WO2005021720A2 - Procede de differentiation in vitro de cellules souches neuronales, de neurones moteurs et de neurones de dopamine provenant de cellules souches embryonnaires de primates - Google Patents

Procede de differentiation in vitro de cellules souches neuronales, de neurones moteurs et de neurones de dopamine provenant de cellules souches embryonnaires de primates Download PDF

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WO2005021720A2
WO2005021720A2 PCT/US2004/027841 US2004027841W WO2005021720A2 WO 2005021720 A2 WO2005021720 A2 WO 2005021720A2 US 2004027841 W US2004027841 W US 2004027841W WO 2005021720 A2 WO2005021720 A2 WO 2005021720A2
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cells
neurons
population
neural
cell
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WO2005021720A3 (fr
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Su-Chun Zhang
James A. Thomson
Ian David Duncan
Xue-Jun Li
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Wisconsin Alumni Research Foundation
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Priority to CA2536588A priority Critical patent/CA2536588C/fr
Priority to JP2006524872A priority patent/JP2007503811A/ja
Priority to GB0605851A priority patent/GB2421029B/en
Priority to AU2004269361A priority patent/AU2004269361B2/en
Priority to EP04782339A priority patent/EP1670901A4/fr
Publication of WO2005021720A2 publication Critical patent/WO2005021720A2/fr
Publication of WO2005021720A3 publication Critical patent/WO2005021720A3/fr
Priority to IL173832A priority patent/IL173832A/en
Priority to IL198450A priority patent/IL198450A/en

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Definitions

  • Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of pre-implantation embryos (Thomson, J. A., et al., Science 282:1145-1147, 1998). Similar to mouse ES cells, they can be expanded to large numbers while maintaining their potential to differentiate into various somatic cell types of all three germ layers (Thomson, J. A., et al., supra, 1998; Reubinoff, B.E., et al.. Nat. Biotech. 18:399. 2000; Thomson, J.A. and Odorico, J.S.. Trends Biotech 18:53-57, 2000; Amit, M., et al., De ⁇ BjoL 227:271-278, 2000).
  • mice ES cells have been found to differentiate in vitro to many clinically relevant cell types, including hematopoietic cells (Wiles, M.V. and Keller, G., Development 111 :259-267. 1991), cardiomyocytes (Klug, M.G., et al.. J. Clin. Invest. 98:216-224. 1996), insulin-secreting cells (Soria, B., et aj., Diabetes 49:157-162. 2000), and neurons and glia (Bain, G.. et al.. Dev. Biol.
  • the present invention is a method of creating a population of cells comprising a synchronous population of cells cultured from embryonic stem cells which are characterized by an early rosette morphology and are Sox1 " , Pax6 + .
  • the method comprises the steps of: (a) propagating embryonic stem cells into embryoid bodies and (b) propagating embryoid bodies into a synchronous population of neural stem cells in the form of neural tube-like rosettes, wherein this propagation is in the presence of FGF2,
  • the total time period between the propagation of embryonic stem cells to development of early rosettes is preferably 8 - 10 days.
  • the total population of Pax6 + /Sox1 " cells is at least 70% of the total cell population.
  • the present invention is also a population of cells created by this method.
  • the invention is a method of creating a population of synchronized neural stem cells wherein the cells are characterized by a neural tube-like rosette morphology and are Pax6 + /Sox1 + , the method comprising the step of culturing cells that are characterized by an early rosette morphology and are Sox1 " , Pax6 + for 4-6 days in the presence of FGF2, FGF4, FGF8, or RA.
  • the invention is also a population of cells created by this method.
  • the early rosette cells were cultured with FGF8, preferably for 4 - 7 days, and are EN1 + .
  • the cells were cultured with FGF2, preferably for 4 - 7 days, and are Bf1 + .
  • the cells were cultured with RA, preferably for 4 - 7 days, and are Hox + .
  • the invention is a method of isolating a population of midbrain dopamine neurons, comprising the step of culturing the cells described above in the presence of FGF8 with SHH, wherein the resulting cells express TH, AADC, EN-1 , VMAT2 and DAT, but do not express DbH and PNMT.
  • the invention is also a population of cells created by this method.
  • the invention is a method of isolating a population of spinal motor neurons comprising the step of culturing the cells above described above in the presence of RA with SHH, wherein the resulting cells express HB9, HoxB1 , HoxB6, HoxC5, HoxC ⁇ , ChAT and VAChT.
  • the invention is also a population of cells created by this method.
  • the present invention is a method of isolating a population of forebrain dopamine neurons comprising the step of culturing the cells described above with SHH.
  • the invention is also a population of cells created by this method.
  • the present invention is also a method of testing the cell populations described above to screen agents for the ability to affect normal human neural development.
  • FIG. 1A-I Differentiation and isolation of neural precursors from ES cells.
  • FIG. 1 A An attached EB grown in the presence of FGF2 for five days shows flattened cells at the periphery and small elongated cells congregated in the center.
  • Fig. 1 B By seven days, many rosette formations (arrows) appeared in the
  • the upper-right inset is the 1- ⁇ m section of the rosette
  • rosette center
  • Fig. 1C Musashi-1
  • Fig. 1 D Musashi-1
  • Fig. 1 D Musashi-1
  • Fig. 1 E A combined image of Fig. 1C and Fig. 1 D with all cell nuclei labeled with DAPI.
  • Fig. 1 F After treatment with dispase for 20 minutes, the rosette formations retracted whereas the surrounding flat cells remained attached.
  • Fig. 1G-I Isolated cells are positively stained for nestin in a filamentous pattern (Fig. 1G), Musashi-1 in cytoplasm (Fig. 1 H), and PSA-NCAM
  • Fig. 2A-G Characterization of ES cell-derived neural precursors in vitro.
  • Fig. 2B Differentiation of a cluster of ES cell-derived neural precursors for three weeks shows neurite bundles with cells migrating along them.
  • FIG. 2C Immunostaining after three weeks of differentiation indicates that the majority of cells are ⁇ m-tubulin + neurons (red) and that only a few cells are GFAP + astrocytes (green).
  • FIG. 2D After forty-five days of differentiation, many more GFAP + astrocytes (green) appear along with NF200 + neurites (red, yellowish due to overlapping with green GFAP).
  • FIG. 3A-K Incorporation and differentiation of ES cell-derived neural precursors in vivo. Grafted cells are detected by in situ hybridization with a probe to the human a/t/-repeat element (Fig. 3A-E, G) or an antibody to a human-specific nuclear antigen (Fig. 3F).
  • Fig. 3A Individual donor cells in the host cortex of an eight-week-old recipient (arrows).
  • Fig. 3B Extensive incorporation of ES cell- derived neural precursors in the hippocampal formation. Cells hybridized with the human alu probe are labeled with red dots (pseudo-colored).
  • FIG. 3C Incorporated human cells in the vicinity of the hippocampal pyramidal layer at P14.
  • FIG. 3D ES cell-derived cells in the septum of a four-week-old recipient mouse.
  • FIG. 3E High power view of an individual donor cell in the hypothalamus. Note the seamless integration between adjacent unlabeled host cells.
  • Fig. 3F Donor cells in the striatum of a four-week-old host, detected with an antibody to a human-specific nuclear antigen.
  • FIG. 3G Extensive migration of transplanted cells from the aqueduct into the dorsal midbrain.
  • FIG. 3H Human ES cell-derived neuron in the cortex of a two-week-old host, exhibiting a polar morphology and long processes. The cell is double labeled with antibodies to a human-specific nuclear marker
  • Fig. 3J Donor-derived multipolar neuron, double labeled with an antibody recognizing the a and b isoforms of MAP2.
  • Fig. 3K ES cell-derived astrocyte in the cortex of a four- week-old animal, double labeled with the human-specific nuclear marker (green) and an antibody to GFAP (red). Note that all the double labelings are confocal images
  • Fig. 3C Fig. 3D 100 ⁇ m
  • Fig. 3E Fig. 3F
  • Fig. 4 Generation and regional specification of neuroectodermal cells.
  • Fig. 4A Columnar cells appeared in the differentiating ES cell colony at day nine in the presence of 20 ng/ml of FGF2.
  • Fig. 4B The columnar cells formed neural tubelike rosettes at day fourteen.
  • Fig. 4C The cells in the rosettes with columnar morphology were positive for Sox1 (red).
  • Fig. 4D The neural rosette cells in FGF2 treated cultures expressed Bf1 (red), but not En-1 (green).
  • Fig. 4E The neural rosette cells in FGF2 treated cultures expressed Bf1 (red), but not En-1 (green).
  • En-1 (green) expression was observed in the nestin 4" (red) neuroectodermal cells that were treated by six days with fibroblast growth factor 8 (FGF8) (100 ng/ml) at day nine, expanded in FGF8 for four days and then treated with sonic hedgehog (SHH) (200 ng/ml) for another six days on laminin substrate).
  • FGF8 fibroblast growth factor 8
  • SHH sonic hedgehog
  • Fig. 5A Differentiation of DA neurons.
  • Fig. 5B All TH + cells (red) in the culture were positively stained with a neuronal maker (green).
  • Fig. 5C-E All TH + cells (d, green) in the culture were positively stained with aromatic acid decarboxylase (AADC) (d and e, red), but some AADC + cells were TH " (e, arrowheads).
  • Fig. 5F Differentiation of DA neurons.
  • Fig. 5A About one third of the differentiated cells were tyrosine hydroxylase (TH) positive in the cultures that were treated with FGF8, SHH and ascorbic acid (AA) at three weeks of differentiation.
  • Fig. 5B All TH + cells (red) in the culture were positively stained with a neuronal maker (green).
  • Fig. 5C-E All TH + cells (d, green) in the culture were positively stained with aromatic acid decarboxylase (AADC) (d and e, red), but
  • TH + cells were negative for noradrenergic neuron marker dopamine /?-hydroxylase (D/?H) (green).
  • the inset indicated that D ?H positively stained cells in the section of adult rat brain stem.
  • Fig. 6A The differentiated DA neurons expressed genes characteristic of midbrain fate revealed by RT-PCR.
  • EB embryoid body
  • NE neuroectodermal cells
  • 3w the DA culture differentiated for three weeks
  • NC negative control.
  • Fig. 6B The majority of TH + cells (red) in the cultures expressed midbrain marker En-1 (green).
  • Fig. 6C GABA expressing cells (red) were present in the culture but very few TH + cells (green) co-expressed GABA (red, inset).
  • Fig. 7A-C All TH + cells (a, green) expressed c-Ret (red).
  • Fig. 7D-F. TH + cells (d, green) co-expressed VMAT2 (e and f; red).
  • Fig. 8. Functional characteristics of the in vitro generated DA neurons.
  • Fig. 8A Spontaneous and depolarization (56 mM KCI in HBSS)-induced DA release in the control and the treated cultures at three weeks of differentiation. Data were presented as means ⁇ SD from three experiments. *p ⁇ 0.05 vs. control by the unpaired student t test.
  • Fig. 8B Action potentials evoked by depolarizing current steps (0.2 nA) in two neurons differentiated for thirty days. Passive membrane properties: (i) V rest -49 mV, C m 15.5 pF, R m 5.0 G ⁇ ; (ii) V res t-72 mV, C m 45 pF, R m 885 G ⁇ .
  • Fig. 8C Passive membrane properties: (i) V rest -49 mV, C m 15.5 pF, R m 5.0 G ⁇ ; (ii) V res t-72 mV, C m 45 pF, R m 885 G ⁇ .
  • Fig. 8C
  • Fig. 9 Neuroectodermal cells induced by FGF2 display rostral phenotypes. ES cells, differentiated in FGF2 for ten days, displayed small, columnar morphology in the colony center, and organized into rosette formations. The columnar cells in the rosettes, but not the surrounding flat cells were positive for Pax6 and negative for Sox1 (A). By fourteen days, the columnar cells formed neural tube-like rosettes (B) and were positive for both Pax6 (C) and Sox1 (D).
  • Fig. 10 Generation of motor neurons from neuroectodermal cells.
  • FIG. 11 Effect of RA, FGF2 and SHH on neuroectodermal cells.
  • RT-PCR analyses indicated changes of rostrocaudal genes from early rosettes cells that were cultured with RA or 20 ng/ml of FGF2 for one week in the neural induction medium.
  • B Comparison of homeobox gene expression in early and late neuroectodermal cells treated with RA 0.1 ⁇ M for one week. The early neuroectodermal cells, treated with RA and then differentiated for twelve days, became mostly negative for Otx2 (C) but positive for HoxC8 (D). All the HoxC8 + cells were ?w-tubulin + (E). The Pax6-expressing neuroectodermal cells were negative for Olig2 (F).
  • FIG. 12 Maturation of motor neurons in culture. ChAT-expressing cells were localized mainly in the cluster (A), and were large multipolar cells (B). Confocal image showed co-localization of ChAT in the soma and processes and HB9 in the nuclei in a three-week culture (C). Most cells in the cluster expressed VChAT (D). Many ChAT + cells were also positive for synapsin in somas and processes after five weeks in culture (E). (F) AP's evoked by depolarizing current steps (0.15 nA) in neurons maintained for 42 DIV. Resting membrane potential (Vm) -59 mV (fi) and -70 mV (fii).
  • Fig. 13 Electrophysiological characterization of in vitro generated motoneurons.
  • A AP's evoked by depolarizing current steps (0.15 nA) in neurons maintained for 42 DIV. Resting membrane potential (Vm) -59 mV (ai) and -70 mV (aii).
  • B Spontaneous AP's in a neuron maintained for 42 DIV, Vm -50 mV.
  • C Spontaneous inward and outward synaptic currents at - 0 mV using K-gluconate- based pipette solution under control conditions (ci).
  • Applicants disclose a method of differentiating early rosettes (Pax6 + /Sox1 " ) from ES cells through an embryoid body intermediate. By differential treatment, Applicants can differentiate these early rosettes into three different forms of neural tube-like rosettes that are then suitable for development into forebrain dopamine neurons, midbrain dopamine neurons, or motor neurons.
  • FGF8 Neural Tube- SHH Midbrain * Like Rosettes
  • Dopamine 4-6d (Pax6+/Sox1+) Neurons En-1 + (midbrain)
  • Neural Tube- SHH Forebrain 4-d 4-6d (Pax6+/Sox1-) 4-6d Like Rosettes
  • Table 2 describes Phases 1 and 2 for generating dopamine and motor neurons. Table 2 also describes various intermediate products that Applicants consider to be markers of suitable development.
  • this invention includes two main embodiments.
  • One embodiment is the procedure for generating a synchronized population of neural stem cells (or neuroepithelial cells) in the form of neural tube-like rosettes and expression of neuroepithelial markers Pax ⁇ , Sox1 , nestin, Musashi-1.
  • synchronize means a population of cells that are at the same developmental stage, as opposed to those induced by RA which results in heterogeneous differentiation, i.e., the culture contains cells in developmental stages from progenitors to differentiated neurons.
  • the second embodiment is a method of further differentiation of the neuroepithelial cells to specialized neurons, such as midbrain dopamine neurons, forebrain dopamine neurons and spinal motor neurons.
  • Table 3 below describes a preferred method of obtaining cells of the present invention.
  • Table 3 includes both general culture broth components, that can be replaced by similar culture broths, and critical growth factor and timing components. When applicants refer to neural cell culture medium, many culture components are suitable. The sections below emphasize the culture components necessary for correct differentiation.
  • a suitable medium is any medium used for growing neural cells.
  • the following references (Bain, G., et al., supra. 1995; Okabe, S., et al., supra. 1996; Mujtaba, T., et al., supra. 1999; Housele, O., et J., supra. 1999; Zhang, S.-C, et aL, J. Neurosci. Res. 59:421-429, 2000; Zhang, S.-C, et a]., Proc. Natl. Acad. Sci. USA 96:4089-4094, 1999; Svendsen, C.N., et a]., Exp, Neurol.
  • neuroepithelial cells neural stem cells
  • the generation of neuroepithelial cells involves formation of embryoid bodies in suspension culture for 4-6 days, followed by adherent culture in the presence of growth factors, preferably FGF2 or FGF8, for 4-5 days when cells in the center of each colony become columnar and organize into a rosette form (Fig. 1 A, Fig. 4A, Fig. 9A, B).
  • FGF4 and FGF9 are also suitable growth factors.
  • the columnar cells in these rosettes express a neural transcription factor Pax6 but do not express another neural transcription factor Sox1 (Fig. 9C, D).
  • Sox1 a neural transcription factor
  • We call these rosettes "early rosettes” because they appear early and form by monolayer of columnar cells without a lumen. Every single colony possesses early rosettes. The total population of early rosette cells is at least 70% of the total cells.
  • Further culture of these early rosettes for 4-6 days will lead to formation of neural tube-like rosettes (Fig. 1 B, Fig. 4B, Fig. 9E).
  • the neural tube-like rosettes are formed by multiple layers of columnar cells with a clear lumen.
  • the cells in the rosettes express Sox1 in addition to Pax6 (Fig. 4C, Fig.
  • the functional significance of these cells is relevant to the present invention in that the Pax6+/Sox1- neuroepithelial cells in the early rosettes, but not the Pax6+/Sox1+ neuroepithelial cells in the neural tube-like rosettes, can be efficiently induced to become neurons carrying positional identities other than forebrain such as midbrain dopamine neurons and spinal motor neurons (Table 1 , see above).
  • the neuroepithelial cells represent at least 70-90% of the total differentiated cells.
  • the neuroepithelial cells in the form of neural tube-like rosettes can be purified through treatment with a low concentration of dispase and differential adhesion (described in U.S. Serial No. 09/960,382). 2. Generation of midbrain dopamine neurons
  • a functional neuron with potential therapeutic application must possess at least two additional characteristics in addition to being a neuron: a specific positional identity and the capacity to synthesize, release, and uptake a neural transmitter.
  • the first step in generating midbrain dopamine neurons is the induction of a midbrain identity.
  • Pax6 + /Sox1 + neural tube-like rosette cells results in efficient differentiation of the cells to progenitors that express midbrain transcription factors Engrailed 1 (En-1) and Pax 2 (Fig. 4E, F) and down regulation of forebrain marker Bf-1 (Fig. 4D).
  • the second step is to culture the midbrain progenitors in the presence of sonic hedgehog (SHH, 50-250 ng/ml) for 6-7 days, then in the regular neuronal differentiation medium (such as that described in Table 3) for additional 2 weeks until dopamine neurons develop.
  • SHH sonic hedgehog
  • the regular neuronal differentiation medium such as that described in Table 3
  • at least 35% of the total differentiated cells will become dopamine neurons.
  • a preferred differentiation medium is described in Table 3.
  • the dopamine neurons express TH, AADC, but not DbH and PNMT
  • the dopamine neurons express En-1 , ptx3, Nurrl , and Lmx1 b (Fig.
  • the dopamine neurons do not express GABA (Fig. 6C). Coexpression with GABA is the feature of dopamine neurons in the olfactory bulb. [0048] The dopamine neurons do not express calbindin (Fig. 6D).
  • Coexpression with calbindin is the feature of dopamine neurons in the tegamental area of the midbrain.
  • the dopamine neurons generated in our culture system are midbrain dopamine neurons, more closely resembling those in the substantial nigra, the dopamine neurons that are lost in Parkinson's disease.
  • the dopamine neurons also express VMAT2 (Fig. 7D, E, F), a transporter required for storage and release dopamine. They also express DAT (Fig. 7G, H, I), a transporter necessary for dopamine uptake after release.
  • VMAT2 Fig. 7D, E, F
  • DAT Fig. 7G, H, I
  • the dopamine neurons express synaptophysin (Fig. 7) for formation of synapses. They can fire action potentials and can secrete dopamine in response to stimulation (Fig. 8). Therefore, the dopamine neurons are functional.
  • the first step in generating spinal motor neurons is the induction of a spinal cord (caudal) identity.
  • Treatment of the Pax6+/Sox1- early rosette cells, but not the Pax6+/Sox1+ neural tube-like rosette cells (Fig. 10A), with RA (0.001-1 uM) for 6-7 days results in efficient differentiation of the cells to progenitors that express spinal cord transcription factor Hox genes such as HoxB1 , HoxB6, HoxC5, HoxC8, but not forebrain markers Otx2 and Bf-1 or midbrain marker En-1 (Fig.
  • the second step is to culture the spinal cord progenitors in the presence of sonic hedgehog (SHH, 50-250 ng/ml) for 6-7 days to induce a ventralized progenitor character, as evidenced by expression of Olig2, (Fig. 11 F, G,
  • a preferred differentiation medium is described in Table 3.
  • At least 22% of the total differentiated cells become spinal motor neurons.
  • the motor neurons express HB9, islet1/2, and Lim3
  • Fig. 10 transcription factors that are specifically expressed by spinal cord motor neurons.
  • the motor neurons also express HoxB1 , HoxB6, HoxC5, HoxC ⁇ , but not forebrain markers Otx2 and Bf-1 or midbrain marker En-1 (Fig. 11 A, C, D, E), indicating that they are spinal motor neurons.
  • the motor neurons express ChAT (Fig. 12A, ⁇ , C, D), an enzyme necessary for synthesizing the motor neuron transmitter acetylcholine.
  • the motor neurons also express VAChT (Fig. 12E), suggesting that the motor neuron can store and uptake the transmitter acetylcholine.
  • the motor neurons express synapsin (Fig. 12F) for formation of synapses. They can fire action potentials (Fig. 13). Therefore, the motor neurons are functional. We have data showing that the motor neurons release acetycholine, as analyzed by HPLC.
  • the present invention is a method of differentiating primate ES cells (preferably human ES cells) into forebrain dopamine neurons, preferably transplantable neural precursors suitable for nervous system repair.
  • primate ES cells preferably human ES cells
  • forebrain dopamine neurons preferably transplantable neural precursors suitable for nervous system repair.
  • the Pax6 + /Sox1 " cells are treated for an additional 4 - 6 days with FGF2 and are then treated with SHH.
  • the steps in generating forebrain dopamine neurons and the analyses for determining the dopamine neuron characters are similar to those described for midbrain dopamine neurons. The main difference is the use of morphogens at a particular period and the features of dopamine neurons.
  • the first step in generating forebrain dopamine neurons is the induction of a midbrain identity.
  • Treatment of the Pax6+/Sox1- early rosette cells with FGF2 (10-20 ng/ml) for 6 - 7 days results in efficient differentiation of the cells to progenitors that express forebrain transcription factors Bf-1 and Otx2.
  • the second step is to culture the forebrain progenitors in the presence of sonic hedgehog (SHH, 50-250 ng/ml) for 6 - 7 days, then in the regular neuronal differentiation medium for additional 2 weeks until dopamine neurons develop. 35% of the total differentiated cells become dopamine neurons.
  • SHH sonic hedgehog
  • a primate ES cell line preferably a human ES cell line, is first obtained and propagated.
  • the embryonic stem cell line will also retain the ability, throughout the culture, to form trophoblasts and to differentiate into tissue derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm).
  • the cells are then cultured.
  • the cells are propagated on a feeder layer of irradiated mammalian, preferably mouse, embryonic fibroblasts, preferably as disclosed below and in Thomson, J.A., et a]., supra. 1998 and U.S. Patent Nos. 5,843,780 and 6,200,806.
  • a feeder layer of irradiated mammalian, preferably mouse, embryonic fibroblasts, preferably as disclosed below and in Thomson, J.A., et a]., supra. 1998 and U.S. Patent Nos. 5,843,780 and 6,200,806.
  • the ES cell colonies are typically removed intact from adherent cultures by treatment with dispase and grown in a suspension as free-floating ES cell aggregates called embryoid bodies (EBs), preferably for four days as described below.
  • EBs embryoid bodies
  • the EBs are then cultured in medium containing FGF2, preferably at
  • a suitable medium is any medium used for growing neural cells.
  • the following references (Bain, G., et al., supra, 1995; Okabe, S., et a]., supra. 1996; Mujtaba, T., et al., supra. 1999; Housele, O., et al., supra. 1999; Zhang, S.-C, et a]., j ⁇ Neurosci. Res. 59:421-429, 2000; Zhang, S.-C, et a]., Proc. Natl. Acad. Sci.
  • neural precursors may confirm the presence of neural precursors by morphology or by immunofluorescence analysis using neural marker antigens such as nestin and Musashi I, as described below.
  • the neural precursors comprise at least 72%, and most preferably at least 84%, of the total cells.
  • treatment with dispase will lead to the preferential detachment of the central neuroepithelial islands.
  • the differentiating EBs cultured for eight to ten days are preferably incubated with 0.1-0.2 mg/ml dispase (Gibco BRL, Lifetechnologies, Rockville, MD) at 37°C for 15-20 minutes. Alternatively, 0.2 mg/ml of dispase may be used. The rosette clumps retract whereas the surrounding flat cells remain adherent.
  • the rosette clumps may be dislodged by swaying the flask, which leaves the flat cells adherent.
  • the clumps are pelleted, gently triturated with a 5 ml pipette and plated into a culture flask for 30 minutes to allow the contaminating individual cells to adhere.
  • the floating rosette clumps are then transferred to a new flask, preferably coated with poly-(2-hydroxyethyl-methacrylate) to prohibit attachment, and cultured in a medium used for human neural precursors with the presence of FGF2 (typically 20 ng/ml).
  • Neural precursor cells typically comprise at least 72-84% of the total cells.
  • the present invention is a cell population comprising at least 72%, and preferably 84%, neural precursor cells. These neural precursor cells can be defined by being nestin and Musashi I positive.
  • Fig. 1 B illustrates the rosette formation characterizing these cells. By rosette formation, we mean that cells are columnar in shape and are arranged in a tubular (rosette) structure, resembling the neural tube (developing brain) in the body. The columnar cell morphology and tubular structures are shown in the insert of Fig. 1B.
  • the present invention is a cell population of at least 90% and preferably at least 96% neural precursor cells. One would preferably obtain these cells after differential enzymatic treatment and adhesion, as described below in the Examples.
  • the described system can be readily modified to mimic pathological processes that lead to death of dopamine neurons (such as in Parkinson's disease) or motor neurons (such as in ALS), which may be effectively used to screen therapeutic agents that are designed to treat these diseases.
  • dopamine neurons such as in Parkinson's disease
  • motor neurons such as in ALS
  • the described system can be readily modified to mimic pathological processes that lead to death of dopamine neurons (such as in Parkinson's disease) or motor neurons (such as in ALS), which may be effectively used to screen therapeutic agents that are designed to treat these diseases.
  • dopamine neurons such as in Parkinson's disease
  • motor neurons such as in ALS
  • Human ES cells differentiate to form neural tube-like structures in the presence of FGF2.
  • Human ES cell lines, H1 , H9 and a clonal line derived from H9, H9.2 were propagated on a feeder layer of irradiated mouse embryonic fibroblasts (Thomson, J. A., et al., supra, 1998).
  • ES cell colonies were detached and grown in suspension as embryoid bodies (EBs) for four days. The EBs were then cultured in a tissue culture treated flask in a chemically defined medium (Zhang, S.-C, et al., J. Neurosci. Res.
  • FGF2 was obtained from Peprotech, Inc., Rocky Hill, NJ. After five days of culture in FGF2, the plated EBs had generated an outgrowth of flattened cells. At the same time, an increasing number of small elongated cells was noted in the center of the differentiating EBs (Fig. 1 A). By seven days in the defined medium, the central, small, elongated cells had generated rosette formations (Fig. 1 B) resembling the early neural tube as shown by toluidine blue-stained sections (inset in Fig. 1 B).
  • Neural tube-like rosettes can be isolated by differential enzymatic treatment and adhesion. With continued exposure to FGF2, the columnar rosette cells expanded and formed multiple layers. They frequently made up most of the EB and were sharply demarcated from the surrounding flat cells. Treatment with dispase led to the preferential detachment of the central neuroepithelial islands, leaving the surrounding cells largely adherent (Fig. 1 F). Contaminating single cells were separated by short-term adhesion to cell culture dishes. Cell counts performed immediately after this isolation and enrichment procedure showed that cells associated with the isolated neuroepithelial clusters made up 72-84% of the cells in the differentiated EB cultures.
  • Human ES cell-derived neural precursors generate all three CNS cell types in vitro.
  • the isolated neural precursors were expanded as free-floating cell aggregates in a suspension culture, similar to "neurosphere" cultures derived from human fetal brain tissues (Zhang, S.-C, et al., supra, 2000; Svendsen, C.N., et a ., supra, 1996; Carpenter, M.K., et al., supra, 1999; Vescovi, A.L., et al., supra, 1999).
  • BrdU incorporation studies revealed that stimulation of precursor cell proliferation was dependent on FGF2 and could not be elicited by either EGF or LIF alone.
  • NF high molecular weight NF was observed by seven to ten and ten to fourteen days after plating, respectively (Fig. 2D).
  • Antibodies to various neurotransmitters were used to further characterize the ES cell-derived neurons. While the majority of the neurons exhibited a glutamatergic phenotype (Fig. 2E), a smaller proportion was labeled with an antibody to GABA. Frequently, these neurons showed a polar morphology (Fig. 2F). A small number of neurons were found to express TH (Fig. 2G), the rate-limiting enzyme for dopamine synthesis. GFAP + astrocytes were rarely found within the first two weeks after growth factor withdrawal (Fig. 2C), but became more frequent after prolonged in vitro differentiation.
  • ES cell-derived neural precursors generate all three major cell types of the CNS.
  • Human ES cell-derived neural precursors migrate, incorporate, and differentiate in vivo.
  • DNA in situ hybridization with a human-specific probe and immunohistochemical detection of a human nucleus-specific antigen revealed the presence of grafted cells in numerous brain regions. Gray matter areas exhibiting widespread donor cell incorporation included cortex (Fig. 3A), hippocampus (Fig. 3B,C), olfactory bulb, septum (Fig. 3D), thalamus, hypothalamus (Fig. 3E), striatum (Fig. 3F) and midbrain (Fig. 3G).
  • Fig. 3H, J Frequently, they displayed polar morphologies with long processes (Fig. 3H).
  • Fig. 3J neurons with multipolar and immature unipolar morphologies were found (Fig. 3J).
  • the donor-derived neurons generated numerous axons projecting long distances into the host brain, which were detected in both gray and white matter. They were particularly abundant within fiber tracts such as the corpus callosum, the anterior commissure and the fimbria hippocampi where they could frequently be traced for several hundred micrometers within a single section (Fig. 31).
  • a small number of ES cell-derived astrocytes was detected within the host brain tissue.
  • ES cells Exploiting growth factor-mediated proliferation/differentiation and differential adhesion of neural precursor cells, the in vitro differentiation procedure described herein provides a new platform for the study of neural development and the generation of donor cells for nervous system repair.
  • a key finding of this study is the observation that the differentiation of neural precursors from human ES cells appears to recapitulate early steps of nervous system development with the formation of neural tube-like structures in vitro. This phenomenon can now be exploited to study and experimentally manipulate the initial stages of human neural development under controlled conditions.
  • the chemically defined culture system provides a unique opportunity to explore the effects of single factors on human neuroepithelial proliferation and specification in vitro. Similar to precursors derived from the developing human brain, human ES cell-derived precursors show a strong proliferative response to FGF2 (Flax, J.D., et al., supra, 1998). However, no additive or synergistic effects on proliferation can be elicited by EGF or LIF.
  • the ES cell- derived neural precursors Following transplantation into the neonatal mouse brain, the ES cell- derived neural precursors incorporated into a large variety of brain regions where they differentiated into neurons and glia.
  • the failure to detect mature oligodendrocytes in vivo is likely due to the low oligodendroglial differentiation efficiency of human neural precursors as opposed to their rodent counterparts (Svendsen, C.N., et al.. Brain Pathol. 9:499-513. 1999).
  • donor-derived neurons were not restricted to sites exhibiting postnatal neurogenesis but were also found in many other regions of the brain. Similar data were obtained in studies involving transplantation of human CNS-derived precursors into the adult rodent brain (Tropepe, V.. et al..
  • ES cells were cultured on a feeder layer of irradiated mouse embryonic fibroblasts with a daily change of a medium that consisted of Dulbecco's modified Eagle's medium
  • DMEM fetal calf serum
  • Gibco serum replacement
  • 0.1 mM ⁇ -mercaptoethanol 2 ⁇ g/ml
  • ES cell cultures were incubated with dispase (Gibco BRL, 0.1 mg/ml) at 37°C for 30 minutes, which removed ES cell colonies intact.
  • the ES cell colonies were pelleted, resuspended in ES cell medium without FGF2, and cultured for four days in a 25-cm 2 tissue culture flask (Nunc) with a daily medium change.
  • ES cell colonies grew as floating EBs whereas any remaining feeder cells adhered to the flask.
  • the feeder cells were removed by transferring the EBs into a new flask. EBs ( ⁇ 50/flask) were then plated in a 25-cm 2
  • tissue culture flask (Nunc) in DMEM/F12, supplemented with insulin (25 ⁇ g/ml),
  • transferrin 100 ⁇ g/ml
  • progesterone 20 nM
  • putrescine 60 ⁇ M
  • Isolation and culture of neural precursor cells To separate the clusters of rosette cells from the surrounding flat cells, the cultures were incubated with 0.1 mg/ml dispase at 37°C for 15-20 minutes. The rosette clumps retracted whereas the surrounding flat cells remained adherent. At this point, the rosette clumps were dislodged by swaying the flask, which left the flat cells adherent. The clumps were pelleted, gently triturated with a 5-ml pipette and plated into a culture flask for 30 minutes to allow the contaminating individual cells to adhere.
  • the floating rosette clumps were then transferred to a new flask coated with poly-(2-hydroxyethyl- methacrylate) to prohibit attachment and cultured in a medium used for human neural precursors (Zhang, S.-C, et al., supra, 2000) with the presence of FGF2 (20 ng/ml).
  • a medium used for human neural precursors Zhang, S.-C, et al., supra, 2000
  • FGF2 20 ng/ml
  • freshly separated cell clusters and the flat cells left behind were dissociated with trypsin (0.025% in 0.1% EDTA) and counted.
  • the percentage of putative neural precursors (rosette cells) among the total cells differentiated from ES cells was obtained based on 3 independent experiments on H9 and H9.2 lines.
  • ES cell-derived neural precursors For analyses of the differentiation potential of the ES cell-derived neural precursors, cells were cultured on ornithine/laminin substrate in a medium consisting of DMEM/F12, N2 supplement (Gibco), cAMP (100 ng/ml), and BDNF (10 ng/ml, PeproTech) without the presence of FGF2.
  • DMEM/F12 N2 supplement
  • cAMP 100 ng/ml
  • BDNF 10 ng/ml, PeproTech
  • anti-GFAP polyclonal, Dako, 1 :1 ,000
  • anti-human GFAP Sternberg monoclonals, 1 :10,000
  • O4 mouse IgM, hybridoma supernatant, 1 :50
  • anti-tyrosine hydroxylase TH, Pel Freez, 1 :500.
  • NF neurofilament 68 (mouse IgG, 1 :1 ,000); anti-NF 200 (polyclonal, 1 :5,000); anti-NF 68 (mouse IgG, 1 :1 ,000); anti-NF 200 (polyclonal, 1 :5,000); anti-NF 68 (mouse IgG, 1 :1 ,000); anti-NF 200 (polyclonal, 1 :5,000); anti-
  • MAP2ab mouse IgG, 1 :250
  • GABA anti- ⁇ -aminobutyric acid
  • mice were perfused transcardially with Ringer's followed by 4% paraformaldehyde. Brains were dissected and post-fixed in the same fixative at 4°C until use. Donor cells were identified in 50- ⁇ m coronal vibratome-sections by in situ
  • MAP2ab Sigma, clones AP-20 and HM-2, 1 :300
  • phosphorylated medium molecular weight human neurofilament (clone HO-14, 1 :50, a gift of J. Trojanowski).
  • Primary antibodies were detected by appropriate fluorophore- conjugated secondary antibodies. Sections were analyzed on Zeiss Axioskop 2 and Leica laser scan microscopes.
  • a first step toward potential application of stem cell therapy in neurological conditions is the directed differentiation of neural cells with correct positional and transmitter phenotypes.
  • DA dopaminergic
  • ES human embryonic stem
  • FGF8 treatment of human ES-derived neuroectodermal cells at an early stage, before the expression of Sox1 , with FGF8 is essential for specification of DA neurons with correct midbrain DA projection neuronal phenotypes.
  • the in vitro generated DA neurons may be used for toxicological and pharmaceutical screening and for potential cell therapy in
  • Parkinsons' disease results from progressive degeneration of DA neurons in the midbrain, especially the substantia nigra.
  • Current therapy for PD relies primarily on symptom relief by systemic administration of DA precursors such as levadopa. Such therapy is effective for the first few years but almost invariably loses its efficacy and produces serious side effects.
  • Administration of growth factors such as glial cell line-derived neurotrophic factor (GDNF) has been shown to be effective in a small clinical trial (Gill, S.S., et aj., Nat Med. 9:589-595, 2003). This therapy would depend on a sufficient number of surviving DA neurons, and its long- term therapeutic potential remains to be investigated.
  • GDNF glial cell line-derived neurotrophic factor
  • DA neurons can be efficiently generated from mouse ES cells, which are derived from the inner cell mass of pre-implantation embryos at the blastocyst stage (Evans, M.J. and Kaufman, M.H., Nature 292:154- 156, 1981 ; Martin, G.R., Proc. Natl. Acad. Sci. USA 78:7634-7638, 1981 ).
  • Mouse ES cells are first induced to neuroectodermal cells by FGF2 (Lee, S.H., et aj., Nat- Biotech nol.
  • ES cell colonies detached from a feeder layer, were cultured in suspension as aggregates for four days in ES cell growth medium, and then grown in an adhesive culture dish in a chemically defined neural medium containing FGF2
  • FGF8 For differentiation to DA neurons, neuroectodermal cells in the neural tube-like rosettes were enriched through differential enzymatic and adhesion treatment (Zhang, S.C, et al., supra, 2001 ), expanded for four days as aggregates in suspension with FGF2, and were then plated onto a laminin substrate and treated with SHH (50-200 ng/ml) and FGF8 (20-100 ng/ml) for six days. Immunocytochemical analyses revealed that the vast majority of the neuroectodermal cells remained positive for Bfl but not for En-1 (not shown). [0097] The failure of FGF8 to induce Sox1 + neuroectodermal cells to express
  • Sox1 -expressing neuroectodermal cells may be refractory to patterning signals. Since the Sox1 -expressing cells are generated two weeks after differentiation of human ES cells (equivalent to a six-day-old embryo (Thomson, J.A., et aj., supra, 1998)) and formed neural tube-like structures, they may correspond to the neuroectodermal cells at neural tube closure during which neuroectodermal cells express Sox1 and are regionally specified (Lumsden, A. and Krumlauf, R.. Science 274:1109-1115, 1996). This led us to hypothesize that FGF8 may promote midbrain specification before neuroectodermal cells express Sox1.
  • FGF8 100 ng/ml
  • Regionalized neuroectodermal cells differentiate into DA neurons [0098] The neuroectodermal cells were dissociated and differentiated in a neural differentiation medium. They did not express stage specific embryonic antigen 4 (SSEA4), a glycoprotein highly expressed by undifferentiated human ES cells. The disaggregated neuroectodermal cells, initially distributed evenly, reformed rosettes three to five days after plating. They then extended processes and exhibited polar morphology. At three weeks after differentiation, about one third of the total differentiated cell population (31.8 ⁇ 3.1 % TH + cells of 17,965 cells counted from four experiments) were positive for tyrosine hydroxylase (TH) (Fig. 5A).
  • SSEA4 stage specific embryonic antigen 4
  • TH + cells were 10-20 ⁇ m in diameter. They exhibited multipolar morphology, with differentiable axons and dendrites (Fig. 5A). All the TH + cells were stained positively with a neuronal marker ? ⁇ n-tubulin + neurons, about 50% were TH + (Fig. 5B, 6,383 TH + cells of 12,859 #n-tubulin + neurons from four experiments).
  • DOPA which is subsequently decarboxylated to become DA by AADC.
  • D ?H and phenylethanolamine N-methyltransferase (PNMT) transform DA to norepinephrine and catalyze norepinephrine to epinephrine, respectively.
  • PNMT phenylethanolamine N-methyltransferase
  • Immunostaining showed that all TH + cells were AADC (Fig. 5C-E) although some AADC + cells were negative for TH (Fig. 5E).
  • TH + cells were negative for D ⁇ H (Fig. 5F) and PNMT (not shown), although D ?H strongly stained noradrenergic neurons in the adult rat and embryonic monkey brainstem (inset in Fig. 5F).
  • ES cell-generated DA neurons display midbrain phenotypes [00100]
  • RT-PCR analyses indicated that Nu ⁇ , Limxl b, En-1 and Ptx3, which are involved in midbrain DA neuron development (Zetterstrom, R.H., et al., Science 276:248-250, 1997; Smidt, M.P.. et al.. Proc. Natl. Acad. Sci. USA 94:13305-13310, 1997; Saucedo-Cardenas, O., et aj., Proc. Natl. Acad. Sci.
  • DA neurons generated using the above approach possess a midbrain positional identity.
  • DA neurons in the olfactory bulb often co-express -arninobutyric acid
  • GABA GABA
  • Double immunostaining of TH and GABA indicated that most of the DA neurons were negative for GABA although GABA* neurons were found in the culture (Fig. 6C).
  • TH + cells 8% (8.7 ⁇ 3.9%, 6,520 TH + cells counted from four experiments) of TH + cell co-expressed GABA. Most of these double positive cells were small bipolar cells (inset in Fig. 6C).
  • Some midbrain DA neurons especially those in the ventral tegmental area, co-express cholecystokinin octapeptide (CCK8) or calbindin along with TH (McRitchie, D.A., et al.. J. Comp. Neurol. 364:121-150, 1996; Hokfelt. T.. et al.. Neurosci. 5:2093-2124. 1980).
  • Immunohistochemical analyses indicated that the TH + neurons were observed (Fig. 6D). These calbindin neurons were mostly small cells. No CCK8 positive cells were detected in the cultures.
  • ES cell-generated DA neurons are biologically functional
  • Immunostaining showed that all TH + neurons expressed c-Ret, a component of the receptor for GDNF (Fig. 7A-C).
  • the majority of the TH + cells especially those with branched neurites, expressed vesicular monoamine transporter 2 (VMAT2, Fig. 7D-F), which is responsible for packaging dopamine into subcellular compartments in monoamine neurons (Nirenberg, M.J., et al., J. Neurosci. 16:4135- 4145, 1996).
  • TH + neurons expressed synaptophysin, a membrane glycoprotein essential to synapse formation (Calakos, N. and Scheller, R.H., J. Biol. Chem. 269:24534-24537. 1994) (Fig. 7A-I).
  • Dopamine release is a functional hallmark of DA neurons.
  • High performance liquid chromatography (HPLC) analyses revealed the presence of dopamine in the medium of DA differentiation cultures, with 230.8 ⁇ 44.0 pg/ml in the cultures treated with ascorbic acid (AA), FGF8 and SHH and 46.3 ⁇ 9.2 pg/ml in the control cultures without the treatment of AA, FGF8 and SHH (Fig. 8A).
  • AA ascorbic acid
  • FGF8 and SHH 46.3 ⁇ 9.2 pg/ml in the control cultures without the treatment of AA, FGF8 and SHH
  • Action potential (AP) threshold ranged from -26 to -5.2 mV (-17.4 ⁇ 2.1 mV), and peaked at -9.6 to 30 mV.
  • AP's up to 50.2 mV were observed (32 ⁇ 2.8 mV).
  • AP duration ranged from 3 to 20.6 ms (7.2 ⁇ 1.3 ms).
  • Spontaneous firing was observed in two cells (Fig. 8C).
  • In voltage clamp mode both inward and outward currents were observed in all cells (not shown), but their relative magnitudes varied considerably. Inward currents were activated rapidly ( ⁇ 1 ms), and peaked within 1-3 ms.
  • TTX tetrodotoxin
  • DA neurons with midbrain neuronal projection characteristics can be efficiently generated from human ES cells through three simple non-genetic steps: induction of neuroectodermal cells with FGF2, specification of ventral midbrain identity by FGF8 and SHH during neuroectodermal formation, and differentiation of the regionally specified progenitors to DA neurons.
  • induction of neuroectodermal cells with FGF2 specification of ventral midbrain identity by FGF8 and SHH during neuroectodermal formation
  • differentiation of the regionally specified progenitors to DA neurons Unlike the findings obtained from mouse ES cell studies in which DA neurons with midbrain characteristics can be generated from expanded neuroectodermal cells (Lee, S.H., et aj., supra. 2000), we have found that specification or regionalization with FGF8 before precursor cells become Sox1 + neuroectodermal cells is essential for a robust generation of human DA neurons with correct midbrain and functional phenotypes.
  • the projection neurons including midbrain DA neurons, are differentiated from neuroectodermal cells in the neural tube at the early stage and these neuroectodermal cells are already regionally specified during the process of neural tube closure (Lumsden, A. and Krumlauf, R., supra. 1996). This may explain why the human ES cell-generated Sox1 -expressing neuroectodermal cells that possess forebrain phenotypes are not responsive to morphogens for generating DA neurons with midbrain phenotypes.
  • FGF8 may instruct the early precursors to adopt a midbrain identify is confirmed by the generation of DA neurons that have characteristics of projection neurons such as large cell bodies with complex processes and expression of midbrain makers En1 , after the Sox1 " columnar cells in the early rosettes are treated with FGF8.
  • DA neurons are present in several areas of the brain, including midbrain, hypothalamus, retina, and olfactory bulbs.
  • the human ES cell-generated DA neurons in this study resemble midbrain projection DA neurons.
  • Most of the DA neurons do not co-express GABA, whereas co-expression of GABA and TH is a major feature of olfactory DA interneurons (Kosaka, T., et aj., supra, 18987; Gall, CM., et al., supra. 1987).
  • DA neurons In the midbrain, there are at least two major groups of DA neurons, those in the substantia nigra (A9) and in the ventral tegmental area (A10), each having different targets (Bjorklund, A. and Lindvall, O., Handbook of Chemical Neuroanatomy, Vol. 2: Classical Transmitters in the CNS (Bjorklund, A., Hokfelt, T., eds), Amsterdam, Elsevier Science Publishers, pp. 55-111 , 1984). Most DA neurons in the ventral tegmental area express calbindin or CCK, whereas few in the substantial nigra do (McRitchie, DA. et al.. J. Comp. Neurol.
  • ES cell cultures Human ES cell lines, H9 (p21-56) and H1 (p35-40), were propagated weekly on irradiated mouse embryonic fibroblasts (MEF) with a daily change of an ES cell growth medium that consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco), 20% serum replacer (Gibco), 1 mM glutamine (Sigma), 0.1 mM non-essential amino acids (Gibco), 2 ⁇ g/ml of heparin (Sigma), 0.1 mM S-mercaptoethanol (sigma), and 4 ng/ml of FGF2 (R & D Systems), as described by Thomson (Thomson, J.A., et al., supra, 1998).
  • DMEM Dulbecco's modified Eagle's medium
  • F12 serum replacer
  • 1 mM glutamine Sigma
  • 0.1 mM non-essential amino acids Gibco
  • a neural medium consisting of DMEM/F12 (2:1), supplemented with N2 (Gibco), 0.1 mM non-essential amino acids, 2 ⁇ g/ml heparin with a medium change every other day.
  • the ES cell aggregates attached and formed individual colonies at around day six.
  • Neuroectodermal cells exhibited by columnar cells organizing into neural tube-like rosettes, were developed at around day fourteen (Zhang, S.C, et al., supra, 2001 ).
  • the neural rosettes were isolated through differential enzymatic response (Zhang, S.C, et al., supra, 2001). Growth factors were added during the course of differentiation to influence regionalization (see results).
  • DA neuron differentiation The enriched neuroectodermal cells were dissociated by 0.025% trypsin and 0.27 mM EDTA in PBS at 37°C for 10-15 minutes and plated onto 12-mm coverslips (pre-coated with 100 ⁇ g/ml polyomithine and 10 ⁇ g/ml laminin) at a density of 40,000-50,000 cells/coverslip.
  • the neuronal differentiation medium consisted of neurobasal medium (Gibco) supplemented with N2, 0.1 mM non-essential amino acids, 0.5 mM glutamine, 1 ⁇ g/ml laminin, 1 ⁇ M cAMP, 200 ⁇ M AA (Sigma), 10 ng/ml BDNF (R & D Systems) and 10 ng/ml GDNF (R & D Systems).
  • the cells were cultured for three to four weeks with medium change every other day.
  • mouse anti-SSEA4 (1 :40), mouse anti-En-1 (1 :50) and mouse anti-Pax6 (1 :5000, all from Developmental studies hybridoma bank
  • rabbit anti-Sox 1 (1 :500), rabbit anti-human nestin (1 :200), rabbit anti-AADC (1 :1000), sheep anti D ⁇ H, (1 :400), mouse anti-synaptophysin (1 :500) and rabbit anti-CCK8 (1 :2000, all from Chemicon
  • mouse anti-TH (1 :1000), mouse anti- ⁇ lll tubulin (1 :500), rabbit anti-GABA (1 :5000) and mouse anti-calbindin (1:400, all from Sigma
  • Goat anti-c-Ret (1 :400) and mouse anti-Oct4 (1 : 1000, both from Santa Cruz
  • rabbit anti-Bfl (1 :5000; gift from Lorenz Studer
  • Antibody-antigen reaction was revealed by appropriate fluorescence-conjugated secondary antibody.
  • Cell nuclei were stained with Hoechst 33342. Staining was visualized with a Nikon fluorescence microscope. Brain sections from adult rats and E38 embryonic monkeys were used as positive controls for many of the antibodies against neuronal types and neurotransmitters. Negative controls were also set by omitting the primary or secondary antibodies in the immunostaining procedures.
  • Cell counting was achieved blindly by using a reticule on eyepiece and a 40x objective. The cells in ten visual fields were randomly selected and counted from each coverslip.
  • Negative control was achieved by omitting transcriptase during reverse transcription or cDNA sample during PCR.
  • the primers and product lengths were as follows: GAPDH (5'-ACCACAGTCCATGCCATCAC-3 ⁇ 5'-TCCACCACCCTGTTGCTGTA-3', 450 bp); Nurrl (5'-
  • En-1 (5'-CCCTGGTTTCTCTGGGACTT-3', 5'-GCAGTCTGTGGGGTCGTATT-3', 162 bp).
  • HBSS Hank's balanced salt solution
  • Electrophysiological properties of the DA neurons differentiated from human ES cells were investigated using whole-cell patch-clamp recording technigues (Hammill, P.P.. et al.. Pflugers Arch. 391 :85-100, 1981 ). Pipettes were filled with intracellular solutions containing (mM) KC1 140 or K-gluconate 140, Na + - HEPES 10, BAPTA 10, Mg 2+ -ATP 4, (pH 7.2, 290 mOsm, 2.3-5.0 M ⁇ ). Biocytin (0.5%, Sigma) was added to the recording solution and subsequent labeling with streptavidin-Alex Flur 488 (1 : 1000, Molecular Probes) and an antibody against TH was used to identify DA neurons.
  • the bath solution contained (in mM) NaC1 127, KH 2 PO 4 1.2, KCI 1.9, NaHCO 3 26, CaCI 2 2.2, MgSO 4 1.4, glucose 10, 95% O 2 /5% CO 2 (pH 7.3, 300 mOsm).
  • TTX (1 ⁇ m) was applied in the bath solution to block voltage-gated sodium currents.
  • Synaptic events were detected using a template detection algorithm (Mini Analysis Program 4.6.28, Synaptosoft) and deactivation phase was fitted to a biexponential function using the Levenberg-Marquardt algorithm. Data are presented as mean ⁇ SE.
  • Generation of motor neurons in vertebrate animals involves at least three steps: neuralization of ectodermal cells, caudalization of the neuroectodermal cells, and ventralization of the caudalized neural progenitors (Jessell, T.M., Nat. Rev.
  • the first sign of neural differentiation was the appearance of columnar cells forming rosettes in the center of colonies 8-10 days after ES cells were removed from feeder cells for differentiation.
  • Soxl (Fig. 9A), which is expressed by neuroepithelial cells during neural tube formation (Pevny, L.H., et al., Development 125:1967-1978, 1998). With further culturing in the same medium for another four to five days, the columnar cells organized into neural tube-like rosettes with lumens (Fig. 9B) and expressed both
  • Pax6 and Sox1 (Fig. 9C, D).
  • differentiation of neuroectodermal cells from hES cells involves at least two distinctive stages, the Pax6 + /Sox1 " columnar cells in the early rosettes eight to ten days after neural induction, and the Pax6 + /Sox1 + cells forming neural tube-like late rosettes fourteen days after induction.
  • Immunocytochemical analyses revealed that the rosette cells, which expressed Pax6 (Fig. 9E), Sox1 , and nestin, were positive for Otx2 (Fig. 9F, H), a homeodomain protein expressed by fore- and mid-brain cells; but negative for HoxC8 (Fig. 9H), a homeodomain protein produced by cells in the spinal cord.
  • retinoic acid RA, 0.001-1 ⁇ M
  • caudalizing reagent Blumberg, B., et al., Development 124:373-379, 1997)
  • SHH 50-500 ng/ml
  • a ventralizing morphogen Jessell, T.M., Nat. Rev. Genet. 1 :20-29, 2000; Briscoe, J. and Ericson, J.. Curr. Opin. Neurobiol. 11 :43-49. 2001 ).
  • the Sox1 -expressing cells may correspond to neuroectodermal cells in the neural tube given the formation of neural tube-like rosettes and expression of Sox1 at a time equivalent to a three-week-old human embryo.
  • the neuroectodermal cells in the neural tube are regionally specified (Lumsden, A. and Krumlauf, R., Science 274:1109-1115, 1996). This consideration led us to hypothesize that RA may promote caudalization and/or motor neuron specification before neuroectodermal cells express Sox1.
  • RA 0.001-1 ⁇ M
  • HB9-expressing cells first appeared at day six and reached a high proportion around day ten to twelve after the neural rosettes were plated for differentiation. They were largely localized to the cluster, with about 21 % of the total cells in the cluster and few cells in the outgrowth area (Fig. 10A, D). The highest proportion of HB9 + cells was induced in the presence of 0.1-1.0 ⁇ M of RA. RA at the dose over 1.0 ⁇ M resulted in degeneration of some cells in our chemically defined adherent cultures. In the absence of RA, or SHH, or both, there were very few HB9 + cells (Fig. 10D). All the HB9-expressing cells were stained with ? ⁇ -tubulin (Fig. 10C). Thus treatment with RA on early neuroectodermal cells is required for efficient induction of motor neurons.
  • RA choline acetyltransferase
  • the ChAT-expressing cells were largely localized to the cluster (Fig. 12A), corresponding to the localization of the HB9 + cells. These cells were mainly multipolar cells and had large somas of 15-20 ⁇ m in diameter, with some being as big as 30 ⁇ m (Fig. 12A, B). Co-expression of HB9 in the nuclei and ChAT in the soma and processes was observed after three weeks of culture (Fig. 12C). Most of the neurons were also positively stained for vesicular acetylcholine transporter (VAChT, Fig. 12D), which is essential for storage and release of acetylcholine.
  • VAChT vesicular acetylcholine transporter
  • Mouse ES cells have been first directed to neuroectodermal cells which are then treated with morphogens such as FGF8 and SHH for differentiation into dopaminergic neurons (Barberi, T., et al., Nat Biotechnol. 21 :1200-1207. 2003; Lee, S.H., et al.. Nat. Biotechnol. 18:675-679, 2000) or RA and SHH for motor neuron differentiation (Wichterie, H., et al., supra. 2002). These observations seem to fit the notion that neurons are specified from epithelium in the neural tube.
  • the Sox1 -expressing cells generated from hES cells in our culture system resemble those in the neural tube, as they form neural tube-like structures and express Sox1 after two weeks of differentiation from hES cells which are equivalent to a six-day-old human embryo (Zhang, S.C, J_, Hematother. Stem Cell Res. 12:625-634, 2003).
  • the neural tube forms at the end of third week of human gestation (Wood, H.B. and Episkopou, V., Mech. Dev.
  • Sox1 is expressed by the neuroectoderm during the formation of the neural tube in animals (Pevny, L.H., et aj., Development 125:1967- 1978, 1998; Wood, H.B. and Episkopou, V., supra, 1999).
  • Cur finding suggests that the specification of a class of neurons, at least large projection neurons such as motor neurons, begins before stem cells become Sox1 -expressing neuroectodermal cells and may thus explain why brain-derived neuroepithelial cells fail to generate projection neurons of a different regional identity.
  • the functional motor neurons from the renewable source of hES cells offer generic human motor neurons for screening pharmaceuticals designed for treating motor neuron-related disorders such as ALS. These cells also provide a useful source for experimental cell replacement for motor neurons, which may someday lead to applications in patients with motor neuron diseases or spinal cord injury.
  • Human ES cells (lines H1 and H9, passages 19 to 42) were cultured and passaged weekly on a feeder layer of irradiated embryonic mouse fibroblasts as described (Thomson, J.A., et al., supra. 1998). The undifferentiated state of ES cells were confirmed by typical morphology and expression of Gct4 and SSEA4.
  • hES cells were aggregated for four days and then cultured on an adhesive plastic surface for ten days in F12/DMEM supplemented with N2, heparin (2 ng/ml), and FGF2 (20 ng/ml) or RA (Zhang, S.C, et aj., supra, 2001 ).
  • the morphogen-treated neuroectodermal cells were plated onto omithine/laminin-coated coverslips in a neuronal differentiation medium, which consisted of Neurobasal medium (Gibco), N2 supplement, and cAMP (Sigma, IgM) in the presence of RA (0.1 ⁇ M) and SHH (10- 500 ng/ml, R&D) for one week. After that, BDNF, GDNF, and insulin-like growth factor-1 (IGF1) (10 ng/ml, PeproTech Inc.) were added to the medium and the concentration of SHH was reduced to 50 ng/ml.
  • a neuronal differentiation medium which consisted of Neurobasal medium (Gibco), N2 supplement, and cAMP (Sigma, IgM) in the presence of RA (0.1 ⁇ M) and SHH (10- 500 ng/ml, R&D) for one week. After that, BDNF, GDNF, and insulin-like growth factor-1 (IGF1) (10
  • VAChT (Chemicon, 1:1000), lsll/2 (S. Pfaff), Gtx2 (F. Vaccarino), and Plig2 (M.
  • RT-PCR amplifications were performed from hES cell-derived neuroectodermal cells at different stages and motor neuron differentiation cultures.
  • the following primers were used: HoxC ⁇ , 5'-TTTATGGGGCTCAGCAAGAGG-3 ⁇ 5'- TCCACTTCATCCTTCGGTTCTG-3', 318 bp; HoxC ⁇ , 5'-
  • TGAGGGCTGTGTCTGTTCGG-3 4 ⁇ 9 bp; SHH, ⁇ '-CCAATTACAACCCCGACATC- 3', 5'-CCGAGTTCTCTGCTTTCACC-3', 339 bp; Nkx6.1 , 5'- ACACGAGACCCACTTTTTCCG-3', ⁇ '-TGCTGGACTTGTGCTTCTTCAAC-3', 33 ⁇ bp.
  • Electrophysiological properties of hES cell-derived motor neurons were investigated in cultures differentiated for five to six weeks using whole-cell patch- clamp recording techniques (Gao, B.X., et al., J. Neurophvsiol. 79:2277-2287, 1998).
  • Tetrodotoxin I ⁇ M, Sigma
  • bicuculline 20 ⁇ M, Sigma
  • strychnine ⁇ ⁇ M, Sigma
  • D-2-amino- ⁇ -phosphonovaleric acid AP- ⁇ , 40 ⁇ M, Sigma
  • 6-cyano-7- nitroquinoxaline-2,3-dione CNPX, 20 ⁇ M, RBI, Natick, MA
  • 1% biocytin was added to the recording solution.
  • Current- and voltage- clamp recordings were performed using a MultiClamp 700A amplifier (Axon Instruments, Union City, CA).

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Abstract

L'invention concerne un procédé pour différentier des cellules souches embryonnaires dans des cellules neuronales et motrices. Dans un mode de réalisation, l'invention comprend la culture d'une population de cellules comprenant une majorité de cellules caractérisées par une morphologie de rosette précoce et sont représentées par Sox1-/Pax6+ en présence de FGF2, FGF4, FGF8, FGF9, ou RA, les cellules étant caractérisées par une morphologie de rosette précoce et représentées par Pax6+/Sox1+.
PCT/US2004/027841 2003-08-29 2004-08-27 Procede de differentiation in vitro de cellules souches neuronales, de neurones moteurs et de neurones de dopamine provenant de cellules souches embryonnaires de primates WO2005021720A2 (fr)

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JP2006524872A JP2007503811A (ja) 2003-08-29 2004-08-27 霊長類胚幹細胞からの神経幹細胞、運動ニューロン及びドーパミンニューロンのｉｎｖｉｔｒｏでの分化の方法
GB0605851A GB2421029B (en) 2003-08-29 2004-08-27 Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
AU2004269361A AU2004269361B2 (en) 2003-08-29 2004-08-27 Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
EP04782339A EP1670901A4 (fr) 2003-08-29 2004-08-27 Procede de differentiation in vitro de cellules souches neuronales, de neurones moteurs et de neurones de dopamine provenant de cellules souches embryonnaires de primates
IL173832A IL173832A (en) 2003-08-29 2006-02-20 Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
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AU2004269361B2 (en) 2010-05-20
WO2005021720A3 (fr) 2005-05-19
KR20080109943A (ko) 2008-12-17
GB2421029B (en) 2008-04-09
CA2536588C (fr) 2018-04-24
EP1670901A4 (fr) 2007-09-05
GB0605851D0 (en) 2006-05-03
JP2007503811A (ja) 2007-03-01
JP2010162024A (ja) 2010-07-29
JP5529561B2 (ja) 2014-06-25
AU2004269361A1 (en) 2005-03-10
GB2421029A (en) 2006-06-14
IL198450A (en) 2011-01-31
KR20060115351A (ko) 2006-11-08
EP1670901A2 (fr) 2006-06-21
CA2536588A1 (fr) 2005-03-10
IL173832A (en) 2011-02-28
IL173832A0 (en) 2006-07-05

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