JP2006521807A - Induction of terminally differentiated dopaminergic neurons derived from human embryonic stem cells - Google Patents

Abstract

The disclosure of the present invention has been improved to efficiently produce neural progenitor cells and differentiated neurons (eg, dopaminergic and serotonergic neurons) from pluripotent stem cells (eg, human embryonic stem cells). Regarding the method. By using the disclosed method, a cell population containing a high percentage of cells positive for tyrosine hydroxylase (a specific marker of dopaminergic neurons) was isolated. Since the neural progenitor cells and terminally differentiated cells disclosed in the present invention can be generated in large quantities, they can be used as an excellent source for cell replacement therapy in neurological disorders such as Parkinson's disease.

Description

Cross-reference to related applications N / A Federal government commissioned research or development statement N / A Microfiche attachment N / A Background of invention The present invention relates to an improved method for producing terminally differentiated neurons, such as dopaminergic and serotonergic neurons, from pluripotent embryonic stem cells such as human embryonic stem cells. The dopaminergic and serotonergic neurons resulting from the disclosure of the present invention can be used as an excellent source for cell replacement therapy in neurodegenerative disorders and neurological diseases.

2. Description of Related Art Neurodegenerative disorders and neurological diseases such as Parkinson's disease, Alzheimer's disease, and schizophrenia are becoming increasingly prominent in our society. Many of these neuropathies involve dopaminergic or serotonergic neurons. Dopaminergic neurons are on the ventral and ventrolateral sides of the midbrain and control postural reflexes, movements, and reward-related behavior. These neurons innervate multiple structures in the forebrain, and degeneration or abnormal function of the nerves is associated with Parkinson's disease, schizophrenia, and drug addiction (Hynes et al., 1995, Cell 80:95). -101). Serotonergic neurons are concentrated on the ventral and ventrolateral sides of the hindbrain and innervate most of the central nervous system, including the cerebral cortex, limbic system, and spinal cord. These neurons control levels of consciousness, arousal, behavioral characteristics, and feeding behavior, and abnormal functions have been linked to aggressiveness, depression, and schizophrenia (Jacobs and Gelperin, 1981, Serotonin Neurotransmission). and Behavior. The MIT Press, Cambridge, Mass.). Serotonergic dysfunction can be attributed to a variety of mental, neurological and other disorders (eg, mental depression (Asberg et al., 1986, J. Clin. Psychiatry 47: 23-35), suicide ( Lester, 1995, Pharmcopsychiatry 28 (2): 45-50), and the pathophysiology of aggressive aggressive behavior) may also play a role (Brown et al., J. Clin. Psychiatry, 1990). , 54: 31-41; Eichelman, 1990, Anon. Rev. Med. 41: 149-158).

  Parkinson's disease is a progressive neurological disease caused by alteration of nerve cells (neurons) in the brain region that controls behavior. This alteration reduces the brain signaling chemical known as dopamine, thereby causing motor dysfunction that characterizes the disease. Pathological studies indicate that the loss of substantia nigra dopaminergic neurons is involved in Parkinson's disease. For example, in experimental animals, the syndrome caused by symmetrical lesions of the nigrostriatal tract is quite similar to the motor dysfunctions observed in Parkinson's disease: resting tremor, stiffness, immobility, and postural abnormalities is doing. Symmetrical lesions of the nigrostriatal tract due to 6-hydroxydopamine (OHDA) resulted in significant inability to move, lack of thirst, anorexia, and neglect of sensation in rodents (Ungerstedt, 1971) , U. Acta Physiol.Scan.Suppl.367: 95-121; Yirek and Sladek, 1990, Anon.Rev.Neurosci.13: 415-440).

  In Parkinson's syndrome, changes in dopaminergic receptor status may depend on the stage of disease progression. A prominent feature of Parkinson's syndrome is a significant decrease in dopamine contained in all components of the basal ganglia (Homykiewicz, 1988, Mt. Sinai J. Med. 55: 11-20). When dopamine is reduced, various other areas of the brain, such as the thalamus, pallidum, and subthalamic nucleus, begin to fail. Because these areas send signals to other parts of the brain, dysfunction in these small areas leads to extensive brain dysfunction.

  The prevalence of Parkinson's disease varies widely from 82 / 100,000 in Japan to 108 / 100,000 in the UK to about 1% of the North American population (about 1 million). In India, the prevalence of Parkinson's disease is 14 / 100,000 in North India, 27 / 100,000 in South India, 16 / 100,000 in East India, and 363 in the West Indian Parcy region. People / 100,000 people. While Parkinson's disease is currently considered incurable, various drug therapies are available, such as levodoba, bromocriptine, pergolide, selegiline, anticholinergics, and amantadine that relieve the symptoms of Parkinson's disease. Even though these drugs can alleviate the symptoms of Parkinson's disease, it often has significant side effects. In addition, these drugs do not treat the disease or delay the progressive loss of nerve cells, but only relieve symptoms, and the efficacy is often exhausted over time. In some patients, responsiveness to drug therapy begins to decline, in others it becomes irritable, resulting in movement disorders (dyskinesia).

  These unsatisfactory results include, for example, pallidus amputation, deep brain stimulation (DBS) of the pallidus, attempts to destroy sensitive areas of the brain, or placing electrodes in the DBS to calm the area. It has led to the development of other strategies to treat this disease, such as surgical approaches including attempts to block network abnormalities and dopa receptor agonist therapy. Despite these and other types of surgery on patients with Parkinson's disease have some beneficial results, the long-term effects of such surgery are not known. These treatments also have certain limitations and side effects.

  Another strategy being explored for this intractable disease is gene therapy. The discovery of the molecular basis of neurological diseases and advances in gene transfer systems have enabled local and global therapeutic gene delivery for a wide variety of central nervous system diseases. However, gene therapy has certain limitations, for example, stability and regulation of transgene expression and safety of both the vector and the expressed transgene (Cosantini et al., 2000, Gene Therapy 7: 93-109). ). Vectors known to be used for gene therapy include, but are not limited to, herpes simplex virus type 1 (HSV-1) (During et al., 1994, Science 266: 1399-1403). Adeno-associated virus vector (AAV) (During et al., 1998, Gene Therapy 5: 820-827), retrovirus, HSV / Epstein Barr virus (HSV / EBV) hybrid vector, and HSV / AAV hybrid vector Is mentioned. A gene therapy has been found useful in treating animal models of Parkinson's disease. An encapsulated and genetically modified cell line that releases a neuroprotective molecule, a glial cell line-derived neurotrophic factor gene (GDNF), and a lentiviral vector encoding the GDNF gene, Improved differentiation, resulting in accelerated behavioral recovery in animal models (Zurn et al., 2001, Brain Res Rev. 36: 222-229; Date et al., 2001, Cell Transplant 10: 397- 401). Gene therapy using neural stem cells has also been shown to be effective in expressing therapeutic levels of GDNF in vivo (Akerud et al., J. Neurosci. 21: 8108-8118).

  Cell transplantation is another therapeutic strategy that gives hope to replace nerve cells lost in Parkinson's disease, as well as other neurodegenerative disorders and diseases. Clinical trials with fetal tissue transplantation (still in progress) have developed a method to put cells into the brain, which has shown the feasibility of this idea and has shown promising results for at least some patients . Attempts have also been made to transplant dopaminergic neuron precursors directly into the striatum of patients with Parkinson's disease, and transplantation of human fetal or embryonic dopaminergic neurons is beneficial for Parkinson's disease patients (Freed et al., 2001, N. Engl. J. Med. 344: 710-719) 719).

  However, the data suggests that anatomical modification of the pathway rather than ectopic placement of the graft is required to obtain a complete recovery (Winkler et al., 2000, Prog. Brain Res. 127: 233-265). Fetal substantia nigra transplant tissue treatment also requires human fetal tissue from at least 5-10 fetuses in order to obtain clinically reliable improvements in patients, which is very large Raise ethical, legal and safety issues. Thus, there is an urgent need for alternative sources of neurons, such as dopaminergic neurons, to treat neurodegenerative disorders and diseases.

  Recently, a renewable source of neural stem cells has been discovered in the adult human brain. Neural stem cells with the ability to self-renew and form all cells potentially provide an unlimited source of dopamine-producing brain cells, promising a completely new therapeutic approach to neurodegenerative disorders and neurological diseases (Eriksson et al., 1998, Nature Medicine 4: 1313-1317). It has been reported that the culture of neural stem cells derived from embryonic human forebrain can be expanded up to 1 million times in vitro. These adult neural stem cells were transplanted into adult rats, a well-characterized model of Parkinson's disease. In this animal model, the cells survived for up to 1 year after transplantation, differentiated into nerves, and were able to reduce motor dysfunction in several experimental animals (Svendsen et al., 1997, Exp. Neurol. 148). : 135-146). Unfortunately, adult neural stem cells have a limited life span in tissue culture (Kukekov et al., 1999, Exp. Neurol. 156: 333-344).

  One viable surrogate source of dopaminergic and other neurons that may be used to treat various neurodegenerative disorders and diseases is pluripotent embryonic stem (ES) cells, particularly humans ES cells. ES cells can grow indefinitely in an undifferentiated state and are pluripotent, which means that ES cells can differentiate into almost any cell type present in the body. . ES cells have the potential to generate replacement cells for a wide variety of tissues and organs (eg, heart, spleen, nervous tissue, muscle, and cartilage) because of the ability of ES cells to become nearly all of the differentiated cells in the body Has the ability. ES cells can be derived from the inner cell mass (ICM) of the blastocyst, which is a stage of embryo growth that occurs prior to transplantation. Human ES cells may be derived from human blastocysts at an early stage of a growing embryo that lasts from day 4 to day 7 after fertilization. ES cells derived from ICM can be cultured in vitro and grown indefinitely under appropriate conditions.

  ES cells have been successfully established in a number of species, such as mice (Evans et al., 1981, Nature 292: 154-156), rats (Inanaccon et al., 1994, Dev. Biol., 163: 288). -292), pigs (Evans et al., 1990, Therogenology 33: 125-128; Notarianni et al., 1990, J. Reprod. Fertil. Suppl. 41: 51-6), sheep and goats (Meinecken-Tillm) 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). ), Mink (Sukoyan et al., Mol. Reprod. Dev. 1992, 33: 418-431), hamster (Doetscbman et al., 1988, Dev. Biol. 127: 224-227), chicken (Pain et al. , 1996, Development 122 (8): 2339-48), primates (US Pat. No. 5,843,780), and humans (Thomson et al., 1998, Science 282: 1145-1). . 47; Reubinoff et al, 2000, Nature Biotech.18: 399-403), and the like. As with other mammalian ES cells, when human ES cells are injected into immune-deficient mice, human ES cells differentiate to form tissues of all three germ layers, demonstrating the pluripotency of human ES cells. According to published reports, human ES cells can be maintained in culture for over a year, during which human ES cells retain pluripotency, self-renewal ability and normal karyotype (Thomson et al., 1995, PNAS 92: 7844-7848).

  Studies have shown that ES cells can differentiate into neural progenitor cells (Zhang et al., 2001, Nature Biotech. 19: 1129-33; WO 01/88104; US patent application 09 / 872,183, 09 / 888,309, and 10 / 157,288; WO 03/000868; the contents of each of which are specifically incorporated as part of this specification). Therefore, these cells can be further differentiated into dopaminergic neurons (Rollettsch et al., 2001, Mech. Dev. 105: 93-104). The initial stage of ES cell differentiation is embryoid body differentiation, for example, 1 μM retinoic acid promotes neuronal differentiation in the embryoid body (Bain et al., 1995, Dev. Biol. 168: 342-357). . Although retinoic acid can be used to generate neurons, retinoic acid is a powerful teratogen. By using stromal cell inducing activity (SIDA) (Kawasaki et al., 2000, Neuron 28: 1-20), by the expression of nuclear receptor associated-1 gene (Nurr-1) (Kim et al., 2002). , Nature 418: 50-56), or by transplanting undifferentiated ES cells directly into a mouse model (Bjorklund et al., 2002, Proc. Natl Acad. Sci. 99: 2344-2349). Several reports have been published on differentiating into functional neurons. Lee et al. (Lee et al., 2000, Nat. Biotechnol. 18: 675-79) to differentiate ES cells into neural progenitor cells in vitro and further to dopaminergic and serotonergic neurons. Reported the method. However, all of these experiments were performed using mouse ES cells and the differentiation protocol produced 5-50% dopaminergic neurons. About 20% of mouse ES cells differentiated into dopaminergic neurons in the study of Lee et al. (WO 01/83715) and in the study of Studer et al. (WO 02/086073). While dopaminergic neurons also differentiate from human ES cells, the yield of dopaminergic neurons obtained is only about 5-7% as a percentage of total cells in the population. (WO 03/000868).

  Parkinson's disease is considered a particularly suitable clinical target for cell transplantation strategies because it is characterized by selective and gradual loss of dopaminergic neurons in the substantia nigra of the midbrain. Loss of dopamine-producing nerves within specific brain regions results in abnormal firing of neurons that results in the patient being unable to control or direct their own movements. However, a large number of dopaminergic neurons are required for cell replacement therapy. Therefore, there is a need for an alternative protocol for inducing dopaminergic neurons from human ES cells. This increases the effectiveness of such treatment against Parkinson's disease and increases the success rate of treatment. Furthermore, the use of these dopaminergic neurons in vitro can help identify substances that prevent or reduce the death of dopaminergic brain cells in neurodegenerative disorders and neurological diseases.

SUMMARY OF THE INVENTION The present disclosure relates to an improved method for producing neural progenitor cells, and ultimately differentiated nerves, from pluripotent stem cells (eg, pluripotent stem cells such as human stem cells). For example, the present disclosure makes a high proportion of human stem cell populations differentiate into neurons that are positive for tyrosine kinase (TH), a specific marker of dopaminergic neurons (eg, at least about 60%). The present disclosure also shows that a high proportion of human stem cell populations differentiate into serotonergic neurons, the proportion of dopaminergic and serotonergic neurons resulting from the disclosed method is already The methods disclosed herein are also similar to astrocytes and oligodendrocytes from human embryonic stem cells, and they are colonic and sensitive. It can be used to generate cells with phenotypic characteristics of the optic nerve.

  The disclosure of the present invention is a population comprising differentiated cells in an in vitro culture state obtained by differentiating primate pluripotent stem cells, wherein at least 60% of the differentiated cells are tyrosine hydroxylase A cell population is provided that is a dopaminergic neuron that expresses or a dopaminergic neuron that expresses tyrosine hydroxylase. In other embodiments, at least about 30%, 40%, 50%, 70%, 80%, 90%, 95% or 99% of the differentiated cells are dopaminergic neurons. The present invention is a group of differentiated cells in an in vitro culture state obtained by differentiating primate pluripotent stem cells, and at least 30% of the differentiated cells are serotonergic neurons. . In another embodiment, at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the differentiated cells are serotonergic neurons. In a preferred embodiment, the neural progenitor cells or neuronal differentiated primate pluripotent stem cells are human embryonic stem cells.

The present disclosure also provides a method for generating a population of differentiated neurons from primate pluripotent stem cells, comprising:
(A) expanding a culture of primate pluripotent stem cells;
(B) culturing pluripotent stem cells and selecting nestin-positive neural progenitor cells;
(C) selecting nestin-positive neural progenitor cells for enrichment of NCAM-positive cells;
(D) culturing nestin-positive NCAM-positive cells in a differentiation medium to differentiate the nestin-positive NCAM-positive cells to generate a differentiated neuronal cell population;
A method for generating a differentiated neural cell population is provided.

  In a preferred embodiment, said primate pluripotent stem cell is a human embryonic stem cell. In another preferred embodiment, the pluripotent stem cells used in the above method are preferably obtained using a laser ablation method.

  In another embodiment, the method further comprises culturing the pluripotent stem cells of step (b) to form embryoid bodies. Preferably, these embryoid bodies are cultured under conditions that select neural progenitors that are positive for nestin, eg, by culturing pluripotent stem cells or embryoid bodies in a serum-free medium. In a preferred embodiment, the serum-free medium is an ITSFn serum-free synthetic medium and one or more soluble factors selected from the group consisting of insulin, sodium selenite, basic fibroblast growth factor, transferrin, and fibronectin. Is preferably included. In preferred embodiments, these methods produce at least about 60-75% nestin positive cells, more preferably about 80-90% nestin positive cells, and most preferably about 95-99% nestin positive cells.

  The nestin-positive neural progenitor cells are then stored and appropriate immunological techniques such as immunolabeling and fluorescence sorting (sorting) such as magnetic cell separation (MACS), solid phase adsorption, fluorescence activated cell sorting (FACS). ), NCAM positive cells can be enriched by flow immunocytochemistry against cell surface markers, or by flow cytometry assays. According to preferred embodiments, these methods comprise at least about 40-70% NCAM positive cells, more preferably about 50-60% NCAM positive cells, and most preferably about 80-99% NCAM positive cells. Generate nestin positive cells containing. In some embodiments, the method further comprises the step (c) of growing nestin positive NCAM positive neural progenitor cells in a growth medium, preferably for 6-10 days. Preferably, the growth medium is insulin, sodium selenite, transferrin, laminin, putrescine, progesterone, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), sonic hedgehog (SHH), One or more soluble factors selected from the group consisting of fibroblast growth factor-8 (FGF-8) and brain-induced neurotropic factor (BDNF).

  The nestin-positive NCAM-positive neural progenitor cells are preferably cultured and serially passaged to double one or more populations. These cells are also stored frozen in liquid nitrogen. NCAM positive neural progenitor cells are preferably grown in differentiation medium for 30-50 days as described in step (d) of the method above. In a preferred embodiment, the differentiation medium comprises a neural basal medium supplemented with fetal calf serum, B27, ascorbic acid, and N-acetyl cysteine. In another preferred embodiment, the differentiation medium further comprises TGF-β3 or interleukin-1β or both. Preferably, the differentiation medium is ascorbic acid, N-acetyl, cysteine glial cell line-derived neurotrophic factor (GDNF), dibutyryl cyclic AMP (db-cAMF), brain-induced neurotrophic factor (BDNF), Neuturin. And one or more differentiating agents selected from the group consisting of Sonic hedgehog protein (SHH), and fibroblast growth factor-8 (FGF-8).

  In a preferred embodiment, the method disclosed above is used to generate a differentiated neuronal population, which is preferably about 40-60% more dopaminergic neurons. Preferably it contains 70-80% dopaminergic neurons, most preferably 90-99% dopaminergic neurons. In some embodiments, these methods are used to generate a differentiated neuronal population, which is preferably about 20-50% serotonergic neurons, more preferably 30-70% serotonergic neurons, most preferably 60-99% serotonergic neurons. In other embodiments, these methods are used to generate a differentiated neuronal population, which is preferably about 15-40% oligodendrocytes, more preferably about 25-25. 50% oligodendrocytes, most preferably about 60-99% oligodendrocytes.

  The disclosure of the present invention provides a method of generating dopaminergic neurons from neural progenitor cells, which enriches the neural progenitor cells as nestin-positive cells and produces TGF-β3 or interleukin- Culturing the cells in the presence of 1β or both includes differentiating the nestin positive cells to produce dopaminergic neurons. Preferably, at least 40-99% of nestin positive cells differentiate into dopaminergic neurons using these methods. In another embodiment, these methods further enrich the neural progenitor cells as NCAM positive cells and preferably differentiate these nestin positive NCAM positive cells to produce dopaminergic neurons (e.g., at least 60-99% of cells are differentiated into dopaminergic neurons). The present disclosure also provides a method of generating nerves from neural progenitor cells, which enriches neural progenitor cells into cells that are positive for nestin and NCAM and differentiates nestin positive NCAM positive cells. Then, by culturing the cells in the presence of TGF-β3 and / or interleukin-1β, serotonergic neurons are generated. Preferably, using these methods, about 30-99% of nectin positive NCAM positive cells differentiate into serotonergic neurons.

The present disclosure also treats a subject with a neurodegenerative disorder or neurological disease by administering to a differentiated neural cell of interest derived from a primate multicerebral stem cell as described herein. Provide a way to do that. For example, a population composed of differentiated nerve cells may be induced as follows. That is,
(A) proliferating a culture of primate pluripotent stem cells;
(B) culturing pluripotent stem cells and selecting neural progenitor cells that are positive for nestin;
(C) selecting the nestin positive neural progenitor cells for enrichment of NCAM positive cells;
(D) culturing nestin-positive NCAM-positive cells in a differentiation medium and differentiating the cells to generate a population of differentiated neurons,
(E) Administering a population of differentiated neurons comprising a therapeutically effective amount to the central nervous system of a subject Preferably, the differentiation medium comprises TGF-β3 or interleukin-1β or both. In a preferred embodiment, the subject is a patient, more preferably a human patient, and the primate pluripotent stem cells are human embryonic stem cells. Preferably, human embryonic stem cells are tissue compatible with the patient. For example, the multicerebral stem cells used have essentially the same genome as that of the patient. In certain embodiments, dopaminergic, serotonergic, cholinergic, sensory neurons, or alternatively astrocytes, or oligodendrocytes are isolated from a population consisting of differentiated neurons, Administered to patients. These cells include, for example, a population of differentiated nerve cells and can be administered to a subject to cause various neurodegenerative disorders or neurological diseases such as, but not limited to, Parkinson's disease, Alzheimer's disease. , Huntington's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, cerebral infarction, and ischemia. Preferably, the cells are administered by transplantation, for example, by transplanting the desired cells into the subject's brain.

  In another embodiment, as described herein, screening of compounds (eg, small molecules and drugs) is performed using differentiated neurons derived from primate pluripotent stem cells. Alternatively, the effect of such compounds on the activity of the cells can be performed. Screening for such compounds can also be done for neurotoxicity or modulation. A compound is added to a population of differentiated neurons and cultured under similar conditions, except that the cell viability, morphology, phenotype, functional activity, or other characteristics are not exposed to the compound. By comparing with differentiated nerve cells, the compound can be evaluated. For example, the compounds are screened to determine whether they affect neurotransmitter synthesis, release, or changes in cellular uptake.

Detailed Description of the Invention The present disclosure provides an effective method of generating cells of neuronal lineage that differentiate from pluripotent stem cells. Cells produced herein include, but are not limited to, neural progenitor cells, dopaminergic, serotonergic, cholinergic, and sensory neurons, and also astrocytes and oligodendrocytes Cells with phenotypic characteristics. The cells generated herein are identified by phenotypic characteristics, morphological characteristics, and / or cell markers that are readily understood by one of ordinary skill in the art of evaluating such cells. As used herein, the term “neuroprogenitor cell” is used interchangeably with the term “neural progenitor cell” or “neural progenitor cell”. Progeny that are either neural cells such as neural progenitors or neurons, or glial precursors, astrocytes, or glial cells such as oligodendrocytes can be generated. The methods disclosed herein include culturing cells in a combination of environmental conditions and soluble factors that promote the differentiation of the cells into cells of the neural lineage. In addition, physical separation or manipulation techniques can be used to further enrich for the desired neuronal cell type.

  These precursors and differentiated neurons can be used in a number of applications, including therapeutic and experimental applications, as well as in vitro drug development and screening (eg, compounds related to neurotoxicity or neuronal function). Ability to modify).

  Progenitors and differentiated neurons derived from pluripotent stem cells (eg, dopaminergic and serotonergic neurons, as well as other specialized neuronal types) are associated with neurodegenerative disorders and diseases For affected individuals, it provides a potentially non-limiting source of these nerves, with considerable potential benefits. Since the precursors and differentiated neurons described herein are generally descendants of the cell population from which they are derived, they are essentially parental populations (genetically modified, transformed, or transfected). Have the same genome).

  One embodiment of the present disclosure provides for pluripotent stem cells, preferably primate embryonic stem (ES) cells or primate embryonic reproductive (EG) cells with mesencephalic nerve characteristics such as dopaminergic neurons. It relates to a method of generating cells. Another embodiment relates to a method of generating neurons from primate ES cells or primate EG cells having hindbrain neural characteristics such as pluripotent stem cells, preferably serotonergic neurons. Primate ES cells or EG cells that can be used in these methods are most preferably human ES cells or EG cells. These nerves are derived from pluripotent stem cells by culturing the cells in the presence of certain soluble factors and environmental conditions.

  As used herein, the term “dopaminergic neurons” refers to neurons that express tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. Preferably, the dopaminergic neurons secrete the neurotransmitter dopamine and express little dopamine-β-hydroxylase. Dopaminergic neurons innervate the striatum, limbic system, and neocortex in vivo, along with several other types of neurons, including motor neurons, in the ventral midbrain. Dopaminergic neurons are specifically located in the substantia nigra of the midbrain and control postural reflexes, movements, and reward-related behavior. The loss of normal functional dopaminergic neurons results in Parkinson's disease, whose abnormal function is associated with schizophrenia and drug addiction. As used herein, “serotonergic neurons” refers to neurons that secrete the neurotransmitter serotonin (5-hydroxytryptamine). Serotonergic neurons generally have a slow firing, periodic pattern, and are concentrated in vivo on the ventral and ventrolateral sides of the hindbrain, and in the cerebral cortex, limbic system, and spinal cord. It innervates most of the central nervous system including it. These neurons control levels of cognition, excitement, behavior, and feeding behavior. Abnormal functions of serotonergic neurons are linked to aggression, depression (including suicide), and schizophrenia.

The present disclosure relates to an improved method that differentiates pluripotent stem cells into neural progenitor cells and also phenotypes, molecules, and / or cells for cells of the neural lineage. To differentiate into a differentiated population of neurons with the same characteristics. Pluripotent stem cells are human ES cells, and differentiated neurons are dopaminergic or serotonergic neurons. The present disclosure also relates to cells and cell populations produced by the disclosed methods. In some embodiments, the disclosed method includes the following steps. That is,
1. A population of pluripotent stem cells is isolated. The pluripotent stem cells are preferably ES cells derived using a novel laser ablation method.

  2. Proliferate pluripotent stem cells to provide sufficient starting material.

  3. Pluripotent stem cells are cultured in suspension to produce embryoid bodies.

  4). Embryoid bodies are re-plated on the substrate and incubated in serum-free medium that selects for neural progenitor cells that are positive for nestin.

  5). Nestin positive cells are sorted to isolate an enriched population of NCAM positive cells.

  6). Nestin-positive and / or NCAM-positive neural progenitor cells are grown in a growth medium containing soluble factors associated with the nervous system.

7). Nestin-positive and / or NCAM-positive neural progenitor cells are differentiated into mature neurons in a neural basal medium containing a combination of soluble factors associated with the nervous system.
Sources of Pluripotent Stem Cells The methods disclosed herein for differentiating neural lineage cells from pluripotent stem cells can differentiate a significantly higher proportion of pluripotent stem cells into a particular neuronal cell type. It involves using specific culture conditions of interest. Pluripotent stem cells are derived from pre-embryo, embryo, or fetal tissue at any time after fertilization and can differentiate into several different cell types under appropriate conditions, The cell type is a derivative of a total of three germ layers (endoderm, mesoderm, and ectoderm). Neural lineage cells can be derived from stem cells isolated from fetal or adult tissues that have the ability to differentiate, or can be reprogrammed into neural lineage cells. Pluripotent stem cells include, but are not limited to, mammalian ES cells and EG cells (desirably primate or human ES cells and EG cells). Desirably, undifferentiated pluripotent stem cells have the ability to divide and grow indefinitely in the medium. As used herein, the term “differentiation” refers to the process by which undifferentiated pluripotent stem cells or precursor cells acquire a more specialized fate. For example, differentiated cells have a phenotype that is characteristic of a particular cell type or tissue.

  In preferred embodiments, the ES cells and ES cell lines used herein are derived from the inner cell mass of a blastocyst. These blastocysts may be isolated from the recovered whole embryos in vivo fertilized or in vitro fertilized (IVF) (eg, conventional insemination, intracytoplasmic sperm injection, or egg transplantation). . Human blastocysts are obtained from couples or donors who voluntarily donate surplus embryos. After obtaining written voluntary consent from these couples or donors, these embryos are used for research purposes. Alternatively, blastocysts can be induced in human or non-human enucleated oocytes by migration of somatic cells or cell nuclei and are thereby stimulated to grow to the blastocyst stage. The blastocysts used may be cryopreserved, or may be cryopreserved at an early stage and continue to grow into embryos at the blastocyst stage. The growth of both blastocysts and inner cell mass varies from species to species and is well known to those skilled in the art.

  Primate or human ES cells can be derived from blastocysts using standard immunosurgery techniques, such as those described in US Pat. Nos. 5,843,780 and 6,200. 806, Thomson et al. , (Science 282: 1145-1147, 1998), and Rebinoff et al. (Nature Biotech 18: 399-403, 2000), the contents of each of which are specifically incorporated as part of this specification. Although ES cells derived by some of the methods known to those skilled in the art can be used in the disclosed methods, preferred embodiments are directed to human ES cells induced by a unique laser ablation method. (U.S. Patent Application No. 10 / 226,711, the contents of which are specifically incorporated herein by reference). Briefly, this method involves isolating cells from the inner cell mass of a blastocyst through laser ablation of a portion of the zona pellucida and the trophectoderm of the blastocyst. The formation forms an aperture or hole in the blastocyst, and the inner cell mass can be aspirated through this aperture or hole. Next, by further culturing these cells, an ES cell line can be established. This technique is advantageous. This is because it is possible to isolate cells of the inner cell mass without performing the difficult procedure of conventional immunosurgery. In addition, ES cell lines generated using this technology can be isolated in the absence of any animal-derived antibodies and sera in certain human ES cell lines, which means that animal microorganisms Can minimize the risk of transmission. In another embodiment, human EG cells derived from protogerm cells present in human fetal material are used (US Pat. No. 6,090,622, and Shamblott et al., 1998, Proc. Natl. Acad Sci. USA). .95: 13726-13731, the contents of each of which are specifically incorporated as part of this specification).

  Preferably, the ES cell line can be maintained in the culture in an undifferentiated state for an extended period of time, for example for a year or more, and can maintain a normal euploid karyotype. Identification of human ES cells is often flatter than mouse ES cells and is often morphologically characterized by a high nucleus to cytoplasm ratio, prominent nucleoli, and compact colonization, with different cell boundaries and colonies Can be done scientifically. Human ES cells are described in Thomson et al. , (1998), Rebinoff et al. , (2000), and Bühr and McLaren (1993), each of which is specifically incorporated by reference herein, preferably a marker for human pluripotent ES cells (E.g., SSEA-3, SSEA-4, GCTM-2 antigen, and TRA2-60). Desirably, human ES cells also express alkaline phosphatase, similar to OCT-4. In another embodiment, human ES cells are capable of forming embryoid bodies under non-adherent culture conditions (US Pat. No. 6,602,711, as part of this specification). Support). These embryoid bodies can be used to induce differentiated derivatives of endoderm, mesoderm, and ectoderm germ layers, as well as other desired cell lineages.

  Pluripotent stem cells (especially human ES or EG cells) can be grown continuously under culture conditions that maintain the cells in a substantially undifferentiated state. ES cells should be kept at the appropriate cell density and repeatedly separated and passaged while frequently changing the medium to prevent differentiation. For general techniques on cell culture and cultured ES cells, practitioners can refer to standard textbooks and reviews. For example, E.I. J. et al. Robertson, “Teratocarcinomas and embronic stem cells: A practical approach” ed. , JRL 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 Arathon, 1990, Bioprocess Technol. 10: 251-70; Xu et al. ,. , 2001, Nat. Biotechnol. 19 (10): 971-4; and Lebkowski et al. ,. 2001, Cancer J .; 7 Suppl. 2: S83-93 is mentioned. The contents of each are specifically incorporated as part of this specification.

  Traditionally, ES cells are cultured in ES medium on a layer of feeder cells. A feeder cell layer is a tissue type cell that is co-cultured with ES cells and provides an environment in which ES cells can grow without substantial differentiation. Methods of culturing ES cells on feeder cell layers are well known to those skilled in the art (US Pat. Nos. 5,843,780 and 6,200,806, WO 99/20741, US patent application 09/530). , 346 and 09 / 849,022, and WO 01/51616, the contents of each of which are specifically incorporated as part of this specification). The feeder cell layer desirably reduces, inhibits or suppresses ES cell differentiation. A feeder cell layer is generally a fetal fibroblast feeder cell layer of human or mouse origin, eg, mouse embryonic fibroblasts, human embryonic fibroblasts, human fibroblast-like cells or mesenchymal cells (human embryos). Stem cells or STO cells).

  ES cells are preferably cultured in the presence of ES medium to reduce, inhibit or suppress the differentiation of ES cells. Preferably, the ES medium used for ES cell culture is supplemented with nutrient serum, for example serum or serum-based solutions that provide effective nutrients to maintain ES cell growth and viability. ing. Nutritional serum is animal serum. For example, fetal calf serum (FBS) or fetal calf serum (FCS) can be used (US Pat. Nos. 5,453,357, 5,670,372, and 5,690,296, herein). As a part of the system). ES media may also be serum-free (WO 98/30679, WO 01/66697, and US patent application Ser. No. 09 / 522,030, each of which specifically forms part of this specification. As a). Examples of suitable ES media with serum for culturing ES cells are Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO) (without sodium pyruvate) with high glucose content (70-90%) Yes, the medium contains FBS or FCS (10-30%), β-mercaptoethanol (0.1 mM), non-essential amino acids (1%), and L-glutamine 2 mM, 4 ng / ml basic fibroblast growth factor (BFGF), 50 U / ml penicillin, 50 μg / ml streptomycin, and 1000 U / ml leukemia inhibitory factor (LIF) are added. Examples of suitable serum-free ES media for culturing ES cells are 80% “KnockOut” Dulbecco's Modified Eagle Medium (DMEM) (GIBCO), 20% KnockOut SR (instead of serum-free) , GIBCO), β-mercaptoethanol (0.1 mM), non-essential amino acids (1%), and L-glutamine 1 mM.

ES cells can also be cultured under unsupported cell line medium conditions. Methods of culturing ES cells in a non-supporting cell line medium are well known to those skilled in the art (US 2002/0022268, WO 03/020920, and US patent application 10 / 235,094, the contents of each). In particular, it is incorporated as part of the present description). ES cells in unsupported cell line media preferably grow on a suitable culture substrate, such as extracellular matrix, Matrigel® (Becton Dickenson) or laminin. Unsupported cell line media also preferably uses conditioned media to support the growth of ES cells. Culturing ES cells without substantial differentiation by preparing a conditioned medium by culturing a first population of mouse fetal fibroblasts or human fetal fibroblasts in the medium for a sufficient period of time. Produce “conditional” media to support. Alternatively, unsupported cell line media can be combined with the extracellular matrix in an effective medium that is freshly added to cultures that are not conditioned by another cell type (US 2003/0017589, this content is In particular, it is incorporated as part of the present description).
Preparation of neural progenitor cells Isolated pluripotent stem cells can be proliferated and subjected to culture conditions that allow the stem cells to differentiate into neural progenitor cells. To advance pluripotent stem cells along the neural differentiation pathway, cells are cultured according to the differentiation protocol disclosed herein. The pluripotent stem cells are cultured on a suitable substrate in a differentiation nutrient medium containing differentiation agents (eg, soluble factors and growth factors). Suitable substrates include, but are not limited to, positively coated solid surfaces (eg, poly-L-lysine or polyornithine), substrates coated with extracellular matrix components (eg, fibronectin, laminin or Matrigel ( Registered trademark)), or a combination thereof. Preferred differentiation nutrient media support the growth, differentiation and survival of the desired neuronal cell type and may contain one or more suitable differentiation agents. As used herein, the term “growth factor” refers to a protein that binds to a receptor on the cell surface that results primarily in activation of cell growth and differentiation. Suitable soluble factors include, but are not limited to, neurotrophins, mitogens (stem cell factors), growth factors, differentiation factors (eg, TGF-β superfamily), TGF-β superfamily agonists, neurotrophic factors , Antioxidants, neurotransmitters, and survival factors. Many soluble factors are quite versatile, while others are specific for a particular cell type, while stimulating cell division in many different cell types.

  In particular, suitable differentiation agents for promoting neuronal cell type differentiation include, but are not limited to, progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, newturin, sonic hedgehog (SHH), noggin , Follistatin, epidermal growth factor (EGF), any type of fibroblast growth factor (eg FGF-4, FGF-8, basic fibroblast growth factor (bFGF)), growth and differentiation factor 5 (GDF- 5), members of the neurotrophin family (nerve growth factor (NGF), neurotrophin 3 (NT-neurotrophin 4 (NT-4), brain-derived neurotrophic factor (BDNF))), transformed growth Factor α (TGF-α), transforming growth factor β-3 (TGFβ3), platelet-derived growth factor (PDGF- A), insulin-like growth factor (IGF-1), bone morphogenic protein (BMP-2, BMP-4) (glial cell-derived neurotrophic factor (GDNF), retinoic acid (RA), midkine, ascorbic acid, dibutyryl cAMP , Dopamine, and ligands for receptors that form a complex with gp130 (eg, LIF, CNTF, SCF, IL-11, and IL-6) Differentiated nutrient media is supplemented with neurons (eg, N2 and B27) (Gibco)) may be included.

  Pluripotent stem cells are first induced to form embryoid bodies. Embryoid bodies are plated directly onto an appropriate substrate (eg, fibronectin or laminin) with or without extracellular matrix components and are suitable for promoting differentiation into neural progenitor cells (eg, nestin-positive neural progenitor cells) It is cultured in a suitable differentiation nutrient medium. Nestin is a cell marker unique to neural progenitor cells. In another embodiment, pluripotent stem cells initially aggregate into heterogeneous cell populations generated by forming embryoid bodies, eg, by culturing pluripotent stem cells in suspension. These cells can be cultured in a nutrient medium with or without serum along with one or more of the differentiation agents listed above to promote cell differentiation in the embryoid body. As used herein, the term “embryoid bodies” refers to differentiation that occurs when pluripotent stem cells grow in suspension culture or grow excessively in monolayer culture. This refers to the aggregation of type cells. The embryoid body may also be an aggregate of cells and have undifferentiated cells. Preferably, such cell aggregation is surrounded by primitive endoderm. Embryoid bodies generally contain cells derived from all three types of germ layers: ectoderm, mesoderm, and endoderm. In mature human embryoid bodies, it is possible to distinguish cells with markers of various cell types (eg, neuronal cells, hematopoietic cells, hepatocytes, and cardiomyocytes). Some cells contained in mature embryoid bodies can behave functionally like differentiated cells. For example, embryonic bodies can be pulsated by active cardiomyocytes. Preferably, the differentiation of pluripotent stem cells is controlled so that specific cell types are obtained for therapeutic purposes.

  The embryoid body is cultured until the embryoid body reaches a sufficient size or a desired differentiation. For example, the embryoid body is plated on a substrate after 3 to 10 days of culture. Preferably, the substrate is coated with an extracellular matrix component, including but not limited to poly-1-lysine, poly-1-ornithine, laminin, collagen, fibronectin, Matrigel (registered) Trademark), or a combination thereof. Preferably, the embryoid bodies are plated directly on the substrate without dispersing the cells. Next, the embryoid bodies are further cultured under conditions that promote the differentiation of the plated cells, for example, in an ITSFn (nestin selection) serum-free synthetic medium that is selective for nestin positive cells. Nestin is an intermediate filament protein expressed in the sensory epithelium. Preferably, cell selection is performed on nestin positive cells over a period of 5-16 days. Preferably, the ITSFn medium used for the growth of nestin positive cells is a group consisting of progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, bFGF, SHH, EGF, FGF-2, FGF-8, and BDNF DMEM: F-12 supplemented with one or more growth factors selected from

  This heterogeneous cell population containing nestin positive cells is then expanded or sorted to enrich for neural cell adhesion factor (NCAM) positive cells. NCAM is a surface marker characteristic of nerve cells. NCAM-positive cells can be sorted immediately after nestin-positive cells are selected or after nestin-positive cells have grown in the medium. In some embodiments, the cells are sorted for NCAM positive cells by contacting the cells with an antibody or ligand that binds to NCAM, followed by separation of the specifically recognized cells by appropriate immunology. Techniques, such as solid phase adsorption, fluorescence activated cell sorting (FACS), flow immunocytochemistry for cell surface markers, flow cytometry assays Or magnetic cell separation (MACS). Other methods for isolating NCAM positive cells include, but are not limited to, differential plating, immunospecific lysis of contaminating cells, or collection techniques. Well-known to vendors. In a preferred embodiment, selection of nestin positive cells (eg, by MACS) enriches the population of viable value float positive cells, NCAM is about 40-70%, preferably about 60% -80%, more preferably Is expressed from about 85% to 90%, more preferably from about 95% to 99%.

  In one embodiment, the embryoid bodies are generated from human ES cells by culturing human ES cells on bacteriological plates in the presence of supporting cells in a suitable medium. Preferably, human ES cells are separated into clusters and then plated on non-adherent plates to promote embryoid body expression. A suitable medium preferably contains high glucose DMEM and is supplemented with 10-20% FCS. Other adjuvants (supplements) may be added to this medium, for example 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin. After the embryoid bodies are grown, preferably for 4-8 days, the embryoid bodies are again grown on a culture plate coated with ITSFn serum-free medium containing 0.1% gelatin for selection of nestin positive cells. Plate in Preferably, the ITSFn serum-free medium is a basal medium DMEM: F-12 (1: 1) supplemented with growth factors, insulin, sodium selenite, transferrin, and fibronectin to select nestin-positive cells. Or contains IMDM medium.

In a preferred embodiment, nestin positive cells are sorted by MACS to isolate NCAM positive cells. NCAM-positive cells substantially grow in media that promotes an increased percentage of neural progenitor cells and induces the cells to adapt to a more differentiated phenotype. Preferably, a culture of NCAM positive cells is pre-coated with extracellular matrix components (eg, poly-1-lysine, poly-1-ornithine, laminin, collagen, fibronectin, and Matrigel®, or combinations thereof ( The pre-coating of the culture dish with extracellular matrix allows better attachment and growth in the growth medium and gives good results for dopaminergic neuronal differentiation. Cultured in growth medium, cells are grown in growth medium such as progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, bFGF, SHH, EGF, FGF-2, FGF-8, BDNF, PDGF, IGF -1, CTNF, and NT-3 Cultured in DMEM / F12 medium supplemented with one or more growth factors that do not want to be bound by any particular mechanism, but these various factors present in the growth medium are responsible for the overall proportion of neurons. In a preferred embodiment, nestin-positive NCAM-positive progenitor cells proliferate over a period of 3 to 10 days. These cells can also be subcultured over at least 30 population doublings and frozen for further use without any loss of differentiation potential.
Differentiation of dopaminergic and serotonergic neurons Neural progenitor cells prepared by the methods disclosed herein are further differentiated to a higher percentage of mature neurons, such as dopaminergic and serotonergic cells Become a sex neuron. Neural progenitor cells can also be further differentiated into astrocytes and oligodendrocytes. Preferably, nestin-positive and / or NCAM-positive neural progenitor cells are grown for 5-60 days in a neural basal medium that promotes differentiation of the progenitor cells into terminally differentiated or mature neurons. In a preferred embodiment, the neural basal medium (Gibco) is 10% FBS or DCS, B27, and interleukin-1β, dibutyryl cyclic AMP (db-cAMP), glial cell-derived neurotrophic factor (GDNF), transformed growth. Factor β3 (TGF-β3), transforming growth factor α (TGFα), neuturin, SHH, ascorbic acid, BDNF, FGF-2, FGF-8, N-acetyl cysteine, C-kit ligand, retinoic acid, NT-3, BMP-2, and BMP-4. In a preferred embodiment, the neural basal medium includes one or more of the following factors: interleukin-1β, db-cAMP, GDNF, TGF-β3, Neuturin, SHH, ascorbic acid, BDNF, FGF-8, and Contains N-acetyl cysteine. In addition, the differentiation may be promoted by withdrawing some or all of the factors that promote neural progenitor cell differentiation, proliferation, or both.

Preferably, a high proportion of neural progenitor cells differentiate into dopaminergic neurons, serotonergic neurons, or oligodendrocytes, eg at least 20%, 25%, 30%, 35%, 40% of the cells, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Also, methods known to those skilled in the art, such as immunolabeling and fluorescence sorting (eg, solid phase adsorption, FACS, and MACS), from a population of differentiated neurons, from one neuronal type (eg, dopaminergic neurons). Can be further purified. In one preferred embodiment, a population enriched by immunoadsorption of NCAM-positive cells is grown and differentiated in a suitable medium, resulting in a high percentage (about 60%) of dopaminergic neurons.
Uses of neural progenitor cells and differentiated neural cells The neural progenitor cells and differentiated neural cells described herein (eg, mature neural cells, top cells, and oligodendrocytes) can be used in various applications, eg, therapeutic applications. Furthermore, it can be used for in vitro evaluation and screening of various compounds such as small molecule drugs that act on these cells. These cells can be expressed live using techniques well known to those skilled in the art, as well as preparing monoclonal or polyclonal antibodies specific for the markers for the particular cells used to analyze the expression pattern of the cells. It can also be used to prepare a rally. These cells can also be used as a treatment for the benefit of individuals suffering from debilitating neurodegenerative disorders and neurological diseases.

  The present disclosure discloses the use of neural progenitor cells and differentiated neural cells as described herein to repair central nervous system (CNS) function in a subject in need of such treatment. provide. For example, these cells could be used as a treatment by transplanting directly to the parenchyma or intrathecal site of the CNS, depending on the disease or condition being treated. These cells can be used to treat acute or chronic damage to the nervous system, as well as debilitating neurodegenerative disorders and neurological diseases such as Parkinson's disease, Alzheimer's Diseases, Huntington's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, cerebral infarction, and ischemia.

  One embodiment of the present disclosure relates to a method of treating a neurodegenerative disorder or neurological disease, the method comprising a dopaminergic neuron derived from a pluripotent stem cell, preferably a human pluripotent stem cell. Is characterized by the alteration or destruction of dopaminergic neurons by administering or transplanting a therapeutically effective amount. In another embodiment, the present disclosure relates to a method of treating a neurodegenerative disorder or neurological disease, wherein the method is serotonergic from a pluripotent stem cell, preferably a human pluripotent stem cell. Characterized by alteration or destruction of serotonergic neurons by administering or transplanting a therapeutically effective amount of neurons. Preferably, treatment of a human patient suffering from a neurodegenerative disease or neurological disorder is performed by transplanting the patient with a therapeutically effective amount of neural progenitor cells and differentiated neural cells of the present disclosure. As used herein, a “therapeutically effective amount” of cells is a differentiated neuron, such as a mature neuron (eg, a dopaminergic or serotonergic neuron). An amount sufficient to inhibit or ameliorate a physiological effect in a subject caused by loss, damage or alteration of astrocytes and oligodendrocytes.

  The therapeutically effective amount of cells used depends on subject requirements, subject age, physiological state and health, desired therapeutic effect, size of target tissue area for treatment, lesion size Depends on the extent, as well as the delivery route chosen. For example, treatment of a disease affecting a larger area of the brain should require more cells to achieve a therapeutic effect compared to a smaller target site. It is also possible to administer cells to multiple sites within a given target tissue using multiple small implants with low cell doses. The cells of the present disclosure may be completely separated prior to transplantation, for example, to make a single cell suspension, or may be almost completely separated prior to transplantation to create small clumps of cells. The cells may be administered such that the cells can be transplanted or migrated to the intended tissue site and that the functionally deficient areas have been reconstructed or re-established.

  A suitable range of cells administered to achieve a therapeutic effect is from about 100 to about 1,000,000 neurons, preferably from about 500 to about 500,000 neurons, or from about 1,000 to about 1,000 cells. 100,000 neurons. The therapeutic concentration of nerve cells administered to the subject is also about 10, 100, 500, 1000, 5000, 10,000, 15,000, 20,000, 25, per μl of pharmaceutically acceptable carrier. 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, Within the range of 250,000, 300,000, 350,000, 400,000, 450,000 to about 500,000 cells. The concentration range of the cells in the carrier is, for example, 100 to 50,000 cells / μl, 1000 to 10,000 cells / μl, 5000 to 25,000 cells / μl, 15,000 to 45,000 cells / μl, 20 50,000 to 50,000 cells / μl, 55,000 to 200,000 cells / μl, 100,000 to 40,000 cells / μl, 150,000 to 50,000 cells / μl. The number of cells transplanted to the transplanted tissue site also affects therapeutic efficacy.

For therapeutic applications, it is often preferred that the population of precursors or differentiated neurons is substantially pure with any undifferentiated pluripotent stem cells.
One strategy for removing pluripotent stem cells from a therapeutic preparation is to transfect the cells with a vector, which has a gene that is preferentially expressed in undifferentiated cells, Expression is selected for pluripotent stem cells. Suitable promoters that are preferentially expressed in undifferentiated cells are the telomerase reverse transcriptase (TERT) promoter and the OCT-4 promoter. The gene expressed in the vector may be, for example, a lytic drug for cells (eg, toxins), or can be selected by applying an external substance.

  As disclosed herein, the ability to generate dopaminergic and serotonergic neurons from pluripotent stem cells is useful for transplantation treatment of various neurodegenerative disorders and neurological diseases. There is a great clinical relevance. For example, neurodegenerative disorders and neurological disorders characterized by abnormalities in the regulation of postural reflexes, movements, and reward-related behaviors (eg Parkinson's disease, schizophrenia, and drug addiction, as well as dopaminergic neurons) Traumatic or other illnesses that cause Parkinson-like symptoms (eg, restless tremor, stiffness, immobility, and postural abnormalities such as immobility, lack of thirst, anorexia, neglect of sensation) Can be used to treat. In addition, serotonergic neurons are responsible for feeding behavior, hormone secretion, stress response, pain and immune function, sexual activity, cardiovascular function, temperature control (eg various psychiatric, neurological, and other Neurodegenerative disorders and neurological diseases characterized by abnormal regulation of diseases such as mental decline, suicide, intense aggressive behavior, compulsive behavior, eating disorders / bulimia, and schizophrenia) Can be used to treat.

  In another embodiment, the present disclosure provides for the differentiation of neural progenitor cells of the present disclosure derived from one or more neuronal survival factors and pluripotent stem cells to treat a neurodegenerative disorder or disease. To the co-administration of the treated neurons. Nerve survival factors can be administered prior to, simultaneously with, or in combination with, or subsequent to administration of the desired cells. As used herein, “neurosurvival factor” refers to a nerve (in vitro or in vivo) that is in contact with a factor to live for a longer period of time than in the absence of the factor. Nerve survival factors that can be used in therapeutic embodiments of the present invention include, but are not limited to, glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF) Ciliary neurotrophic factor (CDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), FGF, IL-1β, TNFα, Insulin-like growth factors (IGF-1, IGF-2) and transforming growth factor β (TFG-β, TFG-β1) can be mentioned.

  It is known that GDNF has in vitro trophic activity for embryonic mesencephalic ventral mesencephalic dopaminergic neurons (Lin et al., 1993, Science 260: 1130-1132; Lin et al., 1994, J. Neurochem. 63: 758-768). Recombinant human GDNF also induces rapid growth of dopaminergic fibers in vivo (Hudson et al., 1993, Soc. Neurosci. Absir. 19: 652), increasing rat substantia nigra dopamine turnover (Miller) et al., 1994, Soc.Neirosci.Abstr.20: 535-7), protecting fetal neurons from 6-OHDA lesions and in vivo substantia nigra tissue rat fetal transplant Increase in growth and fiber formation (Stromberg et al., 1993, Exp. Neurol. 124: 401-412). BDNF is a trophic factor for peripheral sensory neurons, dopaminergic neurons, and retinal ganglia (Henderson et al., 1993, Restor. Neurol. Neurosci. 5: 15-28) and in vitro and live. It has been shown to prevent cell death that normally occurs in the body (Hofer and Barde, 1988, Nature 331: 161-262).

  As used herein, the term “treatment” or “to treat” refers to therapeutic treatment and prophylactic or preventative measures. Accordingly, those in need of treatment include those already having a neurodegenerative disorder or neurological disease, as well as those in which a neurodegenerative disorder or neurological disease is prevented. The disclosed methods of the present invention can also be used to treat any mammal in need of treatment, including but not limited to humans, primates, and households, farms, pets, or Sports animals such as dogs, horses, cats, sheep, pigs, and cows. A “disorder” is any condition that would benefit from treatment with the disclosed neural progenitor cells, differentiated neural cells, or both types of cells. Examples of diseases that benefit from transplantation of dopaminergic neurons are disorders associated with inappropriate posture reflexes, exercise, reward-related behaviors such as Parkinson's disease, schizophrenia, and drug addiction. Examples of disorders that benefit from serotonergic neuron transplantation include, but are not limited to, aggression, depression (including suicide), and schizophrenia. Characterized by abnormalities, including dysphagia, eating disorders / bulimia. Other disorders that may also benefit from treatment with the cells of the present disclosure are Alzheimer's disease, Huntington's disease, and the giant colon.

  The disclosed method of the present invention can be advantageously carried out by transplanting the “neural progenitor cells or differentiated neurons” of the present disclosure directly into the lesion. Methods relating to nerve transplantation and cell culture are well known to those skilled in the art (eg, US Pat. No. 5,514,552; Yurek and Sladek, 1990, Annu. Rev. Neurosci. 13: 415-440; Rosenthal, 1998). , Neuron 20: 169-172; Veskovi et al., 1999, J. Neurotrauma 16 (8): 689-93; Veskovi et al., 1999, Exp. Neuro. 156 (1): 71-83; et al., 1999, Science 285: 754-56; the contents of each of which are specifically incorporated as part of this specification). In one embodiment, the disclosed dopaminergic neurons may be transplanted into the substantia nigra or striatum of a patient suffering from Parkinson's disease. The cells may be delivered alone or in combination with other factors (eg, nerve survival factors) and may be delivered with a pharmaceutically acceptable vehicle. Ideally, such a vehicle enhances cell stability and delivery properties.

  The present disclosure also provides a pharmaceutical composition comprising cells administered using a suitable vehicle (liposome, microparticle, or microcapsule). The cells of the present disclosure can also be supplied in the form of a pharmaceutical composition, which contains an isotonic excipient and is prepared to be sufficiently sterile for human administration. The general principles of pharmaceutical formulations of cell compositions are described in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, G. et al. Morstyn & W. Sheridan eds, Cambridge University Press, 1966, and Hematopoietic Stem Cell Therapy, E .; Ball. J. et al. Lister & P. Law, Churchill Livingstone, 2000, the contents of each of which are specifically incorporated as part of this specification. Moreover, it may be preferable to administer a pharmaceutical composition containing a nerve survival factor locally in the area in need of treatment, e.g., local infusion during surgery, injection, catheter means, or implant It can be achieved by means. Such implants are porous, non-porous, or jelly-like materials and can be, for example, membranes (eg, silastic membranes or fibers).

The neural progenitor cells and differentiated neural cells of the present disclosure can be transplanted into a subject for either substantially allogeneic, almost allogeneic, or heterogeneous cell populations. The substantially allogeneic cell population is greater than 75% single cell type, such as dopaminergic or serotonergic neurons, more preferably about 90%, most preferably 95% to 99%. A heterogeneous cell population is a single cell population such as dopaminergic neurons, serotonergic neurons, Schwann cells, oligodendrocytes, astrocytes, GABA neurons, and glial cells, eg, two or more mixed types Cell types. The cells can also be genetically modified by methods well known to those skilled in the art so that trophic factors, growth factors, at the site of damage to the brain, central nervous system, peripheral nervous system, or other tissues, Nerve survival factors, or other therapeutic compounds are expressed or released. The use of promoter and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology, see, for example, Sambrook, et al. , 1989, Molecular Cloning: A Laboratory Manual 2 nd Ed. See Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the contents of each of which are specifically incorporated as part of this specification.

  In order to achieve expression of trophic factors, growth factors, neural survival factors, or other therapeutic compounds in the neural progenitors and differentiated neurons of the present disclosure, suitable regulatory elements are available in various sources. Can be derived and easily selected by those skilled in the art. Examples of regulatory elements include transcription promoters, enhancers, RNA polymerase binding sequences, as well as ribosome binding sequences that include a translation initiation signal. Other additional genetic factors, such as selectable markers, can also be incorporated into the recombinant molecule. Recombinant molecules can be introduced into pluripotent stem cells, or pluripotent stem cell-derived neural progenitor cells or differentiated neurons using in vitro delivery vehicles or in vivo techniques. Examples of delivery techniques include retroviral vectors, adenoviral vectors, DNA viral vectors, liposomes, physical techniques (eg, microinjection, electroporation, or calcium phosphate precipitation), or metastasis to create recombinant cells Other methods known to those skilled in the art may be mentioned. Genetically modified cells can be encapsulated in microspheres and transplanted into or near diseased or damaged tissue. The protocols used are well known to those skilled in the art and are described, for example, in Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, New York, N .; Y. 1997, the contents of which are specifically incorporated as part of this specification.

  Clinical experiments involving nerve transplantation in patients with Parkinson's disease were performed by using various animal models of Parkinson's syndrome as recipients of fetal embryonic nerve cells or paraneuron tissue grafts to the site of brain injury. In animal experiments with fetal dopaminergic neuronal grafts, such grafts can reverse dopaminergic deficits and restore locomotor function in animals in experimental lesions of the nigrostriatal dopaminergic system I was able to. For example, normal mesencephalic cells obtained from 14-16 day old embryos are advantageously used as allografts or xenografts without immunosuppressive treatment, rather than those collected from late pregnancy donors. (Yurek and Sladek, 1990, Annu. Rev. Neurosci. 13: 415-440). Interestingly, these ages correspond to the gestational age at which dopaminergic neurons undergo their final cell division (Lauder and Bloom, 1974, J. Comp. Neurol. 155: 469-82). Solid grafts of embryonic mesencephalic tissue can be dissociated into cell suspension for transplantation. However, the typical suspension viability was limited to about 10% of transplanted dopaminergic neurons (Brudin et al., 1987, Ann. NY Acad. Sci. 495: 473). 96). Since embryonic mesencephalic tissue is not a pure source of dopaminergic neurons, only about 0.1-1.0% of all viable cells are surviving dopaminergic neurons and dissociated midbrain. Any graft of cells preferably contains a minimum of 100,000 to 150,000 live cells, in order to effectively compensate for the loss of dopaminergic activity.

  Preferably, the cell transplant tissue treatment of the present disclosure is also differentiated from neural progenitor cells for use in transplantation surgery, eg, long-term storage by cryopreservation methods or short-term storage in preservation media Incorporate several means for storing and storing Cryopreserved embryonic mesencephalic tissue can be successfully stored for up to 70 days and can be transplanted as an allograft in rodents (Collier et al., Progress in Brain Research, Vol. 78, New York, Elsevier ( 1988), pp. 631-36, specifically incorporated herein by reference and primates (Collier et al., 1987, Brain Res. 436: 363-66, the contents of which are specifically incorporated herein. ). It has been demonstrated that embryonic mesencephalic cells can be successfully cultured after cryopreservation. Short-term (2-5 days) midbrain tissue can also be stored in 4 ° C. storage medium, which is then transplanted with a viable graft volume similar to that of fresh tissue (Sauer et al.,. 1989, Restor.Neurol.Neurosci. (Suppl.:3.sup.rd Int..Symp.NeuralTranplan.): 56, the contents of which are specifically incorporated as part of this specification).

  Both graft implantation and the extent to which the graft re-innervates the striatum are important factors for the functional recovery of the mesencephalic system dopaminergic system. In animals with unilaterally removed dopaminergic nerves, cortically placed dopaminergic grafts showed reduced motor asymmetry but had little effect on sensory nerve neglect (Bjorkland et al , 1980, Brain Res. 199: 307-33; Dunnett et al., 1981, Brain Res. 215: 147-61). In contrast, a substantia nigra graft placed in the lateral striatum in an animal with bilaterally removed dopaminergic nerves repaired sensory damage (Dunnett et al., 1983, Acta Physiol. Scan). Suppl.522: 39-47). Only when the nucleus accumbens is reinnervated by dopaminergic grafts, reversal of immobility in bilaterally impaired animals is observed (Nadaud et al., 1984, Brain Res. 304: 137). -41).

  While transplant sites for dopaminergic grafts often exist near the area of the ventricle, because of the good cerebrospinal fluid (CSF) environment of those areas to provide graft survival, Parkinson's disease The degree of dopaminergic alteration is more evident in the putamen than in the caudate nucleus. Putamen dopamine levels are lower in the caudate nucleus, often 10-15% (Bemheimer et al., J. Neurol. Sci. 20: 415-55; Nyberg et al.,., 1983, Neurochem. Pathol. 1: 93-202). Moreover, the tail putamen has a more severe reduction in dopaminergic-neuronal cells than the snout putamen (Kish et at, 1986, Ann. Neurol. 20: 26-31). Of the two components of the striatum (the caudate nucleus and the putamen), the putamen undergoes most motor inputs via the cortical thalamic putamen (DeLong & Georgepoulos, 1983, Handbook of Physiology). , Section 1: The Nervous System, Vol.2, ed.Brookhard, Mountcastle, Geiger, pp. 1017-61, Bethesda, Md .: Am.Physiol.Soc.). Thus, the putamen are considered to be a more advantageous site for dopaminergic grafts targeting the dyskinetics associated with Parkinson's disease.

  Neural progenitor cells and differentiated neural cells described herein can be used to screen compounds (eg, pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides), as well as phenotypes or these The same can be used for environmental factors such as culture conditions or operations that affect cell characteristics. In addition, these cells can be used in evaluating candidate growth factors or differentiation factors. For example, a candidate pharmaceutical compound can be added to neural progenitor cells or mature neurons, alone or in combination with other agents, and any changes in cell morphology, phenotype or functional activity can be assessed and Evaluation can be performed.

  In addition, the neural progenitor cells and differentiated neurons described herein can be further modified at any stage of differentiation. For example, in a transient or stable manner, these cells may be genetically modified to have single or multiple genetic recombination. Altering the genes of these cells may be necessary for a number of reasons, such as providing modified cells for gene therapy or replacing tissues for transplantation or implantation. The cells of the present disclosure can be genetically repaired through the introduction of a vector that expresses a selectable marker under the control of a neuron-specific promoter well known to those skilled in the art. These cells can also be modified at any stage and used to further generate differentiated cells derived from pluripotent stem cells, or certain types that can induce differentiation into specific cell lineages. It may be one that expresses a marker or gene. These cells can also be modified to reduce or prevent immune rejection after transplantation (ie, histocompatibility with the intended transplanter).

In order to increase the replication capacity of the cells generated using the present disclosure, the telomerase catalytic component (TERT) is expressed by telomerizing these cells by genetic modification with an appropriate vector. The TERT sequences used are derived from humans or mice (WO 98/14592 and WO 99/27113, specifically incorporated herein), as well as other mammalian species. Alternatively, transcription of the endogenous TERT gene can be increased. Methods used in genetically modifying cells are well known to those skilled in the art. These methods utilize a variety of molecular biological techniques, many of which are generally described in Sambrook, et al. , 1989, Molecular Cloning: A Laboratory Manual 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the contents of which are specifically incorporated as part of this specification.

  It should be understood by those skilled in the art that the following examples are included to demonstrate preferred embodiments of the invention. The technology disclosed in the following examples represents the technology discovered by the present inventor and is intended to fully function the practice of the present invention and constitutes a preferred modification for its implementation. Can do. However, one of ordinary skill in the art appreciates that many changes can be made in the particular embodiments disclosed and that similar or similar results can be obtained without departing from the spirit and scope of the invention in light of the present disclosure. It will be appreciated by those skilled in the art that it can still be obtained.

Example 1
The following examples illustrate the induction of functional dopaminergic and serotonergic neurons from human embryonic stem cells by the applicant of the present disclosure.
(1) Human embryonic stem cells First, human ES cells were isolated from the inner cell mass of human blastocysts. Human pluripotent ES cells were derived from excess human blastocysts after obtaining consent from individual patients for the experimental use of blastocysts. The human ES cell line used herein is a human blastocyst inner cell mass using a novel laser ablation method as disclosed in US patent application Ser. No. 10 / 226,711. Derived from cells. Briefly, human blastocysts with zona pellucida, trophectoderm, and inner cell mass were isolated and a 1.48 μm non-contact diode laser was used to An opening through the trophectoderm was formed. Next, a suction pipette was introduced through the opening, and the cells of the inner cell mass were isolated by suction. These cells were then cultured on 0.1% gelatinized plates with ES medium containing mitomycin C-treated mouse feeder cells to form a cell mass derived from the inner cell mass. ES cell medium consists of Dulbecco's modified EglES medium (DMEM) or knockout DMEM (Gibco), 10-20% ES cell restricted fetal calf serum (FCS) (Hyclone) or serum replacement knockout serum (Gibco), 1 % MEM non-essential amino acid solution, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 4 ng / ml basic fibroblast growth factor (bFGF) (Sigma), 50 U / ml penicillin, and 50 μg / ml streptomycin Made up. The cell mass derived from these inner cell masses is dissociated and re-plated in an ES medium containing a mouse feeder cell layer, which is used to induce a human ES cell line.

Morphologically, induced human ES cells have a high nucleus / cytoplasm ratio and are cell surface markers (eg, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA- 1-81, OCT-4, alkaline phosphatase, telomerase, karyotype analysis, CD-30, crypto-1, GCNF, c-kit, and CD-90).
(2) Culture and expression of human embryonic stem cells Human ES cells were cultured and grown from frozen stocks under standard growth conditions. Undifferentiated human ES cells obtained from frozen stock vials were resuspended in ES medium containing LIF. The ES cell medium consists of Dulbecco's modified Eaglel medium (DMEM), 20% ES cell restricted fetal bovine serum (FBS) (Hyclone), 1% non-essential amino acid (NEAA) solution, 2 mM L-glutamine, 0. Supplemented with 1 mM β-mercaptoethanol, 50 U / ml penicillin, and 1,000 U / mL LIF. Cells were pelleted and plated on mouse feeder cell layers on gelatin-coated plastic culture dishes using ES medium. Composed of high glucose DMEM, 10-20% FCS, 2 mM L-glutamic acid, 1% MEM non-essential amino acid solution, 0.1 mM β-mercaptoethanol, 50 U / ml penicillin, 50 μg / ml streptomycin, 1,000 U / ml LIF, and 4 ng / MlbFGF supplemented with ES cell medium, Thomson et al. , 1998, Science 282: 1145-1147; Shamblott et al. 1998. Proc. Natl. Acad. Sci. USA. 95: 13726-13731; and Rebinoff et al. 2000. Nature Biotech. 18: 399-403.

Human ES cells were grown by culturing, and ES cells were periodically passaged to inhibit differentiation. ES cells were passaged by first washing the cells with Dulbecco's phosphate buffer without Ca ⁺⁺ and Mg ⁺⁺ for 10 seconds and then treating the cells with 0.05% trypsin for 5 seconds. It was. After 5 seconds, trypsin assay was inhibited by serum-containing ES medium. Next, the cells were scrapped and destroyed into small clusters. The clusters were seeded in two 0.1% gelatin coated 100 mm Petri dishes (covered with feeder cells contained in ES medium containing LIF and bFGF as described above). Cells were grown for 4-6 days.
(3) Generation of embryoid bodies Undifferentiated human ES cells were expanded and expanded, and then the cells were cultured to form embryoid bodies. First, ES cells were separated with 0.05% trypsin-EDTA and then scraped to break the cells into small clusters. These clusters were then seeded at about 1 × 10 ⁵ cells / ml on bacteriological dishes without feeder cells. The bacteriological dish used has a non-sticky surface that prevents attachment, thus stimulating ES cell differentiation and embryoid body formation. These cells were cultured as suspension cultures in ES medium without LIF and bFGF. The ES medium used to culture these cells is high glucose DMEM or knockout DMEM, 10-20% FCS or knockout serum replacement, as well as other supplements (eg, β-mercaptoethanol, L-glutamic acid). , And antibodies). No bFGF has been added to the ES medium. Embryoid bodies were grown for 4-8 days. During this time, the ES medium was changed every 2 days by the precipitation method. In this precipitation method, the suspension of aggregates was transferred to a centrifuge tube, the aggregates were allowed to settle to the bottom of the centrifuge tube, the medium was aspirated, and it was replaced with fresh medium. Aggregates contained in fresh medium were then transferred to culture dishes. After 4-8 days, embryoid bodies were collected, centrifuged at low speed, and resuspended in ES cell medium. About 20-30 embryoid bodies were seeded on tissue culture plates coated with 0.1% gelatin-containing ES medium without LIF and incubated for an additional 24 hours.
(4) Selection and proliferation of nestin positive neural progenitors After 24 hours, nestin positive cells (neural progenitor cells) were selected by replacing ES medium with ITSFn (nestin selected) serum-free defined medium.
After 24 hours, nestin positive cells (neural progenitor cells) were selected by using ITSFn (nestin selection) serum-free synthetic medium instead of ES medium. ITSFn medium consists of DMEM: F12 medium (Gibco), growth factor insulin (5-25 μg / ml) (Sigma), sodium selenite (10-50 nM) (Sigma), transferrin (1-10 gg / ml) ( Gibco) and fibronectin (1-5 μg / ml).
This medium allowed selection of nestin positive cells and was performed on an ITSFn medium supplemented every 2 days for 6-10 days, usually 8-9 days. After selection was complete, the neural progenitor cells were characterized for nectin expression using immunofluorescence techniques. As a result, about 95% of cells expressed nectin (FIG. 1A).

Nestin positive cells were subsequently expanded and sorted using magnetic cell separation (MACS) to enrich for neural cell adhesion molecule (NCAM) positive cells.
(5) Selection of NCAM positive cells by magnetic selection Next, in order to enrich the neural cell adhesion molecule (NCAM) positive cells, nestin positive cells were isolated and subjected to proliferation or magnetic cell separation (MACS). NCAM is a nerve cell specific surface marker. To sort nestin positive cells by MACS using NCAM, nestin positive cells were collected from tissue culture plates by first incubating with 0.05% trypsin-EDTA. Next, the separated living cells were washed with PBS and stained with an antibody against the surface marker MCAM (Chemicon) for 30 minutes. Cells were then incubated with MACS ghost anti-rabbit IgG microbeads (Miltenyi Biotec) for 15 minutes. Cells were carefully washed with PBS and subjected to magnetic sorting. To sort cells, pipette a magnetically labeled cell suspension (approximately 2 × 10 ⁸ cells) onto a MACS MS separation column (Miltenyi Biotec), flow the cells through the column, and collect the eluate (negative fraction) did. Recovered by elution with NCAM positive cells.

  Prior to immunosorting, nestin positive cells were analyzed by FACS and approximately 50-60% expressed the neurological surface marker NCAM (FIGS. 2A and 2B). Following immunosorting by MACS, the viable population of nestin positive cells was enriched to approximately 80-85% (FIGS. 3A and 3B).

(6) Proliferation of NCAM positive neural progenitor cells:
Isolated NCAM positive cells were first detached with 0.05% trypsin-EDTA and then the cells were seeded onto poly-L-ornithine / laminin coated plates containing growth media. By culturing NCAM-positive cells in this way, better adhesion and growth are performed in the growth medium. The growth media are DME: F12, insulin (10-100 μg / ml), sodium selenite (10-50 nM), transferrin (1-10 mg / ml), putrescine (50-200 μM), progesterone (5-40 nM), And laminin (10-50 μg / ml). Two or more growth factors such as bFGF (10-50 ng / ml), EGF (10-50 ng / ml), BDNF (50-200 ng / ml), FGF-8 (50-200 ng / ml) and / or SHH ( 200-400 ng / ml) was incubated in serum-free growth medium. Various factors present in the growth medium are involved in increasing the overall proportion of neurons and also induce these midbrain neural progenitors to adopt a dopaminergic phenotype. Growth medium was replenished every 2 days and nestin-positive NCAM-positive cells were grown for 6-10 days (FIG. 1B).

Under these culture conditions, about 50-60% of cells proceeded to the neurite process, and NCAM staining was positive, which revealed that they were capable of forming neurons (FIG. 3). These cells were also immunoreactive with β-tubulin, an early neuronal marker (FIG. 4B). These neural progenitor cells are then passaged and Applicants have been able to pass these neural progenitor cells up to 10 times, to the cell's potential for differentiation to commit mature dopaminergic neurons. It can be seen that it still retains the ability to differentiate into dopaminergic neurons without any apparent effect.
(7) Differentiation of dopaminergic neurons:
The expression of dopaminergic neurons in vivo depends on the regulated behavior of extracellular signal molecules. These molecules activate a cascade of transcriptional regulators with lineage restricted expression. These transcription factors play a role in increasing the expression of a specific set of genes involved in the development of the dopaminergic phenotype. Using the above protocol for functional dopaminergic neurons, the growth factors bFGF and EGF were first withdrawn from the cells to promote proliferating neural precursor cells. Next, differentiation of neural precursor cells into dopaminergic neurons was induced by incubating the cells in neural basal medium for 30-50 days. Neural basal medium contained Neural basal A medium (Gibco), FCS (10-20%) (HYCLONE), and B27 (2-10%) (Gibco) as well as various growth factors as well. Growth factors contained in this medium are interleukin-1β (IL-1β) (1-2 μg / ml) (Sigma), ascorbic acid (50-150 mM) (Sigma), N-acetyl cysteine (50-150 nM) (ICN), dibutyryl cyclic AMP (500-1000 [mu] M) (Sigma), GDNF (1-5 [mu] g / ml) (Sigma), TGF- [beta] 3 (1-5 [mu] g / ml) (Sigma), Neuturin (100-500 [mu] g / ml) ) (Chemicon), BDNF (50-200 ng / ml) (Sigma), FGF-8 (50-200 ng / ml) (R & D Systems) and / or SHH (200-400 ng / ml) (R & D Systems). Nerve basal medium was sterile filtered through a 0.22 μmillipore syringe filter.

  Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine synthesis. Certain strain-restricted growth factors promote the induction of TH synthesis. By adding these factors to the medium, the proportion of cells adopting the dopaminergic phenotype increased. As shown in Table 1, the addition of these factors occurred at different times during neuronal differentiation. IL-1β (present in neural basal media) appears to play an important role in the differentiation of neural progenitors into dopaminergic transcells. Despite wishing not to be limited to any particular mechanism, IL-1β appears to increase the amount of TH-positive neurons (as well as the cellular response to media signal molecules). The application of these growth factors is combined with changing the neural basal medium every 3 days during a 30-50 day tissue culture period.

The mixture of growth factors in the neural basal medium added on different days during differentiation of neural progenitor cells is represented in Table 1 below.
Table 1: Differentiation conditions for neural progenitor cells

ＥＳ細胞が神経基本培地を使用して分化が誘導される場合、ＥＳ細胞は、ドーパミン作動性神経細胞、セロトニン作動性神経細胞、およびオリゴデンドロサイトを含む種々の神経細胞の細胞型で出現する（図５、６、および７）。標準培養条件でドーパミン作動性神経細胞に分化する細胞の割合が比較的低いままである一方で、表１に示すように増殖因子を含む培地を補充することで、生じたドーパミン作動性神経細胞の割合が増加した。例えば、６０％を上回るＥＳ細胞がＴＨ（ドーパミン作動性神経細胞のための特異的マーカー）陽性細胞に分化した。
（８） 分化した神経細胞の特徴付け
本発明の開示によって生ずる分化した神経細胞の型を、細胞の全体的な形態と免疫蛍光検査法によって同定される表現型とによって評価した。当業者に周知の標準的なプロトコールを用いて、免疫蛍光分析を、神経前駆体増殖段階（ＮＣＡＭ陽性細胞の増殖）と、分化の種々の他の時点とでおこなった。第一に、単離された細胞を、細胞外マトリックスによってプレコーティングされた２−ウェル・チャンバー・スライドで増殖させ、ＰＢＳでリンスし、さらに１０分間、室温で４％パラホルムアルデヒドによる固定をおこなった。次に、細胞を０．２％トリトンＸ−１００含むＰＢＳで５分間透過化処理し、１％牛血清アルブミン（ＢＳＡ）／ＰＢＳで２時間阻害し、さらに一次抗体（抗体希釈は１％ＢＳＡ／ＴＢＳ）で４℃一晩おこなった。細胞を以下の一次抗体で染色した。すなわち、初期の神経マーカーであるβチューブリン、ＮＣＡＭ、神経繊維、遅延神経マーカー、微小管結合タンパク質２（ＭＡＰ−２）、神経細胞表面抗原Ａ２Ｂ５、Ｎｕｒｒ−１、チロシン・ヒドロキシラーゼ、ドーパミン輸送体ＤＡＴ、ドーパミンβ−ヒドロキシラーゼ（ＤＢＨ）、星状膠細胞マーカー・グリア線維酸性蛋白（ＧＦＡＰ）、ＧＡＢＡ、オリゴデンドライト、セレトニン、およびシナプトフィシン（すべてＣＨＥＭＩＣＯＮから入手）。最後に、細胞をＦＩＴＣ標識二次抗体とインキュベートした。上記のステップの各々の後、細胞をＰＢＳで３回洗った。

When ES cells are induced to differentiate using neural basal media, ES cells appear in a variety of neuronal cell types including dopaminergic neurons, serotonergic neurons, and oligodendrocytes ( Figures 5, 6 and 7). While the proportion of cells that differentiate into dopaminergic neurons under standard culture conditions remains relatively low, supplementation with a medium containing growth factors as shown in Table 1 reveals that the resulting dopaminergic neurons The percentage increased. For example, over 60% ES cells differentiated into TH (specific markers for dopaminergic neurons) positive cells.
(8) Characterization of differentiated neuronal cells Differentiated neuronal cell types resulting from the present disclosure were evaluated by the overall morphology of the cells and the phenotype identified by immunofluorescence. Using standard protocols well known to those skilled in the art, immunofluorescence analysis was performed at the neural progenitor growth stage (NCAM positive cell growth) and at various other times of differentiation. First, the isolated cells were grown on 2-well chamber slides precoated with extracellular matrix, rinsed with PBS, and fixed with 4% paraformaldehyde at room temperature for an additional 10 minutes. . Next, the cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 minutes, inhibited with 1% bovine serum albumin (BSA) / PBS for 2 hours, and further the primary antibody (antibody dilution was 1% BSA / TBS) at 4 ° C. overnight. Cells were stained with the following primary antibodies. That is, early neuronal markers β-tubulin, NCAM, nerve fiber, delayed nerve marker, microtubule binding protein 2 (MAP-2), nerve cell surface antigen A2B5, Nurr-1, tyrosine hydroxylase, dopamine transporter DAT, dopamine β-hydroxylase (DBH), astrocyte marker glial fibrillary acidic protein (GFAP), GABA, oligodendrites, seretonin, and synaptophysin (all from CHEMICON). Finally, the cells were incubated with a FITC labeled secondary antibody. After each of the above steps, the cells were washed 3 times with PBS.

  The chamber slide was observed under a fluorescence microscope to evaluate the immunopositive area. This immunofluorescence analysis was immunoreactive with the neuronal cell specific markers NCAM (FIG. 3, MAP-2 (FIG. 4A), and β-tubulin (FIG. 4B) in a significant proportion of differentiated cells. Expression of these key antigens resulted in increased incubation time in the differentiation medium, and immunofluorescence analysis showed that expression of these key antigens increased with greater latency in the differentiation medium Lesser proportions of the cells are serotonin (FIG. 7) (non-neural marker), glial fibrillary acidic protein (GFAP) also present in astrocytes, GABA present in oligodendrocytes (FIG. 6) and It was proved to express with glutamate.

  Differentiated cells were also analyzed by double labeling with a primary antibody against TH (green) along with MAP-2, DAT, Nurr-1, β-tubulin, FITC labeled secondary antibody (Texas red) (FIG. 8). This analysis indicated a high proportion of MAP-2 positive cells expressing TH. Cell density of dopaminergic neurons is quantified by counting the number of TH positive and MAP-2 positive cells per field in a randomly selected field using a 40x objective. did. The percent ratio of TH positive cells was calculated (Figure 9). TH-positive neurons also expressed Nurr-1 and DAT, but not in the presence of TH and DBH, confirming the dopaminergic phenotype of the cells. Synapse formation was also identified by immunostaining with synaptophysin.

Immunofluorescence analysis also showed that about 30% expressed serotonin, while the proportion of sorted NCAM positive cells expressing TH was about 60% (FIG. 10). In addition, about 40% of nestin positive cells were stained TH positive, about 30% stained were seretonin positive, and about 28% stained oligodendrocyte positive (FIG. 11). Immunological markers used at different stages of differentiation are shown in Table 3 and show the phenotypic characteristics of undifferentiated and differentiated ES cells.
Table 3: Phenotypic characteristics of undifferentiated and differentiated ES cells

（９）遺伝子発現プロフィール
異なる段階で集められる細胞の遺伝子発現プロフィールも、分析した。開示した方法の以下のステップの各々から細胞を逆転写ポリメラーゼ鎖反応（ＲＴ−ＰＣＲ）について分析するために細胞を回収した。未分化ＥＳ細胞、胚様体、ネスチン陽性神経前駆細胞、ＮＣＡＭ陽性細胞、および分化した細胞を、神経基本培地での分化の１２、１７、２２、２７、および３７日後に単離した。細胞を回収した後、ペレットにして、全細胞ＲＮＡをこの細胞ペレットからＲＮアーゼＱｉａｇｅｎキットを用いて抽出した。単離ＲＮＡを−２０℃に保存した。

(9) Gene expression profile Gene expression profiles of cells collected at different stages were also analyzed. Cells were harvested for analysis of cells from each of the following steps of the disclosed method for reverse transcription polymerase chain reaction (RT-PCR). Undifferentiated ES cells, embryoid bodies, nestin positive neural progenitor cells, NCAM positive cells, and differentiated cells were isolated after 12, 17, 22, 27, and 37 days of differentiation in neural basal medium. Cells were harvested and pelleted, and total cellular RNA was extracted from the cell pellet using the RNase Qiagen kit. The isolated RNA was stored at -20 ° C.

cDNA was synthesized from isolated total RNA using Moloney leukemia virus superscript II reverse transcriptase and oligo (dT) 12-18. CDNA synthesized by reverse transcriptase reaction was used for PCR amplification using different sets of specific primers to determine gene expression in the recovered cells. The PCR reaction was performed using platinum Taq polymerase and cDNA as template under standard PCR conditions well known to those skilled in the art. The general cycling parameters used to amplify the DNA product are as follows: That is,
Denaturation of template cDNA template by 1.4 ° C. for 30 minutes,
2. Primer annealing at 55-65 ° C. for 1 minute depending on the primer used;
3. Incubate the reaction at 72 ° C. for minutes and repeat steps 1-3 (cycle) 25-40 times.

After the PCR reaction, the product was run on a 1.5% agarose gel with an electrophoresis apparatus along the DNA size ladder. The expression of TH, D2RL, DBH, En-1, Nurr-1, and β-tubulin were all analyzed by RT-PCR using the primers listed in Table 5.
Table 4: Primer sets used to amplify dopamine specific genes

ＲＴ−ＰＣＲによる上記の分析は、ドーパミン作動性表現型特性遺伝子（ＴＨ）の発現がドーパミン作動性神経細胞（図１２）に最終分化が生ずるまで、ＥＳ細胞の神経分化の後で見られることを証明した。期待どおりに、βチューブリン（遍在して発現された遺伝子）が全ての細胞試料で見られたが、ＤＢＨは、いかなる細胞試料においても発現されなかった。
（１０）ドーパミン検出のための逆相ＨＰＬＣ
ドーパミン作動性神経細胞の１つの限定的な特徴は、ドーパミンの生産である。したがって、ドーパミンを生産する胚幹細胞由来ドーパミン作動性神経細胞の機能的能力（ｆｕｎｃｔｉｏｎａｌ ｃａｐａｃｉｔｙ）を、逆相ＨＰＬＣ（ＲＰＨＰＬＣ）を用いて細胞内ドーパミンレベルを直接測定することによって評した。各々の試料で検出されるドーパミンの濃度は，各々の実験の直前または直後のカラムに注入されたドーパミン標準液による比較によって決定した。

The above analysis by RT-PCR shows that dopaminergic phenotypic trait gene (TH) expression is seen after neural differentiation of ES cells until terminal differentiation occurs in dopaminergic neurons (FIG. 12). certified. As expected, β-tubulin (a ubiquitously expressed gene) was found in all cell samples, but DBH was not expressed in any cell sample.
(10) Reversed phase HPLC for dopamine detection
One limiting feature of dopaminergic neurons is the production of dopamine. Therefore, the functional capacity of embryonic stem cell-derived dopaminergic neurons producing dopamine was assessed by directly measuring intracellular dopamine levels using reverse phase HPLC (RPHPLC). The concentration of dopamine detected in each sample was determined by comparison with a dopamine standard injected into the column immediately before or after each experiment.

Initially, cells were harvested at different stages of the disclosed method. Undifferentiated ES cells, embryoid bodies, nestin positive neural progenitor cells, NCAM positive cells, and differentiated cells were isolated after 7, 22, and 37 days of differentiation in neural basal medium. Prior to harvesting, cells that differentiate in neural basal medium were first stimulated with HBSS supplemented with 56 mM KCl for 15 minutes to induce dopamine secretion. Approximately 5 × 10 ⁶ cells were trypsinized and pelleted by centrifugation. The cells were then sonicated with cold 1N perchloric acid containing an antioxidant (0.2 g / l sodium metabisulfite) and centrifuged (15,000 rpm / min) at 4 ° C. for 20 minutes. The supernatant was extracted and stored at −70 ° C. for subsequent measurement of intracellular dopamine concentration n by RP-HPCL. The level of dopamine was measured by cell lysate and culture supernatant (48 hours after final media change). The culture supernatant was immediately stabilized with 7.5 orthophosphoric acid and sodium metabisulfite.

Cell lysates (eg, undifferentiated ES cells, embryoid bodies, nestin-positive neural progenitor cells, and NCAM-positive cells) analyzed by RP-HPLC from an early stage by the disclosed method can detect any detectable dopamine. The level was not included. However, after the first week of differentiation in neural basal medium, all cell lysates analyzed by RP-HPLC contained dopamine. The level of intracellular dopamine increased markedly as the number of dopaminergic neurons in the cell lysate increased over time in the basal neurobasal medium and eventually matured. Intracellular levels of dopamine were high even in cell cultures treated with growth factors versus untreated media at the time points shown in FIG. In addition, confirmation of dopamine production by these cell cultures was confirmed with N-methyl-4-phenyl 1,2,3,6-tetrahydropyridine hydrochloride (MPTP) (a neurotoxin that targets dopaminergic neurons). Shown after incubation. In cell cultures incubated with MPTP, dopamine was not detected in cell lysates by RP-HPLC analysis. In contrast, dopamine release into conditioned media increased with cell media stimulated with 56 mM KCL (FIG. 14). Addition of an array of growth factors to differentiated neural basal media also increased the intracellular dopamine levels of cell lysates (Table 5).
Table 5: Dopamine concentration (μg / ml) in cell lysate by HPLC

開示されて、本明細書中に請求される組成物と方法の全ては、作られることができて、本発明の開示を考慮して過度の実験なしで実行されることができる。本明細書で開示およびクレームした組成物および方法の全ては、本発明の開示を考慮して実験することなしに生成および実行することができる。本発明の組成物および方法が好ましい実施形態に関して記載される一方で、本発明の概念、精神、および範囲から逸脱することなく、本明細書に記載された組成物および／または方法ならびに該方法のステップまたは一連のステップに変更を加えることが可能であることは、当業者にとって、明らかである。より詳しくは、化学的または物理的に関連したある種の薬剤を本明細書に記載された薬剤と置き換えて、その一方で同一または類似の結果が得られるであろうことは、明瞭である。当業者にとって明らかなそのような類似の置換および変更はすべて、添付した特許請求の範囲に定義される本発明の精神、範囲、および概念の内にあると考えられる。

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. All of the compositions and methods disclosed and claimed herein can be made and executed without experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, the compositions and / or methods described herein and methods of the methods described herein can be used without departing from the concept, spirit, and scope of the present invention. It will be apparent to those skilled in the art that changes can be made to a step or series of steps. More specifically, it is clear that certain drugs that are chemically or physically related may be substituted for the drugs described herein, while obtaining the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

The following drawings form part of the present specification and are included to further illustrate certain aspects of the present disclosure, the disclosure of which is provided by the specific embodiments presented herein. A better understanding can be obtained by reference to one or more of these drawings in combination with the detailed description.
FIG. 1 shows neural progenitor cells derived from human embryonic stem cells that are positive for nestin. (A) is a neural progenitor cell that is immunoreactive with a nestin marker, and (B) is a phase contrast micrograph of nestin-positive cells grown under serum-free conditions in the presence of a selected growth factor. It is. FIG. 2 shows FACS analysis of nestin positive cells derived from human embryonic stem cells and labeled with NCAM-FITC. (A) shows analysis of unlabeled cells treated with anti-rabbit FITC, and (B) shows analysis of cells treated with primary antibody against NCAM and labeled with anti-rabbit FITC (secondary antibody). In this study, 50-60% of nestin positive cells were immunopositive for NCAM. 3A and 3B are fluorescence micrographs of MCAM positive cells derived from human embryonic stem cells that have been selected using MACS and re-plated on tissue culture plates. 4A and 4B are fluorescence micrographs of neurons labeled with MAP-2 and β-tubulin derived from human embryonic stem cells. FIG. 5 shows the presence of neurons positive for tyrosine hydroxylase (TH). (A) shows that about 60% of the neurons were enriched for NCAM positive cells using MACS and were positive for TH after proliferation and differentiation of unsorted nestin positive cells. Fluorescence analysis. (B) is an immunofluorescence analysis showing that about 40% of the nerves were positive for TH after growth and differentiation of unsorted nestin positive cells. FIG. 6 is a representative fluorescence micrograph of oligodendrocytes. According to immunofluorescence analysis, about 25-30% of isolated nestin positive cells stained positive as oligodendrites. FIG. 7 is a representative fluorescence micrograph of nerve cells expressing the neurotransmitter serotonin. About 30% of nestin positive cells and similarly about 20% of NCAM positive cells are immunoreactive with serotonin. FIG. FIG. 8 is a trend photomicrograph showing dopaminergic neurons immunolabeled against TH (green) and another nerve specific antibody (red), (A) is the coexistence of tubulin and TH, (B) shows the coexistence of MAP-2 and TH, (C) shows the coexistence of Nurr1 and TH, and (D) shows the coexistence of DAT and TH. FIG. 9 is a bar graph showing the ratio of MAP-2 positive neurons (dopaminergic neurons) positive for TH. As shown in the figure, the percentage of neurons positive for TH increases with further differentiation beyond 7, 22, and 37 days. FIG. 10 is a bar graph showing quantitative analysis of different neuronal populations with NCAM positive enriched cells. Approximately 60% of NCAM positive cells were also immunopositive for TH. About 30% were immunopositive for serotonin. In addition, about 15% were immunopositive for GABA and glutamate. FIG. 11 is a bar graph showing quantitative analysis of different neuronal populations after proliferation and differentiation of nestin positive cells as analyzed by immunofluorescence. About 40% of nestin positive cells were immunopositive for TH. About 30% were immunopositive for serotonin. About 28% were immunopositive for oligodendrocytes. In addition, about 2% were immunopositive for glial fibrillary acidic protein (GFAP, marker for astrocytes). FIG. 12 shows the gene expression profile of human embryonic stem cells during in vitro terminal differentiation of human embryonic stem cells under the conditions disclosed in Example 1. UD = undifferentiated, EB = embryoid body, NS = nestin positive cells, NE = nestin proliferating cells, and residual time points, as disclosed in Example 1, cells were cultured in neural basal medium and grown with growth factors Indicates the number of days selected. Factors specific to dopaminergic neurons (eg, Nurr1, En-1, and D2RL) are expressed during the early stages of differentiation. Expression of the dopaminergic neuron-specific gene TH was observed at all stages except undifferentiated stem cells. The absence of any expression of DBH confirmed the midbrain phenotype of these cells. FIG. 13 shows intracellular dopamine levels in cell lysates measured by RP-HPLC on days 7, 22, and 37 of differentiation. In the presence of growth factors, dopamine levels were significantly higher (4-6 μg / ml) than untreated cells (1-4 μg / ml). No dopamine was detected in MPTA-treated cells on days 7 and 22, but a decrease in dopamine levels was seen on day 37. FIG. 14 is a bar graph showing that dopamine release is caused by KCl in differentiated cells cultured in conditioned medium after 7, 22, and 37 days of differentiation. Cells were stimulated with 56 mM KCl for 15 minutes to increase dopamine secretion. The culture suspension was stabilized with 7.5% orthophosphoric acid and sodium metabisulfite (sodium metabisulfite).

Claims (44)

A cell population obtained by differentiating primate pluripotent stem cells, wherein the population is composed of differentiated cells in an in vitro culture state, wherein at least 60% of the differentiated cells are dopaminergic neurons . The cell population of claim 1, wherein the primate pluripotent stem cell is a human embryonic stem cell. A cell population obtained by differentiating primate pluripotent stem cells and comprising differentiated cells in an in vitro culture state, wherein at least 60% of the differentiated cells express tyrosine hydroxylase . 4. The cell population of claim 3, wherein the primate pluripotent stem cell is a human embryonic stem cell. A method of generating a population of differentiated neurons from primate pluripotent stem cells,
(A) expanding a culture of primate pluripotent stem cells;
(B) culturing the pluripotent stem cells and selecting neural progenitor cells positive for nestin;
(C) selecting the nestin-positive neural progenitor cells for enriching NCAM-positive cells;
(D) culturing nestin-positive NCAM-positive cells in a differentiation medium containing TGF-β3 or interleukin-1β or both to differentiate the nestin-positive NCAM-positive cells to generate a differentiated neuronal cell population;
A method for generating a differentiated neural cell population, comprising: 6. The method according to claim 5, wherein the pluripotent stem cells are obtained using a laser ablation method. 6. The method of claim 5, wherein the pluripotent stem cell is a human embryonic stem cell. The method according to claim 7, wherein the human embryonic stem cells are obtained using a laser ablation method. 6. The method of claim 5, wherein the population of differentiated neurons comprises at least about 60% dopaminergic neurons. 6. The method of claim 5, wherein the population of differentiated neurons comprises at least about 30% serotonergic neurons. 6. The method of claim 5, wherein the population of differentiated neurons contains at least about 25% oligodendrocytes. 6. The method of claim 5, further comprising culturing the pluripotent stem cells of step (b) to form embryoid bodies. 13. The method of claim 12, wherein the embryoid body is cultured to select neural progenitor cells that are positive for nestin. 6. The method of claim 5, wherein the neural progenitor cells that are positive for nestin are selected by culturing pluripotent stem cells in serum-free medium. The method according to claim 14, wherein the serum-free medium is an ITSFn serum-free synthetic medium. The method according to claim 14, wherein the serum-free medium comprises one or more soluble factors selected from the group consisting of insulin, sodium selenite, transferrin, and fibronectin. The method according to claim 16, wherein the serum-free medium comprises insulin, sodium selenite, transferrin, and fibronectin. 14. The method of claim 13, wherein the neural progenitor cells that are positive for nestin are selected by culturing the embryoid body in a serum-free medium. The method according to claim 18, wherein the serum-free medium is an ITSFn serum-free synthetic medium. The serum-free medium according to claim 18, wherein the serum-free medium comprises one or more soluble factors selected from the group consisting of insulin, sodium selenite, basic fibroblast growth factor, transferrin, and fibronectin. . The serum-free medium of claim 20, wherein the serum-free medium comprises insulin, sodium selenite, transferrin, and fibronectin. 24. The method of claim 21, wherein the neural progenitor cells comprise at least about 95% nestin positive cells. 6. The method of claim 5, wherein the nestin positive neural progenitor cells of step (c) are sorted to enrich for NCAM positive cells by magnetic cell separation (MACS). 24. The method of claim 23, wherein the nestin positive neural progenitor cells comprise at least about 50-60% NCAM positive cells. 6. The method of claim 5, further comprising growing the nestin-positive NCAM-positive neural progenitor cell in step (c) in a growth medium. The growth medium is insulin, sodium selenite, transferrin, laminin, putrescine, progesterone, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), sonic hedgehog (SHH), fibroblast 26. The method of claim 25, wherein the method is one or more soluble factors selected from the group consisting of growth factor-8 (FGF-8) and brain-derived neurotropic factor (BDNF). 27. The method of claim 26, wherein the cells are grown in the growth medium for 6-10 days. 27. The method of claim 26, wherein the cells are cultured and serially passaged to double one or more populations. 27. The method of claim 26, wherein the cells are stored frozen in liquid nitrogen. 6. The method of claim 5, wherein the differentiation medium comprises a neural basal medium supplemented with fetal calf serum, B27, ascorbic acid, and N-acetyl cysteine. The differentiation medium comprises ascorbic acid, N-acetyl, cysteine glial cell line-derived neurotrophic factor (GDNF), dibutyryl cyclic AMP (db-cAMF), brain-induced neurotrophic factor (BDNF), Neuturin, Sonic 6. The method of claim 5, further comprising one or more differentiation agents selected from the group consisting of hedgehog protein (SHH) and fibroblast growth factor-8 (FGF-8). 6. The method of claim 5, wherein the nestin positive NCAM positive cells are grown in a differentiation medium for 30-50 days. A method of generating dopaminergic neurons from neural progenitor cells, comprising:
As a nestin-positive cell, the neural progenitor cell is enriched, and the nestin-positive cell is cultured in the presence of TGF-β3 and / or interleukin-1β to differentiate the nestin-positive cell, thereby producing dopaminergic activity. A method for generating dopaminergic neurons, which generates neurons. 34. The method of claim 33, wherein at least about 40% of the nestin positive cells differentiate into dopaminergic neurons. 34. The method of claim 33, further comprising enriching the neural progenitor cells as NCAM positive cells. 35. The method of claim 34, wherein the nestin positive NCAM positive cells are differentiated to generate dopaminergic neurons. 38. The method of claim 36, wherein at least about 60% of the nestin positive NCAM positive cells differentiate into dopaminergic neurons. A method of generating serotonergic neurons from neural progenitor cells, comprising:
Enriching the neural progenitor cells as cells that are positive for nestin and NCAM, differentiating the nestin-positive NCAM-positive cells, and the nestin-positive NCAM-positive cells in the presence of TGF-β3 or interleukin-1β or both A method for producing a serotonergic neuron, wherein the nestin-positive NCAM-positive cell is differentiated by culturing to produce a serotonergic neuron. 40. The method of claim 38, wherein at least 30% of the nestin positive NCAM positive cells differentiate into serotonergic neurons. A method of treating a patient having a neurodegenerative disorder or a neurological disease, comprising:
(A) expanding a culture of primate pluripotent stem cells;
(B) culturing the pluripotent stem cells and selecting neural progenitor cells positive for nestin;
(C) selecting the nestin-positive neural progenitor cells for enriching NCAM-positive cells;
(D) culturing nestin-positive NCAM-positive cells in a differentiation medium containing TGF-β3 or interleukin-1β or both to differentiate the nestin-positive NCAM-positive cells to generate a differentiated neuronal cell population;
(E) transplanting a therapeutically effective amount of the differentiated neuronal population into the central nervous system of a patient;
A treatment method comprising: 41. The method of claim 40, wherein the primate pluripotent stem cell is a human embryonic stem cell. 41. The method of claim 40, further comprising isolating dopaminergic neurons from the differentiated neuronal population and administering the dopaminergic neurons to the patient. 41. The neurodegenerative disorder or disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, stroke, and ischemia. The method described in 1. 41. The method of claim 40, wherein the differentiated neural cell population is transplanted into the patient's brain.

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