HK1075673B - Dopaminergic neurons and proliferation-competent precursor cells for treating parkinson's disease - Google Patents

Description

Dopaminergic neurons and proliferative precursor cells for treating parkinson's disease

Reference to related applications

This application claims priority to U.S. utility patent application 09/888,309 filed on 21/6/2001 and 10/157,288 filed on 28/5/2002. Both of the above-referenced priority applications, as well as international patent applications WO01/51616 and WO 01/88104, are incorporated herein by reference in their entirety for the purpose of filing in the licensed United states and other jurisdictions.

Background

New studies on the derivation and expansion of cell lines suitable for human administration hold promise to lead to a nice new medical world. Given the scientific continuing benefit from important new discoveries in the cell biology of neurons and neuronal precursor cells, devastating and previously difficult to treat conditions may lead to the hope of obtaining regenerative medicine.

Among the disorders that require clinical treatment are those related to neurological dysfunction. Near the top of the list of these diseases is parkinson's disease, a spontaneous, slowly developing, degenerative disease of the central nervous system characterized by slow and reduced movements, muscle stiffness, resting tremor and postural instability. Symptoms resulting from the persistent deterioration of pigmented neurons in the substantia nigra, locus ceruleus and other brainstem dopaminergic cells, result in the loss of the neurotransmitter dopamine. Parkinson's disease is the fourth most common neurodegenerative disease in the elderly population, affecting 0.4% of people aged 40 years, and 1% of people aged 65 years. Regardless of the proposed age, the disease often causes devastating results for those afflicted patients.

The cause of the nervous system is so difficult to deal with that the often suffered damage is irreversible. The main hope for these diseases is to develop cell populations that can reconstruct neural networks and reconcile the functional recovery of the nervous system. Interesting evidence suggests that transplantation of fetal dopaminergic neurons can restore the chemical abnormalities of parkinson's disease. But very lack of proper organization.

For this reason, there is a great deal of interest in neural progenitor cells. Various types of lineage-restricted precursor cells renew themselves and reside at selective sites in the central nervous system (Kalyanl et al, biochem cell Biol, 6: 1051, 1998). The putative neural restricted precursor (Mayer-Proschel et al, Neuron, 19: 773, 1997) cells expressed the polysialylated isoform of the neural cell adhesion molecule (PS-NCAM). They are said to have the ability to produce various types of neurons, but not glial cells. On the other hand, the putative glial restricted precursor (Rao et al, Dev. biol, 188: 48, 1997) apparently has the ability to form glia rather than neurons. Inferred neural precursors from fetal or adult tissue are further described in U.S. patent 5,852,832; 5,654,183, respectively; 5,849,553, respectively; and 5,968,829; and WO09/50526 and WO 99/01159.

Unfortunately, progenitor cells isolated from neural tissue have not been shown to have sufficient replicative capacity to produce the number of cells necessary for human clinical therapy.

Another source is pluripotent cells isolated from early embryonic tissue. Embryonic Stem (ES) cells were originally isolated from mouse embryos 25 years ago (g.r.martin, proc.natl.acad.sci.u.s.a.78: 7634, 1981). It is believed that ES cells are capable of producing progeny of virtually any tissue type of the same species. Li. Smith et al (Cur. biol. 8: 971, 1998) reported the generation of neuronal precursors from mouse ES cells by lineage selection. Bjorklund et al reported the production of functional dopaminergic neurons from mouse ES cells (Proc. Natl. Acad. Sci. U.S.A.19: 2344, 2002).

Only recently have human ES cells been isolated (Thomson et al, Science 282: 114, 1998). Human ES cells require very different conditions to maintain them in an undifferentiated state, or to direct them to differentiate along specific differentiation pathways (U.S. patents 6,090,622 and 6,200,806; australian patent AU 729377, and PCT publication WO 01/51616). For this reason, it is much less understood how to prepare relatively homogeneous cell populations from human ES cells.

PCT publication WO 01/88104(Carpenter, Geron Corporation) describes a population of neural progenitor cells obtained by differentiating human ES cells. More than 90% of the population obtained was positive for NCAM, 35% was positive for β -tubulin, and 75% was positive for A2B 5. Zhang et al (Nature Biotech.19: 1129, 2001) subsequently reported the differentiation of neural precursors from human ES cells.

There is an urgent need for techniques to produce further optimized populations of neural cells for the treatment of certain clinical conditions.

Overview

The present invention provides a system for efficiently producing primate cells that have been differentiated from pluripotent cells into cells of the neural lineage. The precursors and terminally differentiated cells of the invention are useful in a number of important applications, including drug testing and the production of drugs to restore nervous system function.

One aspect of the invention is a cell population comprising a high proportion of cells having a neural lineage characteristic, e.g., neuronal cells and their precursors. These cells can be identified based on phenotypic markers such as A2B5, NCAM, MAP-2, drin, β -tubulin III, and other markers listed later in the invention, and by characteristic morphological and functional criteria.

Another aspect of the invention is a method of preparing a population containing neural cells from pluripotent cells, such as embryonic stem cells, embryonic germ cells, primary embryonic tissue, or stem cells from fetal or adult tissue having the ability to differentiate (or be adapted) into cells containing a neural phenotype. The method includes culturing the cells with a combination of soluble factors and environmental conditions conducive to the growth of neural cells having certain desired characteristics. The present invention includes strategies for optimizing differentiation methods for differentiating pluripotent stem cells into neural cells, wherein candidate factors are grouped by function, and the stem cells or their progeny are cultured with various combinations of factor sets. Groups important for producing the desired cell type are identified and then the individual components of each group are removed one by one to determine the minimum amount of composition required.

By way of illustration, pluripotent stem cells may be produced by direct differentiation on a solid surface containing one or more antagonists of the TGF- β superfamily to which are added, for example, noggin and follistatin. In addition, pluripotent stem cells may be cultured as clumps or embryoid bodies. Enrichment of neural cells to various degrees of maturity involves culturing in media containing added mitogens or growth factors (e.g., EGF and FGF) with or followed by addition of neurotrophins (e.g., NT-3 or BDNF) and other factors (e.g., EPO) in various optimized combinations. A list of differentiation factors used in certain cases is set forth in the description and illustrative examples summarized below. Optionally, the practitioner can also use physical separation techniques or manipulation techniques that further facilitate cell enrichment.

Mature neurons and their precursors prepared according to the invention can be characterized as progeny of a cell population or an established cell line from which the mature neurons and their precursors are derived. This can be confirmed by some appropriate technique such as standard DNA fingerprinting, which shows the genome of the neural cell to be substantially identical to the parent population. In addition, this relationship can be established by examining records in the neural cell derivation process. The characteristics of neural cells derived from a parental cell population are important in some respects. In particular, the undifferentiated cell population may be used to produce additional cells having a common genome-one or another set of neural cells, or another cell type useful for therapy-for example, a population of a histocompatibility type that enables patients to pre-tolerate (pretolerize) neural xenografts.

In one embodiment of the invention, neural cells are prepared from human pluripotent cells that differentiate into neuronal precursor cells as described, and then passaged in culture. The use of embryonic stem cells as the starting cell type promotes the generation of a rapidly developing population that nevertheless retains all of the activity that ultimately differentiates into functional neurons-either when cultured with neurotrophins lacking mitogens, or when administered to an appropriate subject. Certain precursor cell populations have the ability to undergo at least 10, 20 or 40 fold population doubling in culture without losing their ability to form highly enriched neuronal populations when further differentiated. Depending on the conditions used, precursor populations may be produced that have the ability to differentiate into a high proportion of tyrosine hydroxylase positive cells. This phenotype is consistent with dopaminergic neurons and is desirable for the treatment of parkinson's disease.

The cells of the invention are useful for screening compounds for neurocytotoxicity, the ability to modulate neuronal cell function, or the ability to assist in neuronal derivation and proliferation.

The cells of the invention may also be used to reconstitute or augment nervous system function in an individual to which the isolated cells or cell populations of the invention are administered. For this purpose, the isolated cell or cell population is formulated as a medicament for the treatment of a disease affecting the nervous system.

These and other embodiments of the invention will be apparent from the description which follows.

Drawings

FIG. 1 is a fluorescence photomicrograph showing neuronal cells obtained by directly differentiating ES cells on a solid substrate using a mixture of differentiation factors. The three regions shown are all taken from a treatment containing neurotrophins and the TNF-beta superfamily antagonists noggin and follistatin. Neuronal processing and staining was observed for the neuronal marker β -tubulin-III by a number of cells. MAP-2 positive cells, and also cells positive for tyrosine hydroxylase, a dopaminergic neuron marker, were present in a proportion of up to about 15%.

FIG. 2 shows the preparation of neurons from hES cells by direct differentiation. The yield of β -tubulin positive neurons was high when undifferentiated cells were plated on laminin and cultured with the TGF- β superfamily antagonists noggin (N) and follistatin (F) (group a). Yields were further improved in the presence of stem cell factor but in the absence of mitogen (treatment F, group B). Retinoic acid increased the number of neurons produced (group C), but decreased the proportion of neurons staining positive for Tyrosine Hydroxylase (TH) (group D).

FIG. 3 shows the case of the preparation of neurons, where differentiation is initiated by culturing hES to form embryoid bodies. The cells are then cultured in mitogens, subjected to differential trypsinization, and then passaged multiple times in media containing mitogens or neurotrophic factor mixtures. When mitogens and neurotrophins are used together, the cells can be passaged approximately 40-fold (group A), maintaining the ability to proliferate and differentiate into mature neurons (group B).

FIG. 4 shows that passaging cells in a mixture of Epidermal Growth Factor (EGF), basic fibroblast growth factor (FGF-2), brain-derived neurotrophic factor (BDNF), and neurotrophin 3(NT) results in a neural precursor population that upon differentiation yields a cell population in which TH-positive cells comprise about 7% of all the cells in the population (group A). The mixture used for terminal differentiation of precursor cells may also improve the production of TH-positive cells (group B).

Detailed Description

It has been found that when pluripotent stem cells are cultured in the presence of a selected differentiating agent, a cell population is derived that contains a significant proportion of cells having the phenotypic characteristics of mature neural cells or their precursors. These cells are suitable for use in drug screening and treatment of disorders associated with neurological abnormalities.

The system encompassed by the present invention is illustrated by a cell population obtained from an established human embryonic stem (hES) cell line. Differentiation can be initiated by a number of techniques described below, such as embryoid body formation, or by culturing hES cells on an appropriate matrix with one or more TGF- β superfamily antagonists present. Obtaining a precursor cell, which belongs to the neuronal lineage, and which can further differentiate into mature neurons.

As shown in fig. 3(a), neuronal precursors formed from hES cells can be passaged approximately 40-fold in culture. Notably, as shown in fig. 3(B), these cells retained the full capacity to differentiate into mature neurons even after multiple passages. Such a strong combination of proliferation and differentiation has not been utilized in the culture of human neural cells in the past.

Mature neurons obtained according to the invention extend the characteristics of this cell type, show staining for neuron-specific markers such as neurofilaments and MAP-2, and show evidence of synapse formation as detected by staining for synaptic vesicle proteins. These cells respond to various neurotransmitter substances and have action potentials as measured in a standard patch clamp system. In all of these aspects, the cell apparently has full neural function.

Of particular importance is the ability of the system to be tuned to optimize the proportion of precursors that are capable of producing neurons with medically important properties. Figure 1 shows neurons that stained positive for tyrosine hydroxylase, a characteristic of dopaminergic neurons. This type of cell is particularly desirable for the treatment of parkinson's disease, but other sources have not previously been described as providing the appropriate species of cell in sufficient abundance. As shown in FIG. 4, passaging the precursor cells in medium containing mitogens EGF and FGF-2, and neurotrophins BDNF and NT-3, resulted in a proliferating cell population capable of producing TH-positive cells in about 7% of the total cells of the population.

Since the pluripotent stem cells and some lineage-restricted precursors of the invention proliferate in large amounts in culture, the system described in this disclosure provides an unlimited supply of neuronal cells. Extensive expansion can occur at the level of undifferentiated pluripotent stem cells, or at the level of neural precursors. The cells of the invention have important uses in research, drug development and treatment of CNS disorders.

Definition of

For the purposes of this disclosure, the term "neural progenitor cell" or "neural precursor cell" refers to a cell that is capable of producing either a neuronal cell (e.g., a neuronal precursor or mature neuron) or a progeny of a glial cell (e.g., a glial precursor, mature astrocyte or mature oligodendrocyte). Typically, when they are cultured in vitro on their own, they do not produce progeny of other embryonic germ layers unless somehow dedifferentiated or reprogrammed.

A "neuronal progenitor cell" or "neuronal precursor cell" is a cell capable of producing a mature neuronal progeny. These cells may or may not also have the ability to produce glial cells. A "glial progenitor" or "glial precursor" cell is a cell that is capable of producing mature astrocytes or mature oligodendrocyte progenies. These cells may or may not also have the ability to produce neuronal cells.

A "differentiation agent," as used in this disclosure, refers to a collection of compounds that are used in the culture systems of the present invention to generate differentiated cells of the neural lineage (including precursor cells and terminally differentiated cells). There is no intended limitation on the mode of action of the compounds. For example, the agent may assist the differentiation process by inducing or assisting a change in phenotype, promoting the growth of cells with a particular phenotype or delaying the growth of other cells, or acting in concert with other agents through an unknown mechanism.

The prototype "primate pluripotent stem cells" (pPS cells) are pluripotent cells derived from pre-embryonic stage, embryo or fetus at any time after fertilization and are characterized by the ability to produce progeny of several different cell types, derivatives of all three germ layers (endoderm, mesoderm and ectoderm), according to standard art-accepted assays, such as the ability to form teratomas in 8-12 week old SCID mice. Included in pPS cells are various types of embryonic cells, such as human embryonic stem (hES) cells, and human embryonic germ cells (hEG), as defined. The pPS cells are preferably not derived from a malignant source. It is desirable (but not always necessary) that the cells be euploid.

pPS cell cultures are described as "undifferentiated" when the majority of stem cells and their derivatives in the population exhibit morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryonic or adult origin. It will be appreciated that colonies of undifferentiated cells within a population are often surrounded by adjacent differentiated cells.

"feeder cells" or "feeder" are terms used to describe one type of cell co-cultured with another type of cell to provide an environment in which a second type of cell can grow. The pPS cell population is said to be "substantially free" of feeder cells if these cells are grown at least one round after division without the addition of fresh feeder cells to support the growth of the pPS cells.

The term "embryoid body" refers to aggregates of differentiated and undifferentiated cells that appear when pPS cells are overgrown on monolayer cultures or maintained in suspension cultures. Embryoid bodies are a mixture of different cell types, typically from several germ layers, that can be distinguished by morphological criteria and the use of immunocytochemically detectable cell markers.

A "growth environment" is an environment in which a cell of interest can proliferate, differentiate, or mature in vitro. The characteristics of the environment include the medium in which the cells are cultured, the presence of any growth or differentiation inducing factors, and, if present, a support structure (e.g., a substrate on a solid surface).

When a polynucleotide is transferred to a cell by any suitable means of manual manipulation, or wherein the cell is the progeny of an originally altered cell that has inherited the polynucleotide, the cell is said to be "genetically altered", "transfected" or "genetically transformed". The polynucleotide often comprises a transcribable sequence that encodes the protein of interest, which enables the cell to express the protein at elevated levels. A genetic alteration is "heritable" if progeny of the altered cell have the same alteration.

General techniques

General methods of Molecular genetics and genetic engineering are described in recently published handbook of Molecular Cloning: A Laboratory Manual (Sambrook et al, Cold Spring Harbor); mammalian Gene Transfer Vectors (Gene Transfer Vectors for Mammalian Cell), eds (Miller & Calos); and Current Protocols in molecular biology (F.M. Ausubel et al eds., Wiley & Sons). Cell biology, Protein chemistry, and antibody technology can be found in Current protocols in Protein Science (J.E. Colligan et al, Wiley & Sons); current Protocol in cell Biology (J.S. Bonifacino et al, Wiley & Sons) and Current Protocol in Immunology (J.E. Colligan et al, Wiley & Sons).

Cell culture methods are generally described in recently published animal cell cultures: basic technical Manual (published of Animal Cells: A Manual of Basic technical) (eds., R.I. Freshney, Wiley & Sons); general Techniques of Cell Culture (General technologies of Cell Culture) (m.a. harrison & i.f. rae, Cambridge univ. press), and embryonic stem cells: methods and procedures (Embryonic StemShell: Methods and Protocols) (K.Turksen, eds., Humana Press).

For a detailed description of the abnormalities of the nervous system, as well as the characterization of various types of neural cells, markers and associated soluble factors, the reader is referred to "CNS regeneration: basic Science and Clinical Advances (CNS Generation: Basic Science and Clinical Advances), M.H.Tuszynski & J.H.Kordower, Academic Press, 1999. The management and feeding of neural cells is described in neuron: third edition in Cell and Molecular Biology (The Neuron: Cell and Molecular Biology), i.b. levitan & l.k.kaczmark, Oxford u.press, 2001; and Neuron in tissue culture (The Neuron in tissue culture), L.W.Haynes eds, John Wiley & Son Ltd, 1999.

Sources of stem cells

The present invention can be carried out using various types of stem cells. Particularly suitable for use in the present invention are primate pluripotent stem (pPS) cells derived from tissue formed after pregnancy, such as a blastocyst, or a fetus or embryonic tissue taken at any time during pregnancy. Non-limiting examples are primary cultures or established cell lines of embryonic stem cells or embryonic germ cells, as described below. The techniques of the present invention can also be performed directly with primary embryonic or fetal tissue, deriving neural cells directly from primary embryonic cells, without the need to initially establish an undifferentiated cell line.

Embryonic stem cells can be isolated from blastocysts of primate members (U.S. Pat. No. 5,843,780; Thomson et al, Proc. Natl. Acad. Sci. USA 92: 7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al (U.S. Pat. No. 6,200,806; Science 282: 1145, 1998; Curr. Top. Dev. biol. 38: 133ff., 1998) and Reubinoff et al (Nature Biotech.18: 399, 2000). Cell types equivalent to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, described in WO 01/51610 (Bresagen).

Human embryonic germ (hEG) cells can be prepared from primordial germ cells present in human fetal material taken about 8-11 weeks after the last menstruation. Suitable methods of preparation are described in Shamblott et al, proc.natl.acad.sci.usa95: 13726, 1998 and us patent 6,090,622.

PPS cells can be propagated continuously in culture using culture conditions that promote proliferation, rather than differentiation. A typical serum-containing ES medium is prepared with 80% DMDM (e.g., knockout DMEM (knock-out DMEM), Gibco), 20% or defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO 98/30679), 1% nonessential amino acids, 1mM L-glutamine and 0.1mM beta-mercaptoethanol. Human bFGF was added to 4ng/ml before use (WO 99/20741, Geron Corp.).

Conventionally, ES cells are cultured on a layer of feeder cells, typically a mixed cell population derived from embryonic or fetal tissue (U.S. Pat. No. 6,200,806). Scientists at Geron found that pPS cells remained undifferentiated even without feeder cells. Can be on an extracellular matrix (e.g., Matrigel)Or laminin) are supported in a feeder cells-free culture and cultured in a nutrient medium containing factors that support proliferation of cells but not differentiation. Typically, conditioned medium is obtained by pre-culturing cells secreting such factors, e.g., irradiated primary mouse embryonic fibroblasts (or derived from human embryonic stem cells)Fibroblast-like cells of (4), 8ng/ml of basic FGF is added before and after the adjustment. Under microscopy, ES cells exhibit high nuclear/cytoplasmic ratios, significant nucleoli and compact population formation, typically expressing characteristic phenotypic markers such as SSEA 3 and 4. Further details regarding the management and feeding of embryonic stem cells are provided in international patent applications WO 99/20741 and WO 01/51616.

Some of the techniques described in the present invention may also be used to maintain or advance the differentiation of nerve cells or nerve precursors obtained from fetal or adult tissue (U.S. Pat. Nos. 5,852,832; 5,654,183; 5,849,553; and 5,968,829; and WO09/50526 and WO 99/01159). Unless otherwise specified, the invention can be practiced using cells of any vertebrate species, including humans, non-human primates, livestock animals and other non-human mammals.

Materials and procedures for preparing neural precursors and terminally differentiated cells

The neural progenitor cells and mature neurons of the invention can be exemplified by differentiated stem cells using appropriate differentiation.

Typically, the differentiation process is carried out in a culture environment containing a suitable matrix and a nutrient medium to which differentiation agents are added. Suitable substrates include solid surfaces coated with a positive charge, examples being poly-L-lysine and polyornithine. The matrix may be coated with extracellular matrix components, examples being fibronectin and laminin. Other permissible extracellular matrices include Matrigel(extracellular matrix from Engelbreth-Holm-Swarm tumor cells). Also suitable are composite substrates, such as poly-lysine binding laminin, laminin or both.

The neural lineage cells of the invention are cultured on media that supports proliferation or survival of the desired cell type. It is often necessary to use a defined medium that provides nutrients such as free amino acids rather than serum. It is also beneficial to add additives to the culture medium that are developed for the sustained culture of neural cells. Examples are N2 and B27 additives, commercially available from Gibco.

The progression of cells along the neural differentiation pathway is facilitated by inclusion in a culture medium, which is a mixture of differentiation agents that increase the growth of the desired cell type. This may involve directing the cells or their progeny to accept a phenotypic characteristic of the differentiated cell type, promoting the growth of cells with the desired phenotype or inhibiting the growth of other cell types. Understanding the mode of action of the reagents is generally not necessary for the practice of the invention.

Suitable differentiation agents include various types of growth factors such as Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF- α), any type of fibroblast growth factor (examples are FGF-4, FGF-8, and basic fibroblast growth factor bFGF), Platelet Derived Growth Factor (PDGF), insulin-like growth factor 1(IGF-1 and others), high concentrations of insulin, sonic hedgehog, members of the neurotrophin family (e.g., nerve growth factor NGF, neurotrophin 3 NT-3, brain derived neurotrophin BDNF), bone morphogenetic proteins (particularly BMP-2 and BMP-4), Retinoic Acid (RA), and ligands for receptors mixed with gp130 (e.g., LIF, CNTF, and IL-6). Still other suitable differentiating agents are another ligand and antibody that binds to the respective cell-surface receptors for the aforementioned factors. Typically, a number of differentiation agents are used, which may contain 2, 3, 4 or more of the agents listed above or described in the examples below.

In one differentiation method, pPS cells are plated directly on a suitable substrate, such as an adherent glass or plastic surface, e.g., a glass coverslip coated with polylysine, with or without neuron-friendly matrix proteins such as fibronectin and laminin. The cells are then cultured in a suitable nutrient medium suitable for promoting differentiation into neural cells. This is referred to as a "direct differentiation" method and is further described in international patent application WO01/51616 and U.S. patent application 09/888,309 with priority. Antagonists of the TGF-. beta.superfamily, such as noggin and follistatin, are particularly useful in directing neural differentiation and increasing the proportion of cells with phenotypic characteristics of neural cells obtained by direct differentiation (example 4).

In another differentiation method, pPS cells are first pre-differentiated into a heterogeneous cell population by forming cell clusters. In a typical variation, embryoid bodies are formed by culturing pPS cells in suspension. Optionally, one or more of the previously listed differentiation agents (e.g., retinoic acid) may be included in the culture medium to promote differentiation within the embryoid bodies. After the embryoid bodies reach sufficient size or mature (typically 3-4 days), they are plated on a differentiation-cultured substrate. Embryoid bodies can be plated directly onto the substrate without dispersing the cells. This allows neuronal cell precursors to move out of the embryoid body and onto the extracellular matrix. In some steps, the cells are first cultured in a mitogen mixture, such as EGF, bFGF, PDGF, and IGF-1, and then passaged in a mitogen and neurotrophin composition to select neural progenitor cells.

The present invention includes strategies for identifying factor compositions effective to produce a particular neural phenotype. Various factors known or suspected to enhance neural differentiation or growth are classified into various functional classes based on known effects on neural cells from other tissues or species, known receptor binding activity, structural homology to other factors of known function, or other appropriate criteria. The factors in each category are aggregated at the appropriate working concentration. The cells were then cultured with each factor individually in various combinations, and the factors were evaluated for their ability to promote growth of precursor cells or the desired type of mature neuron. The identification of the essential classes of factors, when these factors are missing, results in the admixture losing its ability to promote the desired phenotype. Once the essential factors are identified and the other factors are removed, the individual classes are carefully analyzed by removing individual components until a minimal mixture is identified. The implementation of this strategy is illustrated in example 4.

If desired, the differentiated cells can be sorted to enrich for certain populations. For example, cells can be contacted with an antibody or ligand that binds to a marker feature of neural cells (e.g., NCAM), followed by isolation of specifically recognized cells using appropriate immunological techniques, such as solid phase adsorption or fluorescence-activated cell sorting. Also suitable are differential plating or harvesting techniques in which the desired cell type is used for adherent or releasable separation from other cells of a heterogeneous population.

It has been found that neural precursor phenotypes can be passaged in proliferating cultures using a combination of mitogens (e.g., bFGF and EGF) plus one or more neurotrophins (e.g., BDNF, NT-3, or both). This is illustrated in examples 2, 4 and 5. According to the method, the cells can be passaged up to 40-fold (FIG. 3), while maintaining the ability to proliferate and to make mature neurons.

Committed progenitor cells are assumed to be of particular value in human therapy because they are more flexible to manipulate and will retain a greater ability to migrate to the target tissue and integrate in a functionally compatible fashion. Progenitor cells can be grown on a solid surface or in suspension culture as illustrated in example 5, where they tend to form clusters or spherical structures. By way of illustration, neural progenitor cells are collected using trypsin when fusion is approached. Cells were then seeded at approximately half density in non-adherent wells and cultured in supplemented media containing 10ng/ml BDNF, NT-3, EGF and bGFG, with 3 changes per week.

Judicious selection of other components of the culture medium during the derivation or maintenance of neural progenitor cells can affect the range and characteristics of mature cells that they are capable of producing. As described in example 4, retinoic acid included in the medium during direct differentiation of neural progenitor cells increases the proportion of MAP-2 cells produced upon final differentiation-but decreases the proportion of cells positive for Tyrosine Hydroxylase (TH) associated with dopaminergic neurons. On the other hand, it was found that Erythropoietin (EPO) or an agent for increasing the level of cyclic AMP contained in a medium during the formation of neural progenitor cells is enhancedThe ability to form TH positive neurons is established. Alternatively, the cells may be cultured with certain antibodies or antagonists that activate the EPO pathway, or the cells may be cultured under conditions of moderate hypoxia (low O)₂Level means 3-6%). The use of EPO to increase the formation of the dopaminergic phenotype is described in example 3.

The neural precursor cells prepared according to any of these steps can be further differentiated into mature neurons. Fully differentiated cells are desirable for various applications of the invention, such as in vitro evaluation and screening of the effects of various compounds on neural tissue. It is also useful for fully differentiated cells to characterize the functionality of neural progenitor cells that give rise to fully differentiated cells.

Mature neurons can be formed by culturing neural precursor cells with maturation factors such as forskolin (or other compounds that elevate intracellular levels of cAMP, such as cholera toxin, isobutylmethylxanthine, dibutyladenosine cyclic monophosphate), c-kit ligand (c-kit ligand), retinoic acid, or any factor or combination of factors from the neurotrophin family. Particularly effective is the binding of neurotrophin-3 (NT-3) to Brain Derived Neurotrophic Factor (BDNF). Other candidates are GDNF, BMP-2 and BMP-4. In addition, maturation may be enhanced by removing some or all of the factors that promote proliferation of neural precursors, such as EGF, FGF or other mitogens that were previously used to maintain culture.

Further modifications being feasible

The neural cell precursor population of the present invention has a considerable proliferative capacity. Replication can be further enhanced, if desired, by increasing the level of telomerase reverse transcriptase (TERT) in the cell, or by increasing transcription from endogenous genes, or by introducing transgenes. Particularly suitable is the catalytic component of human telomerase (hTERT) as proposed in international patent application WO 98/14592. Transfection and expression of telomerase in human cells is described in Bodnar et al, Science 279: 349, 1998 and Jiang et al, nat. genet.21: 111, 1999. Genetically altered cells can be assessed for hTERT expression, telomerase activity (TRAP assay), immunocytochemical staining or replicative capacity of hTERT by TR-PCR according to standard methods.

For use in therapeutic and other applications, it is often desirable that the population of precursor or mature neural cells be substantially free undifferentiated pPS cells. One way to reduce undifferentiated stem cells from a population is to transfect them with a vector in which an effector gene under the control of a promoter causes preferential expression by the undifferentiated cells. Suitable promoters include the TERT promoter and the OCT-4 promoter. The effector gene may be directly solubilized in the cell (e.g., encoding a toxin or mediator of apoptosis). In addition, the effector gene may sensitize the cell to the toxic effects of an external agent, such as an antibody or a prodrug. A typical example is the herpes simplex thymidine kinase (tK) gene, which causes guanine sensitivity in cells in which it is expressed. Suitable pTERT-tK constructs are described in International patent application WO 98/14593(Morin et al).

Characteristics of neural precursors and terminally differentiated cells

Cells can be characterized according to a number of phenotypic criteria, such as morphological features, detection or quantification of expressed cellular markers, enzymatic activity, or neurotransmitters and their receptors and electrophysiological functions.

Certain cells included in the invention have morphological characteristics of neural cells or glial cells. These features are readily understood by those skilled in assessing the presence of such cells. For example, neurons are characterized by small cell bodies, most of which have axons and dendrites. The cells of the invention may also be characterized according to whether they express phenotypic markers characteristic of various neural cells.

Markers of interest include, but are not limited to, β -tubulin III, microtubule-associated protein 2(MAP-2), or neurofilament, characteristic of a neuron; glial Fibrillary Acidic Protein (GFAP) present in astrocytes; galactocerebroside (GalC) or Myelin Basic Protein (MBP), a characteristic of oligodendrocytes; oct-4, a characteristic of undifferentiated hES cells; and characteristics of stem proteins, neural precursors and other cells. A2B5 (a glycolipid) and polysialylated (polysialylated) neural cell adhesion molecules (abbreviated NCAM) have been described. While A2B5 and NCAM are indicative markers when studying neural lineage cells, it is understood that these markers may sometimes be displayed on other cell types, such as liver or muscle cells. Beta-tubulin III was previously thought to be specific for neural cells, but a sub-population of hES cells was also found to be beta-tubulin III positive. MAP-2 is a more stringent marker for all types of differentiated neurons. Certain cell populations prepared according to the invention contain at least 30%, 50%, 75%, 90% or more cells that test positive for these markers, either singly or in various combinations.

Tissue-specific markers listed in the present disclosure and known in the art can be detected using any suitable immunological technique-e.g., flow immunocytochemistry for cell surface markers, immunohistology for intracellular or cell surface markers (e.g., immunohistology of fixed cells or tissue sections), western blot analysis of cell extracts, and enzyme-linked immunoassay for cell extracts or products secreted into the culture medium. Expression of an antigen by a cell is referred to as a "detectable antibody" if, optionally after cell immobilization, and optionally with amplification of the label using a labeled secondary antibody or other conjugate (e.g., a biotin-avidin conjugate), a detectable amount of antibody, apparent in standard immunocytochemistry or flow cytometry analysis, will bind to the antigen.

Expression of tissue-specific gene products can also be detected at the mRNA level by northern blot analysis, dot blot hybridization analysis, or polymerase chain reaction (RT-PCR) initiated by reverse transcriptase using sequence-specific primers in standard amplification methods. See U.S. Pat. No. 5,843,780 for a more detailed description. Sequence data for specific markers listed in this disclosure can be obtained from public databases such as GenBank (URL www.ncbi.nlm.nih.gov: 80:/entrez). Expression at the mRNA level is said to be "detectable" if analysis of a cell sample according to standard procedures in a typical control assay yields a clearly identifiable hybridization or amplification product, according to one of the analytical methods described in this disclosure. Expression of a tissue-specific marker is considered positive when measured at the protein or mRNA level if the expression level is at least 2-fold, and preferably 10 or 50-fold greater than that of a control cell, such as an undifferentiated pPS cell, fibroblast, or other unrelated cell.

Also characteristic of neural cells, in particular terminally differentiated cells, are receptors and enzymes involved in biosynthesis, release, and reuptake of neurotransmitters, and ion channels involved in depolarization and repolarization events associated with synaptic transmission. Evidence of synapse formation can be obtained by staining of synaptic vesicle proteins. Evidence for the sensitivity of certain neurotransmitters can be obtained by detecting receptors for gamma-aminobutyric acid (GABA), glutamic acid, dopamine, 3, 4-Dihydroxyphenylalanine (DOPA), norepinephrine, acetylcholine, and serotonin.

Differentiation of particular neural precursor cell populations of the invention (e.g., using NT-3 and BDNF) can result in at least 20%, 30%, or 40% MAP-2 positive cell populations. A substantial proportion, e.g., 5%, 10%, 25% or more, of cells positive for NCAM or MAP-2 (based on cell count) will be able to synthesize neurotransmitters, e.g., acetylcholine, glycine, glutamate, norepinephrine, serotonin or GABA. Certain cell populations of the invention contain NCAM or MAP-2 positive cells, of which 1%, 5%, 10% or more are positive for Tyrosine Hydroxylase (TH) -either as a percentage of NCAM or MAP-2 positive cells, as determined by immunocytochemistry or mRNA expression-or all of the cells present in the population. TH is generally recognized in the art as a marker for dopaminergic cells.

To further elucidate the mature neurons that are present in the differentiated population, cells can be assayed according to functional criteria. For example, calcium flux can be measured using any standard technique, response to neurotransmitters, or other environmental conditions known to affect neurons in vivo. First, neuron-like cells in the population are identified by morphological criteria, or by markers such as NCAM. The neurotransmitter or condition is then applied to the cell, and the response is monitored. Standard patch clamp techniques can also be applied to the cells to determine if there is evidence of an action potential and what the lag time between the applied potential and the reaction is.

The cell populations and isolated cells of the invention herein derived from established pPS cell lines can be characterized as having the same genome as the cell lines from which they are derived. This means that there will be more than 90% identity of chromosomal DNA between pPS cells and neural cells, which can be inferred if the neural cells are obtained from undifferentiated cell lines by normal mitotic processes. Since all non-manipulated genetic elements are retained, neural cells treated by recombinant means to introduce a transgene (e.g., TERT) or knock out an endogenous gene are still considered to have the same genome as the cell line from which they are derived.

Use of neural precursors and terminally differentiated cells

The present invention provides methods for producing large quantities of neural precursor cells and mature neurons and glial cells. These cell populations can be used for important research, development and commercial purposes.

The cells of the invention can be used to prepare a cDNA library that is relatively uncontaminated by cDNA that is preferentially expressed in cells from other lineages. For example, pluripotent neural progenitor cells are collected by centrifugation at 1000rpm for 5 minutes, followed by preparation of mRNA, reverse transcription, and optionally subtraction of cDNA from mature neurons, astrocytes, or oligodendrocytes or undifferentiated astrocytes. The expression pattern of neurons can be compared to other cell types by microarraying analysis, generally referred to Fritz et al, Science 288: 316, 2000; "Microarray Biochip Technology", L Shi, www.Gene-chips.

The differentiated cells of the invention may also be used to prepare antibodies specific for markers of multipotent neural progenitor cells, cells belonging to the neuronal or glial lineage, and mature neurons, astrocytes and oligodendrocytes. Polyclonal antibodies can be prepared in an immunogenic form by injecting a vertebrate with cells of the invention. The production of monoclonal antibodies is described, for example, in Harrow and Lane (1988), U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B: 3 (1981).

Applications of commercial interest include the use of cells for screening small molecule drugs, and the preparation of pharmaceutical compositions containing neurons for clinical therapy.

Drug screening

The neural precursor cells of the 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 affect characteristics of the neural precursor cells and their various progeny.

In some applications, pPS cells (undifferentiated or differentiated) are used to screen for factors that promote neural cell maturation or promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation or growth factors can be assayed by adding them to the cells in different wells, and then determining any phenotypic changes obtained, further culturing and using the cells according to the desired criteria.

Other screening applications of the invention involve testing the effect of a drug compound on neural tissue or neural conduction. The screening may be performed either because the contemplated compound has a pharmacological effect on the nerve cells or because the contemplated compound has an effect elsewhere, possibly with unexpected side effects on the nervous system. Any neural precursor cell or terminally differentiated cell of the invention may be used for screening, for example dopaminergic, 5' -hydroxytryptaminergic, cholinergic, sensory and motor neurons, oligodendrocytes, and astrocytes.

The reader is generally referred to standard textbooks In vitro Methods for drug Research (In vitro Methods In pharmaceutical Research), Academic Press, 1997, and U.S. Pat. No. 5,030,015. Evaluation of the activity of a candidate pharmaceutical compound generally involves combining the differentiated cells of the invention with the candidate compound, either alone or in combination with other drugs. The investigator measures any change in the morphology, marker phenotype or functional activity of the cells attributable to the compound (as compared to untreated cells or cells treated with an inactive compound) and then correlates the effect of the compound with the observed change.

Cytotoxicity was first determined by the effect on cell viability, survival, morphology and expression of certain markers and receptors. The effect of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. In particular, at an irregular number of times in the cell cycle, or at a level exceeding that required for cell replication³H]Incorporation of thymidine or BrdU is consistent with the action of the drug. Side effects may also include abnormal rates of sister chromatid exchange as measured by metaphase diffusion. For further details, the reader is referred to In A.Vickers, In vitro Methods for drug Research (In vitro Methods In Pharmaceutical Research) page 375-.

The effect of cell function can be assessed by observing the phenotype or activity of the neural cell using any standard assay, such as receptor binding, neurotransmitter synthesis, release or uptake, electrophysiology, and growth of neuronal processes and myelin-either in cell culture or in an appropriate model. For example, the ability of a drug to alter synaptic contacts and plasticity can be measured in culture by immunocytochemical staining of synapses or synaptic vesicle proteins. Electrophysiology can be assessed by measuring IPSP and EPSP (inhibition and stimulation of postsynaptic potentials). In addition, using a two-electrode system, one cell is stimulated and the response of a second cell in the system is assessed. The behavior of the system with the candidate drug present is compared to the behavior of the system without the drug present and correlated with the ability of the drug to affect synaptic contacts or cellular plasticity.

Therapeutic uses

The invention also provides for the use of neural precursor cells to restore a degree of Central Nervous System (CNS) function to a subject in need of such treatment, which may be as a result of a congenital disorder of function, the effect of a disease, or trauma.

To determine the suitability of neural precursor cells for therapeutic administration, the cells can first be tested in an appropriate animal model. First, the cells were evaluated for their ability to survive and maintain their phenotype in vivo. Neural precursor cells are administered to an immunodeficient animal (e.g., a nude mouse, or an animal rendered immunodeficient by chemical or radiation) at an observable site, e.g., in the brain cavity or spinal cord. Tissues were collected over a period of days to weeks or more and evaluated for the presence of pPS derived cells.

This can be pre-labeled by administering expression (e.g., with BrdU or [ 2 ]³H]Thymidine), a detectable marker (e.g. green fluorescent protein, or β -galactosidase); or by subsequent detection of constituent cellular markers (e.g., using human-specific antibodies). In testing neural precursor cells in rodent models, the presence and phenotype of the cells administered can be assessed by immunohistochemistry or ELISA using human specific antibodies, or by RT-PCR analysis using primers and hybridization conditions that result in amplification specific to human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in the disclosure.

Various animal models for testing nervous system function are described in CNS regeneration: basic Science and Clinical Advances (CNS Regeneration: Basic Science and Clinical Advances), M.H. Tuszynski and Kordower eds, Academic Press, 1999. Parkinson's disease can model the major dopamine pathway in the brain in rats by trauma-induced impairment of the nigrostriatal striatum. Another standard animal model is chemical injury of dopaminergic neurons in the substantia nigra of mice or non-human primates with MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine). In Furns et al, proc.natl.acad.sci.usa 80: 4546, 1983; free et al, appl. neurophysiol.47: 16, 1984; and Bjorklund et al, proc.natl.acad.sci.usa 19: 2344, 2002.

The differentiated cells of the invention may also be used for tissue reconstruction or regeneration in a human patient in need of treatment. These cells are administered in a manner that allows them to be transplanted or migrated to a predetermined tissue site and to reconstitute or regenerate a functionally deficient region. By way of illustration, neural stem cells are transplanted directly to parenchymal or intrathecal sites of the central nervous system, depending on the disease being treated. Transplantation was performed using either a single cell suspension or small aggregates with a density of 25,000-500,000 cells/. mu.L (U.S. Pat. No. 5,968,829). Certain neural progenitor cells encompassed by the present invention are designed for the treatment of acute or chronic injuries to the nervous system. For example, excitotoxicity is affected by a variety of conditions including epilepsy, stroke, ischemia, and alzheimer's disease. Dopaminergic neurons can be formulated for the treatment of parkinson's disease, gabaergic neurons for the treatment of huntington's disease, and motor neurons for the treatment of spinal cord injury or Amyotrophic Lateral Sclerosis (ALS).

According to the present invention, the neural progenitor cells and terminally differentiated cells may be supplied in the form of a pharmaceutical composition containing an isotonic excipient prepared under substantially sterile conditions for administration to humans. The reader is referred to cell therapy, compiled by g.morstyn and w.sheeridan: stem Cell Transplantation, gene Therapy and Cellular Immunotherapy (Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy), Cambridge university Press, 1996; and Hematopoietic Stem Cell Therapy (hematopoetic Stem Cell Therapy), e.d. ball, j.list and p.law, Churchill Livingstone, 2000.

The composition may optionally be packaged in a suitable container on which instructions for a desired purpose, such as the reconstitution of CNS function to ameliorate certain neurological abnormalities, are written.

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

Examples

Example 1: differentiation of embryonic stem cells into mature neurons

Human embryonic stem cells (hES) were obtained from cultures without feeder cells as previously described (AU 729377; WO 01/51616). Embryoid bodies were produced as follows. Confluent monolayer cultures of hES cells were obtained by incubation in 1mg/ml collagenase for 5-20 minutes, followed by scraping the cells from the plates. Cells were then separated into clusters and plated in non-adherent cell culture plates (Costar) in medium containing 80% KO ("knock-out") dmem (gibco) and 20% non-heat-inactivated fbs (hyclone), supplemented with 1% non-essential amino acids, 1mM glutamine, 0.1mM β -mercaptoethanol. Cells were seeded in 2ml of medium per well (6 well plates) at a ratio of 1: 1 or 1: 2.

After 4 days in suspension, embryoid bodies were plated on fibronectin cell-coated plates in defined medium supplemented with 10ng/mL human EGF, 10ng/mL human bFGF, 1ng/mL PDGF-AA and 1ng/mL human IGF-1. Embryoid bodies adhere to the plate and cells begin to migrate to the plastic, forming a monolayer.

After 3 days, many cells with neuronal morphology were observed. Neural precursors were identified as cells positive for BrdU incorporation, dry protein staining, and lineage-specific differentiation marker deficiency. Putative neuronal and glial progenitor cells were identified as positive for polysialylated NCAM and A2B 5. 41-60% of the cells expressed NCAM and 20-66% of the cells expressed A2B5 as determined by flow cytometry. A sub-population of NCAM-positive cells was found to express beta-tubulin III and MAP-2. There is no co-localization with glial markers such as GFAP or GalC. A2B5 positive cells appear to produce neurons and glia. A sub-population of A2B5 cells expressed beta-tubulin III or MAP-2, while another isolated sub-population expressed GFAP. Some cells with neuronal morphology double stained A2B5 and NCAM. Both the NCAM positive and A2B5 positive populations contained far more neurons than glia.

The cell population was further differentiated by repeated plating of the cells in media without mitogens, but containing 10ng/mL neurotrophin-3 (NT-3) and 10ng/mL Brain Derived Neurotrophic Factor (BDNF). Neurons with a large number of processes were observed after about 7 days. Cultures derived from embryoid bodies preserved in Retinoic Acid (RA) showed more MAP-2 positive cells (approximately 26%) than those derived from embryoid bodies preserved in the absence of RA (approximately 5%). GFAP positive cells were observed to be spotted. GalC positive cells were identified, but the cells were large and flat without complex processes.

Assessing the presence of neurotransmitter synthesis. GABA-immunocompetent cells are identified to co-express beta-tubulin III or MAP-2 and have morphological characteristics of neuronal cells. It was identified that occasionally some GABA-positive cells did not co-express neuronal markers, but had astrocyte-like morphology. Neuronal cells were identified that expressed Tyrosine Hydroxylase (TH) and MAP-2. The formation of the protrusions was identified by staining with an antibody to the protruding vesicle protein.

TH staining was observed in cultures differentiated from the H9 cell line of human ES cells. Embryoid bodies were stored in 10 μ M retinoic acid for 4 days and then plated on fibronectin coated plates in EGF, basic FGF, PDGF and IGF for 3 days. They were then passaged on laminin in N2 medium supplemented with 10ng/mL NT-3 and 10ng/mL BDNF, and allowed to further differentiate for 14 days. Differentiated cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then imaged with antibodies for TH, a marker for dopaminergic cells.

Example 2: multiple purposeEnriched populations of dopaminergic cells

Embryoid bodies were cultured in suspension with 10 μm retinoic acid for 4 days, and then plated in defined medium supplemented with EGF, bFGF, PDGF and IGF-1 for 3-4 days. Next, the cells were isolated as A2B 5-positive or NCAM-positive enriched populations by magnetic bead sorting or immunopanning.

Immuno-selected cells were stored in defined medium supplemented with 10ng/mL NT-3 and 10ng/mL BDNF. After 14 days, 25 ± 4% of NCAM-sorted cells were MAP-2 positive-of which 1.9 ± 0.8% of the cells were GABA-positive, 3 ± 1% of the cells were positive for Tyrosine Hydroxylase (TH): the rate-limiting enzymes used for dopamine synthesis are often considered to be representative of dopamine-synthesizing cells.

In the cell population sorted for NCAM, cells positive for NCAM do not express glial markers, such as GFAP or GalC. These data indicate that populations containing neuronal restricted precursors can be isolated directly from hES cell cultures, substantially free of glial precursor impurities.

On the other hand, cells sorted for A2B5 have the ability to produce neurons and astrocytes. After enrichment, cells were placed in 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 begin to extend synapses. After two weeks, the cells exhibited 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% were GABA immunoreactive. These data indicate that cell populations, including those that synthesize dopamine, can be obtained from hES cells containing astrocyte and neuronal progenitor cells.

Further details regarding the conditions for obtaining TH-expressing neurons are provided below. Embryoid bodies were generated from confluent hES cells of the H7 cell line by culturing in 1mg/mL collagenase (37 ℃, 5-20 min) for 32 passages, scraping the culture dish, and placing the cells in non-adherent culture plates (Costar)). The obtained EBs were cultured in suspension in a medium containing FBS and 10 μ M all-trans retinoic acid. After 4 days, the aggregates were collected and allowed to settle into centrifuge tubes. The supernatant was then aspirated and the aggregates were placed in proliferation medium (DMEM/F121: 1 supplemented with N2, half strength B27, 10ng/mL EGF (R)&D system), 10ng/mL bFGF (Gibco), 1ng/mL PDGF-AA (R and D systems), and 1ng/mL IGF (R and D systems)) on poly-L-lysine and fibronectin coated plates.

EB was allowed to attach and proliferate for 3 days; then collected by trypsinization for about 1 minute (Sigma) and expressed at 1.5X 10⁵Individual cells/well density were plated on poly-L-lysine and laminin coated 4-well chambered slides in proliferation medium for 1 day. The medium was then changed to Neural Basal medium supplemented with B27, and one of the following growth mixtures:

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

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

bFGF of only 10ng/mL

Cells were maintained under these conditions for 6 days, and fed every other day. On day 7, the medium was changed to neuralabasal medium with B27, with the addition of one of the following mixtures:

10ng/mL BDNF, 10ng/Mld NT-3

1 μ M cAMP, 200 μ M ascorbic acid

1 μ M Camp, 200 μ M ascorbic acid, 10ng/mL BDNF, 10ng/mL NT-3

Cultures were fed every other day until day 12, at which time they were fixed and labeled with anti-TH or MAP-2 for immunocytochemistry. Expression of the marker was quantified by counting 4 regions in each of 3 wells using a 40X objective lens.

The results are shown in table 1. The highest proportion of TH-positive cells produced were initially cultured in bFGF, BDNF and NT-3.

Example 3: increasing the proportion of dopaminergic cells by culturing with erythropoietin

In subsequent experiments, embryoid bodies were plated on polylysine fibronectin coated wells and cultured with 10ng/mL EGF, 1ng/mL PDGF-AA, 10ng/mL bFGF and 1ng/mL IGF-1. On the fourth day, 5U/mL EPO, 700. mu.M cAMP, or both, were added to the mixture. Cells were replated and treated with 10ng/mL BDNF, 10ng/mL NT-3, and optionally EPO, cAMP, and 200 μ M ascorbic acid for 7 days. The results are shown in table 2. The proportion of total cells in culture that were MAP-2 positive was abnormally low in this assay.

These data provide initial evidence that the addition of cAMP and EPO during the neural precursor cell derivation increases the percentage of tyrosine hydroxylase expressing neurons that are ultimately obtained. Studer et al report that proliferation or differentiation of mesencephalic precursors in the presence of EPO or low partial pressure of oxygen produces higher numbers of dopaminergic neurons (j. neurosci.20: 7377, 2000). EPO is thought to have a neuroprotective effect under hypoxic conditions, driving multipotent progenitor cells toward neuronal pathways (Shingo et al, J.Neurosci, 21: 9733, 2001). This effect may be Janus kinase-2 and nuclear factor kappa (NF-_KB) Results of communication between, upregulation of Bcl-x (L) expression, or activation of the AP-1(Jun/Fos) pathway. General in pPS-derived neural cellsModulation of these pathways by other means mimics the effects of EPO.

Example 4: direct differentiation of hES cells into dopaminergic neurons

This study evaluated various examples for differentiating human ES cells into neurons without forming embryoid bodies.

A strategy was developed in which the test factors were divided into several groups based on homology and/or functional reproducibility (table 3). Grouping factors increase the likelihood that relevant activity within the group will be triggered on the ES cell population. It is hypothesized that certain factors in the mixture will initiate the differentiation cascade. When differentiation proceeds and the receptor expression characteristics of the cells change, they will begin to respond to other factors in the mixture.

Providing a complex mixture of factors continuously throughout the treatment avoids the need to precisely define how and when to alter the cellular response. When a mixture is identified that causes the desired differentiation process, it can be systematically simplified to obtain the minimum amount of optimal mixture. Upon further examination, the minimal amount of processing may eventually include one, two, three, or more of the listed factors, used simultaneously or sequentially according to empirically determined steps.

The test was performed as follows. Monolayer cultures of human ES cell lines were harvested by culturing in collagenase IV for 5-10 minutes, and the cells were scraped from the plates. Cells were detached by milling and nearly confluent with growth factor reduced MatrrielPretreated 96 well tissue culture plates in knockout DMEM (knock-out DMEN) medium (Gibco BRL) containing 2-regulated mouse embryo feeder cellsA 4 hour Knockout Serum Replacement (knock out Serum Replacement) (Gibco BRL). After 1 day of plating, the medium was replaced with Neural Basal (NB) medium (Gibco BRL) supplemented with 0.5mM glutamine, B27 supplement (Gibco BRL), and a test factor set as described below. Cells were fed daily for 11 days with fresh neural basal medium containing glutamine, B27 and test factor.

After 11 days, cells were harvested by incubation in trypsin for 5-10 minutes, replated at a 1: 6 dilution on 96-well tissue culture plates pretreated with laminin, and re-fed daily for 5 days with fresh neural basal medium containing glutamine, B27, and test factors. Cells were fixed in 4% paraformaldehyde for 20 minutes and stained with antibody for the early neuronal marker-beta-tubulin-III, the late neuronal marker-MAP-2 and tyrosine hydroxylase, an enzyme associated with dopaminergic neurons. Nuclei were labeled with DAPI and quantified by visual inspection. The results are shown in table 4.

In another experiment, cells were cultured as before in neural basal media supplemented with glutamine, B27, and test factor sets, harvested with trypsin on day 8, and replated for 5 days. The results are shown in table 5.

Some treatment paradigms induce direct differentiation of neurons. Treatments including group 5 factors (noggin and follistatin) were most effective.

FIG. 1 shows representative regions of directly differentiated cells obtained using treatments B, D and F and stained for β -tubulin-III. Based on morphology and β -tubulin-III staining, approximately 5-12% of the cells are neurons. Staining according to MAP-2, where approximately 1/3 were mature neurons. Approximately 2-5% of the total number of neurons (5-15% of MAP-2 positive neurons) also stained for tyrosine hydroxylase, consistent with a dopaminergic phenotype.

Subsequent experiments were performed to further illustrate the effect and differentiation kinetics of certain factor mixtures.

FIG. 2(A) shows the results of an assay in which the TGF-. beta.superfamily antagonists noggin and follistatin were used for different time periods. The subconfluent hES cells of the H7 cell line were treated with treatment D (except for cAMP at 700. mu.g) for 15 days. The results indicate that noggin and follistatin both contribute to neuronal differentiation and cooperate with each other. Noggin was clearly important at about 1 week (days 5-8), while follistatin was important at about 2 weeks (days 13-15), maximizing production of mature neurons instead of small axons.

Figure 2(B) shows the time course of neuronal induction using the treatment mixture containing TGF- β superfamily antagonists in table 4. FIG. 2(C) further illustrates the role of noggin and follistatin in direct differentiation. hES cells represented by the first bar were treated with factors of groups 1, 4, 6, 7, 9, 10 and 11 (Table 3) with 700. mu.M Camp, 5U/mL EPO, plus 30ng/mL FGF-8 (group 2). Indeed, β -tubulin positive neurons are not formed in the absence of noggin and follistatin. However, noggin and follistatin alone or in combination with retinoic acid induced hES cells directly by the first step of neuronal induction. It is postulated that the initial noggin/follistatin induces the production of neural progenitor cells, which can subsequently be induced to form neurons by the addition of other factors.

FIG. 2(D) shows the benefit of removing Retinoic Acid (RA) from the mixture where dopaminergic neurons are desired. Cells were differentiated according to treatment F (2 bars on the left) or removal of retinoic acid (2 bars on the right) as described previously. The inclusion of retinoic acid slightly increased the percentage of β -tubulin positive neurons, but decreased the proportion of those neurons that stained positively for tyrosine hydroxylase.

Example 5: proliferative regeneration of neural precursors by serial passaging

The neural progenitor cells of the invention can be passaged and expanded in culture, demonstrating some of their unique and beneficial properties.

In a typical example, human embryonic stem cells are harvested and placed in suspension culture to form embryoid bodies in knockout DMEM containing 20% FBS plus 10 μ M retinoic acid. After 4 days, embryoid bodies were plated on poly-L-lysine/laminin coated plates in DMEM/F12 medium supplemented with N2 supplement, half the usual amount of B27, 10ng/mL human EGF, 10ng/mL human bFGF, 1ng/mL human PDGF-AA and 1ng/mL human IGF-1 in DMEM/F12 medium.

Cells were cultured for 3 days and harvested by simple trypsin treatment as follows. 0.5mL of 0.5% trypsin in 0.53mM EDTA (Gibco #25300-054) was plated in each well of a 6-well plate and immediately removed from the plate. After waiting 15 seconds (room temperature), the neural basal medium supplemented with B27 additive was placed in the wells and then removed and centrifuged to recover the released cells (between 1 and 10% of the cells).

6 well plates were coated with 1 mL/well of 15. mu.g/mL poly-L-lysine (Sigma #1274) followed by 1 mL/well of 20. mu.g/mL human placental laminin (Gibco #23017-015) overnight. Cell particles obtained from different trypsinizations were resuspended in neural basal media containing B27 supplement, 10ng/mL NT-3 and 10ng/mL BDNF and plated at 500,000 and 750,000 cells/well on coated wells.

After 5 days, cells were recovered by complete trypsinization, counted and replated with 100,000-500,000 cells/well in new poly-lysine/laminin coated wells with various factor mixtures. The concentrations used were as follows: NT-3 at 10ng/mL, BDNF at 10ng/mL, human EGF at 10ng/mL, human bFGF at 10ng/mL, or LIF at 10ng/mL for each combination. Medium was changed three times a week, half each time for feeder cells. Every 7 days, cells were trypsinized, counted, and passaged again in fresh medium containing the same factors.

FIG. 3(A) shows the growth curve of this experiment. Cells passaged only in BDNF and NT-3 stopped growing after about 1 week, differentiating mainly into neurons. However, addition of EGF and bFGF to the medium allows the cells to continue to proliferate as precursors. Marker characteristics of these cells are shown in table 5.

Thus, cells passaged in a combination of BDNF, NT-3, EGF and bFGF express the neural progenitor cell markers stem protein and NCAM in large quantities.

FIG. 3(B) shows the results obtained when these cells were induced to undergo final differentiation only in BDNF and NT-3. Cells passaged in a combination of BDNF, NT-3, EGF and bFGF produced more neurons upon final differentiation, consistent with a higher proportion of neural precursors prior to differentiation.

FIG. 4(A) shows the proportion of cells staining positive for tyrosine hydroxylase. Again, the combination of BDNF, NT-3, EGF and bFGF in the tested compositions provided the optimal harvest yields.

FIG. 4(B) shows that even more TH-positive neurons could be produced by inducing terminal differentiation not only by BDNF and NT-3, but also by including added factors such as NT-4, nerve growth factor, ascorbic acid, cAMP and dopamine (at the concentrations shown in Table 3). Cells in the population corresponding to 5% of the total number of cells exhibit the phenotype of the dopaminergic marker.

Neural progenitor cells from the H7 hES cell line were replaced with 30% serum containing B27 supplementNeural basal medium of surrogate and 10% DMSO was frozen (5X 10) at passage 10⁵Cell/cryovial). Approximately 6 cells were thawed after half a month. Thawed cells have many of the same characteristics as they had prior to freezing: 60-80% of beta-tubulin and MAP-2 are positive, and about 5% are positive for tyrosine hydroxylase.

In a related experiment, cells were grown and passaged in clusters, rather than on culture medium. Neural progenitor cells were harvested from 6-well plates (approximately 3 or 4X 10) using trypsin when fusion was approached⁵Cells/well). Then they were mixed at about 2.5X 10⁵Cells/well were seeded in non-adherent wells and cultured in 2mL of neural basal medium containing B27 supplement, 10ng/mL BDNF, 10ng/mL EGF and 10ng/mL bFGF. The cells were fed by changing half of the medium the following day and continued to culture for 4 days. They were then differentiated in medium containing 10ng/mL BDNF and 10ng/mL NT-3 but no mitogen.

Adaptation of the invention described in this disclosure is a matter of routine optimization and can be made without departing from the spirit of the invention or the scope of the claims below.

Claims (10)

1. A method of preparing a population of neuronal precursor cells, the method comprising:

a) culturing a cell population comprising pPS cells in a medium comprising one or more additional antagonists of the TGF- β superfamily, and

b) harvesting a population of cells from the culture, wherein at least 50% of the cells express polysialylated NCAM or β -tubulin III.

2. The method of claim 1, wherein the culture medium further comprises one or more neurotrophins and one or more mitogens.

3. The method of claim 1, wherein the cell population is produced by plating pPS cells onto a solid surface without forming embryoid bodies or cell aggregates.

4. The method of claim 1, wherein the cell population is produced by culturing a cell population comprising pPS cells in a medium comprising noggin and follistatin.

5. A method of producing differentiated cells, comprising culturing a population of neuronal precursor cells prepared by the method of any one of claims 1-4 in a medium comprising one or more factors selected from the group consisting of neurotrophins, cAMP, and ascorbic acid, in the absence of added mitogen.

6. A medicament comprising a population of cells produced by the method of any one of claims 1 to 5 for use in the treatment of the human or animal body by surgery or therapy.

7. Use of a population of cells produced by the method of any one of claims 1-5 in the preparation of a medicament for reconstituting or supplementing Central Nervous System (CNS) function in an individual.

8. Use of a cell population made by the method of any one of claims 1-5 in the manufacture of a medicament for the treatment of parkinson's disease.

9. The method of any one of claims 1 to 5, or the medicament of claim 6, or the use of any one of claims 7 to 8, wherein the pPS cells are isolated from human blastocysts, or are progeny of such cells.

10. The method of any one of claims 1 to 5, the medicament of claim 6 or the use of any one of claims 7 to 8, wherein the pPS cells are human embryonic stem cells.