CN1894401A - Derivation of terminally differentiated dopaminergic neurons from human embryonic stem cells - Google Patents

Abstract

The present invention relates to an improved methods for efficiently producing neuroprogenitor cells and differentiated neural cells such as dopaminergic neurons and serotonergic neurons from pluripotent stem cells, for example human embryonic stem cells. Using the disclosed methods, cell populations containing a high proportion of cells positive for tyrosine hydroxylase, a specific marker for dopaminergic neurons, have been isolated. The neuroprogenitor cells and terminally differentiated cells of the present invention can be generated in large quantities, and therefore may serve as an excellent source for cell replacement therapy in neurological disorders such as Parkinson's disease.

Description

From the human embryo stem cell eventually dopaminergic neuron of end differentiation of deriving

The cross-reference of related application

Inapplicable.

About the research of federal government's patronage or the statement of research and development

Inapplicable.

Quoting of " microfilm annex "

Inapplicable.

Background of invention

1. invention field

The present invention openly relates to a kind of from pluripotent embryonic stem cells, and for example human embryo stem cell prepares the neuronal cell of end differentiation eventually, and for example dopaminergic and serotoninergic neuron improves one's methods.The cell that dopaminergic that produces according to the present invention and serotoninergic neuron can be used as neurodegenerative disorders and neuronal disease is replaced the good source of therapy.

2. the description of pertinent literature

Neurodegenerative disorders and neuronal disease, Parkinson's disease for example, Alzheimer and schizophrenia are the significant more destructive diseases that becomes in our society.Multiple situation in these nervous disorders is relevant with dopaminergic or serotoninergic neuron.Dopaminergic neuron is positioned at the outside of belly and the abdomen outer side of midbrain, control attitudinal reflex, the behavior that motion is relevant with award.A plurality of structures of these neurone domination forebrains, their sex change or dysfunction and Parkinson's disease, schizophrenia and drug habit (Hynes etal., 1995, Cell 80:95-101) are relevant.Serotoninergic neuron concentrates on the outside of belly and the abdomen outer side of hindbrain, and the major part of domination central nervous system comprises pallium, limbic system and spinal cord.These neuron control consciousness, awakening, the level of behavioural characteristic and ingestion of food, their dysfunction and attack, depression and schizophrenia (Jacobs and Gelperin, 1981, Serotonin Neurotransmissionand Behavior.The MIT Press, Cambridge is Mass.) relevant.The serotoninergic neuron dysfunction also may be at various mental disorderes, sacred disease and other diseases, spirit depressing (Asberget al, 1986, J.Clin.Psychiatry 47:23-35) for example, (Lester commits suiside, 1995, Pharmocopsychiatry 28 (2): 45-50) with brute force attack behavior (Brown et al., J.Clin.Psychiatry, 1990,54:31-41; Eichelman, 1990, work in pathologic, physiologic generating process Annu.Rev.Med.41:149-158).

Parkinson's disease is a kind of carrying out property neurological disorder that is caused by neurocyte (neurone) sex change in the motion control district of brain.This sex change makes the brain signal that is called as Dopamine HCL transmit chemical substance (neurotransmitter) and becomes not enough, causes occurring the dyskinesia of this genius morbi.Pathological study shows that the loss of black substance district dopaminergic neuron facilitated Parkinson's disease.For example, the bilateral in nigrostriatum path damage has produced a kind of and the closely similar syndrome of observed dyskinesia in Parkinson's disease in laboratory animal: static tremor, tetanic, motion can not and abnormal posture.The bilateral damage in the nigrostriatum path that is caused by 6-hydroxydopamine (OHDA) has caused that in rodent serious motion can not, adipsia, anorexia and sensory neglect (Ungerstedt, 1971, U.Acta Physiol.Scand.Suppl.367:95-121; Yirek and Sladek, 1990, Annu.Rev.Neurosci.13:415-440).

In Parkinson, the variation of dopaminergic receptor status may depend on the stage of progression of disease.The sign of Parkinson is the serious decline (Hornykiewicz, 1988, Mt.Sinai J.Med.55:11-20) of Dopamine HCL in all parts of basal ganglion.When Dopamine HCL exhausts, many other zones in the brain, thalamus for example, pallidum, subthalamic nucleus begins to occur dysfunction.Because these zones pass the signal to other parts of brain, so the dysfunction of these zonules can cause disordered brain function widely.

Parkinsonian morbidity alters a great deal, and is 82/100,000 people in Japan, is 108/100,000 people at UK, in the state, North America then near 1% (about 100 ten thousand) of population.In India, parkinsonian morbidity is 14/100,000 people in northern India, and southern India is 27/100,000 people, and East India is 16/100,000 people, and the India Parsee community of western India is 363/100,000 people.Although think that at present Parkinson's disease can not cure, the multiple medicine of available comprises levodopa, bromocriptine, and pergolide, selegiline, anticholinergic and amantadine can be alleviated symptoms of Parkinson's Disease.Though these medicines can be alleviated symptoms of Parkinson's Disease, they produce pronounced side effects through regular meeting.And these medicines neither can be cured this disease, neuronic the carrying out property that can not slow down loss, and be merely able to relief of symptoms, the beneficial effect of generation also weakens usually in time gradually.Some patients die down to the reaction of treatment, and other patients then become extremely sensitive and develop into dyskinesia.

These results unsatisfactory cause people begin one's study the treatment this disease other method, for example dopamine 2 receptor agonist treatment and surgical method, comprise pallidotomy, pallidum DBS (DBS) and trial make these zones quiet to stop this network unusual by destructive activity over-drastic brain zone or implantation DBS electrode.Though these and other type of surgery at the parkinsonian have produced some useful result, the long-time effect of this operation is not clear.These treatments also have certain limitation and side effect.

The another kind of carrying out is gene therapy at this method that can not cure diseases.The progress of the discovery of sacred disease molecular basis and gene transfer system allows to carry out part or the whole therapeutic gene that transmits at multiple central nervous system disorders.But gene therapy has certain limitation, for example stability of transgene expression and adjusting, and the genetically modified security of carrier and expression (Costant ini et al., 2000, Gene Therapy 7:93-109).Can be used for Vectors in Gene Therapy and include but not limited to herpes simplex types 1 virus (HSV-1) (During et al., 1994, Science 266:1399-1403), gland relevant viral vector (AAV) (During et al., 1998, Gene Therapy 5:820-827), retrovirus, HSV/EB virus (HSV/EBV) heterozygosis carrier and HSV/AAV heterozygosis carrier.Have been found that a kind of gene therapy method can be used for treating parkinsonian animal model.A kind of genetically engineered cell that can discharge neuroprotective molecule (the glial cell-line deutero-trophic nerve factor (GDNF) gene) that wraps quilt is and the lentiviral vectors of coding gdnf gene can improve survival and the differentiation of graft, therefore the behavior of quickening in the animal model recovers (Zurn et al., 2001, Brain Res Rev.36:222-229; Date et al., 2001, Cell Transplant 10:397-40 ".Gene therapy that also find to use neural stem cell in vivo can the effective expression treatment level GDNF (Akerud et al., 2001.J.Neurosci.21:8108-8118).

Transplanted cells provides the replacement Parkinson's disease, and the another kind of methods of treatment of the hope of the neurocyte that loses in other neurodegenerative disorders and the neuronal disease.The fetal tissue of well afoot transplants clinical trial and has has researched and developed cell is implanted method in the brain, and demonstrates the life of this notion, and has produced result likely on one's body at least some patients.Also attempt the precursor cell of dopaminergic nerve cell is grafted directly in parkinsonian's the striatum, the transplanting of finder's fetus or embryo's dopaminergic neuron has produced beneficial effect (Freed et al. to the parkinsonian, 2001, N.Engl.J.Med.344:710-719).May need repair path on the anatomy yet experimental data shows will obtain to return to one's perfect health, rather than dystopy is settled graft (Winkler et al., 2000, Prog.Brain Res.127:233-265).And in order to make the patient obtain reliable clinically improvement, fetus black substance transplantation treatment need be from the human fetal tissue of 5-10 fetus at least, and this can cause a large amount of morals, law and safety problem.Therefore press for neuronal cell, for example treat the alternative source of the dopaminergic neuron of neurodegenerative disorders and neuronal disease.

In Adult Human Brain, found renewable neural stem cell source recently.Having the neural stem cell of self upgrading and forming all kinds brain cell ability provides potential unlimited supply to produce the mode of Dopamine HCL brain cell, provide brand-new methods of treatment (Eriksson et al. for neurodegenerative disorders and neuronal disease thus, 1998, Nature Medicine 4:1313-1317).Existing report shows that the Culture of neural stem cells thing that derives from people embryo forebrain can be at amplification in vitro to thousands of times.It is in the adult rat of Parkinson disease model of fine sign that these adult animals neural stem cell have been transplanted to.Transplant back cell survival time in animal model and reach 1 year, be divided into neurone, and can in some laboratory animal, alleviate dyskinesia (Svendsenet al., 1997, Exp.Neurol.148:135-146).Yet unfortunately, the lifetime of adult animals neural stem cell in tissue culture limited (Kukekov et al., 1999, Exp.Neurol.156:333-344).

Dopaminergic neuron and other neuronic feasible alternative sources that can be used for treating various neurodegenerative disorders and neuronal disease are pluripotent embryonic stem (ES) cells, particularly people ES cell.The ES cell can be with the undifferentiated state infinite multiplication, and is polyenergic, mean they can break up the adult internal memory nearly all cell type.Because the ES cell almost can become all body specialized cells, so they have and produce extensively tissue and organ, heart for example, pancreas, nervous tissue, muscle, the potential of the alternative cell of cartilage etc.The ES cell can be derived from the inner cell mass (ICM) of blastocyst, and blastocyst is an embryonic development period before transplanting.People ES cell can be derived from people's blastocyst of the 4th day to the 7th day embryonic development commitment of after fertilization.The ES cell that derives from ICM can be cultivated external, under suitable condition infinite multiplication.

Successfully set up and comprised mouse (Evans et al., 1981, Nature 292:154-156), rat (Iannaccone et al., 1994, Dev.Biol., 163:288-292), pig (Evans et al., 1990, Theriogenology 33:125-128; Notarianni et al., 1990, J.Reprod.Fertil.Suppl.41:51-6), sheep and goat (Meinecke-Tillmann and Meinecke, 1996, J.Animal Breeding and Genetics 113:413-426; Notarianni et al, 1991, J.Reprod.Fertil.Suppl.43:255-60), rabbit (Giles et al., 1993, Mol.Reprod.Dev.36:130-138; Graves et al., 1993, Mol.Reprod.Dev.36:424-433), ermine (Sukoyan et al., Mol.Reprod.Dev.1992,33:418-431), hamster (Doetschman et al., 1988, Dev.Biol.127:224-227), poultry (Pain et al., 1996, Development 122 (8): 2339-48), primate (U.S. Patent No. 5,843,780) and people (Thomson et al., 1998, Science 282:1145-1147; Reubinoff et al., 2000, Nature Biotech.18:399-403) in the ES of interior a plurality of species clone.People ES cell is the same with other Mammals ES cells, in the time of in being expelled to the immunodeficient mouse body, can break up and form the tissue of whole three germinal layers, and this has proved its versatility.Disclosed report shows that people ES cell can be cultivated to be kept more than 1 year, during cell keep its versatility, self ability and normal karyotype (Thomson et.al., 1995, PNAS 92:7844-7848).

Studies show that the ES cell can be divided into neural progenitor cell (Zhang et al., 2001, NatureBiotech.19:1129-33; WO 01/88104; U.S.'s sequence number 09/872,183,09/888,309,10/157,288; WO 03/000868; Each file is introduced herein as a reference particularly).These cells can further be divided into dopaminergic neuron (Rolletschek et al., 2001, Mech.Dev.105:93-104).The initial step of ES cytodifferentiation can be to form embryoid, for example the vitamin A acid of 1 μ M can promote Neural Differentiation become embryoid (Bain et al., 1995, Dev.Biol.168:342-357).Though can produce neurocyte with vitamin A acid, vitamin A acid is a kind of strong teratogen.Existing some reports disclose by using stroma cell induced activity (SIDA) (Kawasaki et al., 2000, Neuron28:1-20), by expressing relevant-1 gene (Nurr-1) (the Kimet al. of nuclear receptor, 2002, Nature 418:50-56), or by directly will not breaking up the ES cell value of moving (Bjorklund et al. in the mouse model body, 2002, Proc.Natl Acad.Sci.99:2344-2349) make the ES cytodifferentiation become dopaminergic neuron.Lee etc. (2000, Nat.Biotechnol.18:675-79) reported and a kind ofly external the ES cytodifferentiation has been become neural progenitor cell, the method for dopaminergic or serotoninergic neuron.Yet all these experiments all adopt mouse ES cells to implement, and this differentiation method obtains the dopaminergic neuron of 5-50%.In the research of (WO 01/83715) such as Lee, there is 20% mouse ES cells to be divided into dopaminergic neuron approximately, in the research of (WO 02/086073) such as Studer, has 5-50% to be divided into dopaminergic neuron.Though also can only can obtain the dopaminergic neuron productive rate (accounting for the percentage ratio of all cells sum in the colony) (WO 03/000868) of 5-7% from people ES cytodifferentiation dopaminergic neuron.

Be characterised in that owing to parkinsonian the selectivity of dopaminergic neuron is progressively lost in the substantia nigra of midbrain, so it is considered to be particularly suitable for the clinical disease of Transplanted cells strategy.The loss of the product dopamine neuron in this specific brain regions site causes neurocyte to provide (firing) unusually, and this causes the patient can not control or arrange its motion.But replacing treatment, cell needs a large amount of dopaminergic neurons.Therefore, need more efficiently from the alternative method of people ES cell preparation dopaminergic neuron, it will increase this methods of treatment for parkinsonian suitability, and improve the success ratio of this methods of treatment.In addition, can be at external these dopaminergic neurons that utilize to help to identify the material that in neurodegenerative disorders and neuronal disease, prevents or reduce the death of product Dopamine HCL brain cell.

Summary of the invention

The present invention openly relates to a kind of from multipotential stem cell, and for example human embryo stem cell produces neural progenitor cell (neuroprogenitor cells), and improving one's methods of last differentiated neuron eventually.For example, the open reference's embryonic stem cell group energy of the present invention is divided into a high proportion of tyrosine hydroxylase (TH) (it is the specificity marker thing of dopaminergic neuron) positive neuron (for example about at least 60%).The present invention openly goes back reference's embryonic stem cell group energy and is divided into a high proportion of serotoninergic neuron.Be higher than previously described method according to the dopaminergic neuron of method generation disclosed by the invention and the ratio of serotoninergic neuron.Method disclosed herein also can be used for preparing cell and astroglia cell and the oligodendrocyte with cholinergic neuron and Sensory neurone phenotypic characteristic from human embryo stem cell.

The present invention openly provides the differentiated cell population in passing through the resulting vitro culture thing of differentiating primate animal multipotential stem cell, wherein at least 60% noble cells is a dopaminergic neuron, express tyrosine hydroxylase, or express the dopaminergic neuron of tyrosine hydroxylase.In other embodiments, about at least 30%, 40%, 50%, 70%, 80%, 90%, 95% or 99% noble cells is a dopaminergic neuron.The present invention openly also provides the differentiated cell population in passing through the resulting vitro culture thing of differentiating primate animal multipotential stem cell, and wherein at least 30% noble cells is a serotoninergic neuron.At least about in other embodiments 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% noble cells is a serotoninergic neuron.In preferred embodiments, be divided into neural progenitor cell or neuronic primate pluripotent stem cell is a human embryo stem cell.

The present invention openly also provides from the method for the neural cell group of primate pluripotent stem cell preparation differentiation, comprises the following steps:

(a) culture of amplification primate pluripotent stem cell;

(b) cultivate this multipotential stem cell to select nidogen (nestin) male neural progenitor cell;

(c) sorting nidogen positive neurons progenitor cell is with enrichment of N CAM positive cell;

(d) make the nidogen positive, the differentiation of NCAM positive cell by culturing cell in division culture medium, produce the neural cell group of differentiation.

In preferred embodiments, primate pluripotent stem cell is a human embryo stem cell.In another preferred embodiment, the multipotential stem cell that uses in the aforesaid method preferably obtains by laser ablation (laser ablation technique).

In other embodiments, aforesaid method comprises that further the multipotential stem cell in the culturing step (b) forms the step of embryoid (embryoid bodies).Preferably under the condition of selecting nidogen positive neurons progenitor cell, cultivate these embryoids, for example by in serum free medium, cultivating multipotential stem cell or embryoid.In preferred embodiments, serum free medium is the known serum-free ITSFn of a composition substratum, wherein preferably comprises one or more and is selected from Regular Insulin, Sodium Selenite, Prostatropin, the soluble factor of Transferrins,iron complexes and fibronectin.In preferred embodiments, these methods will produce and preferably comprise at least approximately 60-75% nidogen positive cell, more preferably comprise about 80-90% nidogen positive cell, most preferably comprise the neural progenitor cell of about 95-99% nidogen positive cell.

Can use suitable immunological technique then, for example immune labeled and fluorescence separating method, as magnetic cell sorting art (Magnetic Cell Sorting, MACS), solid phase adsorption, fluorescence activated cell sorting (FACS) comes sorting nidogen positive neurons progenitor cell with enrichment of N CAM positive cell at the streaming immunocytochemistry (flow immunocytochemistry) or the flow cytometry of cell surface marker.In preferred embodiments, these methods will produce and preferably comprise at least approximately 40-70%NCAM positive cell, more preferably comprise about 50-60%NCAM positive cell, most preferably comprise the nidogen positive cell of about 80-99%NCAM positive cell.In certain embodiments, above-mentioned method further is included in the middle nidogen positive of amplification (expanding) step (c), NCAM positive neurons progenitor cell in the amplification culture medium (expansion medium), preferred 6-10 days.Preferably, amplification culture medium comprises one or more and is selected from Regular Insulin, Sodium Selenite, Transferrins,iron complexes, ln, putrescine, Progesterone, Prostatropin (bFGF), Urogastron (EGF), sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8) and neurotrophic factor derived from brain (neurotropic factor) soluble substance (BDNF).

The nidogen positive, NCAM positive neurons progenitor cell be amplification and the one or more population doublings of continuous passage in cultivation preferably.These cells also can freezingly be kept in the liquid nitrogen.Step according to the method described above (d), NCAM positive neurons progenitor cell were preferably grown 30-50 days in division culture medium.In preferred embodiments, division culture medium comprises and has added foetal calf serum, B27, the Neurobasal substratum of xitix and N-acetylcystein (Neurobasal medium).In a further preferred embodiment, division culture medium comprises that TGF-β 3 or il-1 β or both comprise.Preferably; division culture medium further comprises one or more differentiation agents; wherein differentiation agent is selected from xitix; N-acetylcystein; glial cell line derived neurotrophic (neurotropic) factor (GDNF), two butyryl ring AMP (db-cAMP), brain derived neurotrophy (neurotropic) factor (BDNF); neuturin, sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF-8).

In preferred embodiments, top disclosed method is used to preparation and preferably comprises about 40-60% dopaminergic neuron, more preferably comprise about 70-80% dopaminergic neuron, most preferably comprise the differentiation neural cell group of about 90-99% dopaminergic neuron.In specific embodiments, these methods are used to produce and preferably comprise about 20-50%5-hydroxy-tryptamine serotonergic neuron, more preferably comprise about 30-70%5-hydroxy-tryptamine serotonergic neuron, most preferably comprise the differentiation neural cell group of about 60-99%5-hydroxy-tryptamine serotonergic neuron.In other embodiments, these methods are used to preparation and preferably comprise about 15-40% oligodendrocyte, more preferably comprise about 25-50% oligodendrocyte, most preferably comprise the differentiation neural cell group of about 60-99% oligodendrocyte.

The present invention openly provides the method for preparing dopaminergic neuron from neural progenitor cell, comprise enrichment nidogen positive cells from neural progenitor cell, and make the differentiation of nidogen positive cell produce dopaminergic neuron by culturing cell under the situation of TGF-β 3 or il-1 β existence or both's existence.Preferably, 40-99% nidogen positive cell is divided into dopaminergic neuron to use these methods to make at least approximately.In other embodiment, these methods further comprise enrichment of N CAM positive cells from neural progenitor cell, and these nidogen positives, NCAM positive cell are preferably produced dopaminergic neuron (for example at least approximately the cytodifferentiation of 60-99% is a dopaminergic neuron) by differentiation.The present invention openly also provides from neural progenitor cell and has prepared neuronic method, comprise enrichment nidogen and NCAM positive cells from neural progenitor cell, and make nidogen and the differentiation of NCAM positive cells produce serotoninergic neuron by culturing cell under the situation of TGF-β 3 or il-1 β existence or both's existence.Preferably, the 30-99% nidogen positive, NCAM positive cell are divided into serotoninergic neuron to use these methods to make at least approximately.

The method that the present invention openly also provides the neurocyte by using the differentiation that derives from primate pluripotent stem cell as described herein to individuality that neurodegenerative disorders or neuronal disease patient are treated.For example, can prepare the neural cell group of differentiation by following step:

(a) culture of amplification primate pluripotent stem cell;

(b) cultivate this multipotential stem cell to select nidogen male neural progenitor cell;

(c) sorting nidogen positive neurons progenitor cell is with enrichment of N CAM positive cell;

(d) make the nidogen positive, the differentiation of NCAM positive cell by culturing cell in division culture medium, produce the neural cell group of differentiation.

(e) neural cell group that will treat the differentiation of significant quantity is administered in the individual central nervous system.

Preferably, division culture medium comprises that TGF-β 3 or il-1 β or both comprise.In preferred embodiments, individuality is the patient, is more preferably human patients, and primate pluripotent stem cell is a human embryo stem cell.Preferably, human embryo stem cell and patient have histocompatibility, and for example employed multipotential stem cell has the essentially identical genome with the patient.In specific embodiments, from the neural cell group of differentiation, isolate dopaminergic neuron, serotoninergic neuron, cholinergic neuron, or Sensory neurone, or scheme astroglia cell or oligodendrocyte as an alternative, and use to the patient.Can use these to individual subject and comprise many kinds of neurodegenerative disorders of the cell therapy that breaks up neural cell group or neuronal disease, include but not limited to central nervous system (CNS) disorder, Parkinson's disease, Alzheimer, Huntington Chorea, Spinal injury, amyotrophic lateral sclerosis (ALS), epilepsy, apoplexy and local asphyxia.Preferably, for example the purpose cell is transplanted in the individual brain by transplanting dosed cells.

In other embodiments, the neurocyte that derives from the differentiation of primate pluripotent stem cell described herein can be used for SCREENED COMPOUND (for example small molecules and medicine) to breaking up the impact effect of neurocyte or these cytoactives.Neurocyte toxicity that also can the examination compound or to the regulating effect of neurocyte.By compound being joined in the neural cell group of differentiation, then with under simulated condition, cultivate but be not exposed to the neurocyte survival rate relatively of the differentiation of this compound, form, phenotype, other features of functionally active or cell can be estimated compound.For example can determine whether compound can influence the change that cell is synthetic, discharge or absorb neurotransmitter by the examination compound.

The simple description of accompanying drawing

Following accompanying drawing constitutes the part of this specification sheets, is used for particular aspects of the present invention is further described, by reference one or more accompanying drawing, can understand the present invention better in conjunction with the specific embodiments of detailed description given here.

Fig. 1 shows the nidogen positive neurons progenitor cell that derives from human embryo stem cell: (A) show with the nidogen marker immunoreactive neural progenitor cell takes place; (B) be the photo that differs of the nidogen positive cell that when the somatomedin of selecting exists, under serum-free condition, increases.

Fig. 2 has shown the facs analysis of nidogen positive cell of NCAM-FITC that derived from human embryo stem cell and mark: (A) show the analysis with anti--unlabeled cells that rabbit FITC handled; (B) show with NCAM one anti-that handle and with the analysis of the cell of anti--rabbit FITC (two resist) mark.In this research, it is positive that approximately the nidogen positive cell of 50-60% is the NCAM immunity.

Fig. 3 A and 3B are with the MACS sorting and are inoculated into the fluorescence micrograph of the NCAM positive cell that derives from human embryo stem cell in the tissue culture ware.

Fig. 4 A and 4B are the neuronic fluorescence micrographs that derives from human embryo stem cell with MAP-2 and beta tubulin mark.

Fig. 5 has shown the existence of tyrosine hydroxylase (TH) positive neuron: (A) immunofluorescence analysis shows in the NCAM positive cell enriched populations that obtains with MACS that about 60% neurone is the TH positive; (B) about 40% neurone was the TH positive after immunofluorescence analysis was presented at amplification and breaks up unsorted nidogen positive cell.

Fig. 6 is the representational fluorescence micrograph of oligodendrocyte.Immunofluorescence analysis shows that the isolating nidogen positive cell of about 25-30% is the oligodendrocyte positive staining.

Fig. 7 is the representational fluorescence micrograph of expressing the neuronal cell of neurotransmitter serotonin.About 30% nidogen positive cell and about 20% NCAM positive cell and serotonin generation immune response.

Fig. 8 is the fluorescence micrograph that TH (green) and another kind of neuron specific antigen (redness) is carried out the dopaminergic neuron after immune labeled: TH and (A) beta tubulin; (B) MAP-2; (C) Nurr1; (D) DAT locatees altogether.

Fig. 9 is the bar graph that shows TH positive neuron (dopaminergic neuron) per-cent in the MAP-2 positive neuron: as shown in the figure, the TH positive neuron increases when further breaking up 7,22 and 37 days.

Figure 10 is the bar graph that shows different neuronal cell group quantitative analysis in the positive enrichment of cell of NCAM: it is positive that about 60% NCAM positive cell also is the TH immunity; About 30% is the serotonin immunity positive; With about 15% to be the immunity of GABA and L-glutamic acid positive.

Figure 11 is the bar graph of the different neuronal cell groups' of immunofluorescence analysis quantitative analysis after being presented at amplification and breaking up the nidogen positive cell: it is positive that about 40% nidogen positive cell is the TH immunity; About 30% is the serotonin immunity positive; About 28% is the oligodendrocyte immunity positive; About 2% is glial fibrillary acidic protein (glial fibrillar acidic protein, GFAP, the mark of astroglia cell) the immunity positive.

Figure 12 shown under the embodiment 1 disclosed condition at the whole gene expression atlas of human embryo stem cell in the last atomization of cells in vitro: UD=is undifferentiated; The EB=embryoid; NS=nidogen positive cell; NE=nidogen amplifying cells; Remaining time point is presented at the fate of culturing cell in the somatomedin of embodiment 1 disclosed Neurobasal substratum and selection.The dopaminergic neuron atopen, Nurr1 for example, En-1, transcribing in differentiation of D2RL expressed in early days.Except that undifferentiated stem cell, all observe the expression of dopaminergic neuron specific gene TH in all stages.Do not exist any DBH to express and confirmed that these cells have the midbrain phenotype.

Figure 13 is presented at differentiation 7,22 and 37 days, by dopamine level in the born of the same parents of the definite cytolysis thing of RP-HPLC.When somatomedin existed, dopamine level (4-6 μ g/ml) was higher than the level (1-4 μ g/ml) of untreated cell far away.The cell of handling with MPTP did not detect Dopamine HCL in the time of 7 and 22 days, and dopamine level descends in the time of 37 days.

Figure 14 be presented at cultivate in the conditioned medium, Repone K causes the bar graph that Dopamine HCL discharges in the noble cells of differentiation after 7,22 and 37 days.Induced the Dopamine HCL secretion in 15 minutes with 56mM Repone K irritation cell; Before analyzing, stablize culture supernatant with 7.5% ortho-phosphoric acid (orthrophosphoric acid) and sodium metabisulfite with RP-HPLC.

Detailed Description Of The Invention

The present invention openly provides the method for effective generation from the neural pedigree cell of multipotential stem cell differentiation. Here the cell that produces includes but not limited to have neural progenitor cell, dopaminergic neuron, serotoninergic neuron, cholinergic neuron, and sensory neuron, and the cell of the phenotypic characteristic of astroglia or oligodendroglia. Here the cell that produces can pass through phenotypic characteristic, and morphological feature and/or cell sign thing (these are that the technical staff who assesses in the field of this class cell is understandable) are identified. Term used herein " neural progenitor cell " can exchange with term " neural CFU-GM (neural progenitor cell) " or " neural precursor (neural precursor cell) ", finger can produce neuronal cell, for example neuron precursor or neuron, or spongiocyte, spongiocyte precursor for example, the cell of the filial generation that astroglia or oligodendroglia are such. Method disclosed herein comprises being combined with and helps Cell Differentiation and become the soluble factor of neural pedigree and environmental condition that cell is cultivated. In addition, can use the further enrichment purpose of physical separation or operating technology neural cell type.

The nerve cell of these precursors and differentiation can be used for many aspects, comprises being used for the treatment of property and tentative application, and external medicament research and development and screening, and for example screening has neurotoxicity or has the compound of regulating the nerve cell function. Produce the nerve cell of precursor and differentiation from multipotential stem cell, for example dopaminergic and serotoninergic neuron, and the specialization neuronal cell of other types provides potential unlimited supply for these neurons, brought huge potential benefit for suffering from the neurodegenerative disorders that makes people's weakness and the patient of neuronal disease. The nerve cell of precursor described herein and differentiation typically its derived from the daughter cell of cell mass, therefore have the essentially identical genome with parent group (be included on the science of heredity and be changed, transform or the parent group of transfection).

An embodiment disclosed by the invention relates to from multipotential stem cell, dried (ES) cell of preferred primate embryonic or primate embryonic reproduction (embryonic germ) (EG) cell produce the neuron with midbrain neuron feature, and for example dopaminergic neuron improves one's methods. Another embodiment relates to from multipotential stem cell, and preferred primate ES cell or primate EG cell produce the neuron with rear brain neuron feature, and for example serotoninergic neuron improves one's methods. The primate ES cell that can use in these methods or the choosing of EG optimum cell are people ES cell or EG cell. These neurons are derived from multipotential stem cell by cultured cell in the situation of some soluble factor and environmental condition existence.

Term used herein " dopaminergic neuron " refers to express the neuronal cell of tyrosine hydroxylase (TH) (rate-limiting enzyme that dopamine is synthetic). Dopaminergic neuron secretory nerve mediator dopamine preferably, and express hardly or not dopamine β-hydroxylase. Dopaminergic neuron is arranged corpus straitum in vivo, limbic system and neocortex, and it is positioned at Ventral Midbrain with other a few class neurons that comprise motor neuron. Dopaminergic neuron is positioned at substantia nigra of midbrain specifically, and it controls attitudinal reflex, the behavior (reward-associated behaviors) that motion is relevant with award. The loss of normal function dopaminergic neuron can cause Parkinson's disease, and its dysfunction is relevant with schizophrenia and drug habit. Term used herein " serotoninergic neuron " refers to the neuron of secretory nerve mediator serotonin (serotonin). Serotoninergic neuron typically has slow, rhythmical granting (firing) pattern and concentrates in vivo the outside of belly and the abdomen lateral surface of hindbrain and arrange the central nervous system major part of (comprising cerebral cortex, limbic system and spinal cord). These neuron control awareness wake up, behavior and food intake. 5-hydroxytryptamine serotonergic neuron dysfunction and attack, depressed (comprising suicide) is relevant with schizophrenia.

The present invention openly relates to makes multipotential stem cell be divided into neural progenitor cell, and the improving one's methods of neural cell group that is divided into the differentiation with phenotype, molecule and/or the cell characteristic similar to neural pedigree (neural lineage) cell. Multipotential stem cell can be people ES cell, and the nerve cell of differentiation can be dopaminergic neuron or serotoninergic neuron. The present invention openly also relates to cell and the cell mass that produces by disclosed method. In certain embodiments, disclosed method comprises the following steps:

1. separating multipotent population of stem cells; The people ES cell that multipotential stem cell preferably adopts new laser ablation to obtain.

2. the amplification multipotential stem cell is to provide enough parent materials.

3. suspend and cultivate multipotential stem cell to produce embryoid.

4. embryoid is inoculated on the matrix and in the serum free medium of selecting the nestin-positive neural progenitor cell and hatches.

5. sorting nestin-positive cell is to isolate the enrichment of cell group of NCAM positive cell.

6. nestin-positive and/or NCAM positive neurons CFU-GM increase in the amplification culture medium that comprises the relevant soluble factor of nervous system.

7. in the Neurobasal culture medium that comprises the relevant soluble factor combination of nervous system, nestin-positive and/or NCAM positive neurons CFU-GM are divided into mature neuron.

The source of multipotential stem cell

Method of breaking up neural pedigree cell from multipotential stem cell disclosed herein relates to use and can instruct very a high proportion of multipotential stem cell to be divided into the Incubation Condition of specific neuronal cell type. Multipotential stem cell derives from after fertilization preembryonic period, embryo or fetal tissue any time, and under suitable condition, the latter can be divided into several different cell type derived from all three germinal layers (entoderm, mesoderm and ectoderm). The cell of neural pedigree can also derive from having differentiation or reprograming the stem cell that forms neural pedigree cell ability of separating from fetus or adults tissue. Multipotential stem cell includes but not limited to mammal ES cell or EG cell, preferred primate or people ES cell or EG cell. Preferably, undifferentiated multipotential stem cell has the ability of unlimited division and propagation when cultivating. Term used herein " differentiation " refers to not break up multipotential stem cell or precursor obtains the more process of the destiny of specialization. For example, the cell of differentiation has the phenotype for particular cell types or tissue signature's property.

In preferred embodiments, ES cell used herein and ES clone derive from the inner cell mass of blastocyst. These blastocysts can be from the PIE of the internal fertilization of reclaiming, or (IVF) in vitro fertilization, for example by conventional insemination, separates among the embryo that sperm injection technology or archiblast shift and fertilization obtains in the endochylema. People's blastocyst obtains from its superfluous embryo's of voluntary donations/contributions Mr. and Mrs or contributor. After the voluntary written consent that obtains these Mr. and Mrs or contributor, these embryos are used for research purpose. Perhaps, can be by in the enucleation oocyte of body cell or nucleus being transferred to people or inhuman source, stimulate the latter to obtain blastocyst to grow to the blastocyst stage then. Used blastocyst also can freezingly be preserved, or derived from early days freezing preservation of stage and be allowed to continue to develop into blastocyst stage embryo's embryo. The growth of blastocyst and inner cell mass will have according to species different, and this is that those skilled in the art are well-known.

Can use U.S. Patent No. 5,843,780 and No.6,200,806, (the Science 282:1145-1147 such as Thomson, 1998) disclosed standard immunoassay ablation technique prepares primate or people ES cell from blastocyst and in (Nature Biotech.18:399-403,2000) (each file is introduced herein as a reference particularly) such as Reubinoff. Although in disclosed method, can use the by any method known to those skilled in the art ES cell of preparation, but a preferred embodiment is used by unique laser ablation (U.S.'s sequence number 10/226,711, it is introduced herein as a reference particularly) the people ES cell of preparation. In simple terms, this method is by carrying out laser ablation to the oolemma of blastocyst and the part of trophectoderm, forms isolated cell in the hole of the cell that can aspirate inner cell mass or the Kong Laicong blastocyst inner cell mass in blastocyst. Further cultivate then these cells to set up ES clone. This technology is favourable because its conventional loaded down with trivial details program of not carrying out the immunity excision gets final product the cell of separate inner cell mass. In addition, the antibody that can produce no any animal or serum separate the ES clone, particularly human cell line that adopts the method to produce when existing, this so that animal microorganism to the risk minimization of any propagation of people ES clone. In another embodiment, use is from the genitaloid people EG cell (U.S. Patent No. 6 that is present in the human fetal material, 090,622, with Shamblott et al., 1998, Proc.Natl.Acad.Sci.USA. 95:13726-13731 introduces each file herein as a reference particularly).

Preferably, ES clone can be maintained at the long period in the cultivation with undifferentiated state, for example surpass year, and keep its normal euploid caryogram. Can be by high nucleocytoplasmic ratio, the tight formation of conspicuousness kernel and colony (usually having clearly cell boundaries and the colony more smooth than ES cells) and on the form surveyor ES cell. People ES cell also preferably with the mark of human pluripotent stem cells, such as (1998) such as Thomson, Reubinoff etc. (2000), SSEA-3 described in Buehr and the Mclaren (1993) (each file is introduced herein as a reference particularly), SSEA-4, immune response takes place in GCTM-2 antigen and TRA 1-60. Preferably people ES cell is also expressed alkaline phosphatase, and OCT-4. In other embodiments, people ES cell also can form embryoid (U.S. Patent No. 6,602,711 is introduced into herein as a reference) under non-adhere-wall culture condition. These embryoids can be used for deriving entoderm, mesoderm and ectoderm and other required cytophyletic differentiation derivative.

Multipotential stem cell, particularly people ES or EG cell can continue propagation keeping cell and be under the condition of culture of undifferentiated state basically. , must keep suitable ES cell density and repeat disassociation and the subculture cultivation with when preventing the ES Cell Differentiation at the frequent culture medium. Cultivate and the relevant general technology of cultivation ES cell for cell, but the textbook of operating personnel's normative reference and summary, E.J. Robertson for example, " Teratocarcinomas and embryonic stem cells:A practical approach " ed., IRL Press Ltd.1987; Hu and Aunins, 1997, Curr.Opin. Biotechnol.8 (2): 148-53; Kitano, 1991, Biotechnology 17:73-106; Spier, 1991, Curr.Opin.Biotechnol.2:375-79; Birch and Arathoon, 1990, Bioprocess Technol.10:251-70; Xu et al., 2001, Nat.Biotechnol.19 (10): 971-4 and Lebkowski et al., 2001, Cancer is Suppl.2:S83-93 J.7; Each file is introduced herein as a reference particularly.

Traditionally, in the ES culture medium, support the ES cell in feeder layer. Feeder layer is and the ES co-culture of cells, and provides the ES cell to grow therein and a kind of types of organization cell of the environment that do not break up in fact. The method of cultivating the ES cell at feeder cells is the well-known (U.S. Patent No.s 5 of those skilled in the art, 843,780 and No.6,200,806, WO99/20741, United States Patent (USP) sequence number 09/530,346 and 09/849,022, WO01/51616 will introduce herein as a reference particularly for file). Feeder layer preferably can reduce, and suppresses or prevents the ES Cell Differentiation. Feeder layer is the embryo fibroblast feeder layer in people or mouse source typically, for example be MEC, human embryonic fibroblast, HF's like cell, or derive from the mesenchymal cell of human embryo stem cell or STO cell.

Preferably exist and to reduce, suppressing or cultivate the ES cell when preventing the ES culture medium of ES Cell Differentiation. Preferably, be used for cultivating in the ES culture medium of ES cell and added nutrient serum, for example can provide the serum effectively keeping the ES Growth of Cells and survive the desired nutritional material or take the solution of serum as the basis. Nutrient serum can be animal blood serum, for example hyclone (fetal bovine serum, FBS) or hyclone (fetal calf serum, FCS) (U.S. Patent No. 5,453,357,5,670,372, with 5,690,296, be introduced into herein as a reference). The ES culture medium can also not contain serum (WO98/30679, WO 01/66697, United States Patent (USP) sequence number 09/522,030 is introduced each file herein as a reference particularly). The example that contains the suitable ES culture medium that is used for cultivation ES cell of serum is Dulbecco ' s improvement Eagle ' s culture medium (DMEM), do not contain Sodium Pyruvate, glucose content height (70-90%) (GIBCO), FBS or FCS (10-30%) have wherein been added, beta-mercaptoethanol (0.1mM), nonessential amino acid (1%) and 2mM Glu, 4ng/ml basic fibroblast growth factor (bFGF), 50U/ml penicillin, the LIF ELISA (LIF) of 50 μ g/ml streptomysins and 1000 U/ml. The example that does not contain the suitable ES culture medium that be used for to cultivate the ES cell of serum be 80% " knocking out " Dulbecco ' s improvement Eagle ' s culture medium (DMEM) (GIBCO), 20% KnockOut SR (serum-free substitute, GIBCO), beta-mercaptoethanol (0.1mM), nonessential amino acid (1%) and Glu 1mM.

The ES cell also can be cultivated under the condition of culture of no feeder cells. The method of cultivating the ES cell under the condition of culture of no feeder cells is well-known (the open No. 2002/0022268 of United States Patent (USP) of those skilled in the art, WO 03/020920, U.S.'s sequence number 10/235,094 is introduced each file herein as a reference particularly). ES cell preferred growth is at suitable culture matrix in no feeder cells culture, and extracellular matrix for example is on Matrigel  (Becton Dickenson) or laminin. No feeder cells are cultivated the also preferred conditioned medium of supporting the ES Growth of Cells that uses. Conditioned medium is enough to produce and will supports the ES cell to cultivate the time of not having " condition " culture medium that breaks up in fact to prepare by cultivate first group of rat embryo fibroblast cell or human embryonic fibroblast in culture medium. Perhaps, no feeder cells are cultivated can will have effective culture medium to be combined with extracellular matrix, wherein the fresh adding culture of culture medium is not modulated (the open No.2003/0017589 of United States Patent (USP) introduces it herein as a reference particularly) by another kind of cell type.

The preparation of neural progenitor cell

The isolating multipotential stem cell that increases is placed on then and can makes it to be divided in the culture condition of neural progenitor cell.For multipotential stem cell, cultivate according to differentiation method disclosed herein along the development of Neural Differentiation path.Multipotential stem cell is containing differentiation factor, for example cultivates on suitable matrix in the differentiation nutritional medium of soluble factor and somatomedin.Suitable matrix includes but not limited to be coated with positive charge, and for example the solid surface of poly-L-Lysine or poly ornithine is coated with extracellular matrix components, fibronectin for example, ln, Matrigel , or the matrix of its combination.Preferred differentiation nutritional medium is to support required neural cell type propagation, and the substratum of differentiation and survival can contain one or more suitable differentiation factors.The term of Shi Yonging " somatomedin " is meant and can combines with cell surface receptor mainly to cause the protein of activating cells propagation and differentiation herein.Suitable soluble factor includes but not limited to neurotrophin, mitogenic agent, STEM CELL FACTOR, somatomedin, differentiation factor (for example TGF-beta superfamily), TGF-beta superfamily agonist, neurotrophic factor, antioxidant, neurotransmitter and survival factors.Multiple soluble factor is very polyenergic, can stimulate the cell fission of number of different types, other be specific then to particular cell types.

The suitable differentiation factor of differential stimulus neural cell type differentiation includes but not limited to Progesterone, putrescine, ln, Regular Insulin, Sodium Selenite, Transferrins,iron complexes, neurturin, sonic hedgehog (SHH), noggin, follistatin (follistatin), Urogastron (EGF), the fibroblast growth factor of any kind (for example FGF-4, FGF-8, Prostatropin (bFGF)), growth and differentiation factor 5 (GDF-5), neurotrophin family member (nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), neurotrophic factor derived from brain (BDNF)), transforming growth factor-alpha (TGF-α), TGF-(TGF β 3), the platelet-derived long factor (PDGF-AA), rhIGF-1 (IGF-1), Delicious peptide (BMP-2, BMP-4), neurogliocyte derived neurotrophic factor (GDNF), vitamin A acid (RA), midkine, xitix, two butyryl cAMP, Dopamine HCL and form the part LIF for example of the acceptor of complex body with gp130, CNTF, SCF, IL-11 and IL-6).The differentiation nutritional medium also can include the additive that helps keep neuronal cell cultures, for example N2 and B27 additive (Gibco).

At first induced multi-potent stem cells forms embryoid.Directly embryoid is inoculated in and contains or do not contain extracellular matrix components, for example on the suitable matrix of fibronectin or ln, be suitable for promoting cytodifferentiation to become neural progenitor cell, for example cultivating in the suitable differentiation nutritional medium of nidogen (nestin) positive neurons progenitor cell.Nidogen is the distinctive cell sign thing of neural precursor.In another embodiment, at first, for example, multipotential stem cell is gathered into the heterogeneous body cell mass by in suspension, cultivating multipotential stem cell by forming embryoid.Containing or do not containing serum, and containing and cultivate these cells in the nutritional medium of one or more differentiation factors of listing above to promote the cytodifferentiation in the embryoid.The term of Shi Yonging " embryoid " is meant when multipotential stem cell and grows in suspension culture or the aggregate of the noble cells that produced during hypertrophy in monolayer culture herein.Embryoid also can have undifferentiated cell in cell aggregate.Preferably this cell aggregate is surrounded by primitive endoderm.Embryoid generally comprises and derives from all three germinal layers, i.e. entoderm, mesoderm and ectodermic cell.In sophisticated people's embryoid, can pick out cell, neurocyte for example, hematopoietic cell, liver cell and myocardial cell with different cell type marks.Some cell in sophisticated people's embryoid shows the function of similar noble cells.For example, the active myocardium cell can make embryoid beat.Preferably control the differentiation of multipotential stem cell so that obtain to be used for the treatment of the particular cell types of purpose.

Cultivate embryoid until reaching enough big size or required differentiation degree, for example after cultivating 3-10 days, then it is inoculated on the matrix.Preferably matrix is with extracellular matrix components bag quilt, and described composition includes but not limited to poly-L-Lysine or poly--L-ornithine, ln, collagen protein, fibronectin, Matrigel  or its combination.Preferably need not cell dispersion and with the embryoid direct inoculation on matrix.Can stimulate inoculating cell further to cultivate embryoid under the condition of differentiation then, for example the nidogen positive cell had optionally, cultivating in the known serum-free ITSFn of composition (nidogen selection) substratum.Nidogen is the intermediate filament protein of expressing in the neuro epithelium.Preferably, during 5-16 days, from cell, select the nidogen positive cell.Preferably, the ITSFn substratum of the nidogen positive cell that is used to increase is the DMEM:F-12 that has added one or more somatomedins, and wherein somatomedin is selected from Progesterone, putrescine, ln, Regular Insulin, Sodium Selenite, Transferrins,iron complexes, bFGF, SHH, EGF, FGF-2, FGF-8 and BDNF.

Amplification or sorting comprise the heterogeneous body cell mass of nidogen positive cell with enriching nerve cell adhesion molecule (NCAM) positive cell then.NCAM is the distinctive surface marker of neurocyte.Can or cultivate behind the amplification nidogen positive cell sorting NCAM positive cell immediately after selecting the nidogen positive cell.In specific embodiments, by with antibody or the part of cells contacting in conjunction with NCAM, adopt suitable immunological technique such as immune labeled and fluorescence separating method then, solid phase adsorption for example, fluorescence activated cell sorting (FACS), at the streaming immunocytochemistry of cell surface marker, flow cytometry or magnetic cell sorting art (MACS) are isolated by the cell of specific recognition and are sub-elected the NCAM positive cell.Separate the additive method of NCAM positive cell, include but not limited to difference plate streak (differential plating), the immunologic opsonin cracking of contamination of cells, or results technology are that those skilled in the art are well-known.In preferred embodiments, sorting nidogen positive cell (for example passing through MACS) can be enriched to about 40-70% with the nidogen positive cell group alive of expressing NCAM, preferably approximately 60%-80%, more preferably about 85%-90%, most preferably about 95%-99%.

In one embodiment, come from people ES cell preparation embryoid by the cultivation on Micro-Organism Culture Dish in the suitable culture medium of no feeder cell.Preferably that people ES cell breakdown is agglomerating, be seeded in the growth that is beneficial to embryoid in the non-adhesive culture dish then.Suitable medium preferably comprises high sugared DEME and adds 10-20%FCS.Also can add other additive in the substratum, for example 0.1mM 2 mercapto ethanol, 2mM L-glutaminate, 50U/ml penicillin and 50 μ g/ml Streptomycin sulphates.Preferably make embryoid growth 4-8 days, then embryoid is seeded in the culture dish with 0.1% gel pack quilt, in serum-free ITSFn substratum, select the nidogen positive cell.Serum-free ITSFn substratum preferably includes and has added the somatomedin Regular Insulin that is used to select the nidogen positive cell, Sodium Selenite, the basic medium DMEM:F-12 (1: 1) or the IMDM substratum of Transferrins,iron complexes and fibronectin.

In preferred embodiments, by MACS sorting nidogen positive cell to isolate the NCAM positive cell.Helping to increase neural progenitor cell per-cent and further inducing these cells to take amplification NCAM positive cell in the substratum of more phenotypic differentiations then.Preferably, use extracellular matrix components in advance, for example poly-L-Lysine is poly--the L-ornithine, ln, and collagen protein, fibronectin is cultivated the NCAM positive cell on the matrix of Matrigel  or its combination bag quilt.With extracellular matrix culture dish is wrapped in advance and cell to be sticked in amplification culture medium better and breed, also can produce better dopaminergic neuron differentiated result.At the amplification culture medium that has added one or more somatomedins, culturing cell in the DMEM/F12 substratum for example, wherein somatomedin is selected from Progesterone, putrescine, ln, Regular Insulin, Sodium Selenite, Transferrins,iron complexes, bFGF, SHH, EGF, FGF-2, FGF-8, BDNF, PDGF, IGF-1, CTNF and NT-3.Though do not wish to be subjected to any special mechanism to fetter, be sure of that these are present in multiple factors in the amplification culture medium and have facilitated the integral body of neurocyte per-cent to increase, and further induce midbrain neuron precursor cell performance dopaminergic phenotype.In preferred embodiments, make the positive precursor cell propagation of the positive NCAM of nidogen 3-10 days.But these cells are at least three ten population doublings of continuous passage and freezing in order to further use also, and can not lose any differentiation potential.

The differentiation of dopaminergic and serotoninergic neuron

Neural progenitor cell according to method preparation disclosed herein can further be divided into a high proportion of mature neuron, for example dopaminergic neuron and serotoninergic neuron.Neural progenitor cell can also further be divided into astroglia cell and oligodendrocyte.Preferably, can help progenitor cell to be divided in the Neurobasal substratum of the neurocyte of eventually end differentiation or mature neuron the amplification nidogen positive and/or NCAM positive neurons progenitor cell 5-60 days.In preferred embodiments; in Neurobasal substratum (Gibco), add 10%FBS or FCS; B27 and one or more are selected from il-1 β, two butyryl ring AMP (db-cAMP); glial cell-line derived neurotrophic factor (GDNF); transforming growth factor-beta 3 (TGF-β 3), transforming growth factor-alpha (TGF-α), neurturin; SHH; xitix, BDNF, FGF-2; FGF-8; N-acetylcystein, c-kit part, vitamin A acid; NT-3, the somatomedin of BMP-2 and BMP-4.In preferred embodiments, the Neurobasal substratum comprises one or more following factors: il-1 β, db-cAMP, GDNF, TGF-β 3, neurturin, SHH, xitix, BDNF, FGF-8 and N-acetylcystein.In addition, can promote the neural progenitor cell differentiation by cancelling some or all, propagation, or the factor of the two promotes differentiation.

Preferably, the neural progenitor cell of high per-cent is divided into dopaminergic neuron, serotoninergic neuron or oligodendrocyte, for example about at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% cell.In addition, by the well-known method of those skilled in the art, for example immune labeled and fluorescence separating method such as solid phase adsorption, FACS, MACS etc., further a kind of neural cell type of purifying, for example dopaminergic neuron from the neural cell group of differentiation.In a preferred embodiment, amplification and the NCAM positive cell group of differentiation by immune sorting enrichment in appropriate media, thus produce the dopaminergic neuron of high per-cent (about 60%).

The purposes of the neurocyte of neural progenitor cell and differentiation

The neurocyte of neural progenitor cell described herein and differentiation (for example sophisticated neurone, astroglia cell and oligodendrocyte) can be used for many aspects, for example be used for the treatment of, and external assessment and screening all cpds, for example small-molecule drug is to the effect of these cells.The well-known method of also available those skilled in the art is used to prepare the cDNA expression library with the analytical table expression patterns with these cells, and the mono-clonal or the polyclonal antibody of the mark of the used specific cells of preparation specificity.These cells also can use in treatment to benefit and suffer from the neurodegenerative disorders that makes people's weakness and the patient of neuronal disease.

The present invention openly also relates to the function that the neurocyte that uses neural progenitor cell as described herein and differentiation recovers the individual central nervous system (CNS) of the described treatment of needs.For example, use these cells by these cells being grafted directly to treatability ground, site in CNS essence or the sheath (depending on the disease or the illness that need treatment).These cells can be used for treating acute or the chronic neurological damage, and the neurodegenerative disorders and the neuronal disease that make people's weakness, Parkinson's disease for example, Alzheimer, Huntington Chorea, Spinal injury, amyotrophic lateral sclerosis (ALS), epilepsy, apoplexy, local asphyxia, or the like.

An embodiment disclosed by the invention relate to by use or the transplantation treatment significant quantity derive from multipotential stem cell, the dopaminergic neuron of preferred human pluripotent stem cells is treated the neurodegenerative disorders that is characterised in that dopaminergic neuron sex change or damage or the method for neuronal disease.In another embodiment, the present invention openly relate to by use or the transplantation treatment significant quantity derive from multipotential stem cell, the serotoninergic neuron of preferred human pluripotent stem cells is treated the neurodegenerative disorders that is characterised in that serotoninergic neuron sex change or damage or the method for neuronal disease.Preferably, the neurocyte of multipotential stem cell disclosed by the invention by will treating significant quantity and differentiation is implanted in patient's body the human patients of suffering from neurodegenerative disorders or neuronal disease is treated." the treatment significant quantity " of cell used herein is meant and is enough to stop or improves in the subjects body neurocyte by differentiation, mature neuron (for example dopaminergic and serotoninergic neuron) for example, astroglia cell and oligodendrocyte forfeiture, the amount of damage or the physiological effect that sex change produced.

The treatment significant quantity of employed cell depends on individual needs, individual age, physiological condition, state of health, required result of treatment, the size of the target tissue region of being treated, the delivery path of pathology degree and selection.For example, compare with less target region, the treatment of conditions that influences big zone in the brain may need the greater amount cell to obtain result of treatment.Cell can also be applied to more than one site in the given target tissue by a plurality of little graft of low cell dosage.Cell disclosed by the invention can disperse before transplanting fully, for example forms single-cell suspension liquid, or almost completely disperses before transplanting, for example forms little cell aggregation thing.Cell can be with allowing its transplanting or moving to predetermined tissue site and make the insufficiency of function regional reconstruction or the regenerated mode is used.

Can use optimum range with the cell that produces result of treatment and can be about 100 to about 1,000,000 neurone, preferably approximately 500 to about 500,000 neurones or about 1000 to about 100,000 neurones.The treatment concentration range of the neurocyte of using to individuality can also be about 10,100,500,1000,5000,10,000,15,000,20,000,25,000,30,000,35,000,40,000,45,000,50,000,60,000,70,000,80,000,90,000,100,000,150,000,200,000,250,000,300,000,350,000,400,000,450,000 to about 500,000 cells/pharmaceutically acceptable carrier of μ l.The concentration range of cell comprises for example 100-50 in the carrier, 000 cell/μ l, 1000-10,000 cell/μ l, 5000-25,000 cell/μ l, 15,000-45,000 cell/μ l, 20,000-50,000 cell/μ l, 55,000-200,000 cell/μ l, 100,000-40,000 cell/μ l, 150,000-50,000 cell/μ l etc.The cell quantity in implantable graft site also will influence result of treatment.

Use for treatment, preferably be substantially devoid of any precursor cell of multipotential stem cell or neural cell group of differentiation of not breaking up usually.A strategy removing multipotential stem cell from healing potion is that described expression can select to remove multipotential stem cell with the carrier transfectional cell that contains preferential gene of expressing in undifferentiated cell.The preferential suitable promotor of expressing is reverse transcriptase of telomere (TERT) promotor and OCT-4 promotor in undifferentiated cell.The gene of expressing in carrier for example can be the cleavable cell, and for example toxin perhaps can select to remove this gene by using exterior materials.

The ability that produces dopaminergic neuron and serotoninergic neuron by multipotential stem cell has very big clinical correlation for the transplantation treatment of multiple neurodegenerative disorders and neuronal disease as described here.For example, can be characterised in that attitudinal reflex with the dopaminergic neuron treatment, unusual neurodegenerative disorders and neuronal disease appear in the adjusting of motion and award related behavior, Parkinson's disease for example, schizophrenia and drug addiction, and because wound or other cause the class parkinson symptom, for example static tremor, tetanic, motion can not with abnormal posture as the motion can not, adipsia, the pathology that the disease of aphagia and sensory neglect causes.Can treat with serotoninergic neuron in addition and be characterised in that ingestion of food, hormone secretion, stress reaction, pain and immunologic function, sexual activity, the adjusting of cardiovascular function and temperature exists unusual neurodegenerative disorders and neuronal disease, for example various spirit, neural or other disease, as spirit depressing, introgression, brute force attack behavior, compulsive behavior, apositia/bulimia nervosa and schizophrenia.

In another embodiment, the present invention openly relates to and uses one or more neuronal survival factors simultaneously and of the present inventionly come from the neural progenitor cell of multipotential stem cell and the neurocyte of differentiation is treated neurodegenerative disorders or neuronal disease.Can be before using required cell, or associating with it, combination or after using required cell, use neuronal survival factor." neuronal survival factor " of Shi Yonging is meant any material of neuronic survival time when the neuronic survival time is not existed above this factor herein.Can treat the neuronal survival factor that uses in the embodiment in the present invention and include but not limited to neurogliocyte derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophic factor derived from brain (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), FGF, IL-1 β, TNF α, rhIGF-1 (IGF-1, IGF-2) and transforming growth factor-beta (TGF-β, TGF-β 1).

Known ectotrophic effect (Lin etal., 1993, the Science 260:1130-1132 that has embryo's midbrain veutro dopaminergic neuron of GDNF; Lin etal., 1994, J.Neurochem.63:758-768).Recombinant human GDNF also is proved to be (the Hudson et al. of the sprouting of Induction of dopaminergic neurofibril in vivo; 1993; Soc.Neurosci.Absir.19:652); increase conversion (the Miller et al. of Dopamine HCL in the rat substantia nigra; 1994; Soc.Neitrosci.Abstr.20:535-7); the opposing 6-OHDA of neuroprotective unit pathology; the growth and the fiber that can increase rat fetal black substance tissue grafts in eye form (Stromberg et al.; 1993, Exp.Neurol.124:401-412).BDNF is a peripheral sensory neuron, dopaminergic neuron, the nutritional factor of ganglia retinae (Henderson etal., 1993, Restor.Neurol.Neurosci.5:15-28), demonstrate it and can both prevent the normal necrocytosis (Hofer and Barde, 1988, Nature 331:161-262) that takes place in vitro and in vivo.

Term used herein " treatment " or " being used for treating " refer to the measure of therapeutic treatment and preventative or preventing property.Therefore, need the object of treatment to comprise that those have suffered from the individuality of neurodegenerative disorders or neuronal disease, and those need prevent the individuality of neurodegenerative disorders and neuronal disease.Method disclosed by the invention can be used for treating any Mammals that needs treatment, includes but not limited to the mankind, primate, the animal of raising and train, farm-animals, pet or motion animal, dog for example, horse, cat, sheep, pig, ox etc." illness " refers to can be from using neural progenitor cell disclosed by the invention, the neurocyte of differentiation, or obtain any state of benefit in the treatment carried out of this cell of two types.The example that can obtain the illness of benefit from dopaminergic neuron is transplanted is that those and abnormal posture reflect, motion and the relevant state of award related behavior, for example Parkinson's disease, schizophrenia and drug habit.The example that can obtain the illness of benefit from serotoninergic neuron is transplanted is that those have consciousness, arouses the state of the feature that behavior and ingestion of food are unusual, include but not limited to attack, depressed (comprising suicide), schizophrenia, apositia/bulimia nervosa.Other illness that can obtain benefit from the treatment of using cell disclosed by the invention to carry out has Alzheimer, Huntington Chorea and HirschsprungShi disease.

Method disclosed by the invention can be implemented to lesion region easily by the neurocyte of directly transplanting neural progenitor cell disclosed by the invention or differentiation.The method of neuron transplantation and cell cultures is that those skilled in the art are well-known, and for example U.S. Patent No. 5,514,552; Yurek and Sladek, 1990, Annu.Rev.Neurosci.13:415-440; Rosenthal, 1998, Neuron 20:169-172; Vescovi et al., 1999, J.Neurotrauma 16 (8): 689-93; Vescovi et al., 1999, Exp.Neuro.156 (1): 71-83; Brustle et al., 1999, Science 285:754-56; Each file is all introduced herein as a reference particularly.In one embodiment, dopaminergic neuron disclosed by the invention is transplanted in parkinsonian's the black substance or striatum.Separately delivery of cells or and other factors, neuronal survival factor delivery of cells together for example, also can with pharmaceutically acceptable carrier delivery of cells together.More satisfactory situation is that this carrier can strengthen the stability of cell and send character.

The present invention provides also openly that comprise can be by using suitable carrier, liposome for example, the pharmaceutical composition of the cell that particulate or micro-capsule are sent.The also available form that comprises the pharmaceutical composition of isotonic excipient of cell disclosed by the invention provides and is prepared under condition fully aseptic for human administration.The rule of the medical preparation of cell composition is found in Cell Therapy:Stem CellTansplantation, Gene Therapy, and Cellular Immunotherapy, G.Morstyn ﹠amp; W.Sheridan eds, Cambrigge University Press, 1996, and Hematopoietic StemCell Therapy, E..Ball, J.Lister ﹠amp; P.Law, Churchill Livingstone, 2000, it is introduced herein as a reference particularly.In addition, the pharmaceutical composition that may need to comprise neuronal survival factor is locally applied to the zone that needs are treated, it can pass through, local infusion in for example performing the operation, injection uses the mode or the implantation mode of conduit to realize that wherein said implant can be to comprise film, the porous of silicone rubber membrane or fiber for example, non-porous or gelatinous material.

Can be with homogeneous basically, near homogeneous or heterogeneous cell mass form with the nerve cells transplantation of neural progenitor cell disclosed by the invention or differentiation in individuality.Basically the cell mass of homogeneous comprises greater than 75%, more preferably greater than 90%, and most preferably greater than the single cell type of 95%-99%, for example dopaminergic neuron or serotoninergic neuron.The foreign cell group is by two or more cell types that are blended in the cell mass, dopaminergic neuron for example, and serotoninergic neuron, Scs, oligodendrocyte, astroglia cell, GABA neurone and neurogliocyte are formed.Cell can also adopt the well-known method of those skilled in the art to carry out the genetics change so that it is at brain, central nervous system, express in the damage field of peripheral nervous system or its hetero-organization or release nutritional factor, somatomedin, neuronal survival factor or other treatment compound.Using the combination of promotor and cell type is that branch is well-known in the technician of field of biology with expressing protein, for example referring to Sambrook, and et al.1989, Molecular Cloning:ALaboratory Manual 2 ^NdEd.Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY introduces it herein as a reference particularly.

In order to express nutritional factor in the neurocyte that is implemented in neural progenitor cell disclosed by the invention and differentiation, somatomedin, neuronal survival factor or other therapeutic compound, the controlling element that can obtain suiting from multiple source, and those of ordinary skills can select it at an easy rate.The example of controlling element comprises transcripting promoter, enhanser, and RNA polymerase binding sequence, and ribosome binding sequence comprise translation initiation signal.Also can for example select selective marker to be incorporated in the recombinant molecule other additional genetic elements.Can use in external delivery vector or the body technology will weigh ancestral's molecule and import multipotential stem cell, or derive from the neurocyte of the neural progenitor cell of multipotential stem cell or differentiation.The example of delivery technique comprises retroviral vector, adenovirus carrier, dna viral vector, liposome, for example microinjection and of physical technique by the transfection of electroporation or calcium phosphate precipitation, or become known in this area shifting to generate the additive method of reconstitution cell.The cell that genetics can be changed be wrapped in the microballoon and be transplanted to pathology or damaged tissue in or near it.Employed method steps is that those skilled in the art are well-known, is found in for example Ausubel etal., Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, New York, N.Y., 1997, introduce herein as a reference.

From the various animal models that use Parkinson are implanted to the basic scientific research of receptor in brain injury zone as embryo's neurocyte or paraneuron tissue, developed and related to suffering from the clinical trial that the parkinsonian carries out neuron transplantation.Adopt the animal experiment of fetus dopaminergic nerve cell graft that the inspiration result is provided, promptly this graft can be reversed the dopaminergic defective, and recovers the motor function of the animal of the experimental damage of nigrostriatum dopaminergic system.For example, the mouse midbrain cell of taking from 14-16 day instar embryo is used for transplanting and not carrying out immunosuppressant therapy (Yurek and Sladek as autograft or allogeneic better than the cell of taking from later Gestation period donor, 1990, Annu.Rev.Neurosci.13:415-440).Be interestingly, these ages and dopaminergic neuron experience its final cell splitted gestational age corresponding (Lauder and Bloom, 1974, J.Comp.Neurol.155:469-82).The solid graft of cerebral tissue among the embryo can also be dispersed into cell suspension is used for transplanting.But, the general survival rate of this cell suspension be limited to transplanting dopaminergic cell about 10% (Brudinet al., 1987, Ann.N.Y.Acad.Sci.495:473-96).Because cerebral tissue is not pure dopaminergic cell source (in total survivaling cell only approximately the dopaminergic cell for survival of 0.1-1.0%) among the embryo, any dispersive midbrain cell graft preferably comprises minimum 100,000 to 150,000 viable cell is so that effectively remedy the dopaminergic loss.

Preferably, cellular transplantation therapy disclosed by the invention also comprises storage that some is used for transplantation and the method for preserving the neurocyte of neural progenitor cell and differentiation, for example standing storage by freezing preservation, or preserving substratum storage a middle or short term.Cerebral tissue successfully stores and reaches 70 days and migrate to rodent (Collier et al. as autograft among the embryo of freezing preservation, Progress in Brain Research, Vol.78, New York, Elsevier (1988), pp.631-36, it is introduced herein as a reference particularly) and primate (Collier et al., 1987, Brain Res.436:363-66 introduces it herein as a reference particularly) in.Embryo's midbrain cell still can successfully be cultivated also after freezing preservation and be proved to be.Middle cerebral tissue also can be at 4 ℃ in preserving substratum storage a middle or short term (2-5 days), and transplant (Sauer et al. with survival graft volume with the volume similar methods that is used for flesh tissue subsequently, 1989, Restor.Neurol.Neurosci. (Suppl.:3.sup.rd Int.Symp.Neural Tranplan.): 56, it is introduced herein as a reference particularly).

Placement of graft and graft make striatum regain the important factor that innerv degree all is middle brain striatum dopaminergic system functional rehabilitation.In the animal of one-sided removal dopaminergic nerve domination, it is asymmetric to have shown that the dopaminergic graft of placing in the cortex can reduce motion, but sensory inattention is not almost influenced (Bjorklund et al., 1980, Brain Res.199:307-33; Dunnett etal., 1981, Brain Res.215:147-61).On the contrary, remove in the animal of dopaminergic nerve domination at bilateral, being placed near the black substance graft of side striatum can have efficient recovery sensation damage (Dunnettet al., 1983, Acta Physiol.Scan.Suppl.522:39-47).And, in the impaired animal of bilateral, only when making volt nuclear regain innervation, the dopaminergic graft can observe akinetic reverse (Nadaud et al., 1984, Brain Res.304:137-41).

The transplanted sites of dopaminergic graft is most commonly near the district of chamber (condition of graft survival can be provided because of good cerebrospinal fluid (CSF) environment in these zones), but the degree of the interior dopaminergic neuron sex change of Parkinson's disease mesochite nuclear is than remarkable in the caudatum.Dopamine level is often than the low 10-15% in the caudatum (Bemhiemer et al., J.Neurol.Sci.20:415-55 in the shell nuclear; Nyberg et al., 1983, Neurochem.Pathol.1:93-202).In addition, the depletion of shell nuclear afterbody (caudal putamen) dopaminergic neuron than shell nuclear beak portion (rostral putamen) more serious (Kish et al., 1986, Ann.Neurol.20:26-31).In these two striatum parts (caudatum and shell nuclear), shell nuclear is accepted most motion input (DeLong ﹠amp by cortex-thalamus-shell nuclear path; Georgopoulos, 1983, Handbook of Physiology, Section I:The Nervous System, Vol.2, ed.Brookhard, Mountcastle, Geiger, pp.1017-61, Bethesda, Md.:Am.Physiol.Soc.).Therefore, when the target dyskinesia relevant with Parkinson's disease shell to endorse can be the implantation site of dopaminergic graft more suitably.

The neurocyte of neural progenitor cell described herein and differentiation also can be used for for example medical compounds of compound that examination influences the phenotype of these cells or feature, solvent, small molecules, peptide or polynucleotide, and for example culture condition or operation of environmental factor.In addition, these cells can be used for assessing candidate's somatomedin or differentiation factor.For example, the candidate medical compounds can be joined in neural progenitor cell or the mature neuron separately or with other drug together, the pair cell form, any variation of phenotype or functionally active is evaluated and tested and is assessed.

In addition, the neurocyte of neural progenitor cell described herein and differentiation can be done further to modify in any stage of differentiation.For example, can carry out genetic modification to these cells makes it have single or multiple temporary transient or stable genetic modifications.The hereditary change of these cells can be that multiple reason is required, for example for the cell of the modification that is provided for gene therapy or be used to the replacement tissue transplanting or implant.Cell disclosed by the invention can carry out genetic modification by importing those skilled in the art's method well-known, express the carrier of selective key thing under the control of nerve-specific promotor.These cells also can be modified to express some in any stage and can be used for being further purified the noble cells that comes from multipotential stem cell or be used for inducing cell and be divided into the mark or the gene of specific cells pedigree.Can modify these cells to reduce or to prevent to transplant the back immunological rejection, promptly with required recipient's histocompatibility.

In order to improve the replication of using the present invention to disclose prepared cell, can be by make it express telomerase catalytic composition (TERT) with these cells of suitable carriers hereditary change with its telomereization.Used TERT sequence can derive from people or mouse (WO 98/14592 and WO 99/27113 introduce it herein as a reference particularly), and other mammal species.Perhaps, can increase endogenous TERT gene transcription.The method that is used for genetically modified cell is that those skilled in the art are well-known.These methods have been used multiple Protocols in Molecular Biology, and wherein multiple technologies usually are described in Sambrook, et al.1989, Molecular Cloning:A Laboratory Manual 2 ^NdEd.Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY introduces it herein as a reference particularly.

***

Following examples are in order to explanation the preferred embodiments of the invention.Those skilled in the art will be understood that the good technology of function when carrying out an invention that on behalf of the inventor, disclosed technology find in following examples, therefore can think that it constitutes preferred Implementation Modes.Yet those skilled in the art will be understood that (under enlightenment disclosed by the invention) can make multiple change to disclosed specific embodiments under the situation that does not deviate from spirit and scope of the invention, and still can obtain similar or similar result.

Embodiment 1

In following examples, applicant of the present invention has proved can be from human embryo stem cell derivation function dopaminergic and serotoninergic neuron.

1) human embryo stem cell:

At first, from the inner cell mass separation of human ES cell of people's blastocyst.After agreeing tentative use blastocyst, individual patient from supernumerary's blastocyst, obtains people's multipotency ES cell.Disclosed new laser ablation is obtained the people ES clone of using herein in the use United States Patent (USP) sequence number 10/226,711 (introducing herein as a reference) from the inner cell mass cell of people's blastocyst.In brief, separation has zona pellucida, nourishes people's blastocyst of germinal layer and inner cell mass, uses 1.48 μ m non-contact diode lasers to produce the hole that penetrates people's blastocyst zona pellucida and trophectoderm then.Subsequently, use the suction tube isolated at suction that imports through this hole to go out cell in the inner cell mass.The mouse feeder cell of handling with ametycin in the ES substratum on 0.1% gelation culture dish are cultivated these cells subsequently, form inner cell mass deutero-cell mass.The consisting of of ES cell culture medium added the excellent foetal calf serum of 10-20%ES cell (FCS) (Hyclone) or blood serum substituting product Knockout serum (Gibco), 1%MEM non-essential amino acid solution, the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 4 η g/ml Prostatropins (bFGF) (Sigma), 50U/ml penicillin, the Dulbecco ' s improvement Eagles substratum (DMEM) or the Knockout DMEM (Gibco) of 50 μ g/ml Streptomycin sulphates.These inner cell mass deutero-cell masses are disperseed, and are seeded in once more on the mouse feeder layer in the ES substratum, are used for preparing people ES clone.

Deutero-people ES cell has high nuclear/kytoplasm ratio on form, with cell surface marker, and SSEA-1 for example, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, alkaline phosphatase, Telomere terminal transferase, caryogram, CD-30, cripto-1, GCNF, c-kit and CD-90 carry out feature description.

2) cultivation and amplification human embryo stem cell:

Cultivation and propagation derive from the people ES cell of refrigerated storage thing under the standard growth condition.The not differentiation of human ES cell that derives from the refrigerated storage thing bottle is suspended in the ES substratum that contains LIF once more.Consisting of of ES cell culture medium added the excellent foetal calf serum of 20%ES cell (FCS) (Hyclone), 1% non-essential amino acid (NEAA) solution, the 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, the Dulbecco ' s improvement Eagles substratum (DMEM) of 50U/ml penicillin and 1000U/ml LIF.Make cell form precipitation and it is inoculated on the mouse feeder layer on the plastic culture dish of gel pack quilt with the ES substratum.According to Thomson et al., 1998, Science 282:1145-1147; Shamblott et al., 1998.Proc.Natl.Acad.Sci.USA.95:13726-13731; With Reubinoff et al., 2000.Nature the method for describing among the Biotech.18:399-403,10-20%FCS has been added in consisting of of this ES cell culture medium, 2mM L-glutamine, 1%MEM non-essential amino acid solution, 0.1mM beta-mercaptoethanol, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates, the high glucose DMEM of 1000U/ml LIF and 4 η g/ml bFGF.

Adopt amplification people ES cell by cultivating to suppress its differentiation with the ES cell that regularly goes down to posterity.Earlier with not conforming to Ca ⁺⁺And Mg ⁺⁺10 seconds of Dulbecco ' s phosphate buffer saline solution washing cell, then with the 0.05% trypsin treatment cell ES cell that goes down to posterity for 5 seconds.After 5 seconds, the ES substratum that contains serum by adding suppresses tryptic activity.Subsequently, scrape cell and it is dispersed into little group.Then cell mass being inoculated in two scribbles and places the substratum of ES as mentioned above that contains LIF and bFGF on 0.1% gel and the 100mm culture dish with feeder cell bag quilt.Amplifying cells 4-6 days.

3) formation of embryoid:

Behind the breeding and the undifferentiated people ES cell that increases, it is cultivated to form embryoid.At first, with 0.05% trypsinase-EDTA ES cell that dissociates, then scrape cell and cell is dispersed into little group.Then with about 1 * 10 ⁵The density of cell/ml is inoculated in these cell masses on the Micro-Organism Culture Dish of no feeder cell.Employed Micro-Organism Culture Dish has and prevents from the inadhesion surface adhered to stimulate the differentiation of ES cell and the formation of embryoid thus.These cells of suspension culture in not containing the ES substratum of LIF and bFGF.The ES substratum that is used for cultivating these cells contains and has added 10-20%FCS or Knockout serum substitute, and other the interpolation factor, beta-mercaptoethanol for example, L-glutaminate and antibiotic high glucose DMEM or Knockout DMEM.Do not add bFGF in the ES substratum.Make embryoid growth 4-8 days.During this time, changed an ES substratum in per 2 days by settling process.By the suspension of aggregation is transferred in the centrifuge tube, allow aggregation to be deposited to the bottom of pipe, sucking-off substratum and it is replaced with fresh culture implement this settling process.Then the aggregation in the fresh culture is returned culture dish.Collected embryoid when finishing in 4-8 days, low-speed centrifugal is suspended in the ES cell culture medium once more.Then about 20-30 embryoid is inoculated in the ES substratum that does not contain LIF with the tissue culture ware of 0.1% gel pack quilt, hatched 24 hours.

4) selection and amplification nidogen positive neurons progenitor cell:

After 24 hours, select nidogen positive cell (neural progenitor cell) by replacing the ES substratum with ITSFn (selection nidogen) substratum of serum-free principal component.Consisting of of ITSFn substratum added somatomedin Regular Insulin (5-25 μ g/ml) (Sigma), Sodium Selenite (10-50nM) (Sigma), Transferrins,iron complexes (1-10 μ g/ml) is (Gibco) and the DMEM:F12 substratum (Gibco) of fibronectin (1-5 μ g/ml).This substratum can screen the nidogen positive cell, can implement 6-10 days, and 8-9 days usually, per 2 days additional ITSFn substratum.After selection is finished, adopt immunofluorescence technique to express at nidogen and neural progenitor cell is characterized, show about 95% cell expressing nidogen (Figure 1A).

Nidogen positive cell and use magnetic cell sorting art (MACS) to carry out sorting with enriching nerve cell adhesion molecule (NCAM) positive cell subsequently increases.

5) adopt magnetic sorting art sorting NCAM positive cell:

Subsequently, separating the nidogen positive cell also implements amplification or carries out sorting with enriching nerve cell adhesion molecule (NCAM) positive cell with magnetic cell sorting art (MACS).NCAM is a kind of neuronal specificity surface marker.In order to use MACS to pass through NCAM sorting nidogen positive cell, at first from the tissue culture ware, gather in the crops the nidogen positive cell by 0.05% trypsinase-EDTA incubated cell.Then, wash dissociated viable cell, use the antibody (Chemicon) of anti-surface marker NCAM to dye then 30 minutes with PBS.Then cell and MACS goat anti-rabbit igg microballon (Miltenyi Biotec) were hatched 15 minutes together.Pair cell carries out magnetic sorting after carefully washing with PBS.For sorting cells, with the cell suspension (about 2 * 10 of magnetic mark ⁸Individual cell) draws to MACS MS separator column (Miltenyi Biotec), allow stream of cells cross pillar, collect effluent liquid (negative fraction).Collect the positive fraction that contains the NCAM positive cell by the PBS wash-out then.

Before immune sorting, with facs analysis nidogen positive cell, approximately the nidogen positive cell of 50-60% is expressed neuronal cell surface mark NCAM (Fig. 2 A and 2B).After by MACS immunity sorting, the survival nidogen positive cell group of expressing NCAM is enriched to about 80-85% (Fig. 3 A and 3B).

6) amplification of NCAM positive neurons progenitor cell:

By at first use 0.05% trypsinase-EDTA dissociated cells and the isolating NCAM positive cell that then cell inoculation increased on the culture dish of the poly--L-ornithine/ln bag quilt that contains amplification culture medium.Cultivate the NCAM positive cell by this way and make that cell can adherent better and propagation in amplification culture medium.Amplification culture medium comprises DMEM:F12, Regular Insulin (10-100 μ g/ml), Sodium Selenite (10-50nM), Transferrins,iron complexes (1-10mg/ml), putrescine (50-200 μ M), Progesterone (5-40nM) and ln (10-50 μ g/ml).This serum-free amplification culture medium also comprises two or more somatomedins, for example bFGF (10-50ng/ml), EGF (10-50ng/ml), BDNF (50-200ng/ml), FGF-8 (50-200ng/ml) and/or SHH (200-400ng/ml).The multiple factor of these that exist in the amplification culture medium has facilitated neurocyte per-cent integral body to increase, and can further induce these midbrain neuron precursor cells to take the dopaminergic phenotype.Replenished an amplification culture medium in per 2 days, and made the nidogen positive, 6-10 days (Figure 1B) of NCAM positive cell propagation.

Under these culture condition, the cell of about 50-60% has stretched out the spinous process projection and NCAM dyeing is positive, and this has proved that it forms neuronic ability (Fig. 3).These cells also present the immunoreactivity (Fig. 4 B) of early stage neurone mark 'beta '-tubulin.Then with these neural progenitor cell continuous passages, the applicant finds that these neural progenitor cells can go down to posterity nearly 10 times and still keep being divided into the ability of dopaminergic neuron, and pair cell is divided into the potential of the ripe dopaminergic neuron of typing and does not have any obvious influence.

7) differentiation of dopaminergic neuron and maturation:

The growth of dopaminergic neuron depends on the regulating effect of extracellular signaling molecule in the body.These molecules activate the cascade of the transcription factor with the restricted expression of pedigree.These transcription factors are responsible for increasing the expression that the dopaminergic phenotype is grown one group of related specific gene.For the neural progenitor cell of the amplification that promotes to use method for preparing is formed with the dopaminergic neuron of function, at first from these cells, withdraw from somatomedin bFGF and EGF.Then, by culturing cell 30-50 in the Neurobasal substratum days, induce neural progenitor cell to be divided into dopaminergic neuron.The Neurobasal substratum comprises NeurobasalA substratum (Gibco), FCS (10-20%) (HYCLONE) and B27 (2-10%) (Gibco), and multiple somatomedin.The somatomedin that comprises in this substratum has interleukin-1 ' beta ' (IL-1 β) (1-2 μ g/ml) (Sigma), xitix (50-150nM) (Sigma), N-acetylcystein (50-150nM) (ICN), db-cAMP (500-1000 μ M) (Sigma), GDNF (1-5 μ g/ml) (Sigma), TGF-β 3 (1-5 μ g/ml) (Sigma), Neurturin (100-500 μ g/ml) (Chemicon), BDNF (50-200ng/ml) (Sigma), FGF-8 (50-200ng/ml) (R﹠amp; D Systems) and/or SHH (200-400ng/ml) (R﹠amp; D Systems).The Neurobasal substratum carries out sterile filtration with the micropore injection filter of 0.22 μ m.

Tyrosine hydroxylase (TH) is a Dopamine HCL synthetic rate-limiting enzyme.The restricted somatomedin of particular lineage can promote the TH synthetic to induce.These factors are joined the cell of the performance dopaminergic phenotype that causes higher percent in the substratum.These factors can add in the different time points in the neurone atomization, referring to table 1.As if IL-1 β (being present in the Neurobasal substratum) play an important role in neural progenitor cell is divided into the process of dopaminergic neuron.Though do not wish to be subject to any specific mechanism, as if IL-1 β can increase the quantity of TH positive neuron, and these cells are to the reactivity of signaling molecule in the substratum.During the 30-50 that carries out tissue culture days, the application of these somatomedins and to change a Neurobasal substratum in per 3 days linked together.

Below table 1 described neural progenitor cell and joined combinations of. growth factors in the Neurobasal substratum at different time between the differentiation phase:

Table 1: the differentiation condition of neural progenitor cell

The culture condition that is used to break up
				1-4 days	4-7 days	8-50 days	TH positive cell (%)
The Neurobasal substratum, FCS, B27, IL-1 β	The Neurobasal substratum, FCS, B27, Shh, FGF-8, IL-1 β, db-cAMP, GDNF, BDNF	The Neurobasal substratum, FCS, B27, Shh, FGF-8, IL-1 β, db-cAMP, GDNF, BDNF, TGF-β 3, Neurturin	65％
				The Neurobasal substratum, FCS, B27, IL-1 β	The Neurobasal substratum, FCS, B27, Shh, FGF-8, IL-1 β, db-cAMP, GDNF, BDNF	The Neurobasal substratum, FCS, B27, Shh, FGF-8, IL-1 β, db-cAMP, GDNF, BDNF, TGF-β 3, Neurturin	55％
The Neurobasal substratum, FCS, B27, IL-1 β	The Neurobasal substratum, FCS, B27, Shh, FGF-8, IL-1 β, db-cAMP, GDNF, Shh, FGF-8, BDNF, xitix	The Neurobasal substratum, SHH, FGF-8, FCS, B27, IL-1 β, db-cAMP, GDNF, BDNF, TGF-β 3, Neurturin, xitix	42％
				The Neurobasal substratum, FCS, B27, IL-1 β	The Neurobasal substratum, Shh, FGF-8, FCS, B27, IL-1 β, db-cAMP, GDNF, BDNF, N-acetylcystein	The Neurobasal substratum, Shh, FGF-8, FCS, B27, IL-1 β, db-cAMP, GDNF, BDNF, TGF-β 3, Neurturin, N-acetylcystein	64％
The Neurobasal substratum, FCS, B27	The Neurobasal substratum, FCS, B27,	The Neurobasal substratum, FCS, B27,	20％

When using the Neurobasal substratum to induce the ES cytodifferentiation, the ES cell develops into multiple neuronal cell type, comprises dopaminergic neuron, serotoninergic neuron and oligodendrocyte (Fig. 5,6 and 7).The per-cent of cell that is divided into dopaminergic neuron under the type culture condition is relatively low, and the substratum that has added somatomedin as shown in table 1 causes the per-cent of the dopaminergic neuron that produces to increase.For example, surpass 60% ES cytodifferentiation and become TH (the specificity marker thing of dopaminergic neuron) positive cells.

8) sign of differentiated neuron

Entirety morphology by cell and the phenotype that adopts immunofluorescence technique to determine are assessed and are disclosed prepared differentiated neuron cell type according to the present invention.Adopt the well-known standard method of those skilled in the art, implement immunofluorescence analysis in neural progenitor cell amplification stage (amplification of NCAM positive cell) and a plurality of other differentiation time point.At first,, wash cell, at room temperature fix 10 minutes with 4% paraformaldehyde with PBS going up the growth isolated cells with the 2-pore chamber slide (chamber slide) of extracellular matrix bag quilt in advance.Then, changed the processing cell thoroughly 5 minutes, sealed 2 hours, resist (antibody diluent that in 1%BSA/TBS, prepares) overnight incubation with one at 4 ℃ with 1% bovine serum albumin (BSA)/PBS with the PBS that contains 0.2%Triton X-100.Dye with following one anti-pair cell: early stage neurone mark beta tubulin, NCAM, neurofilament, late period neurone mark microtubule-associated protein 2 (MAP-2), neuronal cell surface antigen A 2B5, Nurr-1, tyrosine hydroxylase, dopamine transporter DAT, dopamine (DBH), astroglia cell mark glial fibrillary acidic protein (GFAP), GABA, oligodendrocyte, serotonin and synaptophysin, all these are one anti-all available from CHEMICON.At last, two of cell and FITC mark anti-hatched together.After above-mentioned each step is finished, all use the PBS washed cell three times.

Observation ward's slide is estimated immune positive region under fluorescent microscope.This immunofluorescence analysis proves that the noble cells of big per-cent has neuronal specificity mark NCAM (Fig. 3), the immunoreactivity of MAP-2 (Fig. 4 A) and 'beta '-tubulin (Fig. 4 B).The higher number of times of hatching increases in the foster base with dividing in these crucial antigenic expression.Immunofluorescence analysis also proves the cell expressing serotonin (Fig. 7) of low per-cent, and is present in the non-neuron mark glial fibrillary acidic protein (GFAP) in the astroglia cell and is present in GABA and L-glutamic acid (Fig. 6) in the oligodendrocyte.

Also resist (green) and anti-respectively MAP-2 by one of the anti-TH of employing, DAT, Nurr-1, two anti-(texas Red) double labeling cells with the FITC mark of β-Tubulin have carried out analyzing (Fig. 8) to noble cells.The ratio of the MAP-2 positive cell of this analytical proof expression TH is higher.By TH positive cell and MAP-2 positive cell in every visual field in the visual field of selecting at random being counted, quantitative to the cell density of dopaminergic neuron with 40 * object lens.Calculate the per-cent (Fig. 9) of TH positive cell.The TH positive neuron is also expressed Nurr-1 and DAT, but does not have the co of TH and DBH, and this has confirmed the dopaminergic phenotype of cell.In addition, by identified the formation of cynapse with the synaptophysin immunostaining.

Immunofluorescence analysis also shows about 60% sorting NCAM positive cell expression TH, and about 30% expression serotonin (Figure 10).In addition, about 40% nidogen positive cell is the TH stained positive, and about 30% cell is the serotonin stained positive, and about 28% cell is oligodendrocyte stained positive (Figure 11).Shown the immunological marker thing that uses in the different steps of differentiation in the table 3, its indication is differentiation and the phenotypic characteristic that has broken up the ES cell not.

Table 3: not differentiation and the phenotypic characteristic that has broken up the ES cell

Do not break up the ES colony	Neuronal cell	Dopaminergic neuron	Spongiocyte
				SSEA-1 SSEA-3 SSEA-4 Tra-1-60 Tra-1-81 Oct-4 GCTM-2 CD-30 cripto-1 GCNF c-Kit	NCAM beta tubulin neurofilament A2B5 MAP-2 serotonin GABA synaptophysin	Nurr-1 TH DAT DBH	A2B5 GFAP O4

9) gene expression profile

Also analyzed the gene expression of cells spectrum of collecting in different steps.Analyze by reverse transcriptase-polymerase chain reaction (RT-PCR) from following each stage collecting cell of disclosed method: do not break up the ES cell, embryoid, nidogen positive neurons progenitor cell, NCAM positive cell and in the Neurobasal substratum, break up 12,17, isolated noble cells after 22,27 and 37 days.Behind the collecting cell, make it form precipitation, use RNeasy Qiagen test kit extracting cell total rna from cell precipitation.Isolating RNA is stored in-20 ℃.

With Moloney leukosis virus superscript II reversed transcriptive enzyme and Oligo (dT) _12-18From the synthetic cDNA of isolating total RNA.By this reverse transcriptase reaction synthetic cDNA with different special primers to carrying out pcr amplification, to determine in the cell of collecting, expressing which kind of gene.Under the PCR of standard condition, use cDNA to implement these PCR reactions as template and platinum Taq polysaccharase, described PCR condition is that those skilled in the art are well-known.It is as follows to be used for the general loop parameter of DNA amplification product:

1.94 ℃ sex change template cDNA 30 seconds;

2.55-65 ℃ primer annealing 1 minute, this depends on employed primer; With

3.72 ℃ incubation reaction 1 minute, repeating step 1-3 (circulation) 25-40 time.

After the PCR reaction, product and DNA size ladder are implemented 1.5% agarose gel electrophoresis.TH, D2RL, DBH, En-1, the primer that the expression of Nurr-1 and 'beta '-tubulin is all listed in the employing table 4 is analyzed by RT-PCR.

Table 4: the primer of amplification Dopamine HCL specific gene is right

Gene	Primer sequence
		TH(417bp)	TGT CAG AGC AGC CCG AGG TC(SEQ ID NO：1) CCA AGA GCA GCC CAT CAA AG(SEQ ID NO：2)
D2RL(404bp)	GCA GTC GAG CTT TCA GAG CC(SEQ ID NO：3) TCT GCG GCT CAT CGT CTT AAG(SEQ ID NO：4)
		DBH(447bp)	CAC GTA CTG GTG CTA CAT TAA GGA GC(SEQ ID NO：5) AAT GGC CAT CAC TGG CGT GTA ACA CC(SEQ ID NO：6)
En-1(390bp)	TGG TCA AGA CTG ACT CAC AGC A(SEQ ID NO：7) TCT CGT CTT TGT CCT GAA CCG T(SEQ ID NO：8)
		Nurr1(255bp)	TGA AGA GAG CGG AGA AGG AGA T(SEQ ID NO：9) TCT GGA GTT AAG AAA TCG GAG CT(SEQ ID NO：10)
'beta '-tubulin (317bp)	GGA ACA TAG CCG TAA ACT GC(SEQ ID NO：11) TCA CTG TGC CTG AAC TTA CC(SEQ ID NO：12)

Above-mentioned analytical proof by RT-PCR carries out is divided into dopaminergic neuron in the neurone differentiation back of ES cell until whole end, observes the expression (Figure 12) of dopaminergic phenotype specific gene (TH).As expectedly, in all cells sample, all see 'beta '-tubulin (gene that omnipresence is expressed), and DBH does not express in any cell sample.

10) reversed-phase HPLC carries out the Dopamine HCL detection

A definite feature of dopaminergic neuron is to produce Dopamine HCL.Therefore, by using reversed-phase HPLC (RP-HPLC) directly to measure the functionally active that intracellular dopamine level evaluation comes from the dopaminergic neuron generation Dopamine HCL of embryonic stem cell.By comparing to determine the detected dopamine concentration of every duplicate samples with the standard dopamine solution of injecting post near before each experiment and back.

At first, at the different steps collecting cell of described method: do not break up the ES cell, embryoid, nidogen positive neurons progenitor cell, NCAM positive cell and in the Neurobasal substratum, break up isolated noble cells after 7,22 and 37 days.Before collecting, at first the HBSS that contains 56mM KCl by adding to the cytositimulation that in the Neurobasal substratum, breaks up 15 minutes with the secretion inducing Dopamine HCL.With trypsin treatment about 5 * 10 ⁶Individual cell is by the centrifugal throw out that obtains.Supersound process cell in the cold 1N perchloric acid that contains antioxidant (0.2g/l sodium metabisulfite) then, 4 ℃ down 15,000rpm/ divided centrifugal 20 minutes.Collect supernatant liquor and be stored in-70 ℃ and be used for determining intracellular dopamine level by RP-HPLC subsequently.Measure the dopamine level in cell lysate and the culture supernatant (change for the last time substratum after 48 hours).Culture supernatant is stable with 7.5% ortho-phosphoric acid and sodium metabisulfite immediately.

Derive from the cell lysate of disclosed method commitment, for example do not break up the ES cell, embryoid, but nidogen positive neurons progenitor cell and NCAM positive cell are analyzed the Dopamine HCL of finding not contain any detection level by RP-HPLC.But after first week of differentiation, all cells lysate of analyzing with RP-HPLC all contains Dopamine HCL in the Neurobasal substratum.Along with the increase in differentiation Neurobasal substratum and final ripe in time of dopaminergic neuron quantity in the cell lysate, dopamine level significantly increases in the cell.Compare dopamine level even higher in the cell in the cell culture that time point was as shown in figure 13 handled with somatomedin with untreated culture.With cell culture and N-methyl-4-phenyl 1,2,3, after 6-tetrahydropyridine hydrochloride (MPTP) (neurotoxin of target dopaminergic neuron) is hatched together, demonstrate further confirmation to these cell cultures deposits yields Dopamine HCLs.In the cell culture of hatching, in cell lysate, do not detect Dopamine HCL by the RP-HPLC analysis with MPTP.On the contrary, increase (Figure 14) with the Dopamine HCL that is discharged in the 56mM KCl stimulated cells culture in the conditioned medium.One group of somatomedin is joined dopamine level (table 5) in the cell that has also increased cell lysate in the differentiation Neurobasal substratum.

Table 5: by dopamine concentration (μ g/ml) in the definite cell lysate of HPLC

#	The differentiation culture condition	The differentiation fate
					7 days	22 days	37 days
		1	There is not somatomedin	0.5				1.3	1.1
2	Add somatomedin				5.0	21.5	5.5
		3	Stimulate with 56mM KCl	5.5				22	7.2
4	The N-acetylcystein combined growth factor				4.93	20.9	9
		5	Stimulate with 56mM KCl	8.8				30.9	10
6	The xitix combined growth factor				6.2	3.7	9.7
		7	Stimulate with 56mM KCl	6.9				5.5	10

Disclosed herein and claimed all compositions and method need not undo experimentation and just can prepare and implement under enlightenment disclosed by the invention.Though the compositions and methods of the invention are described by the mode of preferred embodiment, but for a person skilled in the art apparently, do not deviating from notion of the present invention, under the situation of spirit and scope, can implement to change to the step or the sequence of steps of composition described herein and/or method and method.More specifically, obviously can replace reagent described herein and obtain same or analogous result simultaneously with some reagent that is associated on chemistry or the physiology.All this conspicuous for a person skilled in the art similar substitute or modify all should considered to be in the spirit of the present invention that claims limit, within scope and the notion.

Claims (44)

1. the differentiated cell population in the vitro culture thing that obtains by differentiating primate animal multipotential stem cell, wherein at least 60% noble cells is a dopaminergic neuron.

2. the cell mass of claim 1, wherein primate pluripotent stem cell is a human embryo stem cell.

3. the differentiated cell population in the vitro culture thing that obtains by differentiating primate animal multipotential stem cell, wherein at least 60% noble cells is expressed tyrosine hydroxylase.

4. the cell mass of claim 3, wherein primate pluripotent stem cell is a human embryo stem cell.

5. the method from the neural cell group of primate pluripotent stem cell generation differentiation comprises the following steps:

(a) culture of amplification primate pluripotent stem cell;

(b) cultivate this multipotential stem cell to select nidogen male neural progenitor cell;

(c) sorting nidogen male neural progenitor cell is with enrichment of N CAM positive cell;

(d), make the nidogen positive, the differentiation of NCAM positive cell to produce the neural cell group of differentiation by comprising TGF-β 3 or interleukin-1 ' beta ' or containing culturing cell in both division culture medium.

6. the method for claim 5 wherein adopts laser ablation to obtain multipotential stem cell.

7. the method for claim 5, wherein multipotential stem cell is a human embryo stem cell.

8. the method for claim 7 wherein adopts laser ablation to obtain human embryo stem cell.

9. the method for claim 5, wherein Fen Hua neural cell group comprises about at least 60% dopaminergic neuron.

10. the method for claim 5, wherein Fen Hua neural cell group comprises about at least 30% serotoninergic neuron.

11. the method for claim 5, wherein Fen Hua neural cell group comprises about at least 25% oligodendrocyte.

12. the method for claim 5 comprises that further multipotential stem cell in the culturing step (b) is to form embryoid.

13. the method for claim 12 is wherein cultivated embryoid and is selected nidogen male neural progenitor cell.

14. the method for claim 5 is wherein selected nidogen male neural progenitor cell by cultivate multipotential stem cell in serum free medium.

15. the method for claim 14, wherein serum free medium is the serum-free ITSFn substratum of principal component.

16. the method for claim 14, wherein serum free medium comprises one or more and is selected from Regular Insulin, Sodium Selenite, the soluble factor of Transferrins,iron complexes and fibronectin.

17. the method for claim 16, wherein serum free medium comprises Regular Insulin, Sodium Selenite, Transferrins,iron complexes and fibronectin.

18. the method for claim 13 is wherein selected nidogen male neural progenitor cell by cultivate embryoid in serum free medium.

19. the method for claim 18, wherein serum free medium is the serum-free ITSFn substratum of principal component.

20. the method for claim 18, wherein serum free medium comprises one or more and is selected from Regular Insulin, Sodium Selenite, Prostatropin, the soluble factor of Transferrins,iron complexes and fibronectin.

21. the method for claim 20, wherein serum free medium comprises Regular Insulin, Sodium Selenite, Transferrins,iron complexes and fibronectin.

22. the method for claim 21, wherein neural progenitor cell comprises about at least 95% nidogen positive cell.

23. the method for claim 5, wherein by the nidogen positive neurons progenitor cell in magnetic cell sorting art (MACS) sorting step (c) with enrichment of N CAM positive cell.

24. the method for claim 23, wherein nidogen positive neurons progenitor cell comprises at least approximately NCAM positive cell of 50-60%.

25. the method for claim 5 further is included in the nidogen positive, NCAM positive neurons progenitor cell in the amplification step in the amplification culture medium (c).

26. the method for claim 25, wherein amplification culture medium comprises one or more and is selected from Regular Insulin, Sodium Selenite, Transferrins,iron complexes, ln, putrescine, Progesterone, Prostatropin (bFGF), Urogastron (EGF), Sonic hedgehog (SHH), the soluble factor of fibroblast growth factor-8 (FGF-8) and neurotrophic factor derived from brain (BDNF).

27. the method for claim 26, wherein cell was grown in amplification culture medium 6-10 days.

28. the method for claim 26, the wherein one or more population doublings of culturing cell and continuous passage.

29. the method for claim 26, wherein cell is frozen and is deposited in the liquid nitrogen.

30. the method for claim 5, wherein division culture medium comprises and has added foetal calf serum, B27, the Neurobasal substratum of xitix and N-acetylcystein.

31. the method for claim 5; wherein division culture medium further comprises one or more differentiation factors; described differentiation factor is selected from xitix; N-acetylcystein; glial cell line derived neurotrophic factor (GDNF), two butyryl ring AMP (db-cAMP), neurotrophic factor derived from brain (BDNF); neuturin, Sonichedgehog albumen (SHH) and fibroblast growth factor-8 (FGF-8).

32. the method for claim 5, wherein growing nest protein positive, NCAM positive cell 30-50 days in division culture medium.

33. method that produces dopaminergic neuron from neural progenitor cell, comprise enrichment nidogen positive cells from neural progenitor cell, and make the differentiation of nidogen positive cell by culturing cell under TGF-β 3 or il-1 β or situation that both exist, produce dopaminergic neuron.

34. the method for claim 33, wherein about at least 40% nidogen positive cell is divided into dopaminergic neuron.

35. the method for claim 33 wherein further comprises enrichment of N CAM positive cell from neural progenitor cell.

36. the method for claim 34 is wherein broken up the nidogen positive, NCAM positive cell to produce dopaminergic neuron.

37. the method for claim 36, wherein about at least 60% the nidogen positive, NCAM positive cell are divided into dopaminergic neuron.

38. method that produces serotoninergic neuron from neural progenitor cell, comprise the positive and NCAM positive cells of enrichment nidogen from neural progenitor cell, and make the nidogen positive, the differentiation of NCAM positive cell by culturing cell under TGF-β 3 or il-1 β or situation that both exist, produce serotoninergic neuron.

39. the method for claim 38, wherein about at least 30% nidogen positive, NCAM positive cell are divided into serotoninergic neuron.

40. a method for the treatment of neurodegenerative disorders or neuronal disease patient comprises the following steps:

(a) culture of amplification primate pluripotent stem cell;

(b) cultivate this multipotential stem cell to select nidogen male neural progenitor cell;

(c) sorting nidogen male neural progenitor cell is with enrichment of N CAM positive cell;

(d) by comprising TGF-β 3 or il-1 β or comprising that culturing cell makes the nidogen positive, the differentiation of NCAM positive cell to produce the neural cell group of differentiation in both division culture medium;

(e) the differentiation neural cell group that will treat significant quantity is transplanted in patient's the central nervous system.

41. the method for claim 40, wherein primate pluripotent stem cell is a human embryo stem cell.

42. the method for claim 40, it further comprises from the neural cell group of differentiation and separates dopaminergic neuron and use this dopaminergic neuron to the patient.

43. the method for claim 40, wherein neurodegenerative disorders or neuronal disease are selected from Parkinson's disease, Alzheimer, Huntington Chorea, Spinal injury, amyotrophic lateral sclerosis (ALS), epilepsy, apoplexy and local asphyxia.

44. the method for claim 40, wherein Fen Hua neural cell group is transplanted in patient's brain.

Images