KR20060115351A - Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells - Google Patents

Abstract

A method of differentiating embryonic stem cells into neural and motor cells is disclosed. In one embodiment, the invention comprises culturing a population of cells comprising a majority of cells that are characterized by an early rosette morphology and are Sox1- /Pax6+ in the presence of FGF2, FGF4, FGF8, FGF9, or RA wherein the cells are characterized by an neural tube-like rosette morphology and are Pax6+/Sox1+.

Description

METHODS OF IN VITRO DIFFERENTIATION OF NEURAL STEM CELLS, MOTOR NEURONS AND DOPAMINE NEURONS FROM PRIMATE EMBRYONIC STEM CELLS} In Vitro Differentiation of Neural Stem Cells, Motor Neurons and Dopamine Neurons of Primate Embryonic Stem Cell Origin

Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of embryos prior to transplantation (Thomson, J. A., et al., Science 282: 1145-1147,1998). Similar to mouse ES cells, they can proliferate in large numbers, maintaining the potential to differentiate into various somatic cells with 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., Dev. Biol. 227: 271-278, 2000. In vitro differentiation of ES cells provides a new prospect for studying cellular and molecular mechanisms for the early development and production of donor cells for transplantation treatment. Indeed, mouse ES cells include hematopoietic cells (Wires, MV and Keller, G., Development 111: 259-267, 1991), cardiomyocytes (Klug, MG, et al., J. Clin). . Invest. 98: 216-224, 1996, insulin secreting cells (Soria, B., et al., Diabetes 49: 157-162, 2000) and glial cells (Bain, G., et al., Dev) . Biol. 168: 342-357, 1995; Okabe, S., et al., Mech. Dev. 59: 89-102, 1996; Mujtaba, T., et al., Dev. Biol. 214: 113-127,1999; Many clinically relevant cell types, including Brustle, O., et al., Science 285: 754-756, 1999, have been shown to differentiate in vitro. After transplantation into the rodent central nervous system (CNS), ES cell-derived neural precursors have been found to integrate into host tissues and in some cases improve function. McDonald, J. W., et al., Nat. Med. 5: 1410-1412,1999. Clinical application of human ES cells requires the generation of highly pure donor cells for specific tissues and organs.

There is a need in the art for a simple but efficient strategy to separate transplantable neuronal and motor neuron precursors from differentiated human ES cell cultures.

Summary of the Invention

A method of generating a cell population comprising a cell population from the culture tuning, Pax6 ⁺ of embryonic stem ^cells In one aspect, the invention features an early rosette (rosette) form and Sox1. In one aspect, the invention includes (a) propagating embryonic stem cells into embryoid bodies and (b) propagating embryonic bodies into neural stem syngeneic cell populations in the form of neural tubular rosettes, wherein proliferation is FGF2, FGF8 , In the presence of FGF4 or FGF9. The total time from the proliferation of embryonic stem cells to early rosette development is preferably 8 to 10 days. Preferably, the total population of Pax6 ⁺ / Sox1 ⁻ is at least 70% of the total cell population.

The invention also relates to a cell population produced by the method.

In another aspect, the present invention relates to a method for producing a neural stem syngeneic cell population characterized by a neural tubular rosette form, which is Pax6 ⁺ / Sox1 ⁺ , wherein the method is characterized by an early rosette form and may be FGF2, FGF4, FGF8 or Culturing the cells Sox1 ⁻ , Pax6 ⁺ for 4-6 days in the presence of RA. The invention also relates to a cell population produced by the method.

In one embodiment, the early rosette cells are preferably incubated with FGF8 for 4-7 days and are EN1 ⁺ . In another embodiment, the cells are preferably incubated with FGF2 for 4-7 days and are Bf1 ⁺ . In another embodiment, the cells are preferably incubated with RA for 4-7 days and are Hox ⁺ .

In another aspect, the invention relates to a method for isolating a group of midbrain dopamine neurons, the method comprising culturing the cells described above with SHH in the presence of FGF8, wherein the cells obtained are TH, AADC , EN-1, VMAT2 and DAT but not DbH and PNMT. The invention also relates to a cell population produced by the method.

In another aspect, the invention relates to a method for isolating a group of spinal motor neurons, the method comprising culturing with SHH in the presence of RA, wherein the cells obtained are HB9, HoxB1, HoxB6 , HoxC8, ChAT and VAChT. The invention also relates to a cell population produced by the method.

In another aspect, the invention relates to a method for isolating whole group of brain dopamine neurons and the method comprises culturing the cells described above with SHH. The invention also relates to a cell population produced by the method.

The invention also relates to the aforementioned cell population test method for screening agents for their ability to affect normal human neural development.

Other objects, advantages and features of the invention will become apparent after a review of the specification, claims and drawings.

1A-1 depict differentiation and isolation of neural precursors of ES cell origin. FIG. 1A shows attached EBs grown for 5 days in the presence of FGF2 are squamous cells in the periphery and small elongated cells centrally aggregated. 1B shows that by day 7 many rosette formations (arrows) appear in the center of the erupting EB. The upper right inset shows a 1 μm section of a rosette stained with toluidine blue, which shows columnar cells arranged in a tubular structure. Bar = 20 μm. 1C-E show that the rosette cluster cells (bottom left) and miniature rosettes (center) are Nestin (FIG. 1C) and Musash-1 (FIG. 1D) positive while peripheral squamous cells are negative. FIG. 1E is a composite image of all cell nuclei labeled with FIGS. 1C and 1D and DAPI. 1F shows that after treatment with dispase for 2 minutes, rosette formation retreats while peripheral squamous cells remain attached. In FIG. 1G-I, the isolated cells were stained positively with filament pattern by Nestin (FIG. 1G), positively stained with Musash-1 in the cytoplasm (FIG. 1H) and positively with PSA-NCAM for membranes Stained (FIG. 1I). All nuclei are stained with DAPI. Bar = 100 μm.

2A-G show the properties of ES cell derived neural precursors in vitro. In FIG. 2A BrdU incorporation by dissociated ES cell-derived neural precursors is elevated in the presence of FGF2 (20 ng / ml) but of epidermal growth factor (EGF) (20 ng / ml) or leukemia inhibitory factor (LIF) (5 ng / ml). It does not rise in presence. This is data representative of one of three replicate experiments. ^* Indicates the difference between the experimental and control groups (p <0.01, n = 4, Student's t-test). 2B shows that differentiation of ES cell-derived neural precursor clusters over three weeks forms a bundle of fibrous fibers with cells traveling along the fibrous fiber. In FIG. 2C, immunostaining after 3 weeks of differentiation shows that the majority of cells are β _III -tubulin ⁺ neurons (red) and only a few cells are GFAP ⁺ astrocytes (green). 2D shows that after 45 days of differentiation, more GFAP ⁺ astrocytic cells (green) appear with NF200 ^{+ hard} fibers (red but yellow due to overlapping with green GFAP). 2E-G shows that ES cell-derived neurons of various morphologies express distinct neurotransmitters such as glutamate (FIG. 2E), GABA (FIG. 2F) and enzyme tyrosine hydroxylase (FIG. 2G). O4 ⁺ oligodendrocytes (arrows) are observed after 2 weeks of differentiation in glial differentiation medium. Bar = 100 μm.

3A-K show incorporation and differentiation of ES cell derived neural precursors in vivo. Transplanted cells are detected by in situ hybridization of probes and human alu-repeat elements (FIGS. 3A-E, G) or antibodies to human specific nuclear antigens (FIG. 3F). 3A shows each donor cell in the host cortex of an 8 week old receptor (arrow). 3B shows extensive incorporation of ES cell-derived neural precursors in hippocampus. Human alu probes and hybridized cells are labeled with red dots (virtual colors). 3C shows human cells incorporated adjacent to the hippocampus pyramidal layer at P14. 3D shows ES cell derived cells in the septum of 4 week receptor mice. 3E shows a high quality view of each donor cell in the hypothalamus. Note that seamless integration between adjacent unlabeled host cells. 3F shows donor cells in the ancestors of a four week host detected with antibodies to human specific nuclear antigens. 3G shows that the transplanted cells migrate extensively from the canal to the dorsal midbrain. 3H shows that human ES cell derived neurons exhibit polar morphology and long processes in the cortex of a two week host. Cells are double labeled with antibodies to human specific nuclear markers (green) and β _III -tubulin (red). 3I shows a network of donor-derived axons in the hippocampus, identified as antibodies to human neurofilaments. 3J shows donor-derived multipolar neurons overlabeled with antibodies recognizing the a and b isotypes of MAP2. 3K shows ES cell-derived astrocytic cells in the cortex of four week-old animals overlabeled with human specific nuclear markers (green) and antibodies against GFAP (red). Note that all duplicate markers are confocal images and identified as a single optical cut. Bars: 200 μm in FIGS. 3A, 3B, 3G; 100 μm in FIG. 3C, FIG. 3D; 10 micrometers in FIGS. 3E, 3F and 3H-K.

4 shows the generation and regional specialization of neuroectodermal cells.

4A shows that columnar cells appear in ES cell colonies that differentiate on day 9 in the presence of 20 ng / ml FGF2. 4B shows that columnar cells form neural tubular rosettes on day 14. 4C shows the cells are positive for Sox1 (red) in rosettes with columnar morphology. 4D shows that neural rosette cells express Bf1 (red) but not En-1 (green) in FGF2 treated cultures. In FIG. 4E, En-1 (green) expression was treated with fibroblast growth factor 8 (FGF8) (100 ng / ml) up to 6 days on day 9 and proliferated in FGF8 for 4 days, followed by an additional 6 days for laminin substrate. It is observed in nestin ⁺ (red) neuroectodermal cells treated with Sonic Hedgehog (SHH) (200 ng / ml). 4F shows that En-1 ⁺ cells (green) are negative for Bf1 (red) in cultures treated as in FIG. 4E. Cell nuclei are stained with Hoechst (c, d: blue). Bar: 50 μm.

5 shows the differentiation of DA neurons. 5A shows that about one third of differentiated cells are tyrosine hydroxylase (TH) positive in culture treated with FGF8, SHH and ascorbic acid (AA) at 3 weeks of differentiation. 5B shows that all TH ⁺ cells (red) in culture stain positively with neuronal marker β _III -tubulin (green). 5C-E shows that all TH ⁺ cells in culture are positively stained with aromatic acid decarboxylase (AADC) (a and e, red) but some AADC ⁺ cells are TH ⁻ (e, arrowhead). 5F shows that TH ⁺ cells are negative for noradrenaline neuron marker dopamine β-hydroxylase (DβH) (green). The photo indicates that DβH stains cells positively in adult rat brain stem. Cell nuclei were stained with Hoechst (a, b, f; blue). Bar = 50 μm.

6 shows the properties of human ES cell derived DA neurons. 6A shows that differentiated DA neurons express genes that characterize midbrain fat found by RT-PCR. EB: embryoid body; NE: neuroectodermal cells; 3w: DA culture differentiated for 3 weeks; NC: negative control. 6B shows that the majority of TH ⁺ cells (red) in culture express the midbrain marker En-1 (green). 6C shows that GABA expressing cells (red) are present in the culture but very few TH ⁺ cells (green) co-express GABA (red, photo). 6D shows that TH ⁺ cells (red) are negative for calvindine (green). Bar = 50 μm.

7 shows expression of receptors and transporters in DA neurons derived from human ES cells. 7A-C show that all TH ⁺ cells (a, green) express c-Ret (red). 7D-F show that TH ⁺ cells (d, green) co-express VMAT2 (e and f; red). 7G-I shows that TH ⁺ neurons (j, green) co-express cinnaphtopycin (k and l, red). Bar = 25 μm.

8 shows the functional properties of DA neurons produced in vitro. 8A shows that spontaneous and depolarization (56 mM KCl in HBSS) -induced DA is released in control and treated cultures at 3 weeks of differentiation. Data is shown as mean ± SD from three experiments. ^* P <0.05 vs. unpaired Student's t test. Control. 8B shows the action potential induced by the depolarized current step in two neurons differentiated over 30 days. Passive membrane properties: (i) V _{resting period} -49 mM, Cm 15.5 pF, R _m 5.0 GΩ; (ii) V _{resting period} -72 mV, C _m 45 pF, R _m 885 GΩ. 8C shows spontaneous postsynaptic potential in neurons differentiated for 36 days. 8D shows spontaneous postsynaptic currents in neurons maintained for 30 days in culture. Neurons are bent at -40 mV voltage using a K-gluconate base pipette solution. Outward currents reflect an inhibitory response and inward currents reflect a stimulus response in this low chloride recording solution. (ii) average response of cell origin as described in panel (i). The weight decay time constants are 61.4 ms and 9.9 ms for suppression (n = 17 responses) and stimulus (n = 14 responses) currents. 8E-G shows that immunostaining indicates that the recorded neurons (f, green) are TH ⁺ (e and g, red). Bar = 50 μm.

9 shows neuroectodermal cells induced by FGF2 display beak phenotype. ES cells differentiated from FGF2 for 10 days show small columnar morphology at the colony center and are configured to form rosettes. The columnar but peripheral squamous cells in the rosette are positive for Pax6 and negative for Sox1 (A). By day 14 columnar cells form neural tubular rosettes (B) and are positive for Pax6 (C) and Sox1 (D). Pax6 ⁺ cells (E) in rosettes are also Otx2 ⁺ (F) or En-1 ⁻ (G). In neural tubular rosette, cells are positive for Otx2 and negative for HoxC8 (H). Blue indicates Hoechst stained nuclei. Bar = 50 μm.

10 shows the generation of motor neurons from neuroectodermal cells. (A) shows that for two weeks (upper row) Sox1 ⁺ neuroectodermal differentiation produces a wide range of neurons and expresses lsl 1 in the overgrowth region, but very few HB9 ⁺ cells. Pax6 ⁺ / Sox1 ^- neural processing of ectodermal cells (2 ^nd column) is the projection of the wide variety of hyperproliferative and moving a small number of cells and the expression 1 and lsl high proportion of HB9 ⁺ cells. About 50% of lsl 1/2 ⁺ that differentiates from early neuroectodermal cells is also HB9 ⁺ (B). HB9 ⁺ is also ^positive for β _III -tubulin (C). About 21% of the cells in the cluster are HB9 ⁺ when the culture differentiates in the presence of both retinoic acid (RA) and SHH, while little HB9 ⁺ is observed when cultured in RA alone or SHH alone (D). Not observed. Blue indicates Hoechst stained nuclei. Bar = 50 μm.

11 shows the effect of RA, FGF2 and SHH on neuroectodermal cells. (A) shows changes in the lateral genes of early rosette cell origin incubated with 20ng / ml of RA or FGF2 for one week in neuronal induction media indicated by RT-PCR analysis. (B) compares homeobox gene expression in early and late neuroectodermal cells treated with RA 0.1 μM for one week. Early neuroectodermal cells treated with RA and then differentiated for 12 days are mostly negative for Otx2 (C) but positive for HoxC8 (D). All HoxC8 ⁺ cells are β _III -tubulin ⁺ (E). Pax6-expressing neuroectodermal cells are negative for Olig2 (F). After treatment with RA for 1 week and differentiation for 2 weeks in the presence of SHH (100 ng / ml), many cells express Olig2 (G). Very few Olig2 ⁺ cells are observed when late neuroectodermal cells are treated with RA and then differentiated under the same culture conditions (H). Blue indicates Hoechst stained nuclei. Bar = 50 μm.

12 shows the maturation of motor neurons in culture. ChAT expressing cells are predominantly located in clusters (A) and are large multipolar cells (B). Confocal images show co-location of ChAT and intranuclear HB9 in somatic and process cells after 3 weeks of culture. Most of the cells in the cluster expressed VChAT (D). Many ChAT ⁺ cells are positive for synapsin in somatic cells and processes 5 weeks after culture (E). (F) shows the AP caused by the depolarized current step (0.15nA) in neurons maintained at 42DIV. Resting membrane potential (Vm) -50 mV (fi) and -70 mV (fii). (G) shows spontaneous AP in neurons maintained for 42DIV, Vm -50mV. (H) shows spontaneous inward and outward synaptic currents at −40 mV with K-gluconate-based pipette solution under control conditions (Hi). Bath application of bicuculin (20 μM) and striknin (5 μM) block outward currents (IPSC, Hii). Subsequent application of AP-5 (40 μΜ) and CNQX (20 μΜ) then blocks residual inward currents (EPSC, Hiii). (i) shows the mean sIPSC and sEPSC of cell origin described in Panel H. (J) shows dual immunostaining for biocitin (derived from recording electrode) and ChAT. Blue indicates Hoechst stained nuclei. Bar = 50-μιη.

13 shows the electrophysiological characteristics of motor neurons generated in vitro. (A) Exer shows AP induced by depolarized current step (0.15nA) in neurons maintained for 42DIV. The resting membrane potential (Vm) is -59 mV (ai) and -70 mV (aii). (B) shows spontaneous AP in neurons maintained for 42DIV, Vm-50mV. (C) shows spontaneous inward and outward synaptic currents at −40 mV using K-gluconate base pipette solution under control conditions (ci). Bath application of bicuculin (20 μM) and striknin (5 μM) blocks outward currents (IPSC, cii). Subsequent application of AP-5 (40 μM) and CNQX (20 μM) blocks the residual inward current (EPSC, ciii). (D) shows the mean sIPSC and sEPSC of cell origin described in panel c. In (E-F), after recording, the cover cultures were immunostained with ChAT to show that the biocithin filled neurons are positive for ChAT. Bar = 50 μm.

Here, we describe a method of producing dopamine (pro brain and midbrain) from human embryonic stem cells. Preferred methods are generally described in Tables 1 to 3 below.

In particular, we describe a method of differentiating premature rosettes (Pax6 ⁺ / Sox1 ⁻ ) from ES cells via embryonic intermediates. By differential treatment, we were able to differentiate these early rosettes into three different forms of neural tubular rosettes which were then suitable for development into whole brain dopamine neurons, midbrain dopamine neurons or motor neurons.

We refer to Table 2 below, which describes steps 1 and 2 for generating dopamine and motor neurons. Table 2 also describes the various intermediate products that Applicants consider to be suitable developmental markers.

Generation of Dopamine and Motor Neurons from Human Embryonic Stem Cells Step 1 Proliferation of Embryonic Stem Cells and Their Development into Neural Stem-Sync Cell Groups in Neural Tubular Rosette Form Step 2 Development of Phase 1 Cells into Whole Brain Dopamine Neurons, Midbrain Dopamine Neurons, or Motor Neurons Through Differential Culture Conditions

As noted above, the present invention encompasses two main aspects. One aspect is the process of generating a synaptic neural stem cell population (or neuroepithelial cells) in neural tubular rosette form and expressing neuroepithelial markers Pax6, Sox1, Nestin, Musash-1. As used herein, tuning is in the same developmental stage as opposed to being induced by RA inducing heterologous differentiation (ie, the culture comprises cells in the developmental stage from precursor to differentiated neurons). Means a cell population. In this case we observe Pax6 ⁺ / Sox1 ^- early neuroepithelial cells at an early stage or Pax6 ⁺ / Sox1 ⁺ neuroepithelial cells at a later stage. At any stage, we did not observe any differentiated neurons. This tuning development allows direct differentiation towards specific neurons as described herein.

A second aspect is a method of further differentiating neuroepithelial cells into specific neurons, such as midbrain dopamine neurons, forebrain dopamine neurons, and spinal cord motor neurons.

Table 3 below describes a preferred method for obtaining the cells of the invention. Table 3 contains general culture broth components that can be replaced by similar cultures and important growth factors and timing components. When we refer to nerve cell culture media, many culture components may be suitable. The following section highlights the culture components necessary for proper differentiation.

In general, a suitable medium is any medium used to grow nerve cells. See Bain, G., et al., Supra, 1995; Okabe, S., et al., Supra, 1996; Mujtaba, T., et al., Supra, 1999; Bustle, O., et al., Supra, 1999; Zhang, S.-C. , et al., J. Neurosci. Res. 59: 421-429,2000; Zhang, S.-C. , et al., Proc. Natl. Acad. Sci. USA 96: 4089-4094,1999; Svendsen, C. N., et al., Exp. Neurol. 137: 376-388,1996; Carpenter, M. K., et al., Exp. Neurol. 158: 265-278, 1999; Vescovi, A. L., et al., Exp. Neurol. 156: 71-83,1999, use the same or similar media.

1. Differentiation of neuroepithelial cells (nerve stem cells) from human ES cells

The production of neuroepithelial cells is followed by the formation of embryoid bodies in suspension culture for 4-6 days, followed by the presence of growth factors, preferably FGF2 or FGF8, when cells are columnar and composed in rosette form in the center of each colony. Or adhesion culture for 5 days (FIGS. 1A, 4B, 9A, B) (Zhang, et al., Nature Biotechnol., 2001). FGF4 and FGF9 are also suitable growth factors.

In these rosettes, the columnar cells express the neural transcription factor Pax6 but do not express another neural transcription factor Sox1 (FIG. 9C, D). We call these rosettes "early rosettes" because they appear early and are formed by monolayer columnar cells without lumen. Every single colony owns an early rosette. The total premature rosette cell population is at least 70% of the total cells.

Further culture of these early rosettes for 4-6 days leads to neural tubular rosette formation (FIGS. 1B, 4B, 9E). Neural tubular rosettes are formed by multiple columnar cells with apparent lumens. Cells in rosette express Pax6 as well as Sox1 (Figures 4C, 9F, G, H). Progression from an early rosette to neural tubular rosette takes about 4-6 days in the presence of 10-20 ng / ml of FGF2, FGF4, FGF8, FGF9 or in the presence of 0.001-1 μM.

The process of neural epithelial differentiation from neural tubular rosette formation in ES cells takes 14-16 days. Human ES cells are derived from 5.5 day old human embryos (Thomson, J.A., et al., Supra. 1998). Thus, the development of neuroepithelial cells of human ES cell origin in the culture system of the present invention corresponds to those developed in human embryos for 19 to 21 days. In normal human development, neural tubes form at 20-21 days. Thus, the differentiation of neuroepithelial cells from human ES cells reflects normal human embryonic development. Zhang, S. C., J. Hematother. Stem Cell Res. 12: 625-634, 2003]

Two-stage neuroepithelial development has not been described previously, as evidenced by morphological transformation and overt gene expression patterns. Pax6 and Sox1 have been found to be expressed by neuroepithelial cells when neural tubes form simultaneously in frogs, ornamental fish, chickens and mice (Pevny, et al., Development 125: 1967-1978,1998). Thus, the inventors believe that the discovery of continuous Pax6 and Sox1 expression upon neuroepithelial differentiation in human ES cells is novel and may be unique to humans. Pax6 + / Sox1- neuroepithelial cells are the earliest neuroepithelial cells identified so far. The functional significance of these cells is to ensure that Pax6 + / Sox1 neuroepithelial cells, not Pax6 + / Sox1 + neuroepithelial cells in neural tubular rosettes, are neurons with positional entities other than forebrain such as midbrain dopamine neurons and spinal cord motor neurons in early rosettes. It is suitable for the present invention in that it can be derived (see Table 1 above).

Every ES cell colony that differentiates forms a neural tubular rosette. Neuroepithelial cells comprise at least 70-90% of total differentiated cells.

Neural epithelial cells in neural tubular rosette form can be purified through treatment with low concentrations of dispase and differential adhesion (described in US Patent Application Serial No. 09 / 960,382).

2. Generation of Midbrain Dopamine Neurons

Functional neurons with potential therapeutic applications must not only become neurons but also have two or more additional features: the ability to synthesize, secrete, and absorb specific positional entities and neurotransmitters.

The first step in producing midbrain dopamine neurons is the induction of midbrain entities. Pax6 ⁺ / Sox1 ⁺ non-neural tubular rosette cells Pax6 ⁺ / Sox1 ^- midbrain made early rosette cells FGF8 (50 to 200ng / ml) to the elongated treated for 6 or 7 days a transcription factor 1 (En-1) and Pax2 ( 4E, F) and efficiently differentiate cells that downregulate forebrain marker Bf-1 (FIG. 4D).

The second stage is the midbrain for 6-7 days in the presence of sonic hedgehog (SHH, 50-250 ng / ml), followed by the development of dopamine neurons for 2 weeks in normal neural differentiation medium (as shown in Table 3). It is to incubate the precursors. Preferably, at least 35% of the total differentiated cells will be dopamine neurons.

Preferred differentiation media are listed in Table 3.

Dopamine neurons express TH, AADC but not DbH and PNMT (FIG. 5) so that dopamine can be synthesized but no further metabolism to norepinephrine or nephrin occurs.

Dopamine neurons express En-1, ptx3, Nurr1 and Lmx1b (FIGS. 6A, B), transcription factors required for midbrain dopamine neuron development.

Dopamine neurons do not express GABA (FIG. 6C). Co-expression with GABA is characteristic of dopamine neurons in olfactory bulbs.

Dopamine neurons do not express calvindine (FIG. 6D). Coexpression with calvindine is characteristic of dopamine neurons in the cortical region of the midbrain.

Together, the feature is that the dopamine neurons produced in the culture system of the present invention are midbrain dopamine neurons more similar to neurons in medulla, which are dopamine neurons lost in Parkinson's disease.

Dopamine neurons have growth factors required for the survival and function of c-ret, receptors for GDNF (FIGS. 7A, B, C), dopamine neurons.

Dopamine neurons also express VMAT2 (FIGS. 7D, E, F), the transporters required for storage and secretion dopamine. They also express DAT (Fig. 7G, H, I), the transporter required for dopamine absorption after release. Thus, the dopamine neurons produced in the culture system of the present invention have the necessary apparatus for the synthesis, storage, release and uptake of the transporter dopamine.

Dopamine neurons express synaptopycin (FIG. 7) for synapse formation. They can initiate action potentials and secrete dopamine in response to stimulation (FIG. 8). Thus, dopamine neurons are functional.

3. Generation of Spinal Motor Neurons

The first step in generating spinal motor neurons is the induction of spinal column (coccal) entities. Spinal transcription factor Hox genes such as HoxB1, HoxB6, HoxC5, and HoxC8 were treated by treating Pax6 + / Sox1- early rosette cells with RA (0.001 to 1 μM) for 6-7 days rather than Pax6 + / Sox + neural tubular rosette cells (FIG. 10A). Cells are efficiently differentiated into precursors that express but do not express the forebrain markers Otx2 and Bf-1 or the midbrain markers En-1 (FIGS. 11A, C, D, E).

The second stage only expresses expression of Olig2, a transcription factor expressed by the internal neural precursors (FIG. 11F, G, H), followed by further development of spinal motor neurons in normal neuronal differentiation medium for 7-10 days. As demonstrated by incubating the spinal precursor in the presence of sonic hedgehog (SHH, 50-250 ng / ml) for 6-7 days to induce the characteristics of the internalized precursor.

Preferred differentiation media are listed in Table 3.

In a preferred embodiment, at least 22% of the total differentiated cells become spinal cord motor neurons. Motor neurons express the transcription factors HB9, islet1 / 2 and Lim3 (FIG. 10) specifically expressed by spinal motor neurons. Motor neurons also express HoxB1, HoxB6, HoxC5, HoxC8 but do not express the forebrain markers Otx2 and Bf-1 or the midbrain marker En-1 (Figures 11A, C, D, E), indicating that these are spinal motor neurons.

Motor neurons express ChAT (FIG. 12A, B, C, D), an enzyme required to synthesize motor neuron transporter acetylcholine. Motor neurons also express VAChT (FIG. 12E), suggesting that motor neurons can store and absorb the transporter acetylcholine.

In addition, motor neurons express synapsin (FIG. 12F) for synapse formation. They can initiate action potentials (FIG. 13). Thus, motor neurons are functional. We have data showing that motor neurons release acetylcholine as analyzed by HPLC.

4. Generation of Whole Brain Neurons

In another aspect, the invention relates to a method of differentiating primate ES cells (preferably human ES cells) into forebrain dopamine neurons, preferably implantable neural precursors suitable for nervous system repair. Preferably anyone will disclose a method as described above for midbrain dopamine neuron production. To generate whole brain neurons, Pax6 ⁺ / Sox1 ⁻ cells are further treated with FGF2 for 4-6 days followed by SHH. The steps for generating whole brain dopamine neurons and the assays for measuring dopamine neuron characteristics are similar to those described for midbrain dopamine neurons. The main difference is the use of morphogens and the characteristics of dopamine neurons in certain periods of time.

The first step in producing whole brain dopamine neurons is the induction of midbrain entities. Pax6 + / Sox1-premature rosette cells are treated with FGF2 (10-20 ng / ml) for 6 or 7 days to efficiently differentiate the cells into precursors expressing the whole brain transcription factors Bf-1 and Otx2.

The second step is to cultivate the whole brain precursors in the presence of sonic hedgehog (SHH, 50-250 ng / ml) for 6-7 days, followed by further development of dopamine neurons in normal neuronal differentiation medium for 2 weeks. 35% of total differentiated cells become dopamine neurons. The following description, taken from US Patent Application Serial No. 09 / 970,382, describes a preferred method.

Primate ES cell lines, preferably ES cell lines, are obtained and propagated first. As described in Thomson, JA, et al., Science 282: 1145-1147 (1998) and US Pat. Nos. 5,843,780 and 6,200,806 or with karyotypes normal and at least 11 months and preferably 12 months ES cell lines can be obtained by other methods suitable for obtaining ES cell lines having the ability to proliferate in undifferentiated state after continuous culture. Embryonic stem cells will also retain the ability to form a cortical layer throughout culture and to differentiate into tissues derived from all trioderm layers (endoderm, mesoderm and ectoderm).

The cells are then cultured. In a preferred embodiment of the invention, the cells are preferably irradiated mammals as described in Thomson, JA, et al., Supra, 1998 and US Pat. Nos. 5,843, 780 and 6,200,806. Animals, preferably mice, embryonic fibroblasts are grown on the feeding layer. We also contemplate that the cells can proliferate without the nutrient supply cell layer.

ES cell colonies are typically removed intact from the adhesion culture by treatment with dispase and preferably grown in suspension as free flowing ES cell aggregates (called embryoid bodies) for 4 days as described below.

The EB is then incubated in medium containing preferably 20 ng / ml FGF2 to produce early rosette cells. Other preferred components of the medium are as described in Table 3. However, many other media components may be suitable. In general, a suitable medium is any medium used to grow nerve cells. See Bain, G., et al., Supra, 1995; Okabe, S., et al., Supra, 1996; Mujtaba, T., et al., Supra, 1999; Bustle, O., et al., Supra, 1999; Zhang, S.-C. , et al., J. Neurosci. Res. 59: 421-429,2000; Zhang, S.-C., et al., Proc. Natl. Acad. Sci. USA 96: 4089-4094, 1999; Svendsen, C. N., et al., Exp. Neurol. 137: 376-388,1996; Carpenter, M. K., et al., Exp. Neurol. 158: 265-278, 1999; Vescovi, A. L., et al., Exp. Neurol. 156: 71-83,1999] use the same or similar media.

After incubation in the medium for about 5 days, the aliquots of EB cause hyperproliferation of squamous cells and cells slightly longer in length by 7 days induce rosette formation as shown in FIG. 1B. These formations are similar to the early neural tube (photo in FIG. 1B). The presence of neural precursors can be confirmed by immunofluorescence analysis using neural marker antigens such as Nestin and Musash I as described below. Preferably, the neural precursors comprise at least 72% of the total cells and most preferably at least 84%.

In addition, neural tubular rosettes can be isolated, preferably by differential enzymatic treatment and adhesion, as described in the Examples below. Briefly, treatment with dispase preferentially desorbs the central neuroepithelial islets. To separate clusters of rosette cells from surrounding squamous cells, differentiated EBs cultured for 8-10 days are preferably 0.1 to 0.2 mg / ml of dispase (Gibco BRL, Lifetechnologies, Incubated with Rockville, MD). Also, 0.2 mg / ml of dispase can be used. Rosette Klump retreats while surrounding squamous cells remain adhered. At this point, the rosette clump can be removed by shaking the flask to which the squamous cells are attached. The clumps are pelleted and ground in a weak 5 ml pipette and aliquoted into culture flasks for 30 minutes to adhere each contaminating cell. Floating rosette clumps are then transferred to a fresh flask, preferably coated with poly- (2-hydroxyethyl-methacrylate) to block adhesion and to be used for human neural precursors with the presence of FGF2 (typically 20 ng / ml). Incubate in the medium. As described in the Examples below, treatment with dispase followed by differential adhesion typically results in high integration of neural precursor cell populations, typically at least 90% and most preferably at least 96%. In addition, other methods, such as immune isolation using antibodies against PSA-NCAM, can be used to separate neural progenitor cells.

The following examples demonstrate that human ES cell derived neural precursors can generate all three CNS cell types in vitro.

The following table is a flow chart for various aspects of this aspect of the invention.

Characterization of neural precursor cells in vitro and in vivo

배상체

신경관형 구조로의 분화

다스파제 처리를 사용한 신경 전구체의 분리ES cell

Embryonic body

Differentiation into Neural Tubular Structure

Isolation of Neural Precursors Using Daspase Treatment Treatment with dispase and incubation in ES medium without FGF2 for 4 days followed by incubation in chemically defined medium with FGF2 (10-20 ng / ml). There is a unique structure representing neuroepithelial cells as identified by adhesion culture histology and immunohistochemistry for 7-9 days in chemically defined media containing FGF2. Neural progenitor cells typically comprise at least 72-84% of the total cells. Treatment with dispase yields a highly integrated group of neural precursor cells (preferably at least 95%) by differential adhesion.

In another embodiment, the invention is a cell population comprising at least 72% and preferably 84% neuronal precursor cells. These neural progenitor cells can be identified as being positive for Nestin and Musash I. 1B depicts rosette formation specifying the cells in question. Rosette formation means that the cells are columnar and arranged in a tubular (roset) structure to resemble a neural tube in the body (a developing brain). Columnar cell morphology and tubular structure are shown in the photograph of FIG. 1B.

In another embodiment, the invention is a cell population of at least 90% and preferably at least 96% neural precursor cells. Preferably, the cells are obtained after differential enzymatic treatment and adhesion as described in the Examples below.

5. Use of Cell Population of the Invention

The generation of specific human neuronal cell types with specific transporter phenotypes and unique positional entities is an implantable cell source for treating neurological disorders, for example, midbrain dopamine neurons for Parkinson's disease, whole brain dopamine neurons for mental disorders. And spinal cord motor neurons for motor neuron disease, including spinal cord injury and ALS.

The establishment of a staged and chemically defined culture system for directly differentiating human embryonic stem cells into neural epithelial cells and then to specific neurons also provides an unprecedented system for screening toxic and therapeutic agents. Currently, toxicological and therapeutic drug screening is performed using animal, animal cell cultures or genetically abnormal human cell lines. Its differentiation into human embryonic stem cells and certain neuronal cells represents a normal process for human neural development. Thus, the present invention can be used to screen for agents that affect normal human neurodevelopment or for agents that can stimulate the restoration of nerve types in disease symptoms as well as agents that potentially develop abnormal brains. In addition, the described systems can be readily modified to mimic pathological processes that kill dopamine neurons (eg, Parkinson's disease) or pathological processes that kill motor neurons (eg, ALS), thereby preventing those diseases. It can be used to screen therapeutic agents designed for treatment.

In a preferred method of the invention in this aspect, one of the cell populations of the invention is exposed to the test compound and the results of the exposure are compared with the unexposed control cell population. The nature of the cultures can be investigated and compared to the known developmental properties included herein to understand whether a particular test compound affects the cell population.

Neuroepithelial (neural stem) cell-preferred culture conditions and markers step Cell name Cell-specific markers Culture medium components Culture medium condition Electrophysiological markers Step 1 ES Oct4 +, SSEA4 + Pax6-, Sox1- Irradiated mouse fibroblasts; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercaptoethanol; 4ng / ml FGF2 Daily medium change; 36.5 ° C., 5% CO ₂ N / A Step 1 First dispase treatment N / A 1-2 mg / ml dispase 30 minutes; 37 ° C .; Ambient atmosphere N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercamptoethanol; FGF2 absence 4 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); 25 μg / ml insulin; 100 μg / ml transferrin; 20nM progesterone; 60 μM putrescine; 30 nM sodium selenite; 10-20ng / ml FGF2 2 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Early rosette Oct4-, SSEA4- Pax6 +, Sox1- Nestin + PSA-NCAM- DMDM / F12 (1: 1); 25 μg / ml insulin; 100 μg / ml transferrin; 20nM progesterone; 60 μM putrescine; 30 nM sodium selenite; 10-20ng / ml FGF2 5 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Neural Tubular Rosette Pax 6 +, Sox1 + Nestin +, Bf-1 + DMDM / F12 (1: 1); 25 μg / ml insulin; 100 μg / ml transferrin; 20nM progesterone; 60 μM putrescine; 30 nM sodium selenite; 10-20ng / ml FGF2 3 to 5 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Second dispase treatment N / A 0.1 to 0.2 mg / ml dispase 15 to 20 minutes; 36.5 ° C .; Ambient atmosphere N / A

Midbrain Dopamine Neurons-Preferred Culture Conditions and Markers step Cell name Cell-specific markers Culture medium components Culture medium condition Electrophysiological markers Step 1 ES Oct4 +, SSEA4 + Pax6-, Sox1- Irradiated mouse fibroblasts; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercaptoethanol; 4ng / ml FGF2 Daily medium change; 36.5 ° C., 5% CO ₂ N / A Step 1 ES (first dispase treatment) N / A 1-2 mg / ml dispase 30 minutes; 37 ° C .; Ambient atmosphere N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercamptoethanol; FGF2 absence 4 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-20ng / ml FGF2 2 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Early rosette Oct4-, SSEA4- Pax6 +, Sox1- Nestin + PSA-NCAM- DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-20ng / ml FGF2 5 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Neural Tubular Rosette Pax6 +, Sox1 + Nestin +, En1 +, Pax2 + DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-200ng / ml FGF8 5 or 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Nerve epithelial cells (second dispase treatment) N / A 0.1-0.2mg / ml dispase 15 to 20 minutes; 36.5 ° C .; Ambient atmosphere N / A Step 2 Proliferation of nerve epithelial cells Pax 6 +, Sox1 + Nestin +, En1 +, Pax2 + DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-200ng / ml FGF8 + 50-250ng / ml SHH 5 to 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Midbrain DA Neurons TH +, AADC +, DbH-, PNMT-, EN1 +, Bf-1-, Nurr1 +, ptx3 +, Lmx1b +, VMAT +, DAT +, c-ret +, GABA-, Calvindine-, CCK- Nerve basal medium; N2; 0.1 mM non-essential amino acid; 0.5 mM glutamine; 1 μg / ml laminin; 1 μM cAMP; 200 μM ascorbic acid; 10 ng / ml BDGF; 10 ng / ml GDNF 2-3 weeks; Changing medium once every two days; Culture plate dish; 36.5 ° C., 5% CO ₂ DA secretion, action potential

Spinal Motor Neurons-Preferred Culture Compositions and Markers step Cell name Cell-specific markers Culture medium components Culture medium condition Electrophysiological markers Step 1 ES Oct4 +, SSEA4 + Pax6-, Sox1- Irradiated mouse fibroblasts; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercaptoethanol; 4ng / ml FGF2 Daily medium change; 36.5 ° C., 5% CO ₂ N / A Step 1 ES (first dispase treatment) N / A 1-2 mg / ml dispase 30 minutes; 37 ° C .; Ambient atmosphere N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercamptoethanol; FGF2 absence 4 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-20ng / ml FGF2 2 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Early rosette Oct4-, SSEA4- Pax6 +, Sox1- Nestin + PSA-NCAM- DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-0.01-1M RA 5 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Neural Tubular Rosette Pax6 +, Sox1 + Nestin +, HoxC +, HoxB +, Olig2 +, Otx2-, Bf1-, En1- DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-0.01-1M RA; 10-500ng / ml SHH 5 or 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Nerve epithelial cells (second dispase treatment) N / A 0.1-0.2mg / ml dispase 15 to 20 minutes; 36.5 ° C .; Ambient atmosphere N / A Step 2 Proliferation of Neuroepithelial Cells Pax6 +, Sox1 + Nestin +, HoxC +, HoxB +, Olig2 +, Otx2-, Bf1-, En1- DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 0.01-1 M RA; 50-250ng / ml SHH + 10ng / ml FGF2 5 or 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 1st Harry N / A N / A Accutase (Gibco) N / A Step 2 Spinal Motor Neurons HB9 +, lslet +, Lim3 +, HoxC +, ChAT +, VAChAT + Nerve basal medium; N2; 0.1 mM non-essential amino acid; 0.5 mM glutamine; 1 μg / ml laminin; 50ng / ml SHH; 10 ng / ml BDNF; 10 ng / ml GDNF, 10 ng / ml IFG1 2-3 weeks; Changing medium once every two days; Culture plate dish; 36.5 ° C., 5% CO ₂ Action potential

Whole Brain Dopamine Neurons-Preferred Culture Conditions and Markers step Cell name Cell-specific markers Culture medium components Culture medium condition Electrophysiological markers Step 1 ES Oct4 +, SSEA4 + Pax6-, Sox1- Irradiated mouse fibroblasts; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercaptoethanol; 4ng / ml FGF2 Daily medium change; 36.5 ° C., 5% CO ₂ N / A Step 1 ES (first dispase treatment) N / A 1-2 mg / ml dispase 30 minutes; 37 ° C .; Ambient atmosphere N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); 20% serum substitute; 2 μg / ml heparin; 0.1 mM β-mercamptoethanol; FGF2 absence 4 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 EB Oct4 +, SSEA4 + Pax6-, Sox1- Suspension culture, mouse fibroblasts absent; DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-20ng / ml FGF2 2 days; Changing medium daily; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 1 Early rosette Oct4-, SSEA4- Pax6 +, Sox1- Nestin + PSA-NCAM- DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-20ng / ml FGF2 5 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Neural Tubular Rosette Bf-1 +, Otx2 +, Nestin +, En1 +, Pax2 + 10-20ng / ml FGF2 5 or 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 Nerve epithelial cells (second dispase treatment) N / A 0.1-0.2mg / ml dispase 15 to 20 minutes; 36.5 ° C .; Ambient atmosphere N / A Step 2 Proliferation of nerve epithelial cells Bf-1 +, Otx2 + DMDM / F12 (1: 1); N2 supplement; 2 ng / ml heparin; 10-200ng / ml FGF8 + 50-250ng / ml SHH 5 to 6 days; Changing medium once every two days; 25 cm ² tissue culture flask; 36.5 ° C., 5% CO ₂ N / A Step 2 1st Harry N / A N / A Accutase (Gibco) N / A Step 2 Whole Brain DA Neurons TH +, AADC +, DbH-, PNMT-, Bf-1-, Otx2 +, c-ret +, GABA- Nerve basal medium; N2; 0.1 mM non-essential amino acid; 0.5 mM glutamine; 1 μg / ml laminin; 1 μM cAMP; 200 μM ascorbic acid; 10 ng / ml BDGF; 10 ng / ml GDNF 2-3 weeks; Changing medium once every two days; Culture plate dish; 36.5 ° C, 5% CO ₂ Secretion of DA, Action potential

Example 1 Generation of Whole Brain Dopaminergic Neurons

result

Human ES cells differentiate to form neural tubular structures in the presence of FGF2. Human ES cell lines, H1, H9, and clones H9.2 derived from H9 (Amit, M., et al., Supra, 2000) were propagated on the feeder layer of irradiated mouse embryo fibroblasts. (Thomson, JA, et al., Supra, 1998). To initiate differentiation, ES cell colonies were detached and grown in suspension as embryoid bodies for 4 days. EB was then chemically defined in a medium containing FGF2. Zhang, S.-C., et al., J. Neurosci. Res. 59: 421-429,2000; Zhang, S.-C., et al., Proc. Natl. Acad. Sci. USA 96: 4089-4094, 1999] was cultured in tissue culture treated flasks. FGF2 was purchased from Peprotech, Inc., Rocky Hill, NJ. Five days after incubation in FGF2, the dispensed EB produced overgrown squamous cells. At the same time, an increased number of small long cells appeared in the center of the differentiated EBs (FIG. 1A). Small long cells centered up to 7 days in the identified media formed rosettes (FIG. 1B), which are similar to the early neural tube as shown by the toluidine blue stained portion (photo of FIG. 1B). Immunofluorescence assays showed expression of the neural marker antigens Nestin and Musash-1 (Lendahl, U., et al., Cell 60: 585-595, 1990; Kaneko, Y., et al., Dev. Neurosci. 22: 139-153, 2000] are mostly expressed in squamous cells around EB (Fig. 1C-E) that are localized and differentiated into cells in rosettes. Undifferentiated ES cells are immunonegative for these markers. The formation of neural tubular structures was seen in a large number of EBs (94% in total 350 EBs, 3 separate experiments from H9 and H9.2 weeks) in the presence of FGF2. No well-formed rosette was observed in the absence of FGF2.

Neural tubular rosettes can be separated by differential enzymatic treatment and adhesion. With continued exposure to FGF2, columnar rosette cells proliferated and formed multiple layers. They often make up the majority of the EB and are clearly distinguished from the surrounding squamous cells. Treatment with dispase preferentially detaches the central neuroepithelial islands, leaving most of the surrounding cells attached (FIG. 1F). Contaminated single cells were separated by short-term adhesion into cell culture dishes. Cell counting was performed immediately after this isolation and integration process showed that cells associated with isolated neuroepithelial clusters accounted for 72-84% of cells in differentiated EB cultures. Immunohistochemical analysis showed 96 ± 0.6% of isolated rosette cells stained positive for Nestin based on 13,324 cells examined in four separate experiments. The majority of these cells were also positive for Musash-1 and PSA-NCAM (FIG. 1G, H, I).

Human ES cell derived neural precursors produce all three CNS cell types in vitro. The isolated neural precursors were propagated in suspension cultures similar to "neural bulb" cultures derived from human fetal brain tissue as free-flowing cell aggregates. Zhang, S.-C., et al., Supra, 2000; Svendsen, CN, et al., Supra, 1996; Carpenter, MK, et al., Supra, 1999; Vescovi, AL, et al., Supra, 1999]. BrdU incorporation studies revealed that precursor cell proliferation stimulation was dependent on FGF2 and could not be induced by EGF or UIF alone. In addition, when FGF2 was combined with EGF and / or LIF (FIG. 2A), no additional or synergistic effects were observed.

In vitro differentiation of ES cell derived neural precursors was induced by recovering FGF2 and dispensing on ornithine and laminin substrates. Within a few days, individual cells and a number of growth cones protrude from the sphere to show a star explosion appearance. Dissipation from the spheres by 7-10 days after dispensing produced significant fiber bundles. Frequently, small migration cells were observed in association with the fibers (FIG. 2B). Immunofluorescence analysis of differentiated cultures revealed that the majority of cells in the overgrowth region expressed neuron markers MAP2ab and β _III -tubulin (FIG. 2C). Expression of low molecular weight neurofilament (NF) and high molecular weight NF was observed until 7 days to 10 days and 10 days to 14 days after dispensing (FIG. 2D). Antibodies to various neurotransmitters were further used to characterize ES cell derived neurons. Many neurons showed a glutamate phenotype (FIG. 2E), while smaller proportions were labeled with antibodies to GABA. Frequently, these neurons showed a polar form (FIG. 2F). A few neurons were shown to express TH (FIG. 2G), a rate-limiting enzyme for dopamine synthesis. GFAP ⁺ astrocytes were rarely observed within the first 2 weeks after growth factor recovery, but were more common after prolonged in vitro differentiation. By 4 weeks, they formed a broad layer under differentiated neurons (FIG. 2D). Rare oligodendrocytes were not observed under standard culture conditions, but several O4-immunoreactive cells with typical oligodendrocyte morphology were observed when cultured for more than two weeks in the presence of platelet derived growth factor A (FIG. 2H). . Thus, ES cell derived neural precursors produce all three major types of CNS.

Human ES cell derived neural precursors migrate, incorporate and differentiate in vitro. To assess the differentiation of human ES cell derived neural precursors in vivo, we transplanted them into the lateral ventricles of newly born mice. Flax, JD, et al., Nat. Biotech. 16: 1033-1039,1998. The transplanted cells form clusters in various regions of the ventricular system and are incorporated in large numbers in various host brain regions. Of the 22 brains analyzed between 1 and 4 weeks after transplantation, intraventricular clusters and incorporated cells were found in 19 and 18 recipient brains, respectively. Each animal analyzed after a long time showed that the transplanted cells can be detected for at least 8 weeks after transplantation. Intracluster cells showed strong immunoreactivity against antibodies to nestin, β _III -tubulin and MAP2ab. Only a few cells in the aggregate expressed GFAP. Alkaline phosphatase and cytokeratin, markers typically expressed in undifferentiated ES cells and non-neural epithelial cells, were not detected in this cluster. No teratoma formation was observed.

DNA in situ hybridization using human specific probes and immunohistochemical detection of human nuclear specific antigens revealed the presence of cells transplanted into multiple brain regions. Gray matter regions showing extensive donor cell incorporation include cortex (FIG. 3A), hippocampus (FIG. 3B, C), olfactory bulb, septum (FIG. 3D), hypothalamus, hypothalamus (FIG. 3E), progenitor (FIG. 3F) and midbrain (FIG. 3G). Incorporation into the white matter region is most evident in the corpus callosum, stromal fibrous membrane, and hippocampal fibrous track. Morphologically, incorporated human cells are indistinguishable from surrounding host cells and can only be detected using human specific markers (FIG. 3). Double labeling with cell type specific antibodies revealed that the incorporated cells differentiate into both neurons and glial cells. Human ES cell-derived neurons can be clearly depicted using antibodies against β _III -tubulin and MAP2ab (FIG. 3H, J). Often, they exhibit a polar form with a long process (FIG. 3H). In addition, neurons with multipolar and immature monopolar forms were found (FIG. 3J). Donor-derived neurons produced a number of axons that distantly protrude into the host brain, detected in both gray and white matter. They are particularly abundant in fiber tracks such as cerebral pulmonary, predrill and hippocampus, which can often be traced at several hundred micrometers in a single portion (Figure 3I).

In addition to neurons, few ES cell derived astrocytes were detected in host brain tissue. They exhibit radial morphology and strongly express GFAP (FIG. 3K). In contrast, double labeling of human cells incorporated with antibodies to myelin proteins failed to detect mature oligodendrocytes. Several donor cells that migrated to the host brain had a Nestin positive phenotype up to 4 weeks after transplantation. Many of these cells have been found in locations around blood vessels.

debate

The study indicates that implantable neural precursors capable of producing mature neurons and glia can be produced in high yield from human ES cells. Using growth factor mediated proliferation / differentiation and differential adhesion of neural progenitor cells, the in vitro differentiation process described herein provides a new step toward the development of donor cells for neurodevelopment studies and nervous system repair.

The main finding of this study is the observation that differentiation of neural precursors from human ES cells repeats the early stages of nervous system development with neural tubular structure formation in vitro. This phenomenon can be used to study and experimentally manipulate the early stages of human neural development under current controlled conditions. Chemically defined culture systems offer the only opportunity to use the effect of a single factor on human neuroepithelial proliferation and detail in vitro. Similar to precursors derived from developing human brains, human ES cell derived precursors show a strong proliferative response to FGF2 (Flax, J. D., et al., Supra, 1998). However, no additional or synergistic effects on proliferation can be caused by EGF or LIF. This finding is different from the data obtained with primary cells [Zhang, S.-C., et al., Supra, 2000; Svendsen, C. N., et al., Supra, 1996; Carpenter, M. K., et al., Supra, 1999; Vescovi, A. L., et al., Supra, 1999] It may be suggested that proliferating ES cell derived neural precursors exhibit immature stages than precursor cells derived from the fetal human brain. Practically, studies on rodent cells indicate that neural stem cells isolated from early neurogenesis rely on FGF2 for proliferation and that responses to EGF are obtained in later stages of multidose neural progenitor cell differentiation. Kalyani , AD, et al., Dev. Bioi. 186: 202-223, 1997; Fricker, R. A., et ai., J. Neurosci. 19: 5990-6005, 1999].

The potential for separating these structures based on in vitro production of neural tubular structures and their differential adhesion provides a simple but efficient method for generating high purity of human ES cell derived neural precursors. In particular, strong cell-cell contact in the construct and very low adhesion to tissue culture substrates allows for the isolation of selective neurons without significant contamination of undifferentiated ES cells or cells of another somatic lineage. The high efficiency of this process is reflected by the fact that at least 95% of the isolated cells exhibit a Nestin positive phenotype and no ES cell or non-neural epithelium can be detected at the transplanted receptor. Since precursors to undifferentiated ES cells and other lineages can form tumors and external tissues, the generation of high-purity somatic cell populations is a major prerequisite for developing ES cell-based neuronal transplant strategies.

After transplantation into neonatal mouse brain, ES cell derived neural precursors are incorporated into various brain regions where they differentiate into neurons and glial cells. Failure to detect mature oligodendrocytes in vivo may be due to low efficiency of oligodendrocyte differentiation as opposed to rodents [Svendsen, C. N., et al., Brain Pathol. 9: 499-513, 1999]. Notably, donor derived neurons are not limited to sites showing postnatal neuronal development but are also found in many other areas of the brain. Similar data were obtained in studies involving transplantation of GNS derived precursors into adult rodent brains. Tropepe, V., et al., Dev. Biol. 208: 166-188, 1999. The incorporation of each precursor cell into the brain region after mitosis is particularly associated with cell replacement in the adult brain and spinal column. More detailed studies are needed to determine how much of the incorporated cells acquire region specific properties and how functionally they are active.

No space occupying lesions were detected in the host brain except for intraventricular clusters of mature and immature neuroblasts. Most obviously, no teratoma formation was observed during the postoperative period of less than eight weeks. In particular, although it is clear that stronger long-term stability studies are required before considering potential clinical applications in non-human primates, the data of the present invention suggest that neural precursors isolated from differentiating human ES cell cultures are promising for neural repair. Point out the donor source.

Experimental protocol

Culture of ES Cells. ES cells, H1 (passage 16-33), H9 (passage 34-55) and H9-derived clones H9.2 (passage 34-46) (see Amit, M., et. al., supra, 2000) were cultured on the permanent feed layer of irradiated mouse embryo fibroblasts and Dulbecco's Modified Eagle's Medium (DMEM) / F12, 20% Serum Substitute (Gibco), 0.1 mM β as previously described. -Medium (Thomson, JA, et al., Supra, 1998) consisting of mercaptoethanol, 2 μg / ml heparin and 4 ng / ml FGF2 (PeproTech Inc, Rochy Hill, NJ) is exchanged daily. Karyotyping indicated that the cell line is a drainage at a given passage.

Differentiation Culture of ES Cells. ES cell cultures were incubated with dispase at 37 ° C. for 30 minutes and ES cell colonies were intact. ES cell colonies were pelleted and suspended in ES cell medium without FGF2 and incubated for 4 days in 25 cm ² tissue culture flasks (Nunc) with daily medium change. ES cell colonies grew as floating EB, while any residual feeder cells were attached to the flask. Feeder cells were removed by transferring EB to a new flask. EB (-50 / flask) followed by insulin (25 μg / ml), transferrin (100 μg / ml), progesterone (20 nM), putrescine (60 μM), sodium selenite (30 nM) and heparin (2 μg / ml ) Was dispensed into 25-cm ² tissue culture flasks (Nunc) in DMEM / F12 supplemented in the presence of FGF2 (20 ng / ml). Zhang, S.-C., et al., Supra, 2000; Zhang, S.-C., et al., Supra, 1999].

Isolation and Culture of Neural Precursor Cells: To separate rosette cell clusters from surrounding squamous cells, the cultures were incubated with 0.1 mg / ml dispase at 37 ° C. for 15-20 minutes. Rosette Klump retreats, while surrounding squamous cells remain adhered. At this point, the rosette clump is shaken off the flask and the squamous cells are still attached. The clumps are pelleted and gently ground with a 5 ml pipette and dispensed onto the culture flask for 30 minutes to allow for attachment of each contaminating cell. The floating rosette clumps were then transferred to a new flask coated with poly- (2-hydroxyethyl-methacrylate) that inhibited adhesion and incubated in the medium used for human neural precursors in the presence of FGF2 (20 ng / ml). See Zhang, S.-C., et al., Supra, 2000. To quantify the efficiency of neural differentiation and separation, freshly isolated cell clusters and remaining squamous cells were dissociated with trypsin (0.025% in 0.1% EDTA) and counted. The percentage of putative neural precursors (roset cells) among the total cells differentiated from ES cells was calculated based on three independent experiments on H9 and H9.2 cell lines. To analyze the differentiation potential of ES cell-derived neural precursors, cells were harvested in medium consisting of DMEM / 12, N2 supplement (Gibco), cAMP (100 ng / ml) and BDNF (10 ng / ml, PeproTech) without FGF2. Cultured on chitin / laminin substrate. For oligodendrocyte differentiation, ES cell derived neural precursors are described by N1 (Gibco) and platelet derived growth factors as described in Zhang, -S.-C., et al., Supra, 2000. Cultured in DMEM supplemented with A (PDGFA) (2 ng / ml). Morphological observations and immunostaining with markers for precursors and more mature neurons were performed during differentiation.

Histochemical and immunohistochemical staining. To better visualize rosette formation, the cultures with rosettes are washed with PBS and immobilized in 4% paraformaldehyde and 0.25% glutaraldehyde for 1 hour. Immobilized cells were then embedded and processed in plastic resins as described in Zhang, -S.-C., et al., Supra, 1999. The cultured cells were then cut to 1 μm thick and stained with toluidine blue. For immunostaining, coverslip cultures are described in Zhang, S.-C., et al., Supra, 2000; Zhang, S.-C., et al., Supra, 1999. The following primary antibodies detected by suitable fluorescent secondary antibodies described in detail: anti-nestine (polyclonal, gift of Dr. R. McKay of NINDS, 1: 1,000); Anti-polysialized neuronal cell adhesion molecules (PSA-NCAM, mouse IgM, donated from G. Rougon, University of Marseille, France, 1: 200); Anti-Musash-1 (rat IgG, donated from Dr. H. Okano, University of Tokyo, Japan, 1: 500); Anti-GFAP (polyclonal, Dako, 1: 1,000); Anti-human GFAP (Sternberg monoclonals, 1: 10,000); 04 (mouse IgM, hybridoma supernatant, 1: 50); Immunostaining using anti-tyrosine hydroxylase (TH, Pel freeze, 1: 500). The remaining antibodies were purchased from Sigma: anti-ßx tubulin (mouse IgG, 1: 500); Anti-neural filament (NF) 68 (mouse IgG, 1: 1,000); Anti-NF 200 (polyclonal, 1: 5,000); Anti- MAP2ab (mouse IgG, 1: 250); Anti-y-aminobutyric acid (GABA, polyclonal, 1: 10,000); Anti-glutamate (mouse IgG, 1: 10,000). For bromodeoxyuridine (BrdU) incorporation, 4 coverslip cultures in each group were immobilized in 4% paraformaldehyde, denatured with 1N HCl, 2 μM for 16 hours before processing for immunolabeling and cell counting. Incubated with BrdU of Zhang, S.-C., et al., Supra, 2000; Zhang, S.-C., et al., Supra, 1999].

Cerebral ventricular transplantation and in vivo analysis. A suspension of 100,00 viable cells / μL was prepared in L15 medium (Gibco) after dissociating aggregates of neural precursors with trypsin (0.025% in 0.1% EDTA for 5-10 minutes at 37 ° C.). 2-3 microliters of cell suspension were slowly injected into each of the outer ventricles of cryoanized newborn mice (C3HeB / FeJ) using sub-head illumination. Implanted animals are immunosuppressed by daily injection of cyclosporin A (10 mg / kg, ip). One, two, four and eight weeks after transplantation, mice were perfused with Ringer's solution followed by 4% paraformaldehyde. The brain is dissected and immobilized in the same fixative at 4 ° C. until use. Donor cells were digested with human alu repeat elements using a digoxigenin labeled probe (Brustle, O., et al., Nat. Biotech. 16: 1040-1044, 1998] or by 50 μm coronal vibratom incision by in situ hybridization or using antibodies against human specific nuclear antigens (MAB1281, Chemicon, 1:50). It was. Immunopositive cells include GFAP (1: 100), Nestin, β _III -tubulin (TUJ1, BabCo, 1: 500), MAP2ab (Sigma, clones AP-20 and HM-2, 1: 300) and phosphorylated medium Double labeled with antibody to molecular weight human neurofilament (clone HO-14, 1:50, donated by J. Trojanowski). Primary antibodies were detected with appropriate fluorophore conjugated secondary antibodies. Incisions were analyzed on a microscope (Zeiss Axioskop 2 and Leica laser scan microscopes).

Example 2- Generation of Midbrain Dopaminergic Neurons

The first step for potential applications of stem cell therapy in neurological symptoms is the direct differentiation of neurons with the correct positional and transporter phenotype of neurons. Here we show the vigorous production of functional dopaminergic (DA) neurons from human embryonic stem (ES) cells through a specific sequence of morphogen action. Treatment of human ES-derived neuroectodermal cells with FGF8 at an early stage, prior to Sox1 expression, is essential for the specialization of DA neurons with the correct midbrain DA protruding neuron phenotype. In vitro generated DA neurons can be used for toxicological and pharmaceutical screening and potential cell therapy in Parkinson's disease.

Parkinson's disease (PD) results from the progressive degeneration of DA neurons in the midbrain, especially in melanoma. Current therapies for PD rely primarily on symptomatic relief by systemic administration of DA precursors such as levadopa. This treatment is effective for the first few years but almost permanently loses its efficacy and causes trilogy. Administration of growth factors such as glial cell line derived neurogenic factor (GDNF) has been shown to be effective in small clinical trials. Gill, S. S., et al., Nat. Med. 9: 589-595, 2003. Such treatment relies on a sufficient number of surviving DA neurons and their long-term therapeutic potential remains a challenge. Because of the pathological nature of neurodegeneration, cell transplantation has been proposed as another treatment. Bjorklund, A. and Lindvall, O., Nat. Neurosci. 3: 537-544, 2000]. In some successful cases, transplanted fetal midbrain cells survive for more than 10 years and contribute to alleviation of symptoms [Kowdower, J. H., et al., N. EnL J. Med. 332: 1118-1124, 1995; Piccini, P., et al., Nat. Neurosci. 2: 1137-1140, 1999] However, recently controlled clinical trials doubt the efficacy of fetal tissue transplantation therapy for PD [Freed, C. R., et al., N. Engl. J. Med. 344: 710-719, 2001; Olanow, C. W., et al., Ann. Neurol. 54: 403-414, 2003. These phenomena point to the complex tendency of PD. A reliable source of new functional human midbrain DA neurons is urgently needed for system studies on the development of DA systems, which are pathogenic processes that affect the survival and function of DA neurons, and for the development of long-term therapies for PD.

It has been found that DA neurons can be produced efficiently from mouse ES cells derived from the inner cell mass of pre-transplanted embryos at the blastocyst stage. Evans, MJ and Kaufman, MH, Nature 292: 154- 156,1981; Martin, GR, Proc. Natl. Acad. Sci. USA 78: 7634-7638,1981. Mouse ES cells are first described by FGF2 [Lee, SH, et al., Nat. Biotechnol. 18: 675-679, 2000] or by inducing activity derived from stromal cells [Kawasaki, H., et ai., Neuron 28: 31-40,2000; Barberi, T., et ai., Nat. Biotechnol. 21: 1200-1207, 2003] to neuroectodermal cells. Neuroectodermal cells are then exposed to FGF8 followed by SHH for DA neuron induction. In this study, we induced human ES cells (Thomson, JA, et al., Science 282: 1145-1147,1998) to identify a number of DA neurons with midbrain protrusion properties in response to FGF8 and SHH. To differentiate into the resulting neuroectodermal cells. Zhang, SC, et al., Nat. Biotechnol. 19: 1129-1133, 2001] A strong system was built. The inventors have shown that in order to generate DA neurons with the midbrain pluripotent neuron phenotype, human ES cells must be exposed to FGF8 before the precursor cells become neuroectodermal cells expressing Sox1 ⁺ .

result

Human ES derived neuroectodermal cells exhibit whole brain properties.

ES cell colonies detached from the nutrient supply layer are grown in suspension as aggregates for 4 days in ES cell growth medium and then grown in adherent culture dishes in chemically defined neuronal medium containing FGF2 (20 ng / ml). : Zhang, SC, et al., Supra, 2001]. The cells in the center of the colony form a circumferential form and form a rosette on day 9 (FIG. 4A). These columnar cells are positive for Pax6 but negative for pan-neuronal transcription factor Sox1 (not shown) and indicate that they are early neuroectodermal cells. For 5 or 6 additional days (14-15 days) the circumferential cells proliferate and consist of neural tubular rosettes (FIG. 4B) and express Sox1 (FIG. 4C), and transcription factors are restricted to neuroectodermal cells during neural tube closure. Expressed in Pevny, LH, et al., Development 125: 1967-1978,1998. They are positive for brain factor (Bf1), a transcription factor expressed by forebrain cells (Tao, W. and Lai, E., Neuron 8: 057-966,1992), but are transcription factors expressed by midbrain cells. Negative for human jagged 1 (En-1) (FIG. 4D), Davidson, D., et al., Development 104: 305-316,1988; Wurst, W., et al., Development 120: 2065-2075, 1994] This suggests that the in vitro whole brain produces neuroectodermal cells.

Induction of the midbrain phenotype requires early action of FGF8.

For differentiation into DA neurons, neuroectodermal cells in neural tubular rosettes were accumulated via differential enzyme and adhesion treatment (Zhang, SC, et al., Supra, 2001) and as agglutinates in suspension with FGF2 for 4 days. Growth was followed by dispensing onto laminin substrate and treated with SHH (50-200 ng / ml) and FGF8 (20-100 ng / ml) for 6 days. Immunocytochemical analysis revealed that the majority of neuroectodermal cells remained positive for Bf1 but not for En-1 (not shown) .

The failure of FGF8 to induce Sox1 ⁺ neuroectodermal cells to express En-1 suggests that Sox1 expressing neuroectodermal cells may be resistant to patterned signals. Since Sox1 expressing cells are produced two weeks after differentiation of human ES cells (same as 6-day embryonic bodies (Thomson, JA, et al., Supra, 1998)) and form neural tubular structures, they are neuroectodermal cells May correspond to neuroectodermal cells in neural tube occlusion that express Sox1 and are localized (Lumsden, A. and Krumlauf, R., Science 274: 1109-1115, 1996). This led us to make the assumption that FGF8 can promote midbrain specification before neuroectodermal cells express Sox1. Therefore, the present inventors applied FGF8 (100 ng / ml) at that time when the cells became columnar on the ninth day in the center of the colony. After 6 days, cells in the center of the colony produced neural tubular formation as shown in the presence of FGF2. These neuroectodermal cells similarly accumulate and proliferate in FGF8 for 4 days and then with SHH for 6 days on laminin substrate. Under these culture conditions, En-1 expression was observed in nestin expressing neuroectodermal cells (FIG. 4E) but there are still cells still expressing Bf1 (FIG. 4F). Thus, neuroectodermal cells are efficiently localized before they become Sox1 ⁺ .

Localized neuroectodermal cells differentiate into DA neurons.

Neuroectodermal cells dissociated and differentiated in neural differentiation medium. They did not express stage specific embryonic antigen 4 (SSEA4), a glycoprotein that is highly expressed by undifferentiated human ES cells. Disassembled neuroectodermal cells initially distributed evenly but formed rosettes for 3 to 4 days after dispensing. Three weeks after differentiation, about one third of the total differentiated cell population (31.8 ± 3.1% TH ⁺ cells in 17,965 cells counted from four experiments) was positive for tyrosine hydroxylase (TH) (FIG. 5A). . Similar percentages of TH ⁺ cells were obtained from H9 and H1 human ES cell lines. Most TH expressing cells are 10-20 μm in diameter. They exhibit a multipolar form and have differentiateable axons and dendrites (FIG. 5A). All TH ⁺ cells were stained positive with neuron marker β _III -tubulin ⁺ neurons and about 50% TH ⁺ [FIG. 5B, 6,383 TH ⁺ cells in 12,859 β _III -tubulin ⁺ neurons from 4 experiments] .

In the biosynthesis of monoamines, TH hydroxylates tyrosine to L-DOPA which is then decarboxylated by AADC and becomes DA. Two other enzymes, DβH and phenylethanolamine N-methyltransferase (PNMT), respectively convert DA into norepinephrine and catalyze norepinephrine to epinephrine. Immunostaining shows that all TH ⁺ cells are AADC (FIG. 5C-E) but some AADC ⁺ cells are negative for TH (FIG. 5E). However, TH ⁺ cells are negative for DβH (FIG. 5F) and PNMT (not shown) while DβH strongly stained noradrenergic neurons in adult rats and in embryonic monkey brain stem (photo in FIG. 5F). These data show that TH expressing neurons possess the two enzymes required for dopamine biosynthesis, which neurons are rather DA neurons rather than noradrenaline neurons or adrenergic neurons.

ES cell generated DA neurons exhibit midbrain phenotype.

RT-PCR analysis showed that Nurr1, Limx1b, En-1 and Ptx3 are involved in the development of midbrain DA neurons [see Zetterstrom, RH, et al., Science 276: 248-250,1997; Smidt, MP, et al., Proc. Natl. Acad. Sci. USA 94: 13305-13310, 1997; Saucedo-Cardenas, O., et al., Proc. Natl. Acad. Sci. USA 95: 4013-4018, 1998; Wallen, A., et al., Exp. Cell Res. 253: 737-746, 1999; Smidt, MP, et ai., Nat. Neurosci. 3: 337-341,2000; Simon, HH, et al., J. Neurosci. 21: 3126-3134,2001; Van den Munckhof, P., et al., Development 130: 2535-2542,2003; Nunes, I., et ai., Proc. Nati. Acad. Sci. USA 100: 4245-4250, 2003] indicated that neuroectodermal cells are not expressed at high levels until they differentiate into DA neurons (FIG. 6A). Immunostaining revealed that most TH ⁺ cells with multiple processes coexpressed the midbrain marker En-1 in the nucleus (FIG. 6B). Thus, DA neurons generated using this method possess midbrain positional entities.

DA neurons in olfactory bulbs frequently co-express γ-aminobutyric acid (GABA). Kosaka, T., et al., Exp. Brain Res. 66: 191-210, 1987; Gall, CM, et al., J. Comp. Neurol. 266: 307-318, 1987. Double immunostaining of TH and GABA indicated that most of the DA neurons were negative for GABA but GABA ⁺ neurons were found in culture (FIG. 6C). Of all TH ⁺ cells, 8% of TH ⁺ cells (8.7 ± 3.9%, 6,520TH ⁺ cells counted from four experiments) co-expressed GABA. Most of these double positive cells are small bipolar cells (photo in FIG. 6C). Several midbrain DA neurons, particularly neurons in the ventral overlying region, doi-express the cholecystokinin octapeptide (CCK8) or calvindine along with TH. McRitchie, DA, et al., J. Comp. Neurol. 364: 121-150, 1996; Hokfelt, T., et al., Neurosci. 5: 2093-2124, 1980]. Immunohistochemical analysis indicated that TH ⁺ neurons were observed (FIG. 6D). The calvindine neurons are mostly small cells. No CCK8 positive cells were detected in the culture.

ES cell-generated DA neurons are biologically functional

Immunostaining showed that all TH ⁺ expresses c-Ret, a component of the receptor for GDNF (FIGS. 7A-C). Many TH ⁺ cells, especially those with side chain neurites, expressed porous monoamine transporter 2 (VMAT2, FIG. 7D-F) involved in packaging dopamine into blast compartments in monoamine neurons. Nirenberg, MJ, et al., J. Neurosci. 16: 4135-4145, 1996. In addition, TH ⁺ neurons expressed synaptopycin, a membrane glycoprotein essential for synapse formation. See Calakos, N. and Scheller, RH, J. Biol. Chem. 269: 24534-24537, 1994] (FIG. 7A-I).

Dopamine release is a functional feature of DA neurons. High performance liquid chromatography (HPLC) analysis shows 230.8 ± 44.0 pg / ml in ascorbic acid (AA), FGF8 and SHH treated cultures, 46.3 ± 9.2pg in control cultures without AA, FGF8 and SHH treated. Dopamine was present in DA differentiation culture medium as / ml (FIG. 8A). When cultured cells were washed and incubated in HBSS for 14 minutes, dopamine levels were similar between the two cultures (FIG. 8A). However, depolarization of cultured neurons with 55 mM KCl in HBSS significantly increased the amount of DA (35.8 ± 9.2 and 111.0 ± 15.0 pg / ml in the absence and presence of AA, FGF8 and SHH treatment; FIG. 8A). These observations suggest that DA neurons produced in vitro can secrete DA and the release of DA is activity dependent.

Electrophysiological records were used to determine whether the ES produced DNA neurons were functionally active. In cells continued to be cultured for 30 to 38 days (n = 14), the resting membrane potential (V _{resting period} ) ranges from 32 to -72 mV (-54 ± 2.9 mV) and the cell capacitance (Cm) ranges from 11 to 45 pF (21). ± 2.7 pF) and the input resistance (R _in ) ranges from 480 to 3500 MQ (1506 ± 282 MΩ). Depolarization current steps (0.2 nA x 200-500 ms) generally result in a single action potential but in many cases it was observed that the action potential continues to decrease (Figures 8bi and ii). The threshold of action potentials (AP) ranges from -26 to -5.2 mV (-17.4 ± 2.1 mV) and peaks at -9.6 to 30 mV. APs below 50.2 mV were observed (32 ± 2.8 mV). The AP duration ranges from 3 to 20.6 ms (7.2 ± 1.3 ms). Spontaneous initiation 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 amplification varied considerably. Inward currents are rapidly activated (<1 ms) and peak within 1-3 ms. The activation threshold was -30 ± 1 mV and the maximum peak current amplitude was obtained at an average voltage of -13 ± 1.9 mV and the current was completely blocked by Tetrodotoxin (TTX, n = 3). These properties are consistent with the presence of voltage-gated sodium channels. In three cells, we observed spontaneous transient currents characterized by synaptic currents, including rapid rise and slower decreases. One of these recordings was performed using a K-gluconate base pipette solution and by keeping the cells at -40 mV, we can observe both outward (inhibitory) and inward (stimulatory) currents (FIGS. 8di and dii). . Biocithin was injected into all 14 cells, but only 5 cells recovered after the completion of the immunostaining process. However, all five biocithin filled cells were labeled TH (FIG. 8E-G).

debate

Herein, we present a functional DA neuron with midbrain neuronal protrusion properties in three simple non-genetic steps: induction of neuroectodermal cells using FGF2, specification of ventricular midbrain entities by FGF8 and SHH during neuroectodermal formation, and locally. It has been demonstrated that differentiation of specialized precursors into DA neurons can be produced efficiently from human ES cells. Unlike the findings obtained from mouse ES cell studies that DA neurons with midbrain characteristics can be produced from proliferated neuroectodermal cells (Lee, SH, et al., Supra, 2000), the inventors have found that precursor cells Characterization or localization with FGF8 before becoming Sox1 ⁺ neuroectodermal cells has been shown to be essential for the vigorous generation of human DA neurons with the correct midbrain and functional phenotype.

From a stem cell biology perspective, we associate mouse ES cells with neuroectodermal cells and propagate them and localize or characterize them with FGF8 and SHH and then transfer them to DA neurons using a stepwise protocol developed by McKay and colleagues. Differentiation seems to be very logical (Lee, SH, et al., Supra, 2000). We have assumed that the same principle should apply to human primates. Indeed, we differentiate human ES cells into neuroectodermal cells that express Sox1 in the presence of FGF2 and constitute a neural tubular rosette (Zhang, SC, et al., Supra, 2001). Can be treated with FGF8 and SHH to induce ventral midbrain fate and finally differentiate cells into neurons to generate multiple DA neurons. However, most of the DA neurons produced in this manner lack some of the major properties of midbrain protruding DA neurons, for example, the expression of midbrain transcription factors at large size and protein levels with complex morphology. Sox1 positive neuroectodermal cells are still negative for En-1 but positive for Bf1 after treatment with FGF8 and SHH, suggesting that Sox1 expressing neuroectodermal cells are non-responsive for specification to midbrain fat. The differentiation process of neuroectodermal from human ES cells in the culture system of the present invention is parallel to that seen during in vivo development (Zhang, SC, supra, 2003). In vivo, neural tubes are formed at the end of the third week of human gestation and Sox1 is expressed by neuroectodermals during neural occlusion based on mouse embryo studies. Pevny, LH, et al., Development 125: 1967-1978 , 1998]. In culture, neuroectodermal cells form neural tubular rosettes and express Sox1 two weeks after differentiation from the same human ES cells as 6-day-old human embryos (Thomson, JA, et al., Supra, 1998). Protruding neurons, including midbrain DA neurons, differentiate from neuroectodermal cells in neural tube tubes at an early stage and these neuroectodermal cells are already localized during the neural tube closure process. Lumsden, A. and Krumlauf, R., supra, 1996] This may explain why human ES cell generated Sox1 expressing neuroectodermal cells with a whole brain phenotype do not respond to norogens for producing DA neurons with a midbrain phenotype. The assumption that FGF8 can direct early precursors to use midbrain entities suggests that proliferating neurons, such as large cell bodies, with complex processes and expression of the midbrain marker En1 after Sox1 ^- columnar cells are treated with FGF8 in early rosettes. Confirmed by the generation of DA neurons with characteristics.

Currently, it is not clear why, unlike human ES cell derived neuroectodermal cells, FGF2-derived mouse ES cell derived neuroectodermal cells can be efficiently localized after proliferation. There is recent evidence that the dorsal or ventral entity of neural precursors isolated from the mouse spinal column can be deregulated in culture, especially in the presence of FGF2 [Gabay, L., et al., Neuron 40: 485- 499,2003] This may partly explain the ability of proliferated mouse ES derived neuroectodermal cells to be respecific. Our study of the differentiation of other protruding neurons, such as spinal motor neurons, is consistent with our observation that the generation of large protruding neurons requires early action of morphogens.

DA neurons are present in several areas of the brain, including the midbrain, hypothalamus, retina and olfactory bulb. Human ES cell generated DA neurons in this study are similar to midbrain protruding DA neurons. Most DA neurons do not co-express GABA, while co-expression of GABA and TH is a major feature of olfactory DA interneurons. Kosaka, T., et ai., Supra, 18987; Gall, C. M., et al., Supra, 1987]. In the midbrain there are at least two major groups of DS neurons, one in the black matter (A9) and the other in the ventral overlying region (A10), each with a different target. 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 overlying region express Calvindine or CCK, while they are rarely seen in melanoma (McRitchie, D. A., et al., J. Comp. Neurol. 364: 121-150, 1996; Hokfelt, T., et al., Neurosci. 5: 2093-2124, 1980; Haber, S. N., et al., J. Comp. Neurol. 362: 400-410, 1995. Our observation that human ES cell generated DA neurons do not co-express TH with CCK8 or Calvindine suggests that these DA neurons are more similar to melanocyte DNA neurons.

The vigorous ability of human ES cells to generate large protruding neurons with appropriate local entities, such as midbrain DA neurons, opens up an unprecedented opportunity to dissect the early stages of neural development using human ES cell systems. The data of the present invention demonstrate that morphogens, such as FGF8, must act on unspecified early neuroectodermal cells for the generation of early emergent midbrain DA neurons. This may explain why stem cells or precursors that have been isolated and expanded from already specified embryonic and adult mammalian brains have been unresponsive to producing overhanging neurons. See Svendsen, C. N., etai., Exp. Neurol. 148: 135-146,1997; Daadi, M. M. and Weiss, S., J. Neurosci. 19: 4484-4497, 1999; Storch, A., et al., EXp. Neurol. 170: 317-325, 2001. In vitro generated human DA neurons also provide a system for toxicological and pharmaceutical screening for chemicals and drugs that may affect human DA neurons. Studies are ongoing to determine whether these human DA neurons generated in culture plate dishes are functional in the PD animal model.

Way

ES cell culture. Human ES cell lines, H9 (p21-56) and H1 (p35-40), are described in Dulbecco's Modified Eagle Medium (DMEM) / F12 (Gibco) as described in Thomson, JA, et al., Supra, 1998. ), 20% serum substitute (Gibco), 1 mM glutamine (Sigma), 0.1 M non-essential amino acid (Gibco), 2 μg / ml heparin (Sigma), 0.1 mM β-mercaptoethanol (Sigma) and 4 ng ES cell growth medium consisting of / ml of FGF2 (R & D Systems) was propagated weekly on irradiated mouse embryo fibroblasts (MEF) with daily change. Differentiated colonies were physically removed using a curved Paster pipette and the ES cells in undifferentiated state were identified by immunostaining with typical morphology and Oct4 and SSEA4.

Differentiation and accumulation of neuroectodermal cells. Human ES cell colonies are detached from the MEF layer by treatment of the cultures with 0.2 mg / ml of Roche Diagonostics and grown as floating cell aggregates (embryoderm) for 4 days with daily ES cell medium change. They are then grown to an adhesive substrate in a medium of nerve medium consisting of DMEM / F12 (2: 1) supplemented with N2 (Gibco), 0.1 mM non-essential amino acid, and 2 μg / ml heparin. . ES cell aggregates attached and formed respective colonies about 6 days. Neuroectodermal cells, represented by the columnar cells that make up the neural tubular rosette, developed on day 14 (Zhang, SC, et al., Supra, 2001). Neural rosettes were isolated via differential enzymatic reactions (Zhang, SC, et al., Supra, 2001). Growth factors are added during the differentiation process to influence localization (shows results).

DA neuron differentiation. Accumulated neuroectodermal cells were dissociated by 0.025% trypsin and 0.27 mM EDTA in PBS for 10-15 minutes at 37 ° C. and preliminary with 12-mm coverslips (100 μg / ml polyornithine and 10 μg / ml laminin). Coating) at a density of 40,000-50,000 per coverslip. Neuronal differentiation medium is N2, 0.1 mM non-essential amino acid, 0.5 mM glutamine, 1 μg / ml laminin, 1 μM cAMP, 200 μM AA (Sigma), 10 ng / ml BDNF (R & D Systems) and 10 ng / Neuronal basal medium (Gibco) supplemented with ml of GDNF (R & D Systems). The cells were cultured for 3-4 weeks with changing medium once every two days.

Immunocytochemistry and Cell Quantitation. Coverslip cultures were immobilized in 4% paraformaldehyde in PBS for 10-20 minutes or in methanol (-20 ° C.) for 5 minutes and treated for immunostaining. Zhang, SC, et al., supra, 2001]. The following primary antibodies were used: mouse anti-SSEA4 (1:40), mouse anti-En-1 (1:50) and mouse anti-Pax6 (1: 5000, all from developmental study hybridoma banks) ; Rabbit anti-DβH (1: 400), mouse anti-synaptopycin (1: 5000) and rabbit anti-CCK8 (1: 2000, all obtained from Chemicon); Mouse anti-TH (1: 1000), mouse anti-βIII tubulin (1: 5000), rabbit anti-GABA (1: 5000) and mouse anti-calvindine (1: 400, all obtained from Sigma); Rabbit anti-TH (1: 500) and rabbit anti-VMAT2 (1: 500, both obtained from Pel-Freez); Goat anti-c-Ret (1: 400) and mouse anti-Oct4 (1: 1000, both obtained from Santa Cruz); Rabbit anti-Bf1 (1: 5000; donated by Lorenz Studer). Antibody-antigen responses have been found to be suitable fluorescently conjugated secondary antibodies. Cell nuclei were stained with Hoechst 33342. Staining was visualized with a Nikon fluorescence microscope. Brain sections of adult rat and E38 embryonic monkey origin were used as positive controls for many antibodies to nerve types and neurons. Negative controls were also set up by excluding primary or secondary antibodies in the immunostaining process. Cell counts were performed blindly using a mesh pocket on the alternative and 40 × objectives. Cells in the ten visible regions were randomly selected and counted from each coverslip.

RT-PCT

Total RNA was extracted from cultured cells by treatment using RNA Stat-60 (Tel-Test, Friendswood, TX) followed by DNaseI (DNA free, Ambion). Synthesis of cDNA was performed using a superscript single chain synthesis system for RT-PCR (Invitrogen) according to the manufacturer's instructions. PCR amplification was performed using a standard procedure using Taq polymerase (Promega). The number of cycles varies from 25 to 35 cycles depending on the specific abundant mRNA and is denatured at 94 ° C. for 15 seconds, annealed at 55 ° C. or 60 ° C. temperature for 30 seconds depending on the primer and extended at 72 ° C. for 45 seconds. Negative controls were achieved by excluding cDNA samples during reverse transcriptase or PCR. Primers and product lengths are as follows: GAPDH (5′-ACCACAGTCCATGCCATCAC-3 ′, 5′-TCCACCACCCTGTTGCTGTA-3 ′, 450 bp); Nurr1 (5'- CGATGCCTTGTGTTCAGGCGCAG-3 ', 5'-AGCCTTTGCAGCCCTCACAGGTG-3', 858 bp); Ptx3 (5'-GTGGGTGGAGAGGAGAACAA-3 ', 5'-TTCCTCCCTCAGGAAACAATG-3', 175 bp); Lmx1 b (5'-GGGATCGGAAACTGTTACTGC-3 ', 5'-GTAGTCACCCTTGCACAGCA-3', 218 bp); En-1 (5'-CCCTGGTTTCTCTGGGACTT-3 ', 5'-GCAGTCTGTGGGGTCGTATT-3', 162 bp).

DA measurement

21 days after DA neural differentiation, conditioned media was harvested for 48 hours. Activity dependent dopamine release from cultured cells was determined by conditioning the cells incubated in Hank's homogenate solution (HBSS) for 15 minutes, then replacing them with HBSS containing 56 mM KCl for 14 minutes at 37 ° C. Dopamine in culture medium or HBSS was stabilized by the addition of 20 μl stabilization buffer (900 mg EGTA and 700 mg glutathione in 10 ml 0.1 M NaOH) and the samples were stored at -80 ° C. Monoamine was extracted using HPLC kit (Chromsystems). Monoamine levels were measured using HPLC (Model 508 autosampler and Model 118 pump, Beckman) coupled with an electrochemical detector (Coulochem II, ESA Inc.) using an MD-TM mobile phase. Cultures in each group were divided into three and data was collected from three separate experiments.

Electrophysiological record

The electrophysiological properties of DA neurons differentiated from human ES cells were investigated using whole cell patch clamp recording techniques. Hammill, OP, et al., Pflugers Arch. 391: 85-100, 1981. The pipette was filled with an intracellular solution containing (mM) KCl 140 or K-gluconate 140, Na ⁺ -HEPES 10, BAPTA 10, Mg ²⁺ -ATP 4, (pH 7.2, 290mOsm, 2.3-5.0MΩ). . DA neurons were identified by adding biocithin (0.5%, Sigma) to the recording solution and then labeling with antibodies against streptavidin-Alex flu 488 (1: 1000, Molecular Probes) and TH. The bath solution contained NaCl 127, KH ₂ PO ₄ 1.2, KCl 1.9, NaHCO ₃ 26, CaCl ₂ 2.2, MgSO ₄ 1.4, glucose 10, 95% O ₂ /5% CO ₂ (pH 7.3. 300mOsm). . For some embodiments, TTX (1 μm) was applied to the bath solution to block voltage-gated sodium currents.

Current clamp and voltage clamp recordings were performed using a multiclamp 700A amplifier (Axon Instrument). Signals were filtered at 4 kHz, sampled at 10 kHz using a Digidata 1322A analog-to-digital converter (Axon Instruments) and stored on a computer hard disk using commercially available software (pClamp9, Axon Instruments). Access resistance is typically 818 MΩ and compensated by 50-80% using an amplifier circuit. The voltage was corrected for a liquid connection potential of +13 mV (Neher, E., Methods Enzymol, 207: 123-131, 1992). The V _{resting period} and the action potential were investigated in the current clamp mode. Spontaneous stimulation (inward) and suppression (outward) synaptic currents were characterized at voltage clamp mode and V _hold = 40 mV using a K-gluconate base pipette. Synaptic responses were detected using a template detection algorithm (Small Analysis Program 4.6.28, SynaptoSoft) and the inactivation step was adjusted to a double algebraic function using the Levenberg-Marquardt algorithm. Data were given as mean ± SE.

Example 3- Generation of Motor Neurons

The generation of motor neurons in vertebrates involves three or more stages: neurization of ectoderm cells, caudalization of neuroectodermal cells, and embryonicization of caudalized neural precursors. Jessell, TM, Nat. Rev. Genet. 1: 20-29,2000]. We first based on the principle that neuroectodermals of vertebrates develop in response to FGF and / or anti-BMF (bone morphological protein) signals (Wilson, SI and Edlund, T., Nat. Neurosci. 4: Suppl. : 1161-1168, 2001] using adhesive colony cultures in the presence of FGF2. Zhang, SC, et ai., Nat. Biotechnol. 19: 1129-1133, 2001] A culture system for efficient neural envelope differentiation from hES cells (Thomson, JA, et ai., Science 282: 1145-1147, 1998) (H1 and H9) was established. . The first sign of neuronal differentiation was the appearance of columnar cells forming a rosette in the center of colonies 8 to 1 day after ES cells were removed from trophic feed cells for differentiation. The columnar cells in the rosette are not pan-neroderm transcription factor SoxI (FIG. 9A) expressed by neuroepithelial cells during neural tube formation (Pevny, LH, et al., Development 125: 1967-1978, 1998). Expression of the neuroectodermal marker Pax6 but no squamous cells in the overgrowth region. On further incubation in the same medium for 4 or 5 days, the columnar cells formed neural tubular rosettes with lumens (FIG. 9B) and expressed both Pax6 and Sox1 (FIG. 9C, D). Thus, differentiation of neuroectodermal cells from hES cells comprises at least two characteristic steps: Pax6 ⁺ / Sox1 ^- columnar cells in early rosettes on days 8-10 after nerve induction and neural tubular late rosettes on day 14 after induction Pax6 ⁺ / Sox1 ⁺ cells to form.

Immunocytochemical analysis showed that Rosette cells expressing Pax6 (FIG. 9E), Sox1 and Nestin were positive for Otx2 (FIG. 9F, H), a homeodomain protein expressed by forebrain and midbrain cells, but not by intrathecal cells. It is negative for HoxC8 (FIG. 9H) which is the homeodomain protein produced. They are also negative for En1 expressed by midbrain cells (FIG. 9G). These results indicate that neuroectodermal cells possess a forebrain phenotype similar to that initially obtained by neuroectodermal cells during early in vivo development (Stem, DC, Nat. Rev. Neurosci. 2: 92-98, 2001). Suggests.

In order to differentiate motor neurons from neuroectodermal cells, Sox1 ⁺ neuroectodermal cells in neural tubular rosettes were isolated via enzymatic treatment, see Zhang, SC, et al., Nat. Biotechnol. 19: 1129-1133, 2001] retinoic acid (RA, 0.001-1 μM), caudalization reagents (Blumberg, B., et al., Development 124: 373-379,1997) and SHH (50-500 ng) / ml), embryonic morphogen (see Jessell, TM, Nat. Rev. Genet. 1: 20-29, 2000; Briscoe, J. and Ericson, J., Curr. Opin. Neurobiol. 11: 43 -49, 2001. On the laminin substrate in the presence of up to 14 days after dispensing, a number of cells in the overgrowth region networked through their processes (FIG. 10A). Is positive for neuronal markers β _III -tubulin and MAP2, most of them (> 50%) are also positive for Isl1 (FIG. 10A) and Lim3 (not shown), which are transcription factors associated with motor neuron development [ See Jessell, TM, supra; Briscoe, J. and Ericson, J., supra, 2001; Shirasaki, R. and Pfaff, SL, Annu. Rev. Neurosci. 25: 251-281, 2002. However, 1 Very in culture between 2-3 weeks The small motor neuron cell-specific transcription factors. [Reference:. Arber, S., et al , Neuron 23: 659-674, 1999] was expressed in HB9 (Figure 10A) which Sox1 ⁺ neuroectodermal cell movement Suggests that there may be no response to neuronal induction.

Sox-1 expressing cells may correspond to neuroectodermal cells in the neural tube when neural tubular rosettes are formed and Sox1 is expressed at the same time point as the three week human embryo. Neuroectodermal cells in the neural tube are localized (Lumsden, A. and Krumlauf, R., Science 274: 1109-1115,1996). This consideration allowed us to assume that RA can promote caudalization and / or motor neuron characterization before neuroectodermal cells express Sox1. We therefore treated neuroectodermal cells with RA (0.001-1 μM) at an early stage, ie when the columnar cells began to make up the rosette and expressed Pax6. Cultures treated in this manner for 6 days developed neural tubular rosettes and expressed Sox1, indistinguishable from FGF2 treated culture origin. After detaching the rosette clusters and adhering with laminin substrates, a number of progenitor cells were extended from the clusters as early as 24 to 58 hours after dispensing. By 14 days after dispensing, dendritic cell overgrowth areas covered the entire (11-mm diameter) coverslip, although there was a limited number of neuronal cell bodies in the overgrowth area (FIG. 10A). The majority of cells are positive for lsl 1/2 of which about 50% is also HB9 ⁺ (FIG. 10B) which suggests that these double positive cells are motor neurons. lsl 1/2 ⁺ and HB9 ^_ cells are interneurons.

HB9-expressing cells first appeared 6 days after neural rosettes were dispensed for differentiation and reached high rates after about 10-12 days. They were largely confined to closters and accounted for about 21% of the total cells in the cluster and few cells in the overgrowth region (Figures 10A, D). The highest percentage of HB9 ⁺ cells were induced in the presence of 0.1-1.0 μM RA. At doses greater than 1.0 μM, RA degrades some cells in chemically defined attachment cultures. In the absence of RA or SHH or both, there is a very small number of HB9 ⁺ cells (FIG. 10D). All HB9-expressing cells were stained with β _III -tubulin (FIG. 10C). Thus, treatment with RA for early neuroectodermal cells is required for efficient induction of motor neurons.

To understand why RA induces neuroectodermal cells to differentiate into motor neurons early rather than later, we first examined the effect of RA on caudalization of neuroectodermal cells. Treatment of premature rosette cells (Pax6 ⁺ / Soxl ⁻ ) with RA (0.001 to 1.0 μM) or FGF2 (20 ng / ml) for 7 days reduced the expression of Otx2 and in a dose dependent manner with HoxB1, B6, C5 and C8 Increased expression of the same Hox gene (FIG. 11A). Genes expressed by more caudal cells were induced by higher doses of RA. Treatment of late rosette cells (Pax6 ⁺ / Sox1 ⁺ ) with RA for 1 week did not change (not shown) the pattern of Hox gene expression induced by FGF2. RA treated early rosette cells isolated and cultured in neural differentiation medium expressed HoxC8 protein on the first 6 days and mostly on days 10-12 after differentiation, which was revealed by immunocytochemistry (FIG. 11D). Cells at this stage do not express Otx2 (FIG. 11C). All HoxC8 ⁺ cells were β _III -tubulin ⁺ neurons (FIG. 11E). In contrast, late rosette cells treated with RA for 1 week and then differentiated for 2 weeks produced some HoxC8 ⁺ cells but decreased Otx-2 expressing cells (not shown). Thus, treatment of neuroectodermal cells with RA early rather than later leads to efficient tailing while expressing HoxC proteins associated with spinal neurons. Liu, JP, et al., Neuron 32: 997-1012,2001 ].

We therefore compared the effects of SHH on early and late neuroectodermal cells for embryonicization. No matter whether Pax6 ⁺ or Sox1 ⁺ , hES cell-derived neuroectodermal cells did not express Olig2 (FIG. 11F), a homeodomain protein expressed in ventral neural precursor cells destined to be motor neurons and oligodendrocytes in the spinal column. See Lu, QR, et ai., Cell 109: 75-86,2002; Zhou, Q., et al., Neuron 31: 791-807, 2001. Very few cells expressed Olig2 (not shown) when Pax6 ⁺ / Sox1 ⁻ neuroectodermal cells were cultured in the presence of RA for 1 week, then isolated and differentiated for another 2 weeks in the absence of SHH. However, in the presence of SHH (50-500 ng / ml), many cells expressed Olig2 in the nucleus (FIG. 11G). In contrast, Pax6 ⁺ / Sox1 ⁺ neuroectodermal cells differentiated for 2 weeks under the same conditions produced a few Olig2-expressing cells (FIG. 11H). Thus, neuroectodermal cells treated with RA at an early stage, but not at a later stage, can be efficiently induced into ventral neural precursor fate in response to SHH.

Although FGF2 also induces caudal fate (FIG. 11A), in order to further analyze why early RA treatment is required for motor neuron characterization, the inventors have identified a classic I (Irx3) important for refining the precursor domain in the spinal column. , Pax6) and the expression of Classic II (Olig2, Nkx2.2, Nkx6.1) were examined (Jessell, TM, supra, 2000; Briscoe, J., and Ericson, J., supra, 2001]. RA induces stronger expression of Olig2 and Nkx6.1 earlier, especially in late renal ectoderm cells (FIG. 11B). Thus, early neuroectodermal cells are more sensitive to RA in upregulating the expression of SHH and the expression of Classic II elements, which are essential for motor neuron characterization.

Cells expressing choline acetyltransferase (ChAT) appear three weeks after tailed neuroectodermal cells have been dispensed for motor neuron differentiation and these cells are stable for up to seven weeks, the longest culture period analyzed in this study. Increased (FIG. 12A). ChAT-expressing cells mostly localize into clusters (FIG. 12A) corresponding to localization of HB9 ⁺ cells. These cells are predominantly multipolar cells, large somatic cells 15-20 μm in diameter, and some are as large as 30 μm (FIGS. 12A, B). Co-expression of HB9 in the nucleus and ChAT in somatic cells and processes was observed after 3 weeks of culture (FIG. 12C). Most neurons were also stained positive for the porous acetylcholine transporter (VAChT, Figure 12D) which is essential for the storage and secretion of acetylcholine. In particular, after 5 weeks of culture, many ChAT ⁺ cells were labeled positive for synapsin on cell bodies and processes (FIG. 12E).

We assessed functional maturation using electrophysiological techniques (n = 28 cells). The average resting potential was ˜36.9 ± 2.6 mV and the inrush resistance was 920 ± 57 Ω. Single action potential (AP, FIG. 12Fi) or continuous reduction was caused by depolarizing the current phase (0.15-0.2nA × 1s) in 11 of the 13 neurons tested. Spontaneous AP caused by spontaneous depolarized synaptic acquisition was also observed (FIG. 12G). While not all cells survived during recording and thus immunohistochemical analysis, dual immunostaining with biocitin and ChAT demonstrated that many of the cells we recorded were motor neurons (FIG. 12J).

Voltage clamp analysis revealed time and voltage dependent inward and outward currents consistent with sodium and delayed rectifier potassium currents. Inward current and action potential are blocked by 0.1 μM to confirm the presence of voltage activated sodium channels. The characteristic of the outward current was not further analyzed. We also observed spontaneous synaptic currents (FIG. 12H, n = 21 of 23 cells tested). These frequencies were reduced but not eliminated by 1.0 μM TTX, demonstrating the existence of functionally intact synaptic neurotransmission. Using the Csgluconate base pipette solution, the outward (inhibition) current slowly declines (13.6 ms, n = 10 events) and is blocked by the combination of striknin and bicuculin, while the residual inward (stimulus) current rapidly declines ( 2.1 ms, n = 17 events) and blocked by the combination of D-AP5 and CNQX (FIG. 12H, J), demonstrating that inhibition (GABA / glycine) and stimulation (glutamate) neurotransmission occurs as in intact spinal column [Gao, BX, et al., J. Neurophvsiol. 79: 2277-2287, 1998].

The study of the present invention allows functional motor neurons to differentiate into mitotic post-mitotic motor neurons in the presence of neuroectodermal differentiation by FGF2, characterization by RA and / or caudalization followed by ventralized morphogen SHH during later stages of neurogenesis. Demonstrates that it can be efficiently generated from human ES cells. Thus, the basic principles of neural development learned from animals can be applied to human primates and repeated in vitro. Contrary to recent evidence of motor neuron differentiation from mouse ES cells (Wichterle, H., et al., Cell 110: 385-397, 2002), we have revealed a process of neuroectodermal differentiation and such as spinal cord motor neurons. It was found that precursors must be treated with morphogens before they can become Sox1 expressing neuroectodermal cells for the characterization of early neonatal overhang neurons.

Mouse ES cells were first directed to neuroectodermal cells, which were then 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 with morphogens such as RA and SHH (Wichterle, H., et al., Supra, 2002) for motor neuron differentiation. These observations seem to fit the notion that neurons are specified from the epithelium to neuronal tubes. Observations of the present invention also indicate that hES derived Sox1 expressing neuroectodermal cells with a whole brain phenotype are unresponsive to producing spinal motor neurons. Sox1 expressing cells generated from hES cells in the culture system of the present invention are similar to those of neural tubes by forming neural tubular structures and expressing Sox1 after two weeks of differentiation from hES cells equivalent to 6 day old human embryos. Zhang, SC, J. Hematother. Stem Cell Res. 12: 625-634, 2003. In vivo, neural tubes are formed at the end of the third week of human gestation [Wood, H. B. and Episkopou, V., Mech. Dev. 86: 197-201,1999] Sox1 is expressed by neuroectodermal formation during neural tube formation in animals [Pevny, L. H., et al., Development 125: 1967- 1978, 1998; Wood, H. B. and Episkopou, V., supra, 1999]. The inventors' findings suggest that the specification of at least large protruding neurons, such as a class of neurons, motor neurons, is initiated before the stem cells become Sox1-expressing neuroectodermal cells, thus suggesting that regions of brain-derived neuroectodermal cells differ. The reason for failing to produce overhanging neurons of the entity can be explained.

 Functional motor neurons from new sources of hES cells provide general human motor neurons for screening drugs designed to treat motor neuron related disorders such as ALS. These cells also provide a useful source for experimental cell substitutes of motor neurons, which may one day be applied to patients with motor neuron disease or spinal cord injury.

Way

Culture and Neural Differentiation of ES Cells

Nutrient supplying layer of embryonic mouse fibroblasts cultured with human ES cells (cell lines H1 and H9, passages 19-42) and irradiated as described in Thomson, JA, et al., Supra, 1998 The passages were weekly. ES cells in undifferentiated state were confirmed by typical morphology and expression of Oct4 and SSEA4. For neuroectodermal differentiation, hES cells aggregated for 4 days and then cultured on adhesive plastic surfaces for 10 days in F12 / DMEM supplemented with N2, heparin (2 ng / ml) and FGF2 (20 ng / ml) or RA. See Zhang, SC, et al., Supra, 2001.

For motor neuron induction, morphogen-treated neuroectodermal cells were cultured for 1 week in the presence of neuronal medium (Gibco), N2 supplement and in the presence of RA (0.1 μM) and SHH (10-500 ng / ml, R & D). Dispense onto ornithine / laminin-coated coverslips in neural differentiation medium consisting of cAMP (Sigma, IgM). Thereafter, BDNF, GDNF and insulin type growth factor-1 (IGF-1) (10 ng / ml, PeproTech Inc.) were added to the medium and the concentration of SHH was reduced to 50 ng / ml.

Immunocytochemistry and Microscopy (Zhang, S.C., et al., Supra, 2001)

Primary antibodies used in this study were neuronal class III β-tubulin (Covance Research Products, Richmond, CA, 1: 2000), Nestin (Chemicon, Temecula, CA, 1: 750), Sox1 (Chemicon, 1: 1000), Synapsin I (Calbiochem, Darmstadt, German, 1: 500), ChAT (Chemicon, 1: 50), and VAChT (Chemicon, 1: 1000), lsll / 2 (S. Pfaff), Otx2 (F. Vaccarino), and Olig2 (M. Nakafuku). Antibodies against MNR2 or HB9 (81.5C10), lsletl (40.2D6), Lim3 (67.4E12), Pax6 and Nkx2.2 were purchased from Developmental Studies Hybridoma Bank (DSHB, lowa City, IA) and anti-HoxC8 Was purchased from Coveance Research Products (1: 200). To identify electrophysiologically recorded cells, Biocithin (Molecular Probes) filled cells were labeled with streptavidin-FITC (Sigma, 1: 200) and stained for ChAT. Images were collected using a spot digital camera mounted on a Nikon Fluorescence Microscope 600 (FRYER INC, Huntley, IL) or High Focus Microscope (Nikon, Tokyo, Japan). Specificity of antibodies to motor neuron transcription factors and homeodomain proteins, originally developed for non-primate tissues, has been demonstrated in rhesus monkey spines and embryos of brain tissue (source: Wisconsin Primate Research Center) (E34 or E36). .

dose

Among total differentiated cells (chast labeled), HB9-expressing cell populations were counted by remission unknown to the experimental group, either manually or by stereoscopic measurements using Metamorph software (Universal Imaging Corporation, Downingtown, PA). It became. The area being measured is roughly outlined by the tracker and the number of coefficient frames is preset so that the range can be measured at random using automated step movements operated by stereo investigator software (MicroBrightField Inc, Williston, VT). Sample was taken. Microscopy was pre-set up and down for counting regions with overlapping cells to focus positive cells in different layers and the total number of cells in clusters was evaluated by software. Three to four coverslips in each group were counted and data expressed as mean ± SD.

RT-PCT Analysis

RT-PCR amplification was performed from hES cell derived neuroectodermal cells in different stages and motor neuron differentiation culture.

The following primers were used:

HoxC8, 5'-TTTATGGGGCTCAGCAAGAGG-3 ', 5'-TCCACTTCATCCTTCGGTTCTG-3', 318 bp; HoxC5, 5'-TCGGGGTGCTTCCTTGTAGC-3 ', 5'-TTCGTGGCAGGGACTATGGG-3', 290 bp; HoxB6, 5'-AACTCCACCTTCCCCGTCAC-3 ', 5'-CTTCTGTCTCGCCGAACACG-3', 340 bp; Otx2, 5'-CAACAGCAGAATGGAGGTCA-3 ', 5'- CTGGGTGGAAAGAGAAGCTG-3', 429 bp; HoxBl, 5'-TCAGAAGGAGACGGAGGCTA-3 ', 5'-GTGGGGGTGTTAGGTTCTGA-3', 218 bp; GAPDH, 5'-ACCACAGTCCATGCCATCAC-3 ', 5'-TCCACCACCCTGTTGCTGTA-3', 450 bp; Olig2, 5'- AAGGAGGCAGTGGCTTCAAGTC-3 ', 5'- CGCTCACCAGTCGCTTCATC-3', 315 bp; Nkx2. 2, 5'- TGCCTCTCCTTCTGAACCTTGG-3 ', 5'-GCGAAATCTGCCACCAGTTG-3', 337 bp. Irx3, 5'-AAGAACGCCACCAGGGAGAG-3 ', 5'-TTGGAGTCCGAAATGGGTCC-3', 473 bp; Pax6, 5'-GGCAACCTACGCAAGATGGC-3 ', 5'- TGAGGGCTGTGTCTGTTCGG-3', 459 bp; SHH, 5'-CCAATTACAACCCCGACATC-3 ', 5'-CCGAGTTCTCTGCTTTCACC-3', 339 bp; Nkx6. 1, 5'-ACACGAGACCCACTTTTTCCG-3 ', 5'-TGCTGGACTTGTGCTTCTTCAAC-3', 335 bp.

Electrophysiological record

The electrophysiological properties of hES cell derived motor neurons were studied in cultures differentiated for 5-6 weeks using whole cell patch clamp recording techniques. Gao, B. X., et al., J. Neurophvsiol. 79: 2277-2287, 1998]. Tetrodotoxin (TTX, 1 μM, Sigma), bicuculin (20 μM, Sigma), striknin (5 μM, Sigma), D-2-amino-5-phosphonovaleric acid (AP-5, 40 μM, Sigma) or 6-cyano -7-nitroquinoxaline-2,3-dione (CNQX, 20 μΜ, RBI, Natick, MA) was applied to the bath solution to confirm the identity of the voltage activated or synaptic current. For some experiments, 1% biocithin was added to the recording solution. Current and voltage clamp recordings were performed using a multiclamp 700A amplifier (Axon Instruments, Union City, CA). The signal was filtered at 4 kHz, sampled at 10 kHz using a Digidata 1322A analog-to-digital converter (Axon Instument) and stored on a computer hard disk using commercially available software (pClamp9, Axon Instruments). Access resistance is typically 8-15 MΩ and compensated by 50-80% using an amplifier circuit. Spontaneous synaptic currents were detected using the template detection algorithm (Small analysis program 5.6.28, Synaptosoft, Decatur, GA) and adjusted to a single algebraic function using the Levenberg-Marquardt algorithm. The results are given as mean ± SEM.

Claims (24)

(a) propagating embryonic stem cells into embryoid bodies; and (b) includes the step of proliferation of early rosette form of neural stem tuning cell group the compensation body and the proliferation and characterized in the early rosette form, in the presence of FGF2, FGF8, FGF9, or FGF4, Sox1 ^-, Pax6 ⁺ Method for producing a cell population comprising a tuning cell population cultured from phosphorus embryonic stem cells The method of claim 1 wherein the time between step (a) and early rosette development is between 8 and 10 days. The method of claim 1, wherein the Pax6 ⁺ / Sox1 ⁻ cell population is at least 70% of the total cell population. The method of claim 1 wherein the embryoid body is incubated for 4 to 6 days in the presence of FGF2, FGF8, FGF9 or FGF4. The method of claim 1, wherein the growth factor is FGF-2. A cell population produced by the method according to claim 1. Characterized in the early rosette form and, Sox1 ^-, Pax6 ⁺ in cells, and the features a, nerve tubular rosette form, comprising the step of culturing while FGF2, FGF4, FGF8, or 4 to 6 days in the presence of RA, Pax6 ⁺ / Method for preparing neural stem synchronizing cell population that is Sox1 ⁺ . 8. The method of claim 7, wherein the exposure is 4 to 6 days. A cell population produced by the method according to claim 8. The cell of claim 9 incubated with FGF8 and EN1 ⁺ . The cell of claim 9 incubated with FGF2 and Bf1 ⁺ . The cell of claim 9 incubated with RA and Hox ⁺ . A method for preparing a group of midbrain dopamine neurons comprising the step of culturing the cells according to claim 10 in the presence of FGF8, wherein the obtained cells express TH, AADC, EN-1, VMAT2 and DAT, but DbH And a method that does not express PNMT and produces dopamine. The method of claim 13, wherein the exposure to SHH is 6-7 days. A cell population produced by the method according to claim 13. A method for isolating spinal cord motor neurons comprising culturing the cells according to claim 12 with SHH, wherein the obtained cells express HB9, HoxB1, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine How to. The method of claim 16, wherein the cells are exposed to SHH for 6-7 days. A cell population produced by the method according to claim 16. 12. A method for producing whole brain dopamine neuron groups comprising culturing cells according to claim 11 with SHH, wherein the obtained cells express TH, AADC, Bf1, Otx2 but do not express DBH and PNMT and produce dopamine How to. The method of claim 19, wherein the cells are exposed to SHH for 6-7 days. A cell population produced by the method according to claim 19. A method of investigating a test compound for its ability to interfere with nerve cell development, comprising exposing the test compound to a cell according to claim 15 and investigating the results of the exposure compared to a control cell population not exposed to the test compound. . A method of investigating a test compound for its ability to interfere with nerve cell development, comprising exposing the test compound to a cell according to claim 18 and investigating the results of the exposure compared to a control cell population not exposed to the test compound. . A method of investigating a test compound for its ability to interfere with nerve cell development, comprising exposing the test compound to a cell according to claim 21 and investigating the results of the exposure compared to a control cell population not exposed to the test compound. .

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