HK1055765B - Neural progenitor cell populations - Google Patents

Description

Neural progenitor cell population

Technical Field

The present invention relates generally to the field of cell biology of embryonic cells and neural progenitor cells. In particular, the invention relates to the directed differentiation of human pluripotent stem cells into neural and glial lineages using specific culture conditions and screening techniques.

Information on related applications

This application claims U.S. provisional patent application No. 60/205,600 filed on 5/17/2000; and priority of 60/257,608 filed on 22/10/2000. These prior applications are incorporated herein by reference in their entirety for legal implementation in the united states.

Background

The repair of the central nervous system is a sophisticated science that has not yet been overcome by the medical community. Such as alzheimer's disease, parkinson's disease, epilepsy, hunter's disease and stroke, can have devastating consequences for their patients. Injury to the head or spinal cord can leave a person immediately out of normal life and into the disabled person's line.

Intractable damage to the nervous system is often proven irreparable. In this context, the main desire is to develop cell populations that can reconstruct neural networks and re-confer nervous system function.

For this reason, many studies have generated great interest in neural progenitor cells. To date, it is believed that the determination of multipotent neural progenitor cells into either neuro-restricted cells or glial-restricted cells is made early in the differentiation process. It is believed that they, in turn, may give rise to mature neurons or mature astrocytes and oligodendrocytes. Multipotent neural progenitor cells in the neural crest will also differentiate into neurons, smooth muscle and neural membrane cells. It is hypothesized that the various lineage restricted progenitor cells will self-renew and localize at selected locations in the central nervous system, such as the spinal cord. Cell lineages in developing neural tubes are reviewed in the research literature of Kalyani et al (biochem. cell Biol, 6: 1051, 1998).

Putative pluripotent neuroepithelial cells (NEP cells) have been identified in the developing spinal cord. Kalyani et al (Dev. biol, 186: 202, 1997) reported NEP cells in rats. Mujtaba et al (Dev. biol. 214: 113, 1999) reported NEP cells in mice. It is thought that differentiation of NEP cells will result in the formation of restricted progenitor cells with characteristic surface markers.

Mayer-Proschel et al (Neuron 19: 773, 1997) identified putative neuronal restricted precursor cells (NRPs). These cells express cell surface PS-NCAM (a polysialylated isoform of the neural cell adhesion molecule). They are reported to be capable of producing various types of neurons, but do not form glial cells.

Rao et al (Dev. biol. 188: 48, 1997) identified putative glial-restricted precursor cells (GRPs). These cells are apparently capable of forming glial cells, but not neurons.

Ling et al (exp. neurol.149: 411, 1998) isolated progenitor cells from the midbrain germinal region of a rat fetus. The cells can be grown in EGF and seeded on polylysine-coated plates, on which neurons and glial cells are formed and sometimes lysine hydroxylase positive (dopaminergic) cells, and their production can be increased by adding IL-1, IL-11, LIF, and GDNF to the culture medium.

Wagner et al (Nature Biotechnol.17: 653, 1999) reported cells with an abdominocerebral dopaminergic phenotype induced from an immortalized pluripotent neural stem cell line. Such cells were transfected with the Nurr1 expression vector and then cultured with VM 1 type astrocytes. It is stated that more than 80% of the resulting cells have a phenotype similar to endogenous dopaminergic neurons.

Mujtaba et al (supra) reported isolation of NRP and GRP from mouse embryonic stem (mES) cells. Various NRPs have PS-NCAM immune activity, are self-renewing in defined media, and are capable of differentiating into a variety of neuronal phenotypes. They apparently fail to form glial cells. Various GRPs have A2B5 immunological activity and are reported to differentiate into astrocytes and oligodendrocytes, but not into neurons.

A number of recent findings suggest that embryonic cells may be a potent multipotent source of cells and tissues in human therapy. Pluripotent cells are thought to differentiate into almost all cell types in the body (R.A. Pedersen, scientific. am.280 (4): 66, 1999). Early studies of embryonic stem cells were modeled using inbred mouse strains (according to the Robertson review, meth. cell biol.75: 173, 1997; and Pedersen, Reprod, Fertll.Dev.6: 543, 1994).

Monkey and human pluripotent cells proved to be much more fragile than mouse ES cells and could not be grown under the same culture conditions. It has only recently been discovered that primate embryonic cells can be cultured ex vivo.

Thomson et al (Proc. Natl. Acad. Sci. USA 92: 7844, 1995) successfully cultured primate embryonic stem cells the first one, using rhesus and marmoset as models. They subsequently isolated a human embryonic stem (hES) cell line from human blastocysts (Science 282: 114, 1998). Gearhart and colleagues derived several human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al, Proc. Natl. Acd. Sci. USA 95: 13726, 1998). Both hES and hEG cells have long sought after characteristics of human pluripotent stem (hPS) cells: capable of proliferation in vitro without differentiation, they maintain a normal karyotype, and are capable of differentiation to produce all mature cell types.

Reubinoff et al (Nature Biotechnol.18: 399, 2000) reported somatic differentiation of human blastocysts. These cells can spontaneously differentiate in culture without a uniform tissue structure. When the density increases after 4-7 weeks of culture, multicellular aggregates can form on the monolayer. Different cells in culture express a variety of different phenotypes, including β -actin, desmin, and NCAM.

Spontaneous differentiation of pluripotent stem cells in culture or teratomas can produce phenotypically highly heterotypically mixed cell populations that represent a large number of different cell lineages. For therapeutic purposes, it is desirable that the differentiated cells be relatively uniform-not only in their own phenotypes, but also in the types of progeny that they produce.

Accordingly, there is a need for techniques to generate more homotypic differentiated cell populations from pluripotent cells of human origin.

SUMMARY

The present invention provides a system for efficiently producing primate cells that differentiate from pluripotent cells into neuronal or glial cell lineages. Cell populations containing such lineage precursor cells are described which can provide a source for the production of additional precursor cells and mature cells of the central nervous system (neurons, astrocytes or oligodendrocytes). Certain embodiments of the invention may produce cells of both lineages. The precursor cells and mature cells of the invention have many important uses, including drug testing and treatment to restore nervous system function.

One embodiment of the invention is a population of cells that can be propagated in vitro culture obtained by differentiation of primate pluripotent stem (pPS) cells, wherein at least about 30% of the cells in the population are committed to the formation of neuronal cells, glial cells, or both. A second embodiment is a population of cells that can be expanded in vitro culture comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells are capable of differentiating into neuronal cells and at least 10% of the cells are capable of differentiating into glial cells. A third embodiment is a population of cells that can be expanded in vitro culture comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells express A2B5 and at least 10% of the cells express NCAM.

Certain cell populations of the invention are obtained by differentiation of primate pluripotent stem cells (e.g., human embryonic stem cells). Some are obtained by differentiation of stem cells in culture medium containing at least two or more ligands capable of binding to growth factor receptors. Some are obtained by differentiating pPS cells in a medium containing growth factors, which allows for sorting of differentiated cells expressing NCAM or A2B5 and collecting the sorted cells. After some cell populations are enriched, at least 70% of the cells express NCAM or A2B 5.

Another embodiment of the invention is a cell population comprising mature neurons, astrocytes, oligodendrocytes, or any combination thereof, obtained by further differentiating the precursor cell population of the invention. Some of these populations are obtained by culturing various neural precursor cells in a medium containing a cAMP activating factor, a neurotrophic factor, or a combination of such factors. As shown below, neurons made in this way were able to display action potentials, could display gated sodium potassium channels, and could display calcium flow when administered with neurotransmitters or their equivalents. Including cell populations containing large numbers of dopaminergic neurons that can be detected by, for example, tyrosine hydroxylase staining.

Embodiments of the invention also include neural precursor cells, neurons, astrocytes and oligodendrocytes obtained by selecting cells having a desired phenotype from a population of cells.

The cell populations and isolated cells of the invention isolated from established pPS cell lines generally have the same genome as the cell line being isolated. This means that more than 90% of the chromosomal DNA is identical between pPS cells and neural cells, from which it can be concluded whether neural cells are obtained from undifferentiated cell lines by normal mitotic processes. A neural cell treated by introducing a transgene (e.g., TERT) or knocking out an endogenous gene by recombinant means can still be considered karyotype identical to the isolated cell line, since all the unprocessed genetic elements are still retained.

Another embodiment of the invention is a method of screening for compounds that are cytotoxic or modulatory to neural cells, wherein a culture is prepared containing such a compound and neural cells or cell populations of the invention, and any phenotypic or metabolic changes that occur in the cells as a result of contact with such a compound are also determined.

Another embodiment of the invention is a method of obtaining a polynucleotide comprising a nucleotide sequence that is present in an mRNA that is more highly expressed in a neural progenitor cell or a differentiated cell, as further described and exemplified in the present disclosure. This nucleotide sequence, in turn, can be used to make recombinant or synthetic polynucleotides, proteins, and antibodies (as gene products that are enriched for or suppressed in neural cells). Antibodies can also be obtained by using the cells of the invention as immunogens or adsorbents to identify enriched or suppressed markers in neural cells.

A further embodiment of the invention is a method of reconstituting or repairing Central Nervous System (CNS) function in an individual, wherein an isolated cell or cell population of the invention is administered to the individual. The isolated cells and cell populations may be used in clinical or veterinary therapy in the form of pharmaceutical preparations. Drugs containing the cells of the invention can be processed for use in such treatments.

Other embodiments of the invention are methods of using the techniques described in this disclosure on appropriate stem cell populations to obtain the neural precursor cells and fully differentiated cells of the invention. These include methods for producing a cell population containing dopaminergic cells from primate embryonic stem cells at a frequency of 1%, 3%, or 5%, and a progenitor cell population capable of producing dopaminergic cells at such a frequency. This is particularly important for loss of dopamine neuron function, which can occur in parkinson's disease. The compositions, methods, and techniques described in this disclosure hold considerable promise for diagnostic, drug screening, and therapeutic uses.

These and other embodiments of the present invention will be apparent from the description below.

Drawings

FIG. 1 is a graph relating to the growth of neural-labeled cells isolated from human embryonic stem cells. The upper panel shows the growth of cells maintained in a medium containing CNTF, bFGF and NT3, and then sorted for NCAM expression. The lower panel shows the growth of cells maintained in a medium containing EGF, bFGF, PDGF and 1GF-1, followed by sorting for expression of A2B 5. Four different hES cell lines were used: h1, H7, H9 and H13. The selected population of A2B5 was further differentiated into neuronal and glial cells after 7 passages.

Figure 2 is a flow chart depicting exemplary steps for obtaining A2B5 positive cells. The following abbreviations were used: MEF-CM-medium conditions were adjusted with mouse embryonic fibroblasts; +/-SHH with or without sonic hedgehog; D/F12-DMEM/F12 medium; n2 and B27, culture supplements (Gibco); EPFI ═ differentiating agents EGF, PDGF, bFGF and IGF-1; PLL ═ poly L-lysine matrix; PLL/FN ═ poly L-lysine and fibronectin matrices.

FIG. 3 is a fluorescent micrograph of a halftone reproduction of brain tissue from neonatal rats administered cells expressing green fluorescent protein. Left panel: parental hES cell lines. The middle graph is as follows: the parental cell line forms embryoid body cells. Right panel: differentiated cells expressing NCAM. Undifferentiated hES cells and embryoid body cells remained in the drug area and showed marked necrosis. In contrast, differentiated NCAM⁺The cells appeared as single cells and migrated out of the injection site.

FIG. 4 is a duplicate of a fluorescence micrograph showing cells stained for Tyrosine Hydroxylase (TH), a marker for dopaminergic cells. Embryoid bodies prepared from human ES cells were retained in 10 μ M retinoic acid for 4 days, seeded in a neural support mixture, and then passaged to medium containing 10ng/mL NT-3 and 10ng/mL BDNF. Certain populations of the invention contain > 1% TH positive cells.

FIG. 5 shows a series of graphs of the response of a neuro-restrictive precursor to various neurotransmitters. Panel A shows the ratio of emission data for individual cells on two different coverslips. Both cells respond to GABA, high concentrations of potassium ions, acetylcholine, and ATP. Panel B shows the frequency of the cells tested in response to a particular neurotransmitter. Panel C shows the observed response to neurotransmitter compositions.

Figure 6 shows a series of graphs of the electrophysiological properties of the neuro-restricted precursors. Panel A shows the sodium and potassium currents observed in two cells depolarized to detect a potential between-80 and 80mV starting from a supporting potential of-100 mV. Graph B shows the observed peaks of input (Na +) and output (K +) for the current-voltage relationship. Panel C shows the action potentials generated after n responses of the same cell to depolarizing stimuli. These measurements show that neural precursor cells isolated from human ES cells are capable of generating action potentials that are characteristic of neurotransmission.

Detailed Description

The present invention provides a system for preparing and characterizing neural progenitor cells, which is suitable for therapeutic use and drug screening.

It is known that when pluripotent stem cells are cultured in the presence of selected differentiating agents, a significant proportion of the cells of the newly generated cell population carry the phenotypic characteristics of neural cells. Alternatively, sorting differentiated cells according to cell surface markers can increase the proportion of neural cells. Because certain types of pluripotent stem cells (e.g., embryonic stem cells) can proliferate in culture for a year or more, the invention described in this disclosure provides an almost unlimited supply of neural precursor cells. Certain cell populations of the invention are capable of producing neuronal or glial cells that can be replicated by mass passage in culture.

FIG. 1 shows the growth curve of cells cultured with different differentiating agents and then selected according to whether they carry polysialylated NCAM or A2B5 epitopes. These cell populations can be proliferated by massive multiplication of cells.

The differentiated cells selected positive for A2B5 expression contained cells that clearly expressed A2B5 but not NCAM, and cells that expressed both A2B5 and NCSM. In one of the assays described below, these cells can produce 13% oligodendrocytes and 38% neurons after maturation. This population can provide a reservoir for pluripotent cells since these cells do not lose their phenotype after long-term culture propagation. Upon administration to an individual with CNS dysfunction, the population includes cells that restore both neuronal and glial cell lineages, as desired.

The neural precursor cells can be further differentiated in vitro, if desired, either by culturing with maturation factors (e.g., neurotrophic factors) or by removing one or more factors that maintain progenitor cell renewal. Neurons, astrocytes and oligodendrocytes are mature, differentiated cells of the neural lineage that can be obtained by culturing precursor cells as such. The cell type characteristics of neurons obtained by these methods are amplified, i.e., they are stained by neurofilament and MAP-2, etc., and have significant synapse formation (identified by staining for synaptophysin). FIG. 5 shows that these cells respond to many neurotransmitter substances. Figure 6 shows that these cells were able to show action potentials when measured in a standard membrane clip system. All these aspects suggest that these cells apparently have a complete neural function.

Of particular interest is the ability of this system to produce dopaminergic neurons (figure 4). This type of cell is particularly desirable for the treatment of parkinson's disease, for which the best current treatment is allograft fetal brain tissue. There are many supply and procedural problems associated with the use of fetal tissue in clinical therapy, but none of the sources mentioned previously provides sufficient cells of the correct type. The neural precursor cells of the invention are capable of generating differentiated cells in which a proportion of the neurons have a dopaminergic phenotype. This ratio is believed to be sufficient for cell replacement therapy in Parkinson's disease and also demonstrates the therapeutic utility of the progenitor cell populations of the invention.

Since the pluripotent stem cells of the invention, as well as some lineage-restricted precursor cells, can be proliferated in large quantities in culture, the system described in this disclosure provides an unlimited supply of neuronal cells, as well as glial cells, for research, drug development, and treatment of CNS dysfunction. The following description further illustrates the preparation and use of the cells of the invention.

Definition of

For the purposes of this disclosure, the term "neural progenitor cell" or "neural precursor cell" refers to a cell that produces progeny that are either neuronal cells (e.g., neuronal precursors or mature neurons) or glial cells (e.g., glial cell precursors, mature astrocytes or mature oligodendrocytes). Typically, these cells express certain phenotypic markers characteristic of the neural lineage. Generally, they do not produce progeny of other embryonic germinal layers when cultured in vitro unless differentiation or reprogramming is eliminated in some way.

"neuronal progenitor cell" or "neuronal precursor cell" refers to a cell capable of producing mature neuronal progeny. These cells may or may not be capable of producing glial cells.

By "glial progenitor cell" or "glial precursor cell" is meant a cell that is capable of producing mature astrocytes or mature oligodendrocytes. These cells may or may not be capable of producing neuronal cells.

By "population of multipotent neural progenitor cells" is meant a population of cells that is capable of producing both progeny of neuronal cells (e.g., neuronal precursors or mature neurons) and glial cells (e.g., glial precursors, mature astrocytes or mature oligodendrocytes), and sometimes other cell types. This term does not require that each cell in the population be able to form both types of progeny, although there are individual cells that may be multipotent neural progenitor cells.

In cellular ontogenesis, "differentiated" is a term. By "differentiated cell" is meant a cell that can develop further along a developmental pathway than a control cell. Thus, pluripotent embryonic stem cells can differentiate into lineage restricted precursor cells, such as hematopoietic cells, which are pluripotent for various types of blood cells; a hepatocyte precursor, which is pluripotent for hepatocytes; and various types of neural progenitor cells mentioned above. They may be further differentiated sequentially along developmental pathways into other types of precursor cells, or into terminally differentiated cells which play a specific role in a certain tissue type, with or without the ability to further proliferate. Neurons, astrocytes and oligodendrocytes are all examples of terminally differentiated cells.

As used in this disclosure, "differentiation agent" refers to one of a broad class of compounds that can be used in the culture systems of the present invention to produce differentiated cells of the neural lineage, including precursor cells and terminally differentiated cells. There is no limitation on the mode of action of these compounds. For example, such agents may assist in the differentiation process by inducing or participating in a change in phenotype, promoting the growth of cells with a particular phenotype or slowing the growth of other cells, or by combining with other agents by some unknown mechanism.

Unless specifically stated otherwise, the techniques of the present invention can, without limitation, differentiate various types of progenitor cells into neuronal cells or glial cells.

Included in the definition of pPS cells are various types of embryonic cells, e.g., human embryonic stem (hES) cells as described by Thomson et al (Science 282: 1145, 1998); other primate embryonic stem cells such as macaque stem cells (Thomson et al, Proc. Natl. Acad. Sci. USA 92: 7844, 1995), marmoset stem cells (Thomson et al, biol. reprod.55: 254, 1996), and other types of pluripotent cells are also included in this term. pPS cells are not isolated from malignant sources. The cells preferably have a normal karyotype (but this is not always the case).

When a large number of stem cells and their derivatives in a population have morphological characteristics of undifferentiated cells, cultures of pPS cells are said to be "undifferentiated," which clearly distinguishes them from differentiated cells of embryonic or adult origin. Undifferentiated pPS cells, which are typically present in cell colonies with relatively high nucleus/cytoplasm and significant nuclei in the two-dimensional plane of the microscopic field, can be readily identified by those skilled in the art. It is believed that colonies of undifferentiated cells in a population will typically be surrounded by adjacent differentiated cells.

The terms "feeder cell" or "feeder" generally refer to a cell that is co-cultured with another cell and provides a growing environment for the second cell. For example, certain types of pPS cells can be supported by mouse primary embryonic fibroblasts, immortalized mouse embryonic fibroblasts, or human fibroblast-like cells differentiated from hES cells. A pPS cell population is considered "substantially free" of feeder cells if it has been grown for at least one round without adding fresh feeder cells to support growth after the pPS division.

"embryoid body" is synonymous with "aggregate". They refer to a collection of differentiated and undifferentiated cells that occurs when pPS cells are overgrown in monolayer culture, or maintained in suspension culture. Embryoid bodies are a mixture of different cell types, usually from several germ layers, which can be distinguished by morphological criteria and immunochemically detectable cell markers.

"growth environment" refers to an environment in which the relevant cells can proliferate, differentiate, or mature in vitro. The characteristics of the environment include the medium in which the cells are cultured, various growth factors or various differentiation inducing factors that may be present, and, if present, a supporting structure (e.g., a matrix of solid surfaces).

A cell is said to be "genetically transformed", "transfected" or "genetically transformed" when the polynucleotide is transferred into the cell by any suitable means of artificial manipulation, or the cell is a progeny of a previously altered cell and inherits the polynucleotide. Polynucleotides typically contain a transferable sequence that encodes a protein of interest, which allows the cell to express the protein at high levels. A genetic alteration is said to be "heritable" if progeny of the altered cell retain the same alteration.

The term "antibody" as used in this disclosure refers to both polyclonal and monoclonal antibodies. The scope of this term includes not only intact immunoglobulin molecules, but also fragments and derivatives (e.g., single chain Fv structures, diabodies, and fusion structures) of immunoglobulin molecules that can be prepared by techniques known in the art and which retain the desired antibody binding specificity.

Conventional techniques

To further elaborate on the conventional techniques used in the practice of the present invention, practitioners may refer to standard textbooks and reviews on cell biology, histology, and embryology. Including Teratoccarinomasand and alumina stem cells: a practical propaach (E.J. Robertson, ed. IRL publishing Co., Ltd. 1987); guide to Techniques in Mouse Development (P.M. Wasserman et al eds., academic Press, 1993); embryonic Stem Cell Differentiation in vitro (M.V.Wiles, meth.enzymol.225: 900, 1993); properties and uses of Embryonic Stem Cells: prospectra for Application to Human Biology and Gene therapy (P.D. Rathjen et al, reprod.Fertil.Dev.10: 31, 1998).

To further elaborate on the characteristics of neuronal cell abnormalities and various types of neuronal cells, markers and associated lytic factors, the reader is referred to CNS regeneration: basic Science and Clinical Advances, m.h. tuszynski and j.h. kordawer, academic press, 1999.

Various methods in molecular genetics and genetic engineering are described in: molecular Cloning: ALaborory Manual, second edition (Sambrook et al, 1989); oligonucletoideosynthesis (m.j. pent, eds., 1984); animal Cell Culture (r.i. freshney eds, 1987); methods in Enzymology series (academic Press); gene Transfer Vectors for Mammaliancells (J.M.Miller and M.P.Calos eds., 1987); current Protocols in Molecular Biology and Short Protocols in Molecular Biology, third edition (ed. F.M. Ausubel et al, 1987 and 1995); and Recombinant DNA Methodology II (r. wu eds., academic press, 1995). Reagents, cloning vectors, and genetic manipulation kits referred to in this disclosure are available from commercial suppliers, such as BioRad, Stratagene, Invitrogen, and ClonTech.

Conventional techniques used in antibody culture, purification and modification, as well as the design and implementation of immunoassays including immunohistochemistry, the reader is referred to the Handbook of experimental immunology (ed.m. weir and c.c. blackwell); current Protocols in Immunology (J.E.Coligan et al, 1991); and R.Masseyeff, W.H.Albert and N.A.Staines eds, Methods of Immunological Analysis (Weinhelm: VCH Veriags GmbH, 1993)

Sources of stem cells

The present invention can be practiced with various types of stem cells, including the following non-limiting examples.

Pluripotent neural stem cells obtained from brain tissue are reported in U.S. Pat. No. 5,851,832. The production of neural cells from the cerebral hemisphere of a neonate is reported in us patent No. 5,766,948. U.S. Pat. Nos. 5,654,183 and 5,849,553 report the use of mammalian neural crest stem cells. Us patent No. 6,040,180 reports the in vitro production of differentiated neurons from mammalian pluripotent CNS stem cell cultures. WO 98/50526 and WO 99/01159 report the generation and isolation of neuroepithelial stem cells, oligodendrocyte-astrocyte precursors and lineage restricted neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtained from embryonic forebrains and cultured in a medium containing glucose, transferrin, insulin, selenium, progesterone and other growth factors.

The invention can be practiced with any vertebrate stem cell, unless otherwise specified. Including human stem cells, as well as non-human primates, domesticated animals, livestock, and other non-human mammals.

Stem cells suitable for use in the present invention include primate pluripotent stem (pPS) cells. Non-limiting examples are primary cultures or established cell lines of embryonic stem cells or embryonic germ cells.

Propagation of pPS cells in undifferentiated State

pPS cells can be propagated continuously under culture conditions that promote propagation but do not promote differentiation. An exemplary serum-containing ES medium is prepared from 80% DMEM (e.g., Gibco's knockout DMEM), 20% defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO 98/30679), 1% nonessential amino acids, 1mM L-glutamine and 0.1mM beta-mercaptoethanol. Before use, human bFGF was added to 4ng/mL (WO 99/20741, Geron Corp.).

It is customary to culture ES cells on a feeder cell layer, from which fibroblasts have generally been isolated from embryonic or fetal tissue. Embryos were harvested from CF1 mice 13 days pregnant, transferred to 2mL trypsin/EDTA, minced, and incubated at 37 ℃ for 5 minutes. The residue was precipitated by adding 10% PBS, and the cells were propagated in 90% DMEM, 10% FBS and 2mM glutamine. To prepare the feeder cell layer, the cells are irradiated to inhibit proliferation, but allow synthesis of factors (about 4000 rads of gamma rays) that support the ES cells. Plates were covered with 0.5% gelatin overnight and 375,000 irradiated mEF was inoculated into each well and allowed to stand for 5 hours to 4 days. This medium was changed to fresh hES medium and pPS cells were seeded in-situ.

Geron's scientists found that pPS cells could be maintained in an undifferentiated state even in the absence of feeder cells. The feeder-free culture environment comprises a suitable culture medium, such asOr an extracellular matrix such as laminin. The cells are seeded at a density greater than 15,000 cells per square centimeter (desirably 90,000 and 170,000 cells per square centimeter). Digestion with the enzyme is typically stopped before the cells are completely dispersed (e.g., about 5 minutes with collagenase IV). Clumps of about 10-2000 cells were then seeded directly onto the matrix without further dispersion treatment.

The nutrient medium conditions are typically adjusted to support feeder-free cultures by culturing irradiated primary mouse embryo fibroblasts, terminally pelleted mouse fibroblasts, or fibroblast-like cells isolated from pPS cells. Or about 5-6X 10 per square centimeter⁴Cell Density feeders were inoculated in serum-free medium such as KO DMEM (20% serum was replaced and 4ng/mLbFGF was added) to condition the medium. The medium was further supplemented with bFGF 1-2 days after conditioning to support pPS cell growth for 1-2 days.

Microscopically, ES cells have a high nuclear/cytoplasmic ratio, a prominent nucleus, and a compact colony structure with cell junctions that are difficult to discern. Primate ES cells express stage-specific embryonic antigens (SSEA)3 and 4 and express markers detectable by antibody-labeled Tra-1-60 and Tra-1-81 (Thomson et al, Science 282: 1145, 1998). Mouse ES cells served as positive controls for SSEA-1 and as negative controls for SSEA-4, Tra-1-60, and Tra-1-81. SSEA-4 is consistently present on human embryonic carcinoma (hEC) cells. Differentiation of pPS cells in vitro results in loss of expression of SSEA-4, Tra-1-60 and Tra-1-81 and increases expression of SSEA-1. SSEA-1 was also found on hEG cells.

Materials and processes for preparing neural precursors and terminally differentiated cells

Certain neural precursor cells of the invention are obtained by culturing, differentiating or reprogramming stem cells in a specific growth environment that can enrich for cells of the desired phenotype (either by growing the desired cells or by inhibiting or killing other cell types). These methods can be used with a variety of stem cells, including the primate pluripotent stem (pPS) cells described above.

Generally, the differentiation process takes place in a nutrient medium containing a suitable matrix and added differentiation agents. Suitable substrates include solid surfaces bearing a positive charge (e.g., basic amino acids, e.g., poly-L-lysine and polyornithine). The matrix may be coated with an extracellular matrix component, such as fibronectin. Another extracellular matrix comprises(extracellular matrix from Engelbreth-Holm-Swarm tumor cells) and laminin. Various combinations of matrices are also suitable, such as fibronectin-bound poly-L-lysine, laminin, or both.

Suitable differentiation agents include various types of growth factors such as Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF- α), and various types of fibroblast growth factors (e.g., FGF-4, FGF-8, and basic fibroblast growth factor, bFGF), platelet-derived growth factor (PDGF), insulin-like growth factors (IGF-1 and others), high concentrations of insulin, sonic hedgehog, members of the neurotrophin family (e.g., nerve growth factor, neurotrophin 3, NT-3, brain-derived neurotrophin, nf), bone morphogenetic proteins (particularly BMP-2 and BMP-4), retinoic acid (bdra), and various ligands for receptors complexed with gp130 (e.g., LIF, CNTF, and IL-6). Various alternative ligands or antibodies that bind to the respective cell surface receptors for the above factors are also suitable. Typically, a variety of differentiating agents are used, including 2,3, 4 or more of the agents listed above or in the examples below. For example, a mixture containing EGF, bFGF, PDGF and IGF-1 (examples 1 and 2).

Such factors are provided to the cells in a nutrient medium, which may be any medium that supports the proliferation or survival of the desired cell type. It is generally preferred to have a defined medium that provides free amino acids as nutrients rather than serum. It may also be beneficial to add additives to the culture medium to maintain the neural cell culture. Such as N2 and B27 additives (available from Gibco).

When the stem cells are pPS cells, they may also be differentiated by culturing them in a mixture of suitable differentiating agents (obtained from feeder cells or feeder-free cultures).

In one method of triggering differentiation, pPS cells are seeded directly onto a suitable substrate, such as an adherent glass or plastic surface, for example, with a polyamine coating. The cells are then cultured in a suitable nutrient medium that promotes differentiation of the cells into the desired cell line. This is known as the "direct differentiation" method.

In another approach, pPS cells are first differentiated into a heterogeneous cell population. In one variation, culturing the pPS cells in suspension can allow them to form embryoid bodies. Alternatively, one or more of the above differentiating agents (e.g., retinoic acid) may be added to the medium to promote differentiation within the embryoid body. After the embryoid bodies have reached a sufficient size (usually 3-4 days), they are seeded onto the matrix of the differentiation culture. Embryoid bodies can be directly seeded onto the substrate without dispersing the cells. This allows neuronal cell precursors to migrate out of the embryoid bodies and into the extracellular matrix. These cultures are then passaged on a suitable medium to aid in the selection of neural progenitor cells.

Cells prepared according to this procedure were found to be able to proliferate further (example 1). Up to 30%, 50%, 75% or more of the cells express either the polysialylated NCAM or A2B5 epitope, or both together. Typically, at least about 10%, 20%, 30% or 50% of the cells express NCAM and at least about 10%, 20%, 30% or 50% of the cells express A2B 5-indicating that they are capable of forming cells of the neuronal and glial lineages, respectively.

Alternatively, differentiated cells can be sorted according to phenotypic characteristics to enrich for a population. Typically, this involves binding individual cells to a labeled antibody or ligand specific for nerve cells, and then separating the specifically recognized cells from the other cells in the population. One method is immunopanning, in which specific antibodies are bound to a solid surface, the cells are then contacted with the surface, and cells that do not express the marker are washed away. The bound cells are then recovered with a stronger eluent. This is a complex method of affinity chromatography and antibody-mediated magnetic cell sorting. In a typical sorting process, cells are bound to specific primary antibodies and then captured with secondary anti-immunoglobulin reagents bound to magnetic beads. The magnetic beads are then collected in a magnetic field to recover the adsorbed cells.

Another method is fluorescence-activated cell sorting, in which cells expressing a marker are labeled with a specific antibody, usually by a fluorescently labeled secondary anti-immunoglobulin. The cells are then individually separated according to the amount of bound label using a suitable sorting device. Any of these methods can recover a positively selected cell population with the relevant marker, as well as a negatively selected cell population without the marker that is of sufficient density or readily becomes positively selected. Negative selection can also be effectively performed by sequentially incubating the cells with a specific antibody and a complement preparation that can lyse the cells, to which the antibody is bound. Sorting of differentiated cells can be performed at any time, but it is generally found to be most effective to perform sorting shortly after the differentiation process begins.

It was found that cells positively selected for polysialylated NCAM could provide a population that was 60%, 70%, 80% or even 90% NCAM positive (example 1). This suggests that they are capable of forming certain types of nerve cells, including neurons.

Cells positively selected for A2B5 were found to provide a population that was 60%, 70%, 80% or even 90% A2B5 positive (example 2). This suggests that they are capable of forming certain types of nerve cells, possibly including both neurons and glial cells. A2B5 positive cells were further divided into two independent populations: one was A2B5 positive and NCAM negative, and one was A2B5 positive and NCAM positive.

The differentiated or isolated cells prepared according to this step may be maintained or further propagated in any suitable medium. Typically, the culture medium contains most of the components originally used to differentiate the cells.

The neural precursor cells prepared according to these steps may be further differentiated into mature neurons, astrocytes or oligodendrocytes, if necessary. Culturing the cells in maturation factors (e.g., hair follicle stimulating hormone) or other compounds that increase intracellular cAMP levels (e.g., cholera toxin, isobutylmethylxanthine, dibutyladenosine cyclized monophosphate), or other factors (e.g., c-kit ligand, retinoic acid, or neurotrophin) is effective. Particularly effective are neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF). Other candidate substances are GDNF, BMP-2, and BMP-4. Alternatively, removal of some or all of these factors that promote proliferation of neural precursors (e.g., EGF or FGF) may also enhance the maturation process.

For use in therapy or other applications, it is often desirable that the precursor cells or mature neural cell population be substantially free of undifferentiated pPS cells. One method of removing undifferentiated stem cells from a population is to transfer these cells with a vector in which the effector gene is preferentially expressed in the undifferentiated cells under the control of a promoter. Suitable promoters include the TERT promoter and the OCT-4 promoter. The effector gene may lyse the cell directly (e.g., encoding a toxin or apoptosis mediator). Alternatively, the effector gene may predispose the cell to toxic effects of an external agent (e.g., an antibody or prodrug). For example, the herpes simplex thymidine kinase (tk) gene, which can lead to guanine-susceptibility in cells expressing this gene. A suitable pTERT-tk construct is provided in International patent publication No. WO 98/14593 (Morin et al).

Characteristics of neural precursors and terminally differentiated cells

Cells can be characterized according to some phenotypic criteria. These criteria include, but are not limited to, morphological features observed microscopically, qualitative or quantitative expression of cellular markers, enzymatic activity, or neurotransmitter and its receptor, and electrophysiological function.

Certain cells included in the present invention have morphological characteristics that are characteristic of neuronal cells or glial cells. These characteristics can be readily identified by those skilled in the art who are skilled in assessing the presence of such cells. For example, neurons are characterized by small cell bodies with various forms of axons and dendrites. The various cells of the invention may also be characterized according to whether they express phenotypic markers specific to various types of neural cells.

Some markers of interest include, but are not limited to, β -tubulin III, tubulin-related protein 2(MAP-2), or neurofilaments that are characteristic of neurons; glial Fibrillary Acidic Protein (GFAP) present in astrocytes; galactocerebroside (GalC) or sphingomyelin basic protein (MBP) characteristic of oligodendrocytes; oct-4 specific to undifferentiated hES cells; nestin, which is characteristic of neural precursors and other cells; as well as the already described A2B5 and polysialylated NCAM. When studying cells of the neural lineage, since A2B5 and NCAM are useful markers, it is noted that these markers are sometimes also present on other cell types (e.g., hepatocytes or muscle cells). Beta-tubulin III was previously thought to be neural cell specific, but it was now found that some subpopulations of hES cells were also beta-tubulin III positive. MAP-2 is a relatively stringent marker for various types of fully differentiated neurons.

Various tissue-specific markers mentioned in the present disclosure and known in the art can be detected using suitable immunological techniques-for example, flow immunocytochemistry methods for cell surface markers; immunohistochemistry for intracellular or cell surface markers (e.g., fixed cells or tissue fragments); western blot analysis of cell extracts; and enzyme-linked immunoassay is performed on cell extracts or products secreted in the culture medium. In standard immunocytochemistry or flow cytometry, expression of an antigen by a cell is said to be "antibody-detectable" if a clearly measurable antibody is bound to the antigen, which can be done after cell immobilization, or a labeled secondary antibody or other conjugate pair (e.g., biotin-avidin conjugate pair) can be used to amplify the label.

Tissue-specific gene expression products can also be detected at the mRNA level by Northern blot analysis, dot blot hybridization analysis, or reverse transcriptase initiated-polymerase chain reaction (RT-PCR) using sequence specific primers in standard amplification methods. See U.S. Pat. No. 5,843,780 for further details. Sequence data for specific markers listed in this disclosure are available from a shared database such as GenBank (URLwww.ncbi.nlm.nih.gov: 80/entrez). Expression at the mRNA level of an assay according to the present disclosure is said to be "detectable" if a cell sample is analyzed in a typical controlled assay according to standard procedures, with discernible hybridization or amplification products being observed. Expression of a tissue-specific marker detected at the protein or mRNA level is considered positive if this level is at least 2-fold, preferably 10-fold or 50-fold, greater than that of a control cell (e.g., an undifferentiated pPS cell, fibroblast, or other unrelated cell type).

Also characteristic of neural cells, especially terminally differentiated cells, are receptors and enzymes involved in biosynthesis, release and neurotransmitter re-uptake, as well as ion channels involved in depolarization and repolarization processes associated with synaptic transmission. Staining of synaptic vesicle proteins can provide evidence of synapse formation. Evidence for the uptake of these neurotransmitters can be obtained by detecting receptors for gamma-aminobutyric acid (GABA), glutamate, dopamine, 3, 4-Dihydroxyphenylalanine (DOPA), norepinephrine, acetylcholine, and serotonin.

Differentiation of a particular neural precursor cell population of the invention (e.g., using NT-3 and BDNF) can result in a cell population that is at least 20%, 30%, or 40% MAP-2 positive. Essentially, 5%, 10%, 25% or more of cells that are positive for NCAM or MAP-2 are capable of synthesizing neurotransmitters, such as acetylcholine, glycine, glutamate, norepinephrine, serotonin or GABA.

Certain populations of the invention contain 0.1% NCAM or MAP-2 positive cells, and may be 1%, 3% or 5% or more (based on cell count) Tyrosine Hydroxylase (TH) positive, as measured by immunocytochemistry or mRNA expression. In the art, this is generally considered a marker for dopamine-synthesizing cells.

To further illustrate the presence of mature neurons in the differentiated population, the cells can be detected according to functional criteria. For example, calcium flow can be measured by any standard technique in response to neurotransmitters or other environmental conditions known to affect neurons in vivo. First, neuronal-like cells in the population are determined by morphological criteria or by markers such as NCAM. The cell is then subjected to a neurotransmitter or condition and the response is monitored (example 6). Standard membrane clamps can also be used on cells to determine if there is evidence of action potential and how much lag time between applied potential and response. Differentiation of the neural precursor population of the invention may result in cultures of certain subpopulations with morphological characteristics of neurons, which cultures are positive for NCAM or MAP-2 and respond with the following frequencies: at least about 40%, 60% or 80% of the cells respond to GABA, acetylcholine, ATP and high sodium concentrations; at least about 5%, 10% or 20% of the cells respond to glutamate, glycine, ascorbic acid, dopamine or norepinephrine. In the patch clamp system, a greater proportion (at least about 25%, 50% or 75%) of cells positive for NCAM or MAP-2 can exhibit an action potential.

Some standard methods for determining the quality of a population of cells, and conditions for optimizing proliferation and differentiation of cells according to the present invention, may also exhibit other desirable characteristics consistent with functional neurons, oligodendrocytes, astrocytes, and their precursors.

Terminal granulation of neural precursor

In the screening and treatment of certain drugs, it is desirable that neural precursors be replication competent and that containers be provided for the production of differentiated neuronal and glial cells. Cells of the invention may be telomerised to increase their replicative potential either before or after development into cells of restricted developmental lineage or terminally differentiated cells. The telomerized pPS cells may be differentiated along the aforementioned differentiation pathway, or the differentiated cells may be directly telomerized.

Cells can be made telomeric by genetic transformation, transfection or transduction with a suitable vector, homologous recombination, or other suitable technique, so that they express the telomerase catalytic component (TERT), usually under the action of an exogenous promoter in addition to the endogenous promoter. Particularly suitable is the catalytic component of human telomerase (hTERT) as provided in international patent application WO 98/14592. For some uses, species homologues such as mouse TERT (WO 99/27113) may also be used. Bodnar et al, Science 279: 349, 1998 and Jiang et al, nat. genet.21: transfection and expression of telomerase in human cells is described in 111, 1999. In another example, the cloning of hTERT (WO 98/14592) was used as the source of hTERT coding sequence and spliced into the EcoRI site of the PBBS212 vector under the control of the MPSV promoter or into the EcoRI site of a pBABE retroviral vector (commercially available) under the control of the LTR promoter.

Using a carrier containing the supernatantDifferentiated or undifferentiated pPS cells are genetically transformed for more than 8-16 hours and then replaced with growth medium for 1-2 days. Genetically transformed cells were selected with puromycin at 0.5-2.5. mu.g/mL and re-cultured. And evaluating the expression of hTERT by RT-PCR, telomerase activity (TRAP analysis), immunocytochemical staining or replication capacity of hTERT. The following assay kits for research purposes are commercially available:XL telomerase detection kit (catalog s 7707; Intergen, Inc., from New York); and telomere TAGGG telomerase PCR ELISA plus (catalog 2,013, 89; Roche diagnostics, Indianapolis, Ind.). TERT expression can also be assessed at the mRNA level by RT-PCR. LightCycler telomere TAGGG hTERT quantification kit (catalog 3,012,344; Roche Diagnostics) is commercially available for research purposes. The continuously replicating clones can be further cultured to enrich for cells with the desired phenotype under conditions that support proliferation, and optionally cells with the desired phenotype can also be cloned by limiting dilution.

In certain embodiments of the invention, pPS cells can differentiate into various multipotent or committed neural precursors, which are then genetically transformed to express TERT. In other embodiments of the invention, pPS cells are genetically transformed to express TERT and then redifferentiated into various neural precursors or terminally differentiated cells. The success or otherwise of an improvement in TERT expression can be determined by TRAP analysis or by whether the replicative capacity of the cell is improved.

Still other methods of immortalizing cells are contemplated, such as transformation of cells with DNA encoding myc, SV40 large T antigen or MOT-2 (U.S. Pat. No. 5,869,243, International patent applications WO 97/32972 and WO 01/23555). When cells are used for therapeutic purposes, transfection with oncogenes or products of oncoviruses is not well suited. The terminally pelleted cells are of interest in the use of the present invention, and have the advantage that it enables the cells to proliferate and maintain their karyotype-for example, in drug screening and treatment protocols, differentiated cells can be administered to an individual to enhance the function of their CNS.

Use of neural precursors and terminally differentiated cells

The present invention provides methods for the large scale production of neural precursor cells and mature neuronal and glial cells. These cell populations are useful in many important research, development and for commercial purposes.

The cells of the invention can be used to prepare a cDNA library, which is relatively uncontaminated by cDNA preferentially expressed in cells of other lineages. For example, centrifugation is carried out at 1000rpm for 5 minutes to collect multipotent neural progenitor cells, and then mRNA is extracted from the co-precipitate using standard techniques (Sambrook et al, supra). After reverse transcription into cDNA, such preparations can remove cDNA from any or all of the following cell types: cells targeted to neuronal or glial lineages, mature neurons, astrocytes, oligodendrocytes, or other cells of undesired specificity. This creates a selective cDNA library that reflects transcripts that are preferentially expressed in neuronal precursors over terminally differentiated cells. In a similar manner, transcripts can also be made that can be preferentially expressed in neuronal or glial precursors, or mature neurons, astrocytes and oligodendrocytes.

The differentiated cells of the invention may also be used to prepare antibodies specific for markers of multipotent neural precursors, cells directed to neuronal or glial cell lineages, mature neurons, astrocytes, oligodendrocytes. The present invention provides improved methods for increasing the production of such antibodies, since the cell population of a particular cell type has been enriched compared to pPS cell cultures and neuronal and glial cell cultures extracted directly from CNS tissue.

Polyclonal antibodies can be prepared by injecting cells of the invention into a vertebrate in an immunogenic form. The preparation of monoclonal antibodies is described in the following standard references: harrow and Lane (1998), U.S. patent nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B: 3(1981). Other methods for obtaining specific antibody molecules, preferably in the form of single chain variable regions, include the step of exposing a pool of immunologically active cells or virions to the antigen of interest to allow positive selection of clones. See Marks et al, New eng.j.med.335: 730, 1996; international patent applications WO 94/13804, WO90/02809 and McGuiiness et al, Nature Biotechnol.14: 1449, 1996. The desired specificity can be obtained by positive selection using the pPS cells of the invention, and negative selection using cells with a broader distribution of antigens (e.g., differentiated embryonic cells) or adult derived stem cells. Such antibodies, in turn, can be used to identify or rescue neural cells of a desired phenotype from a mixed population of cells, such as costaining in immunodiagnostics using tissue samples, isolating precursor cells from terminally differentiated neurons, glial cells, and other lineages.

Analysis of Gene expression

The cells of the invention are also of interest in identifying expression patterns of transcripts and newly synthesized proteins that are characteristic of neural precursor cells, which may also help direct differentiation pathways or promote cell-cell interactions. Expression patterns of differentiated cells have been obtained and compared to control cell lineages, such as undifferentiated pPS cells, other types of committed precursor cells (e.g., pPS cells differentiated to other lineages, cells targeted to neuronal or glial cell lineages), other types of putative neural stem cells (e.g., from the neural crest, neurosphere, or spinal cord), or terminally differentiated cells (e.g., mature neurons, astrocytes, oligodendrocytes, smooth muscle cells, and neural membrane cells).

Suitable methods for comparing expression at the protein level include the immunoassays or immunohistochemical techniques described above. Suitable methods for comparing expression at the transcriptional level include differential display of mRNA (Liang, Peng et al, Cancer Res.52: 6966, 1992), whole sequencing of cDNA libraries, and array expression systems (matrix array expression systems).

Fritz et al, Science 288: 316, 2000; "Microarray Biochip Technology", LShi, www.Gene-chips.com reviews the use of microarrays for analyzing gene expression. Systems and reagents for performing microarray analysis can be obtained from Affymetrix corporation, Santa Clara, california; gene Logic, Inc., Columbia, Mass; HySeq corporation, Sunnyvale California; molecular dynamics, Sunnyvale, Calif.; nanogen, San Diego Calif.; and Syntei, Fremont California (Inc Genomics, Palo Alto California).

Solid phase arrays are made by attaching probes to specific sites, either by synthesizing the probes at the desired sites, or by pre-synthesizing probe fragments and attaching them to a solid support (U.S. Pat. Nos. 5,474,895 and 5,514,785). Probe assays typically involve contacting a liquid (which may contain the nucleotide sequence of interest) with an array under conditions suitable for hybridization and detecting the resulting hybrids.

An exemplary method is to use a Genetic Microsystems array generator and Axon GenePix^TMBy a scanner. Microarrays are prepared by first amplifying cDNA fragments encoding the marker sequences to be analyzed and spotting them directly onto a glass slide. To compare mRNAs prepared from two related cells, one was converted to Cy 3-labeled cDNA and the other to Cy 5-labeled cDNA. Both cDNA preparations were simultaneously placed on a microarray slide for hybridization and then washed to remove non-specific binding. Each label on the microarray slide is then scanned at the appropriate wavelength, the resulting fluorescence quantified, and the results formatted to indicate the relative abundance of mRNA for each label on the array.

Identifying expression products for use in characterizing and affecting the differentiated cells of the invention, which process comprises analyzing the expression level of RNA, protein, or other gene product in a first cell type (e.g., a multipotent neuronal precursor cell of the invention, or a cell capable of differentiating along a neuronal or glial pathway); then analyzing the expression level of the homologous product in the control cell type; comparing relative expression levels between the two cell types, (typically normalized to total protein or RNA in the sample, or compared to other gene products expected to be expressed at similar levels in the two cell types, such as housekeeping genes); the relevant products are then identified based on the relative expression levels.

Drug screening

The neural precursor cells of the present invention can be used to screen for factors (e.g., solvents, small molecule drugs, peptides, polynucleotides) or environmental conditions (e.g., culture conditions or manipulations) that can affect characteristics of the neural precursor cells and their progeny.

In some applications, pPS cells (undifferentiated or differentiated) are screened for factors that promote maturation into neural cells, or promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation or growth factors are tested by adding them to cells in different wells, detecting any resulting phenotypic changes, and further culturing and using the cells according to desired criteria.

Another screening aspect of the invention is the use of the test drug compounds for their effect on neural tissue or neurotransmission. Screening is performed either because such compounds are designed to have a pharmacological effect on nerve cells or because various effects designed on the compounds additionally have unexpected side effects on the nervous system. Screening can be performed using any neural precursor cell or terminally differentiated cell of the invention (e.g., dopaminergic neurons, 5-hydroxytryptamine neurons, cholinergic neurons, sensory neurons, motor neurons, oligodendrocytes, and astrocytes).

The reader is generally referred to the standard textbook "In vitro Methods In pharmaceutical research", academic Press, 1997 and U.S. Pat. No. 5,030,015. Assays for the activity of a candidate pharmaceutical compound generally include combining the differentiated cells of the invention with the candidate compound, either alone or in combination with other drugs. The inventors attribute any changes in cell morphology, marker phenotype or functional activity to the compound (as compared to untreated cells or cells treated with an inert compound) and then correlate the effect of the compound with the observed change.

Cytotoxicity in the first instance can be determined by analyzing the effect on cell viability, morphology and expression of specific markers or receptors. The effect of a drug on chromosomal DNA can be determined by detecting DNA synthesis or repair. [³H]Incorporation of thymidine or BrdU (especially at times outside the cell cycle, or above levels required for cell replication) is consistent with the action of drugs. Deleterious effects also include an abnormal rate of interfacial chromatid exchange, which can be exhibited by the intermediate stage. The reader is referred to A.Vickers ("In vitro Methods In pharmaceutical Research", page 375- "academic Press, 1997) for further details.

The effect on cell function can be determined by observing the phenotype or activity of the neural cell, such as receptor binding, neurotransmitter synthesis, release or uptake, and neuronal precursor or sphingomyelin sheath growth, in cell culture or in a suitable model using any standard assay.

Therapeutic uses

The invention also provides neural precursor cells that can restore to some extent Central Nervous System (CNS) function in order to be supplied to an individual in need of such treatment (possibly as a result of a congenital defect in function, the effects of a disease, or as a result of an injury).

To determine the suitability of the neural precursor cells for treatment, these cells can first be tested in a suitable animal model. At one level, cells were tested for viability in vivo and for the ability to maintain their phenotype. The neural precursor cells are administered to an immunodeficient animal (e.g., a nude mouse, or an immunodeficient animal due to a chemical or radiation) at a location that is observable (e.g., in the brain cavity or in the spinal cord). Tissues were extracted after several days to several weeks or more and the presence of pPS-derived cells was examined.

This can be accomplished by administering a cell that expresses a detectable marker (e.g., green fluorescent protein or β -galactosidase), wherein the cell is pre-labeled (e.g., with BrdU or [ beta ], [ beta ] -galactosidase)³H]-thymidine); or by subsequent detection of an inherent cell marker (e.g., with a human specific antibody). When neural precursor cells are tested in rodent models, the presence and phenotype of the cells used can be detected by immunohistochemical methods or ELISA (using human specific antibodies), or by RT-PCR analysis (using primers and hybridization conditions that specifically amplify human polynucleotide sequences). Also provided in the present disclosure are markers that can detect gene expression at the mRNA or protein level.

Various animal models for detecting recovery of nervous system function are described in "CNSRegeneration: basic Science and Clinical Advances ", edited by m.h. tuszynski and kordawer, academic press, 1999.

The differentiated cells of the invention may also be used in human patients in need of such cells to allow tissue reconstruction or regeneration. The cells are administered in a manner that allows the cells to transplant or migrate to the designated tissue site and reconstitute or regenerate the functionally defective area.

Certain neural progenitor cells described herein are designed for the treatment of acute or chronic damage to the nervous system. For example, a excitotoxicity is involved in many symptoms including epilepsy, stroke, ischemia, Hunter's disease, Parkinson's disease, and Alzheimer's disease. Certain differentiated cells of the invention are also useful in the treatment of demyelinating disorders (dysmyelinating disorders), such as familial leukoencephalopathy (Pelizaeus-Merzbacher), multiple sclerosis, leukodystrophy, neuritis, and neuropathy. Cell cultures suitable for these purposes are enriched for oligodendrocytes or oligodendrocyte precursors to promote remyelination.

For example, neural stem cells are transplanted directly into soft tissue sites or intrathecal sites of the central nervous system depending on the disease to be treated. Transplantation was performed with single cell suspensions or small aggregates (density of 25,000-500,000 cells per μ L) (U.S. Pat. No. 5,968,829). The effect of transplanted motor neurons or their precursors can be determined in a rat model of acute spinal cord injury as described by McDonald et al (nat. Med.5: 1410, 1999). In successful transplantation, graft-derived cells appear 2-5 weeks after injury, differentiate into astrocytes, oligodendrocytes and/or neurons, migrate from the end of the injury along the spinal cord, and improve in ion channel gating, coordination, and weight gain.

The neural progenitor cells and terminally differentiated cells according to the invention may be administered to humans in the form of a pharmaceutical composition comprising an isotonic excipient prepared under completely sterile conditions. For the understanding of the rationale in terms of pharmaceutical formulation, the reader is referred to Cell Therapy, compiled by g.morstyn and w.sheeridan: stem CellTrans transplantation, Gene Therapy, and Cellular immunology, Cambridge university Press, 1996; and hematopic Stem Cell Therapy, e.d. ball, j.list and p.law, churchiling, 2000.

Such compositions may be packaged in a suitable container with written instructions for a desired purpose, such as re-establishing CNS function to ameliorate certain neurological abnormalities.

The following examples are provided to further illustrate, without limitation, specific embodiments of the present invention

Examples

Procedure of the test

This section provides details on some of the techniques and reagents used in the examples below.

Maintenance of hES cells in Primary mouse embryo fibroblastsCells or feeder cells-free systems. hES cells were seeded in small clusters in irradiated mouse embryonic fibroblasts, or coated with(in a 1: 10 to 1: 30 medium). The hES cell culture on feeder cells was maintained in medium containing 80% KO DMEM (Gibco) and 20% serum replacement (Gibco) supplemented with 1% non-essential amino acids, 1mM glutamine, 0.1mM beta-mercaptoethanol, and 4ng/mL human bFGF (Gibco). Cultures without feeder cells were maintained in the same medium conditioned by pre-culturing embryonic fibroblasts, followed by the addition of 4ng/mL bFGF (daily replacement).

Cells were expanded by serial passage. The monolayer culture of ES colonies was treated with 1mg/mL collagenase at 37 ℃ for 5-20 minutes. The cells were then carefully scraped from the plate. Small clusters of cells were carefully isolated and re-seeded on fresh feeder cells.

The embryoid body was produced by the following method. Incubation in 1mg/mL collagenase for 5-20 minutes to obtain a monolayer culture of fused hES cells, then scraping the cells from the plate. The cells were then spread into clusters and plated on non-adherent cell culture plates (Costar) in medium containing 80% KO (knock out) dmem (gibco) and 20% non heat inactivated fbs (hyclone) with 1% non essential amino acids, 1mM glutamine and 0.1mM beta-mercaptoethanol. Cells were seeded in 2mL of media per well (6 well plate) at a ratio of 1: 1 or 1: 2. Every two days, an additional 2mL of medium was added to each well to culture EBs. When the volume of medium exceeded 4 mL/well, the EBs were collected and fresh medium was added again. After 4-8 days of suspension, EBs were individually seeded onto the matrix and allowed to further differentiate (in the presence of selective differentiation factors).

Differentiation to neural precursors typically occurred in wells coated with fibronectin (Sigma, final concentration in PBS of 20. mu.g/mL). 1 mL/well (9.6 cm)²) The plates were incubated at 4 ℃ overnightOr incubated at room temperature for 4 hours. Fibronectin was then removed and the plates were washed once with PBS or KO DMEM before use.

Immunocytochemical detection of NCAM and A2B5 expression was performed using the following method: cells were incubated with primary antibodies diluted in medium containing 1% sheep serum for 15 minutes at 37 ℃, the medium was washed once and then incubated with labeled secondary antibodies for 15 minutes. After washing, the cells were fixed in 2% paraformaldehyde for 15-20 minutes. For other markers, in PBS preparation of 4% paraformaldehyde fixed 10-20 minutes, PBS washing three times, in 100% ethanol soaking 2 minutes, and then 0.1M PBS washing. The cultures were then incubated in 0.1M PBS blocking solution containing 5% NGS (normal sheep serum) for at least 1 hour at room temperature. The cultures were then incubated in primary antibody diluted in 0.1M PBS (containing 1% NGS) for at least 2 hours at room temperature. Then washed with PBS and incubated with secondary antibody in the same buffer for 30 minutes. The antibodies used are listed in table 1.

Bead immunopanning was performed with the following reagents and equipment: a magnetic cell separator; midi MACs^TMA column; PBS CMF buffer containing 0.5% BSA and 2 mMEDTA; primary antibodies against NCAM and A2B 5; rat anti-mouse IgG (or IgM) microbeads; a pre-separation filter; rat anti-mouse kappa PE; and a FACScan device. Cells were harvested with trypsin/EDTA (Gibco) and dispersed. After removal of trypsin, cells were resuspended in MACs^TMIn a buffer. Cells were labeled with primary antibody for 6-8 min at room temperature and MACs were used^TMThe buffer was washed twice (cells were spun at 300g for 10 min and buffer aspirated). Then every 10 th⁷The individual cells were resuspended in 80. mu.l (micro volume). At every 10⁷Mu.l (micro volume) of MACs ram was added to each cell^TMIgG microbeads, kept at 6-12 ℃ for 15 minutes. By MACs^TMThe buffer solution is toThe samples were washed twice and then subjected to magnetic separation. The column was placed in a magnetic cell separator and the cell suspension was applied to the column (LS + Midi, stored in about 3-5mL buffer). With 3mL MACs^TMThe buffer was washed three times to pass negative cells. The column was then removed from the magnetic field and 5mL MACs were used^TMThe buffer elutes positive cells.

After isolation, cells of A2B5+ or NCAM + were plated on polylysine-and laminine-coated plates in DMEM/F12(Biowhittaker) supplemented with N2(Gibco 17502-014), B27(Gibco 17504-010) and the various factors mentioned. The sources of the factors are shown in table 2.

Expression at the transcriptional level was analyzed by RT-PCR using the following protocol: using the RNAeasy Kit^TM(Qiagen) RNA was extracted from cells as described in the protocol. The final product was digested with dnase to remove contaminating chromosomal DNA. RNA was incubated at 37 ℃ in RNA guard (RNA guard) (Pharmacia Upjohn) and DNase (Pharmacia Upjohn) in the presence of 10mM Tris ph7.5, 10mM MgCl₂And 5mM DDT in buffer) for 30-45 minutes. Extraction was performed with phenol chloroform to remove proteins from the sample, and then RNA was precipitated with 3M sodium acetate and 100% glacial ethanol. The RNA was washed with 70% ethanol, and the pellet was air-dried and resuspended in DEPC-treated water.

For the Reverse Transcription (RT) reaction, 500ng of total RNA was mixed with 1 × first strand buffer (Gibco), 200mMDDT and 25 μ g/mL random hexamer (Pharmacia Upjohn). The RNA was denatured by maintaining at 70 ℃ for 10 minutes and then annealed at room temperature for 10 minutes. dNTPs were added to a final concentration of 1mM, and 0.5. mu.L of Superscript II RT (Gibco) was added, incubated at 42 ℃ for 50 minutes, and then heat-inactivated at 80 ℃ for 10 minutes. The samples were then stored at-20 ℃ until they were subjected to PCR analysis. By using pair-related marksThe specific primers were subjected to a standard Polymerase Chain Reaction (PCR) in the following reaction compounds: 1.0. mu.L of cDNA, 2.5. mu.L of 10 XPCR buffer (Gibco), 10 XPMgCl₂2.5. mu.L, 2.5mM dNTP 3.0. mu.L, 5. mu.M 3 '-primer 1.0. mu.L, 5. mu.M 5' -primer 1.0. mu. L, Taq 0.4.4. mu.L and DEPC water 13.6. mu.L.

Example 1: NCAM positive cells

The purpose of this assay was to determine whether human embryonic stem (hES) cells could differentiate directly into NCAM positive progenitor cells. hES cells can be obtained from mEF-supported cultures or feeder-free cultures and then differentiated by Embryoid Body (EB) formation in suspension cultures using medium containing 20% FBS. The EBs were then inoculated intact onto fibronectin in DMEM/F12 medium (supplemented with N2 supplement (Gibco) and 25ng/mL human bFGF). After 2-3 days of culture, NCAM-positive and A2B 5-positive cells were identified by immunostaining.

Both magnetic bead sorting and immunopanning were successful in enriching NCAM positive cells. The starting cell population typically contains 25-72% cells that are positive for NCAM. After the immuno-isolation, the proportion of NCAM positivity became 43-72%. The results are shown in table 3.

In the first 10 experiments, NCAM positive cells recovered from the taxonomy were seeded on poly-L-lysine/laminin in DMEM/F12 (supplemented with N2 and B27 supplements, as well as 2mg/mL BSA, 10ng/mL human CNTF, 10ng/mL human bFGF, and 1ng/mL human NT-3). In each subsequent experiment, cells were maintained in DMEM/F12 with N2 and B27 supplements, and 10ng/mL EGF, 10ng/mLbFGF, 10ng/mL PDGF and 1ng/mL IGF-1.

FIG. 1 (upper panel) shows the growth curve of NCAM positive cells. The cells studied in the assay were prepared as follows: the suspension was maintained in 20% FBS for 4 days to form embryoid bodies, which were then seeded on a fibronectin matrix in DMEM/F12 (supplemented with N2 and B27 supplements and 25ng/mLbFGF) for 2-3 days. Cells were then positively sorted for NCAM expression and maintained in media containing CNTF, bFGF and NT 3. Sorted cells did not show an increase in survival relative to the unsorted population. Some NCAM positive cells also expressed β -tubulin III, indicating that these cells were able to form neurons. They also have the phenotypic characteristics of neuronal cells. There are also A2B5 positive cells in this population, which are probably glial progenitor cells. However, few cells are positive for GFAP (a marker for astrocytes). Although the cell population proliferated in culture, the proportion of NCAM positive cells (and the ability to form neurons) decreased after several passages.

Example 2: A2B5 positive cells

Cells in this assay were immunoselected for A2B5 surface markers. hES cells can be induced to form EBs in 20% FBS. After 4 days of suspension treatment, EBs were seeded on fibronectin in DMEM/F12 (containing N2 and B27 supplements, as well as 10ng/mL human EGF, 10ng/mL human bFGF, 1ng/mL human IGF-1 and 1ng/mL human PDGF-AA). After 2-3 days, 25-66% of the cells expressed A2B 5. When this population was enriched by magnetic bead sorting, the purity was 48-93% (Table 4).

Figure 2 shows an exemplary process for obtaining A2B5 positive cells. Abbreviations used: MEF-CM-medium conditions were adjusted with mouse embryo fibroblast cultures; +/-SHH with or without sonic hedgehog; D/F12-DMEM/F12 medium; n2 and B27, culture supplements (Gibco); EPFI ═ growth factors EGF, PDGF, bFGF and IGF-1; PLL ═ poly L-lysine matrix; PLL/FN ═ poly L-lysine and fibronectin matrices.

Figure 1 (lower panel) shows the growth curve of sorted A2B5 positive cells. Cells were maintained on poly-L-lysine coated plates in the same medium. Cells proliferate when serially passaged.

Example 3: maturation of A2B 5-Positive cells

Cells positive for A2B5 were induced to differentiate by the addition of a hair-pin. These cells can be assessed by different culture passages as shown in table 5.

Although the cells were sorted for expression of A2B5, this population was shown to produce not only oligodendrocytes and astrocytes, but also a large proportion of neurons. This result is unexpected: cells expressing A2B5 were previously thought to be glial precursors and to give rise to oligodendrocytes and astrocytes, whereas cells expressing NCAM are neuronal precursors and give rise to mature neurons. This experiment demonstrates that pPS cells are capable of differentiating into cell populations that can be repeatedly propagated in culture, and are capable of producing neurons and glial cells.

Example 4: transplantation of differentiated cells into mammalian brain

Transplantation of neural precursor cells was accomplished with cells isolated from two hES cell lines: a line designated H1, and a genetically transformed line designated H7 NHG. The H7NHG cell line carries an expression cassette (expresscasette) which allows the cells to continue to express Green Fluorescent Protein (GFP).

Neonatal Sprague Dawley rats received a unilateral striated in vivo implant of one of the following cell populations:

undifferentiated hES cells

Embryoid bodies derived from hES cells

Neural precursors sorting for NCAM expression (example 1)

Neural precursors sorting for A2B5 expression (example 2)

Control animals received a graft of irradiated mouse embryonic fibroblasts in which undifferentiated hES cells were preserved. To determine whether cell proliferation occurred after transplantation, some animals were pulsed intraperitoneally with BrdU 48 hours after sacrifice. 14 days after transplantation, rats were perfused cardiotically with 4% paraformaldehyde and the tissues were treated with immunohistochemical analysis.

FIG. 3 shows fluorescence observed on a section of animal tissue to which GFP-expressing cells were administered, the animal being. Viable cells were detected in all transplanted groups. Undifferentiated cells were present as large cell masses, indicating uncontrolled growth of necrotic areas and vacuolization of surrounding tissue (left panel). AFP immunostaining was performed on animals transplanted with HI cells, and the results showed that undifferentiated hES cells were transformed into visceral endoderm after transplantation. Embryoid bodies migrated almost without in the graft core and were surrounded by necrotic regions at the same time (middle panel). In contrast, sorted NCAM positive cells appeared as dispersed cells and showed some degree of migration away from the implantation site.

Example 5: differentiation into mature neurons

To produce terminally differentiated neurons, embryoid body formation in FBS medium with or without 10 μ M Retinoic Acid (RA) induced the first stage of differentiation. After 4 days of suspension, embryoid bodies were seeded on fibronectin-coated plates in defined medium supplemented with 10ng/mL human EGF, 10ng/mL human bFGF, 1ng/mL human PDGF-AA and 1ng/mL human IGF-1. Embryoid bodies are attached to the plate and their cells begin to migrate to the plastic surface and form a monolayer.

After 3 days, many cells with neuronal morphology were observed. Cells positive for incorporated BrdU and mosaic staining but lacking lineage-specific differentiation markers can be identified as neural precursors. If positive for polysialylated NCAM and A2B5, putative neuronal and glial progenitor cells can be identified. When counted by flow cytometry, 41-60% of the cells expressed NCAM and 20-66% expressed A2B 5. A subset of NCAM positive cells can express beta-tubulin III and MAP-2. Glial markers like GFAP or GalC do not co-localize. Cells positive for A2B5 can give rise to neurons and glial cells. One subset of A2B5 cells expressed beta-tubulin III or MAP-2, while another subset expressed GFAP. Some cells with neuronal morphology stained both A2B5 and NCAM doubly. Both the NCAM positive and A2B5 positive populations contained far more neurons than glial cells.

The cell population can be further differentiated by reseeding the cells in a mitogen-free medium containing 10ng/mL neurotrophin-3 (NT-3) and 10ng/mL Brain Derived Neurotrophic Factor (BDNF). After about 7 days, neurons with extensive function were seen. Cultures isolated from embryoid bodies stored in Retinoic Acid (RA) showed more MAP-2 positive cells (about 26%) compared to cultures stored in an RA-free environment (about 5%). GFAP-positive cells were observed in the debris. Cells positive for GalC were identified, but these cells were large and flat without complex function.

Table 6 summarizes the cell types and markers that appear at different stages of differentiation.

The presence of neurotransmitters is also determined. GABA-immunoreactive cells were identified that co-expressed beta-tubulin III or MAP2 and had morphological characteristics of neuronal cells. Occasionally GABA positive cells do not co-express neuronal markers but have astrocyte-like morphology. Neuronal cells were identified which co-expressed Tyrosine Hydroxylase (TH) and MAP-2. Synaptic structures can be identified by staining with a vesicular protein antibody.

FIG. 4 shows TH stained in a culture differentiated from human ES cells of the H9 line. Embryoid bodies were kept in 10 μ M retinoic acid for 4 days and then seeded on fibronectin-coated plates placed in EGF, basic FGF, PDGF and IGF for 3 days. Then passaged to laminin in N2 medium (supplemented with 10ng/mL NT-3 and 10ng/mL BDNF) and allowed to continue to differentiate for 14 days. The differentiated cells were fixed with 2% paraformaldehyde for 20 minutes at room temperature and then developed using an antibody for TH (labeling of dopaminergic cells).

Example 6: calcium imaging

Standard fura-2 imaging of calcium flux was used to study functional properties of hES cell-derived neurons. Neurotransmitters investigated included GABA, glutamate (E), glycine (G), high concentrations of potassium (50mM K + instead of 5mM K +), retinoic acid (control), dopamine, acetylcholine (ACh) and norepinephrine. Solution in ratsRinger (RR) solution contains 0.5mM neurotransmitter (but ATP at a concentration of 10 μ M): 140mM NaCl, 3mM KCl, 1mM MgCl₂、2mM CaCl₂10mM HEPES buffer and 10mM glucose. The pH of the external solution was adjusted to 7.4 with NaOH. Cells were perfused into the recording chamber at a flow rate of 1.2-1.8 mL/min, and the solution was used as a bath with a 0.2mL injection loop (located about 0.2mL upstream of the bath inlet). A transient increase in calcium ion concentration is indicated if the calcium ion level exceeds 10% of baseline within 60 seconds and returns to baseline within 1-2 minutes.

FIG. 5 shows the response of a neuro-restrictive precursor to various neurotransmitters. Panel A shows the ratio of emission values of dispersed cells on two different coverslips. The added neurotransmitter is indicated by a triangle above.

Panel B shows the frequency of the cells tested in response to a particular neurotransmitter. Panel C shows the observed response to neurotransmitter compositions. Of the 53 cells tested, 26 responded to GABA, acetylcholine, ATP and high concentrations of potassium. The number of subgroups of populations responding to other antagonist compositions is smaller. Only two cells failed to respond to the various antagonists applied.

Example 7: electrophysiology

Standard whole cell membrane clamping was used on hES cell derived neurons to record the ionic current generated in voltage clamp mode and the action potential generated in current clamp mode. The external bath was rat ringer solution (example 6). The internal solution was 75mM potassium aspartate, 50mM KF, 15mM NaCl, 11mM EGTA and 10mM HEPES buffer, adjusted to pH 7.2 with KOH.

All 6 cells tested expressed sodium and potassium flux and elicited action potentials. The passive membrane behavior was measured when the voltage was increased from-70 to-80 mV and was characterized by the following data: average capacitance (C)_m) 8.97 ± 1.17 pF; film resistance (R)_m) 487.8 ± 42.0 Μ Ω; contact resistance (R)_a) 23.4 ± 3.62 Μ Ω. Place the cells in-1The ionic current was measured at 00mV, and the following data was measured when the voltage was increased from-80 to 80mV in 10mV increments: average sodium current I_Na-531.8 ± 136.4 pA; average potassium current I_K＝441.7±113.1pA；I_Na(density) — 57.7 ± 7.78 pA/pF; i is_K(Density) ═ 48.2. + -. 10.4 pA/pF.

Figure 6 shows the results of a typical experiment. Panel A shows the sodium and potassium currents observed in two cells (depolarized to detect a potential between-80 and 80mV starting from a fixed potential of-100 mV). Panel B shows the input (Na) of the observed current-voltage relationship⁺) And output (K)⁺) Peak of (2). The sodium current was excited between-30 and 0mV, peaking at-10 or 0 mV. The potassium current is excited above-10 mV, with values between 20 and 40mV being equal to or greater than the sodium current. Panel C shows the action potentials generated after n responses of the same cell to depolarizing stimuli. The cell membrane is placed between a voltage of-60 and-100 mV (current of-80 or-150 pA), with only a short depolarization.

Example 8: dopaminergic cells derived from neural progenitor cells

Embryoid bodies were cultured for 4 days in a suspension supplemented with 10. mu.M retinoic acid, and then placed in defined medium supplemented with EGF, bFGF, PDGF and IGF-1 for 3-4 days. The cells were then separated into A2B5 positive or NCAM positive enriched populations by magnetic bead sorting or immunopanning.

Cells selected by immunopanning were maintained in defined media supplemented with 10ng/mL NT-3 and 10ng/mL BDNF. After 14 days, 25. + -. 4% of the NCAM-sorted cells were MAP-2 positive-of which 1.9. + -. 0.8% were GABA positive and 3. + -. 1% were Tyrosine Hydroxylase (TH) (a restriction enzyme for dopamine synthesis, which is generally considered to represent dopamine-synthesizing cells) positive.

In the cell population sorted for NCAM, cells of NCAM + ve do not express markers for glial cells, such as GFAP or GalC. These data indicate that populations containing neuronal restricted precursors, but essentially no glial precursors, can be isolated directly from hES cell cultures.

On the other hand, cells screened for A2B5 were able to produce neurons and astrocytes. After enrichment, cells were placed on defined medium supplemented with NT-3 and BDNF and allowed to differentiate for 14 days. Within the first 1-2 days after plating, cells in the A2B 5-enriched population began to expand. After two weeks, the cells had the morphology of mature neurons, and 32. + -.3% of the cells were MAP-2 positive. Importantly, 3 + -1% of MAP-2 cells were TH positive, while only 0.6 + -0.3% had GABA immunoreactivity. These data indicate that cell populations containing astrocyte precursors and neuronal precursors, including dopamine-synthesizing cell precursors, can be obtained from hES cells.

Further TH-expressing neurons can be obtained by the following method. Embryoid bodies were generated from fusion hES cells of generation 32 of the H7 line by incubating them in 1mg/mL collagenase (37 ℃, 5-20 minutes), scraping the cells from the culture dish, and placing the cells on a non-adherent culture plate (c.) () The above. The resulting EBs were cultured in suspension in medium containing FBS and 10. mu.M all-trans retinoic acid. After 4 days, the aggregates were collected and placed in centrifuge tubes. Centrifuging, aspirating the supernatant, placing the aggregates in proliferation medium (DMEM/F12 supplemented with N2 at 1: 1, half-concentration B27, 10ng/mL EGF (R)&D System), 10ng/mL bFGF (Gibco), 1ng/mLPDGF-AAA (R)&D System) and 1ng/mL IGF-1 (R)&D system)) on plates coated with poly-L-tyrosine and fibronectin.

EBs were allowed to attach and proliferate for 3 days, then collected by trypsin (Sigma) treatment for about 1 min and treated at 1.5X 10⁵The concentration of cells/well was seeded in 4-well chambers coated with poly-L-lysine and laminin placed on proliferation medium and cultured for 1 day. The medium was then changed to neural basal medium to which B27 and one of the following growth mixtures were added:

10ng/mL bFGF (Gibco), 10ng/mL BDNF and 10ng/mL NT-3

10ng/mL bFGF, 5000ng/mL sonic hedgehog and 100ng/mL FGF8b

·10ng/mL bFGF

Cells were cultured under these conditions for 6 days, and fed every two days. On day 7, the medium was changed to a neural basal medium supplemented with B27 and a mixture of:

·10ng/mL BDNF、10ng/mL NT-3

1 μ M cAMP, 200 μ M ascorbic acid

1. mu.M cAMP, 200. mu.M ascorbic acid, 10ng/mLBDNF, 10ng/mL NT-3

Cultures were fed every two days until day 12, at which time they were fixed and labeled for immunocytochemical analysis against TH or MAP-2. The expression of the marker was quantified by counting 4 regions in each of the three wells with a 40-fold objective lens.

The results are shown in table 7. The highest proportion of TH-positive cells produced in bFGF, BDNF and NT-3 when initially cultured

Certain modifications to the invention described in this disclosure will be readily apparent to those skilled in the art as the best mode contemplated for carrying out the invention, and the invention may be practiced without departing from the spirit of the invention or the scope of the appended claims.

Claims (25)

1. A population of cells propagated in vitro culture obtained by differentiating human embryonic stem cells in vitro, wherein at least about 30% of the cells in the population are capable of committed formation of neuronal cells, glial cells, or both.

2. A cell population propagated in vitro culture obtained by differentiating human embryonic stem cells in vitro comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells can differentiate into neuronal cells and at least 10% of the cells can differentiate into glial cells.

3. A cell population propagated in vitro culture obtained by differentiating human embryonic stem cells in vitro comprising at least about 60% neural progenitor cells, wherein at least 10% of the cells express A2B5 and at least 10% of the cells express NCAM.

4. A cell population according to any of claims 1-3, obtained by differentiating human embryonic stem cells in a culture medium comprising at least two ligands which bind to growth factor receptors selected from the group consisting of EGF, bFGF, PDGF, IGF-1 and antibodies to receptors for these ligands.

5. A cell population according to any one of claims 1 to 3, which is obtained by differentiating human embryonic stem cells in a growth factor-containing medium and sorting the differentiated cells according to the expression of NCAM or A2B5, followed by collecting the sorted cells.

6. The cell population of any one of claims 1-3, which is induced to produce a cell population in which at least 30% of the cells have the morphological characteristics of mature neurons and are NCAM positive.

7. The population of claim 6, wherein the cells having morphological characteristics of mature neurons have at least three of the following characteristics:

a) at least 60% of the cells exhibit calcium flux when acetylcholine is administered;

b) at least 60% of the cells show calcium flux when GABA is administered;

c) at least 10% of the cells exhibit calcium flux when norepinephrine is administered;

d) at least 60% of the cells showed calcium flux at an external potassium concentration of 50 mM; or

e) When stimulated in a whole cell patch clamp, at least 25% of the cells demonstrated an action potential.

8. The population of cells of any one of claims 1-3, which is induced to produce a population of cells at least 1% of which stain positive for tyrosine hydroxylase.

9. A cell population comprising at least 60% neural progenitor cells and/or mature neurons having the same chromosome set as an established human embryonic stem (hES) cell line.

10. The population of claim 9, wherein the neural progenitor cells and/or mature neurons express NCAM, A2B5, MAP-2, or Nestin.

11. A cell population comprising mature neurons, astrocytes, oligodendrocytes or a combination thereof, obtained by further differentiating the cell population according to any of the preceding claims.

12. The population of claim 11, comprising a subpopulation of cells at least 30% of which have the morphological characteristics of mature neurons and are NCAM positive, wherein the subpopulation has the following characteristics:

a) at least 60% show calcium flux when acetylcholine is administered;

b) at least 60% show calcium flow when GABA is administered;

c) at least 10% show calcium flux when norepinephrine is administered;

d) at least 60% showed calcium flux at an external potassium concentration of 50 mM; or

e) When stimulated in a whole cell patch clamp device, at least 25% demonstrated an action potential.

13. The population of cells of claim 11 or 12, wherein at least 1% of the cells stain positive for tyrosine hydroxylase.

14. The cell population of claim 11 or 12, obtained by culturing the cell population of any one of claims 1-9 in a medium comprising the activator cAMP, a neurotrophic factor, or a combination thereof.

15. An isolated human neuronal precursor cell which expresses A2B5 and which is capable of differentiating into a neuron or glial cell in vitro.

16. The cell population of any one of claims 1-3, wherein the cell population comprises cells genetically transformed to express telomerase reverse transcriptase.

17. A method of obtaining neural precursor cells capable of giving rise to a cell population at least 1% of which are tyrosine hydroxylase positive cells, the method comprising differentiating human embryonic stem cells in vitro.

18. The method of claim 17 wherein the differentiation process comprises culturing in a medium containing at least two ligands that bind to a growth factor receptor selected from the group consisting of EGF, bFGF, PDGF, IGF-1 and antibodies to receptors for these ligands.

19. A method of obtaining a population of cells that are at least 1% tyrosine hydroxylase positive cells, comprising differentiating human embryonic stem (hES) cells in vitro.

20. The method of claim 19, comprising differentiating human embryonic stem cells in vitro to obtain neural precursor cells capable of producing a cell population that is at least 1% tyrosine hydroxylase positive cells, and then culturing the neural precursor cells in a culture medium comprising cAMP and ascorbic acid, or a neurotrophic factor, or a combination thereof.

21. The method of claim 20, wherein the neurotrophic factor is nerve growth factor, neurotrophin 3, brain-derived neurotrophic factor, or a combination of two or more of these factors.

22. A method of screening for a compound that is toxic or modulatory to neural cells, comprising preparing a culture comprising the compound, and the population of cells or isolated cells of any one of claims 1-16; determining any phenotypic or metabolic change that occurs in the cell as a result of contact with the compound; and correlating such changes with toxicity or modulation of nerve cells.

23. A method for obtaining a polynucleotide comprising a nucleotide sequence of mRNA that is highly expressed in neural progenitor cells, the method comprising:

a) determining the expression level of a plurality of mrnas in one or more cells in the population of cells of claims 1-14;

b) identifying from the population of cells, mRNA expressed at a higher level in the cells, in comparison to the expression profile in more mature cells; and is

c) Preparing a polynucleotide whose nucleotide sequence consists of at least 30 consecutive nucleotides contained in the mRNA selected in step b).

24. A medicament containing a cell population or isolated cells according to any one of claims 1 to 16 for use in the human or animal body by surgical or medical therapy.

25. Use of the cell population or isolated cells of any one of claims 1-16 in the manufacture of a medicament for reconstituting or compensating Central Nervous System (CNS) function in an individual.