic fibroblasts (MEFs) overnight, then filtered in a 0.1 ⁇ PES filter (Fisher). Hatched blastocysts were monitored daily for attachment to the substrate and presence of trophoblast cells (FIG. 1C). Any trophoblast cells that could be targeted independently of the inner cell mass were destroyed with a microscope-mounted XYClone laser ablator (Hamilton Thorne, Beverly, MA).
  • Trophoblast cells close to inner cell mass were laser ablated while those at a distance simply migrated away (FIG. ID).
  • Adherent inner cell masses were allowed to form the first colony and ablation of trophoblast cells continued for approximately 10 days. Ablated trophoblast cell were no longer active (FIG. IE).
  • SuBi-HDl was characterized by immunocytochemistry for markers of pluripotency, fluorescent in situ hybridization for trisomies 12 and 17 (FIG. 1G), short tandem repeat (STR) profile, karyotype analysis (FIG.1H) and genotype for the mutant Huntingtin gene at passage 2, 6 and 12 using standard protocols (FIG. II).
  • Undifferentiated cells were grown on matrigel-coated flasks to sub-confluence in a feeder-free system. Medium was then transitioned from conditioned medium to a DMEM-F12 based striatal medium (SM) for neural induction. Transition to striatal differentiation was conducted by feeding the cells 100% CM on day 0, day 1, cells were subconfiuent and fed 60% CM, 40% DMEM-F12, day 2 cells were fed 30% CM, 70% DMEM-F12, day 3 cells were fed 100$ DMEM-F12 based striatal medium. However, if it was observed that the cells were growing very fast, then there was only one day of transition at 50/50 CM/SM and then day 2 was 100% striatal medium.
  • SM DMEM-F12 based striatal medium
  • Striatal medium consisted of DMEM-F12, 2% B27, 1% ITS (insulin, transferrin, selenium) and 0.5% GlutaMAX (all Invitrogen).
  • Medium was supplemented with 4ng/ml ⁇ -FGF (Chemicon) and lOOng/ml Noggin (R&D Systems). On day 11, Noggin concentration was dropped to 50ng/ml. Cells were fed daily until day 12, at which point they were fed every other day for the duration of the protocol. On day 18, medium was further supplemented with lOOng/ml DK and 20ng/ml BDNF (R&D Systems).
  • FIG. 2A illustrates quantification rings overlaid upon a DARPP-32 immunocytochemistry image.
  • FIG. 2B depicts a histogram of stage-specific marker expression quantification.
  • FIG. 2C confirms DARPP-32 expression at later stages of differentiation by Western blot. Differentiation was repeated an additional 4 times, with comparable results.
  • Undifferentiated cultures of SuBil (derived previously in the Keirstead Research Group) cells were grown in conditioned medium before striatal differentiation was induced. Undifferentiated cells were grown on matrigel-coated flasks to sub-confluence in a feeder-free system. Medium was then transitioned from conditioned medium to a DMEM-F12 based striatal medium (1/2 SM) plus lOmg/ml ⁇ -Fibroblast growth factor (F10) for neural induction (Day 1). Striatal medium consisted of DMEM-F10, 2% B27, 1% ITS (insulin, transferrin, selenium) and 0.5% GlutaMAX (all Invitrogen).
  • Flasks 4 and 5 on day 5 were supplemented with only 50ng/ml Noggin (R&D Systems). On day 7, F10 was reduced to F4. Cells were fed daily until day 15. On day 15, cells were dissociated with collagenase IV (Invitrogen) for 60 minutes, rinsed in HBSS (Invitrogen), and suspended in fresh medium and the cells transferred onto covers lips. On day 17, the cells were fixed and stained for the MSN differentiation marker GSH2. As shown in Figure 6D and 6E, the majority of the cells stain more intense and uniform GSH2 staining. This duplicate flask study established that SHH was not essential to the differentiation and maturation of MSNs.
  • Chamber slides were fixed at day 19, 25, 42, 45, 48, 60 and day 66 for stage- specific immunocytochemistry. NPC cultures were washed in PBS and fixed in 4%
  • GSH2 was quantified at day 19, FoxPl at day 45 and DARPP-32 at day 66 (FIG. 2D-F, 2G-I, and 2J-L, respectively, merged images, Hoechst and FITC). Nuclear markers were expressed as percentage positive nuclei of total nuclei labeled with Hoechst. Because the final maturation stage required colony morphology, DARPP-32-expressing cells were quantified using a radial ring technique (Fig. 2A). The high-density center of each colony was excluded from quantification and a circle was drawn at the limit of quantifiable area.
  • the cells were lysed on ice in chilled RIP A lysis buffer (Millipore) and supplemented with protease inhibitor cocktails (Roche Applied Science).
  • the cell extracts were centrifuged at 14,000 g for 15 minutes at 4°C. Supernatants were collected after centrifugation. Protein content was quantified using the Bradford assay (Sigma-Aldrich) according to the manufacturer ' s guidelines.
  • the protein samples (50 mg per lane) were electrophoretically separated by SDS-PAGE in 4 " 20% gradient gel (PAGEgel, Inc.). After electrophoresis, proteins were transferred to a Hybond-P polyvinylidene difluoride (PVDF) membrane (GE Healthcare Biosciences).
  • PVDF Hybond-P polyvinylidene difluoride
  • Membranes were blocked for 2 hours in 5% nonfat milk in PBS-0.05% Tween-20 and incubated 2 hours in 5% nonfat milk containing primary antibodies: DARPP-32 (1 :2000, Santa Cruz Biotechnology, Inc.) or Beta-Actin (1 :5000, Millipore). Membranes were washed in PBS-0.05% Tween-20, and then incubated for 1 hour at room temperature with secondary HRP- conjugated antibody goat anti-rabbit (1 : 1000, Millipore). After the membranes were washed, the proteins were visualized using Enhanced Chemiluminescence Plus Western blotting detection system (GE Healthcare Biosciences) according to the manufacturer ' s protocol. Protein bands were detected by exposure to autoradiographic film (Amersham Hyperfilm ECL, GE Healthcare Biosciences).
  • Spontaneous postsynaptic current (sPSC) frequencies were determined from 30 second-long data segments using the MiniAnalysis program (Synatptosoft) with visual inspection of detected events.
  • Drugs were obtained from Tocris bioscience, dissolved in ACSF and bath-applied at the following concentrations: 10 ⁇ D-APV, 5 ⁇ NBQX, 5 ⁇ gabazine (SR95531), ⁇ tetrodotoxin (TTX).
  • Isogenic cell lines have almost the exact same genotype, the difference is the gene of interest.
  • a non-HD skin line vs. HD patient skin line has a mutant htt gene, the htt gene is genetically modify to make a normal and a diseased line from the same person.
  • CAT repeat addition can be accomplished using a CRISPR modification to make isogenic cells (An MC, [28]).
  • the cells are phenotypically characterized using cell death assay. Exposing the cell cultures to stressful conditions induces cell death. Stressful conditions include but are not limited to oxidative stress, glutamatergic cytotoxicity- glutamate is exciting, excited to the point of cell death), all of which can induce cell death. Cell death can be measured by Caspase activity, as is known to one of skill in the art.
  • Cell death can be quantitated in cultures with normal, moderate and high CAG repeat numbers using the Apo3 HTS kit (Cell Technology) for Caspase 3/7 activity, according to the manufacturer ' s directions.
  • the Caspase 3/7 activity isone factor active when cells are going through apoptosis.
  • An increase in 3/7 activity is indicative of more cell death while less 3/7 activity correlates with less death in both normal culture conditions and when cells in culture are challenged with a stressor.
  • a rudimentary survival readout such as LDH assay can be done to access stressor challenge, as is known to one of skill in the art.
  • ME A multi-electrode array
  • Cells differentiated on an MEA are conducive to taking electrophysiological recordings from the cells at multiple time-points along the maturation timeline and after or during administration of agents or conditioned media.
  • the MEA chip has a grid and on each node is an electrode such that as cells grow on the electrode they are constantly read for months and observation as to how the cells react to a drug or stress over time or how the MSN cells are resistant to a stressor is measured by each celf s Electrophysiology Output with respect to growth conditions.
  • Cells are grown in sufficient quantity to do mass spectrometry on multiple conditions (e.g., starting population with different genotypes, or cells with different ages, or grow cells for different amounts of time, e.g., grow cells under a stressful condition and then add a drug to rescue the cells from the stressful condition.
  • Use MS to measure what cells are doing electrophysiology, examining the protein expression profile and compare the protein response by the cells under a stress condition. The results can identify differences in protein level and expression of in cells with normal and expanded CAG repeats in response to altered growth conditions or administration of agents.
  • normal cells can respond to a stressor by making a protein in response to the stressful condition whereas HD cells can produce a different metabolite (protein) in response to the same stressor.
  • the advantage to understanding protein expression to a stressor by comparing a normal and an HD cell under the same stress conditions can lead to the identification of proteins lacking in an HD cell or proteins over expressed in the HD cell vs. normal cell and so identify potential a protein(s) as: a target to commercialize; a protein to exploit for treatment mechanism(s).
  • Co-culturing MSNs and dopaminergic neurons can be done in order to develop an in vitro model for LR K2-associated Parkinson ' s disease.
  • Mutant LR K2 expressed in MSNs is thought to result in DAergic cell death and has resulted in identifying one of the few known genetic causes of Parkinson ' s Disease (PD).
  • the co-culturing of mutant LRRK2 expressed in MSNs and WT MSNs with mutant and WT DAergic neurons is performed.
  • Each cell type is measured for functional connectivity with MEAs, cell death in the two populations and expression of relevant markers. This assay is valued by researchers testing drugs to rescue DAergic neurons, researchers investigating the etiology of DAergic neuronal death, or for small molecule screening to discover new targets or compounds relevant to PD.
  • a high-throughput screening format all of the above assays can be used to identify factors that may decrease or intensify readout.
  • Readout can be measured by a: cell death assay- e.g., Placing healthy cells next to diseased cells and then applying a stress factor, the HD cells die more, but once a potential therapeutic agent is applied the readout changes. The HD cells go back to the same cell death readout as normal cells, the untreated, have a low cell death rate.
  • Levodopa-unresponsive Parkinsonism Levodopa-unresponsive Parkinsonism, Multiple System Atrophy with Parkinsonism (LSA-P), Alzheimer ' s Disease, Dementia with Lewy bodies (second most prevalent form of dementia after Alzheimer's), Spinocerebellar ataxia ' Type 3, Schizophrenia.
  • addiction disorders including but not limited to: Opiate abuse, Cocaine abuse, Alcohol abuse,
  • Sprague-Dawley rats were anesthetized with 2-4% isofluorane gas and monitored for level of anesthesia for the duration of the procedure.
  • the animal was placed on a sterile surface for procedure in a stereotactic apparatus. Ear bars were positioned so that animal was held level and stable.
  • a 1.5 cm long midline incision exposing the skull was made using a scalpel.
  • the injection needle was oriented precisely on Bregma and Bregma coordinates were recorded. Needle was raised and moved laterally according to anterior and lateral coordinates. Coordinates were anterior +0.3, lateral +2.7, ventral -4.7. The needle was moved aside and a 1- 2mm diameter hole was drilled through the skull at the coordinate location, without damaging the dura.
  • Injection needle was returned to the coordinate location and lowered through the dura into the brain over a period of 30 seconds. Once in place, needle was not moved for 2 minutes, then ⁇ of 15nM QA (quinolinic acid) was injected over a 4 minute period. After injection, needle was left in place for an additional 2 minutes, then withdrawn over a period of 30 seconds. Animals were then sutured, allowed to recover and returned to home cages for two weeks. On the 12 th day after QA lesion, animals began receiving lOmg/kg Cyclosporine A in preparation for the second surgical procedure.
  • 15nM QA quinolinic acid
  • mice Two weeks after QA lesion, animals were again anesthetized, and surgical procedures were repeated, except the injection consisted of either vehicle control or a 2 ⁇ 1 cell transplant of 50,000 medium spiny neuron progenitors (made from the SuBil line) at Day 45 of the differentiation protocol. Eight weeks after cellular transplant, animals were euthanized and perfused with 4% paraformaldehyde (PFA). Brains were extracted and processed for immunohistochemistry.
  • PFA paraformaldehyde
  • primary antisera mouse anti-Nestin, 1 :200, (Chemicon); rabbit anti-FoxPl, 1 :200, (Cell Signaling Technology); rabbit anti-DARPP- 32, 1 :500, (Santa Cruz Biotech); rabbit anti-Map2, 1 :200, (Chemicon); rabbit anti-Calbindin, 1 :2000, (Swant) were visualized using AlexaFluor conjugated antisera (goat anti-mouse IgG (H+L) AlexaFluor 594, goat anti-rabbit IgG (H+L) AlexaFluor 488, (Invitrogen). Images were captured using a Nikon Eclipse Ti microscope with 10X and 20X objectives, equipped with a Nikon Digital Sight DS-Fil camera and a Zeiss LSM 780 confocal microscope.
  • Example I Method of differentiation with adding sonic hedgehog (SHH)
  • Undifferentiated cultures of SuBil (derived previously in the Keirstead Research Group) cells were grown in conditioned medium before striatal differentiation was induced. Undifferentiated cells were grown on matrigel-coated flasks to sub-confluence in a feeder-free system. Medium was then transitioned from conditioned medium to a DMEM-F12 based striatal medium (SM) (1/2 SM) plus lOmg/ml ⁇ -Fibroblast growth factor (F10) for neural induction (Day 1). Striatal medium consisted of DMEM-F10, 2% B27, 1% ITS (insulin, transferrin, selenium) and 0.5% GlutaMAX (all Invitrogen).
  • SM DMEM-F12 based striatal medium
  • F10 lOmg/ml
  • F10 lOmg/ml ⁇ -Fibroblast growth factor
  • FIG. 6A there is no darkly stained cells for GSH2, 6A is the negative control.
  • FIG. 6B and 6C differentiated in the presence of SHH and Noggin produced only at best about 60% mature MSN- GSH2 staining cells.
  • Example II Method of differentiation with not adding sonic hedgehog (SHH)
  • Flasks 4 and 5 on day 5 were supplemented with only 50ng/ml Noggin (R&D Systems) and they were not supplemented with sonic hedgehog.
  • F10 was reduced to F4.
  • Cells were fed daily until day 15.
  • cells were dissociated with collagenase IV (Invitrogen) for 60 minutes, rinsed in HBSS (Invitrogen), and suspended in fresh medium and the cells transferred onto coverslips
  • the cells were fixed and stained for the MSN differentiation marker GSH2.
  • the majority of the cells stain more intense and uniform GSH2 staining. This result indicates that the differentiated cells from Flasks 3 and 4, that were not given SHH, were of much higher purity than the differentiated cells from from Flasks 2 and 3 that were given SHH.
  • SuBi-HDl displayed immunocytochemical staining for markers of pluripotency SSEA3/4 and Tral81, normal FISH pattern for chromosome 12 and 17 (Fig. 1G, arrows), a novel STR profile, normal karyotype (Fig. 1H) and a heterozygous HD genotype (Fig. II).
  • the line carries one copy of Htt with 22 CAG repeats and one with 44 CAG repeats, making it a heterozygous disease line.
  • sEPSCs spontaneous excitatory PSCs
  • Example VII Fluorojade labeling of control brains revealed that the QA injection produced a lesion with its epicenter in the left striatum and volume nearly filling the striatum (Fig. 5a). Human cells present in the rat striatum expressed Ku80 (Fig. 5b). A subset of these cells also expressed DARPP-32 (Fig. 5c). The Ku80 and DARPP-32 co-labeling confirmed that transplanted cells survived and were capable of maturing in vivo two months posttransplantation.
  • Induced hPSs can be obtained from several sources. Induced hPSCs can be purchase an iPS line from a vendor such as Cells Direct or Science Exchange. Alternatively, induced hPSCs can be obtained from a sample of cells (fibroblasts, B-lymphocytes, or another cell type) from a stem cell bank such as Coriell or ATCC, then perform reprogramming to make them into an iPS line, or one can obtain a tissue/fluid sample from a patient, then reprogram the cells into an iPS line. Methods of reprogramming cells are well known in the art, and an example of such a method is described in Nature Reviews Genetics 12, 231-242 (April 2011), the contents of which are incorporated by reference in its entirety.

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Abstract

La présente invention concerne un procédé de différenciation de cellules pluripotentes en cellules ectodermiques différenciées, telles que des cellules de neurones épineux moyens (NEM) matures, produites par l'incubation de cellules pluripotentes dans un milieu strié (MS), le milieu MS contenant des facteurs de croissance et des protéines associées à la différenciation cellulaire et pas plus de 0 à 150 ng/ml de sonic hedgehog (SHH) ne pouvant être ajoutés au milieu, et par la surveillance des cellules en ce qui concerne l'expression d'au moins un marqueur sélectionné parmi GSH2, FoxP1 et DARPP32. L'invention concerne en outre des procédés d'utilisation des cellules de NEM dans le traitement d'une maladie neurologique et le criblage d'agents thérapeutiques.
PCT/US2015/043343 2014-08-01 2015-08-01 Caractérisation et différenciation de lignées de cellules souches humaines WO2016019366A1 (fr)

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RU2623646C1 (ru) * 2016-08-18 2017-06-28 Федеральное государственное автономное учреждение "Межотраслевой научно-технический комплекс "Микрохирургия глаза" имени академика С.Н. Федорова" Министерства здравоохранения Российской Федерации Способ лечения глаукомной оптической нейропатии посредством трансплантации 3D-клеточной культуры мультипотентных мезенхимальных стволовых клеток лимба

Non-Patent Citations (52)

* Cited by examiner, † Cited by third party
Title
"Generation of Human Induced Pluripotent Stem Cells (hiPSCs) from Fibroblasts using Episomal Vectors", LIFE TECHNOLOGIES, January 2014 (2014-01-01)
"Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS
"Oligonucleotides and Analogs: A Practical Approach", 1991, OXFORD UNIVERSITY PRESS
A. D. CARRI ET AL: "Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons", DEVELOPMENT, vol. 140, no. 2, 18 December 2012 (2012-12-18), GB, pages 301 - 312, XP055225847, ISSN: 0950-1991, DOI: 10.1242/dev.084608 *
ALBERTS; SINGER: "Developmental Biology", 2006, SINAUER ASSOCIATES INC.
AMY E. REDDINGTON ET AL: "Microtransplantation of whole ganglionic eminence cells ameliorates motor deficit, enlarges the volume of grafts and prolongs survival in a rat model of Huntingtonâ€(TM)s disease", FRONTIERS IN CELLULAR NEUROSCIENCE, vol. 91, 2 December 2014 (2014-12-02), pages 1563, XP055225810, DOI: 10.1002/jnr.23282 *
AN MC; O'BRIEN RN; ZHANG N; PATRA BN; DE LA CRUZ M; RAY A; ELLERBY LM: "PLOS Currents Huntington Disease", 15 April 2014, article "Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System"
AN MC; ZHANG N; SCOTT G; MONTORO D; WITTKOP T ET AL.: "Genetic correction of Huntington's Disease phenotypes in induced pluripotent stem cells", CELL STEM CELL, vol. 11, no. 2, 3 August 2012 (2012-08-03), pages 254 - 63
ANDERSON, SA.: "Determination of Cell Fate within the Telencephalon", CHEM. SENSES, vol. 27, no. 6, 2002, pages 573 - 575
ARBER, C; PRECIOUS SV; CAMBRAY S; RISNER-JANICZEK JR; KELLY C; NOAKES Z; FJODOROVA M; HEUER A; UNGLESS MA; RODRIGUEZ TA: "Activin A directs striatal projection neuron differentiation of human pluripotent stem cells", DEVELOPMENT, vol. 142, 2015, pages 1375 - 1386
AUBRY L; BUGI A; LEFORT N; ROUSSEAU F; PESCHANSKI M ET AL.: "Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in Quinolinic acid-lesioned rats", PROC NATL ACAD SCI U S A, vol. 105, 2008, pages 16707 - 16712
BACHOUD-LEVI AC; GAURA V; BRUGIERES P; LEFAUCHEUR JP; BOISSE MF ET AL.: "Effect of fetal neural transplants in patients with Huntington's disease 6 years after surgery: a long-term follow-up study", LANCET NEUROL., vol. 5, no. 4, 2006, pages 303 - 9
BEAL MF; KOWALL NW; ELLISON DW; MAZUREK MF; SWARTZ KJ ET AL.: "Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid", NATURE, vol. 321, 1986, pages 168 - 171
CARTER RL; CHAN AW: "Pluripotent Stem Cells Models for Huntington's Disease: Prospects and Challenges", J GENET GENOMICS, vol. 39, no. 6, 20 December 2011 (2011-12-20), pages 253 - 9
DICK E; RAJAMOHAN D; RONKSLEY J; DENNING C.: "Evaluating the utility of cardiomyocytes from human pluripotent stem cells for drug screening", BIOCHEM SOC TRANS., vol. 38, no. 4, August 2010 (2010-08-01), pages 1037 - 45
EUNJU SHIN ET AL: "GABAergic Neurons from Mouse Embryonic Stem Cells Possess Functional Properties of Striatal Neurons In Vitro, and Develop into Striatal Neurons In Vivo in a Mouse Model of Huntington's Disease", STEM CELL REVIEWS AND REPORTS, HUMANA PRESS INC, NEW YORK, vol. 8, no. 2, 1 July 2011 (2011-07-01), pages 513 - 531, XP035063420, ISSN: 1558-6804, DOI: 10.1007/S12015-011-9290-2 *
EYRE, MD; ANTAL, M; NUSSER, Z.: "Distiinct deep short-axon cell subtypes of the main olfactory bulb provide novel intra- and extrabulbar GABAergic connections", J. NEUROSCI, vol. 28, no. 33, 2008, pages 8217 - 8229
FEYEUX M; BOURGOIS-ROCHA F; REDFERN A; GILES P; LEFORT N ET AL.: "Early transcriptional changes linked to naturally occurring Huntington's disease mutations in neural derivatives of human embryonic stem cells", HUM MOL GENET., vol. 21, no. 17, 1 September 2012 (2012-09-01), pages 3883 - 95
GABRIEL NISTOR MMS; STEPHANE N. POIRIER; SHARYN ROSSI; ALEKSANDRA J. POOLE MEC; JOHN D. MCNEISH; CHRIS N. AIRRIESS; KEIRSTEAD HS: "Derivation of High Purity Neuronal Progenitors from Human Embryonic Stem Cells", PLOS ONE, 2011
GALLINA P; PAGANINI M; LOMBARDINI L; MASCALCHI M; PORFIRIO B ET AL.: "Human striatal neuroblasts develop and build a striatal-like structure into the brain of Huntington's disease patients after transplantation", EXP NEUROL., vol. 222, no. 1, 2010, pages 30 - 41
GONZALEZ,F. ET AL.: "Methods for Making Induced Pluripotent Stem Cells: Reprogramming A La Carte", NATURE REV. GEN., vol. 12, 2011, pages 231 - 242
GRONDIN R; KAYTOR MD; AI Y; NELSON PT; THAKKER DR ET AL.: "Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum", BRAIN, vol. 135, no. 4, April 2012 (2012-04-01), pages 1197 - 209
GRSKOVIC M; JAVAHERIAN A; STRULOVICI B; DALEY GQ: "Induced pluripotent stem cells opportunities for disease modelling and drug discovery", NAT REV DRUG DISCOV., vol. 10, no. 12, 11 November 2011 (2011-11-11), pages 915 - 29
HARNESS JV; TUROVETS NA; SEILER MJ; NISTOR G; ALTUN G ET AL.: "Equivalence of conventionally-derived and parthenote-derived human embryonic stem cells", PLOS ONE, vol. 6, 2011, pages E14499
IRENE FARAVELLI ET AL: "Motor neuron derivation from human embryonic and induced pluripotent stem cells: experimental approaches and clinical perspectives", STEM CELL RESEARCH & THERAPY, BIOMED CENTRAL LTD, LONDON, UK, vol. 5, no. 4, 14 July 2014 (2014-07-14), pages 87, XP021208904, ISSN: 1757-6512, DOI: 10.1186/SCRT476 *
JENSEN J; HYLLNER J; BJORQUIST P.: "Human embryonic stem cell technologies and drug discovery", J CELL PHYSIOL., vol. 219, no. 3, June 2009 (2009-06-01), pages 513 - 9
KAWAGUCHI, Y; KUBOTA, Y: "GABAergic cell subtypes ad thei synaptic connctions in rat frontal cortex", CEREBRAL CORTEX, vol. 7, September 1997 (1997-09-01), pages 476 - 486
KORNBERG; BAKER: "DNA Replication", 1992, W.H. FREEMAN
KRAUSHAAR U; MEYER T; HESS D; GEPSTEIN L; MUMMERY CL ET AL.: "Cardiac safety pharmacology: from human ether-a-gogo related gene channel block towards induced pluripotent stem cell based disease models", EXPERT OPIN DRUF SAF., vol. 11, no. 2, March 2012 (2012-03-01), pages 285 - 98
L. AUBRY ET AL: "Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 43, 28 October 2008 (2008-10-28), US, pages 16707 - 16712, XP055225840, ISSN: 0027-8424, DOI: 10.1073/pnas.0808488105 *
LEHNINGER: "Biochemistry", 1975, WORTH PUBLISHERS
MA ET AL.: "Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells", PLOS CURR, vol. 2, 2010, pages RRN1193
MA ET AL.: "Human Embryonic Stem Cell-Derived GABA Neurons Correct Locomotion Deficits in Quinolinic Acid-Lesioned Mice", CELL STEM CELL, 2012
MA, L.; HU, B.; LIU, Y; VERMILYEA, SC; LIU, H; GAO, L; SUN, Y; ZHANG, X; ZHANG, S-C: "Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice", CELL STEM CELL, vol. 10, April 2012 (2012-04-01), pages 1 - 10
MACDONALD ME; AMBROSE CM; DUYAO MP; MYERS RH; LIN C ET AL.: "Cell", vol. 72, 1993, THE HUNTINGTON'S DISEASE COLLABORATIVE RESEARCH GROUP, article "A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes", pages: 971 - 983
MARIA IDELSON ET AL: "Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells", CELL STEM CELL, vol. 5, no. 4, 1 October 2009 (2009-10-01), pages 396 - 408, XP055046648, ISSN: 1934-5909, DOI: 10.1016/j.stem.2009.07.002 *
MATTHEW F. BURKHARDT ET AL: "A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells", MOLECULAR AND CELLULAR NEUROSCIENCES., vol. 56, 1 September 2013 (2013-09-01), US, pages 355 - 364, XP055218774, ISSN: 1044-7431, DOI: 10.1016/j.mcn.2013.07.007 *
MENALLED L; EL-KHODOR BF; PATRY M; SUAREZ-FARINAS M; ORENSTEIN SJ ET AL.: "Systematic behavioral evaluation of Huntington's disease transgenic and knock-in mouse models", NEUROBIOL DIS, vol. 35, 2009, pages 319 - 336
NATURE REVIEWS GENETICS, vol. 12, April 2011 (2011-04-01), pages 231 - 242
OZER J; RATNER M; SHAW M; BAILEY W; SHOMAKER S: "The current state of serum biomarkers of hepatotoxicity", TOXICOLOGY, vol. 245, no. 3, 20 March 2008 (2008-03-20), pages 194 - 205
PASCHAKI, M; CAMMAS, L; MUTA, Y; MATSUOKA,Y; MAK, S-S; RATAJ-BANIOWSKA, M; FRAULOB, V; DOLLE, P; LADHER, RK: "Retinoic acid regulates olfactory progenitor cell fate and differentiation", NEURAL DEV., vol. 8, no. 13, 2013, pages 1 - 10, Retrieved from the Internet <URL:http://www.neuraldevelopment.com/content/8/1/13>
PLOS CURR., vol. 2, 28 October 2010 (2010-10-28), pages RRN1193
RAJAMOHAN D; MATSA E; KALRA S; CRUTCHLEY J; PATEL A ET AL.: "Current status of drug screening and disease modeling in human pluripotent stem cells", BIOESSAYS, 8 August 2012 (2012-08-08)
RASBAND, W.S.: "ImageJ", 1997, U. S. NATIONAL INSTITUTES OF HEALTH
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SCHWARCZ R; GUIDETTI P; SATHYASAIKUMAR KV; MUCHOWSKI PJ: "Of mice, rats and men: Revisiting the quinolinic acid hypothesis of Huntington's disease", PROG NEUROBIOL, vol. 90, 2010, pages 230 - 245
SHIBA Y; HAUCH KD; LAFLAMME MA: "Cardiac applications for human pluripotent stem cells", CURR PHARM DES., vol. 15, no. 24, 2009, pages 2791 - 806
SILVA A; PEREIRA J; OLIVEIRA CR; RELVAS JB; REGO AC: "BDNF and extracellular matrix regulate differentiation of mice neurosphere-derived cells into a GABAergic neuronal phenotype", J NEUROSCI RES, vol. 87, 2009, pages 1986 - 1996
STEM CELL REV AND REP
URNOV FD; REBAR EJ; HOLMES MC; ZHANG HS; GREGORY PD: "Genome editing with engineered zinc finger nucleases", NAT REV GENET., vol. L 1, no. 9, September 2010 (2010-09-01), pages 636 - 46
VINKEN M; VANHAECKE T; ROGIERS V.: "Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis?", TOXICOL IN VITRO, vol. 26, no. 3, April 2012 (2012-04-01), pages 541 - 4
XUE-JUN LI ET AL: "Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules", STEM CELLS, vol. 26, no. 4, 1 April 2008 (2008-04-01), pages 886 - 893, XP055177240, ISSN: 1066-5099, DOI: 10.1634/stemcells.2007-0620 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2623646C1 (ru) * 2016-08-18 2017-06-28 Федеральное государственное автономное учреждение "Межотраслевой научно-технический комплекс "Микрохирургия глаза" имени академика С.Н. Федорова" Министерства здравоохранения Российской Федерации Способ лечения глаукомной оптической нейропатии посредством трансплантации 3D-клеточной культуры мультипотентных мезенхимальных стволовых клеток лимба

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