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  Pharmaceutical Patents  

 

Title:  Neural precursor cells, method for the production and use thereof in neural defect therapy
United States Patent: 
7,968,337
Issued: 
June 28, 2011

Inventors: 
Bruestle; Oliver (Meckenheim, DE)
Appl. No.: 
12/048,840
Filed: 
March 14, 2008


 

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Abstract

The invention relates to isolated and purified neural precursor cells, to methods for the generation of such precursor cells in unlimited quantities from embryonic stem cells, and to their use for the therapy of neural defects, particularly in mammals, preferably in human beings, and for the generation of polypeptides.

Description of the Invention

The invention relates to isolated and purified embryonic stem cell-derived neural precursor cells, to methods for the generation of such precursor cells in unlimited quantities, and to their use for I) the therapy of neural defects, particularly in mammals, preferably in human beings, and II) for the generation of polypeptides.

Transplantation of neural cells into the nervous systems of mammals represents a promising method for the treatment of numerous neurological diseases. In animal studies, a variety of cell populations have been grafted into the brain and spinal cord (Bjorklund, in: Molecular and Cellular Approaches to the Treatment of Neurological Disease, Raven Press, New York, 1993; Brustle & McKay, Curr. Opinion Neurobiol. 6:688-695, 1996). Recently, neural transplantation has also been applied for the clinical treatment of selected diseases, for example for the treatment of patients with Parkinsons disease (Lindvall, in: Neural transplantation in Parkinson's disease, Raven Press, New York, 1994; Olanow et al., TINS 19:102-109, 1996).

In contrast to many other organs, the mature mammalian nervous system shows only a very limited regeneration potential. This is due to the fact that precursor cells required for generating neural tissue are, with few exceptions, restricted to nervous system development. The availability of precursor cells is a key prerequisite for a transplant-based repair of defects in the mature nervous system. Thus, donor cells for neural transplants are largely derived from the embryonic brain. For example, brain tissue from up to seven human embryos is required to obtain a sufficient amount of donor tissue for the transplantation of an individual Parkinson patient. This creates enormous ethical problems, and it is questionable whether such an approach can be used for the treatment of a large number patients.

Recently there have been numerous efforts to bypass the limited availability of mammalian embryonic brain cells by in vitro proliferation of precursor cells prior to transplantation. Two major strategies were used. One method comprises the immortalization of precursor cells with oncogenes. Most of the genes used for this approach have been originally isolated from tumor tissue. These "tumor genes" are inserted into the genome of the cells and elicit continuous and ill-controlled growth (Lendahl & McKay, TINS 13:132-137, 1990).

More recent and better controlled variants of this technique employ temperature-sensitive oncogenes. This approach permits the in vitro proliferation of the cells under the "permissive" temperature. The non-permissive temperature is chosen to equal the body temperature, resulting in instability of the gene product and ceasing of proliferation after transplantation (Renfranz et al., Cell 66:713-729, 1991). However, the oncogen remains in the transplanted cells, and low activity or reactivation at a later time point cannot be entirely excluded. Newer strategies have been aiming at the removal of the oncogene after completion of the proliferation phase, using molecular biological tools (Westerman & Leboulch, Proc. Natl. Acad. Sci. USA 93:8971-8976, 1996). As all cell lines, oncogen-immortalized precursor cells exhibit a high susceptibility to chromosomal aberrations.

Another method for the in vitro proliferation of precursor cells prior to transplantation is the stimulation of proliferation with growth factors (Cattaneo & McKay, Nature 347:762-765, 1990; Reynolds & Weiss, Science 255:1707-1710, 1992; Richards et al., Proc. Natl. Acad. Sci. USA 89:8591-8595, 1992; Ray et al., Proc. Natl. Acad. Sci. USA 90:3602-3606, 1993; Kilpatrick & Bartlett, Neuron 10:255-265, 1993; Ray & Gage, J. Neurosci. 6:3548-3564, 1994; Davis & Temple, Nature 372:263-266, 1994; Vicario-Abejon et al., Neuron 15:105-114, 1995; Gosh & Greenberg, Neuron 15:89-103, 1995; Gritti et al., J. Neurosci. 16:1091-1100, 1996). It is currently unclear to what extent growth factors permit an in vitro expansion of neural precursors. First transplant studies using growth factor-treated cells show controversial results. Whereas some scientists observed a decreased ability of these cells to integrate into the host tissue (Svendsen et al., Exp. Neurol. 137:376-388, 1996), there are studies that suggest that growth factor-treated cells can incorporate into the recipient brain (Gage et al., Proc. Natl. Acad. Sci. USA 92:11879-11883, 1995).

In summary, the order of magnitude of growth factor-mediated neural precursor cell proliferation and the biological behavior of these proliferated cells following transplantation into a host nervous system are currently unclear. Oncogene-mediated cell expansion strategies carry a high risk with respect to chromosomal aberrations and potential tumor induction. The most severe disadvantage of these strategies is the fact that they both depend on the availability of brain tissue, mostly derived from embryonic donors.

Embryonic stem cells (ES cells) provide entirely new perspectives for the generation of donor cells for transplantation. ES cells were first described in mice in 1981 (Martin, Proc. Natl. Acad. Sci. USA 78:7634-7638, 1981; Evans & Kaufman, Nature 292:154-156, 1981). They can be derived, for example, from the inner cell mass of 3, 5-day-old embryos. ES cells are pluripotent and can generate all tissues and cell types. This is best reflected by the fact that ES cells injected into another blastocyst can participate in the generation of all tissues including the germ line, thereby yielding chimeric animals (Bradley et al., Nature 309:255-256, 1984). A unique feature of ES cells is the fact that in the presence of leukemia inhibitory factor (LIF) they can be maintained and proliferated in a pluripotent stage (Smith et al., Nature 336:688-690, 1988). Today, this is frequently exploited for the genetic modification of ES cells. Blastocyst injection of these engineered ES cells is then used to generate transgenic animals (Robertson et al., Nature 323:445-448, 1986). Less frequently, ES cells have been used for in vitro differentiation studies. This technique permits the study and experimental manipulation of early tissue development under controlled conditions in vitro. Meanwhile, pluripotent embryonic stem cells have been isolated from a large variety of species including rat (Iannaconne et al., Dev. Biol. 163:288-292, 1994), hamster (Doetschman et al., Dev. Biol. 127:224-227, 1988), birds (Pain et al., Development 122:2339-2348, 1996), fish (Sun et al., Mol. Mar. Biol. Biotechno. 4:193-199, 1995), swine (Wheeler, Reprod. Fertil. Dev. 6:563-568, 1994), cattle (First et al., Reprod. Fertil. Dev. 6:553-562) and primates (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844-7848, 1995). Several months following the submission of the German patent application No. 197 56 864.5 two research teams succeeded in isolating ES cells and ES cell-like stem cells from embryonic human tissue (Thomson et al., Science 282: 1145-1147, 1998; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726-13731, 1998). Other recent studies indicate that embryos and embryonic stem cells can be generated by transplanting nuclei from embryonic and mature mammalian cells into enucleated oocytes (Campbell et al., Nature 380:64-66, 1996; Wilmut et al., Nature 385:810-813, 1997).

During the last couple of years, several research groups succeeded in the in vitro differentiation of ES cells into cells of the nervous system. In most cases neural differentiation was initiated by treating aggregated ES cells with retinoic acid (Bain et al., Dev. Biol. 168:342-357, 1995; Strubing et al., Mech. Dev. 53:275-287, 1995; Fraichard et al., J. Cell Sci. 108:3181-3188, 1995; Finley et al., J. Neurosci. 16:1056-1065, 1996). Some of the cells differentiated in this manner exhibited properties of neurons (Bain et al., Dev. Biol. 168:342-357, 1995; Strubing et al., Mech. Dev. 53:275-287, 1995; Fraichard et al., J. Cell Sci. 108:3181-3188, 1995; Finley et al., J. Neurosci. 16:1056-1065, 1996) and glial cells (Fraichard et al., J. Cell Sci. 108:3181-3188, 1995). Retinoic acid-mediated induction of neural differentiation suffers from two major disadvantages. First, neural differentiation occurs only in a fraction of the cells. A sufficient purification of these neural cells has, so far, not been possible. Second, retinoic acids is a strong inducer of ES cell differentiation. Neurons and glial cells differentiated in the presence of retinoic acid have mostly developed beyond the stage of a precursor and entered a postmitotic phase. Therefore, they are of limited value for cell enrichment strategies and transplantation.

An alternative method for the generation of neural precursor from ES cells was reported recently (Okabe et al, Mech. Dev. 59:89-102, 1996). ES cells aggregated to embryoid bodies are plated and cultured in serum-free media for several days. During this time, massive cell death is observed particularly among non-neural cells. At the end of this stage, more than 80% of the cells express nestin, an intermediate filament typically found in neural precursor cells (Frederiksen & McKay, J. Neurosci. 8:1144-1151, 1988; Lendahl et al., Cell 60:585-595, 1990). These precursor cells can be further expanded as a monolayer culture in the presence of basic fibroblast growth factor (bFGF), and, upon bFGF-withdrawal, differentiate into neurons and astrocytes (Okabe et al., Mech. Dev. 59:89-102, 1996). However, the ability of these cells to proliferate in the presence of bFGF is limited, and increasing astrocytic differentiation is observed after only few passages (Okabe, in: Current Protocols in Neuroscience, John Wiley, New York, 1997). After such short proliferation periods the cultures still contain numerous undifferentiated embryonic cells as well as differentiated non-neural cells. Cell populations containing such contaminants are not suitable for reconstructive transplants. Undifferentiated ES cells may generate tumors (teratocarcinomas), and non-neural donor cells may form non-neural tissue within the graft. Up until now there has been no known procedure which permits the generation of ES cell-derived cells with neuronal or glial properties in a purity required for non-tumorigenic transplants in the nervous system and functional activity in vivo such as, for example, remyelination or replacement of lost neurons with recovery of the abnormal behavior elicited by the neuronal loss.

The generation of sufficient numbers of defined neural precursor cells is currently one of the key problems in neural transplantation At present, precursor cells are isolated from the embryonic mammalian brain. For example, material from up to seven human embryos is required for transplantation of an individual Parkinson patient. Such a strategy is associated with severe problems and cannot be used to treat large numbers of Parkinson patients in the long term. Efforts to proliferate neural cells in vitro prior to transplantation have, so far, not lead to significant improvements. Oncogene-mediated immortalization bears considerable risks due to the introduction of a tumorigenic gene into the donor cells. The order of magnitude of growth factor-mediated proliferation of precursor cells is not sufficient for a potential clinical application. In addition, the ability of expanded cells to incorporate into the host tissue is currently unclear.

ES cells represent an interesting alternative donor source for neural transplants. Their key advantage is that they can be multiplied over long periods of time in an undifferentiated, pluripotent stage (Slack, in: From Egg to Embryo, Cambridge University Press, Cambridge, 1991). During proliferation they maintain their ability to differentiate into all tissues, including neural tissue. However, so far it was not possible to differentiate them selectively into neural precursor cells. Attempts to induce neural differentiation with retinoic acid always yielded mixed cell populations with the neural cells representing only a fraction of the cells (Bain et al., Dev. Biol. 168:342-357, 1995; Strubing et al., Mech. Dev. 53:275-287, 1995; Fraichard et al., J. Cell Sci. 108:3181-3188, 1995; Finley et al., J. Neurosci. 16:1056-1065, 1996). In addition, there have been reports on retinoic acid induced formation of ES cell-derived neurons but not on retinoic acid-induced neural precursors. In a study by Dinsmore et al., such mixed populations were grafted into the brain of quinolinic acid-lesioned rats. Quinolinic acid is a neurotoxin which damages and destroys neurons. Following transplantation some of the grafted cells maintained their neuronal phenotype. However, functional innervation of the host brain or reconstitution of lost brain functions were not observed in these experiments (Dinsmore et al., Cell Transplant. 5:131-143, 1996). The culture method described by Okabe et al. comprises plating of embryoid bodies in ITSFn medium and does not depend on retinoic acid. This method yields up to 85% cells expressing the neural precursor cell marker nestin (Okabe et al., Mech. Dev. 59:89-102, 1996). However, the purity of these cell populations, too, is not sufficient to be used for reconstructive purposes. For example, grafted ES cell-derived precursors cultured in ITSFn medium have been shown to form primitive neuroepithelial structures as well as non-neural tissue such as cartilage and adenoid tissue. It is possible to further proliferate cells cultured in ITSFn in the presence of bFGF. However, the cells rapidly loose their multipotency and, within a few passages, differentiate predominantly into astrocytes. Within this short time span it has not been possible to separate non-neural cells from neural precursor cells. Another major disadvantage of this paradigm is the lack of efficient generation of oligodendrocytes. For example, Okabe et al. observed no oligodendroglial differentiation following plain growth factor withdrawal-induced differentiation. Even after addition of the thyroid hormone T3, oligodendroglial antigens were only detected in 1-2% of the cells (Okabe et al., Mech. Dev. 59:89-102, 1996). As far as neuronal differentiation is concerned, the studies reported by Okabe et al. show no evidence for the generation of neurons expressing tyrosin hydroxylase, cholinacetyl transferase or serotonin--compounds that are of great importance for signal transduction between individual neurons. In addition, these studies show no evidence of neurons expressing peripherin. Peripherin is typically expressed in peripheral neurons and in neurons of the brainstem and the spinal cord.

Thus, the challenge for this invention is to provide isolated, purified, non-tumorigenic ES cell-derived precursor cells with neuronal or glial properties, especially purified neurons and glial cells, as well as methods for the production of these precursor cells in virtually unlimited numbers. The generation of such purified precursor cells permits transplantation into the nervous system without tumor formation as well as functional activity in vivo, e.g., remyelination or replacement of lost neurons with improvement of the abnormal behavior resulting from the neuronal loss, and an improvement of the therapy of neural defects.

The invention further relates to a method for the generation of purified precursor cells with neuronal or glial properties, comprising the following steps: (a) proliferation of ES cells (b) culturing of the ES cells from a) to a neural precursor cell stage (c) proliferation of the neural precursor cells in growth factor-containing serum-free medium (d) proliferation of the neural precursor cells from (c) in another growth factor-containing serum-free medium and isolation of the purified precursor cells. (e) proliferation of the precursor cells from (d) in another growth factor-containing serum-free medium and isolation of the purified precursor cells with neuronal or glial properties.

In a preferred embodiment of the invention, the ES cells from (a) are proliferated to aggregates, especially embryoid bodies. In another preferred embodiment of the invention the growth factor-containing serum-free medium in (c) contains bFGF. In other preferred embodiments the growth factor-containing serum-free media in (d) and (e) contain the growth factor combinations bFGF-EGF and bFGF-PDGF, respectively. In another preferred embodiment the purified neural precursor cells are transferred in a medium suitable for injection.

The invention also relates to a method for the generation of purified precursor cells with neuronal or glial properties, comprising the following steps: (a') proliferation of ES cells (b') culturing of the ES cells from (a') to a neural precursor cell stage (c') proliferation of the neural precursor cells in growth factor-containing serum-free medium (d') proliferation of the neural precursor cells from (c') in another growth factor-containing serum-free medium to neural spheres and isolation of the neural spheres. (e') proliferation of the neural spheres from (d') in a growth factor-containing serum-free medium until they form an outgrowth of glial precursor cells and isolation of the purified precursor cells.

In a preferred embodiment of the invention, the ES cells from (a') are proliferated to aggregates, especially embryoid bodies. In an additional step (f'), the glial precursor cells obtained in (e') are guided towards an astrocytic or an oligodendroglial differentiation by adding single factors to the culture medium; then the astrocytic or oligodendroglial cells are isolated. In a preferred embodiment of the invention the growth factor-containing serum-free medium in (c') contains bFGF. In another preferred embodiment the growth factor-containing serum-free media in (d'), (e') and (f') contain the growth factors bFGF and EGF, either alone or in combination. In another preferred embodiment, ciliary neurotrophic factor (CNTF) and thyroid hormone (T3) are used in step (f') to promote astrocytic and oligodendroglial differentiation, respectively. In another preferred embodiment the purified neural precursor cells are transferred in a medium suitable for injection.

The neural precursor cells obtained by the invention may be used as therapeutic tool for medical treatment. A preferred application of the purified neural precursor cells is the generation of therapeutic tools for the treatment of neural defects. A particularly preferred application is the reconstitution of neuronal cells or the remyelination of demyelinated nerve cells, in particular within demyelinated areas of the nervous system, by cell transplantation into the nervous system.

Preferred applications include the reconstitution of neuronal cells damaged or lost as result of traumatic, ischemic, degenerative, genetic, hypoxic, metabolic, infectious, neoplastic or toxic disorders of the nervous system. Particularly preferred is the reconstitution of neural cells in traumatic lesions of the brain and spinal cord, ischemic and hemorrhagic infarctions, Parkinsons disease, Huntingtons disease, Alzheimers disease, hereditary atrophic disorders of the cerebellum and brain stem, motoneuron diseases and spinal muscular atrophies. Preferred applications further include the reconstitution of neuronal cells lost or damaged due to age-related changes. A particularly preferred application is the remyelination of demyelinated areas of the nervous system, particularly in diseases such as multiple sclerosis (MS), adrenoleukodystrophy and Pelizeaus-Merzbacher disease.

Another preferred application of the ES cell-derived neural precursor cells is cell-mediated gene transfer into the nervous system. Preferred applications for cell-mediated gene transfer include hereditary metabolic disorders due to enzyme deficiencies and neoplastic disorders of the nervous system. The ES cell-derived neural precursor cells obtained through this invention may also be used for the in vitro production of factors, e.g., polypeptides, for clinical and commercial applications.


To start the generation of neural precursor cells, embryonic stem cells, for example of mouse origin, may be proliferated to the desired number in serum-containing medium on a feeder layer of non-mitotic embryonic fibroblasts according to standard methods (Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Press, New York, 1994). In addition to established mouse ES cell lines such as J1 (Li et al., Cell 69:915-926, 1992), R1 (Nagy et al. Proc. Natl. Acad. Sci. USA 90:8424-8428, 1993) and CJ7 (Swiatek & Gridley, Genes Dev. 7:2071-2084, 1993), ES cells may also be obtained from embryos, e.g., from 3 to 4-day-old mouse blastocysts (Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Press, New York, 1994). ES cells may also be obtained from other species such as rat (Iannaconne et al., Dev. Biol. 163:288-292, 1994), hamster (Doetschman et al., Dev. Biol. 127:224-227, 1988), birds (Pain et al., Development 122:2339-2348, 1996), fish (Sun et al., Mol. Mar. Biol. Biotechno. 4:193-199, 1995), swine (Wheeler, Reprod. Fertil. Dev. 6:563-568, 1994), cattle (First et al., Reprod. Fertil. Dev. 6:553-562), primates (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844-7848, 1995) or from embryonic human tissue. Several months following the submission of the German patent application No. 197 56 864.5 two research teams succeeded in isolating ES cells and ES cell-like stem cells from embryonic human tissue (Thomson et al., Science 282: 1145-1147, 1998; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726-13731, 1998).

More recent studies indicate that embryos and embryonic stem cells can be generated by transplanting nuclei from cells of an mature individuum into enucleated oocytes (Wilmut et al., Nature 385:810-813, 1997). For the specialist it is obvious that a combination of such nuclear transfer strategies with the invention described herein permits the generation of autologous neural precursor cells from differentiated cells of the same individuum. The generation of embryos through transfer of nuclei from mature cells into enucleated oocytes has been applied to large mammals such as sheep (Wilmut et al., Nature 385:810-813, 1997) and is, therefore, also applicable to humans. ES cells or ES cell-like cells may also be obtained from embryonic germ cells. Studies published after the priority date of this patent application show that human ES cells can be isolated from human blastocysts (Thomson et al., Science 282: 1145-1147, 1998), and human ES cell-like cells can be obtained from human primordial germ cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726-13731, 1998). These studies indicate that the methods described in this patent application, alone or in combination with nuclear transfer strategies, can also be applied to humans.

Feeder-dependent ES cell lines may be plated in gelatin-coated cell culture dishes and subsequently aggregated in uncoated Petri dishes in the absence of LIF to form embryoid bodies (Okabe et al., Mech. Dev. 59:89-102, 1996). Three to four-day-old embryoid bodies may then be plated in cell culture dishes and grown for four to eight days in serum-free medium (ITSFn medium; Okabe et al., Mech. Dev. 59:89-102, 1996). During this time pronounced cell death can be detected among non-neural cells.

After four to eight days in ITSFn medium the cells may be triturated to a single cell suspension using a small-bore pipette and transferred into N3FL medium. N3FL is a serum-free medium that contains bFGF (Okabe et al., Mech. Dev. 59:89-102, 1996). bFGF is used to promote proliferation of neural precursor cells.

After a 4 to 8-day-period in N3FL medium, the generation of glial and neuronal precursor cell populations is initiated. In contrast to neuronal precursors, glial precursors exhibit a pronounced proliferative potential. The method described herein exploits this proliferative potential. Cells derived from N3FL cultures are sequentially propagated through different serum-free growth factor-containing media. During this stage a strong enrichment of bipotent precursors with astrocytic and oligodendroglial differentiation capacity can be observed. Concomitantly, primitive embryonic cells and non-neural cells are differentiated and eliminated from the cultures. To that end, cells cultured in N3FL medium are harvested mechanically, triturated to a single cell suspension and transferred into a serum-free medium which may contain the growth factors bFGF and EGF (N3EFL medium). In this medium the cells are propagated as monolayer until they become subconfluent. After approximately two passages cells cultured in this medium may be used for transplantation purposes. For further purification, an additional step may be included.

Cells cultured in N3EFL medium may be harvested mechanically, triturated to a single cell suspension and replated in a serum-free medium containing a second growth factor combination, e.g., bFGF and PDGF.

Cells obtained through this protocol may be propagated through further passages in this medium. After approximately two passages, the culture consists of purified neural precursor cells which may be used directly or following isolation, e.g., for remyelinating transplants. At this stage, they form a homogenous layer of cells with a bipolar to multipolar morphology. They may be frozen and thawed at this stage or at earlier stages without loosing their precursor cell properties. If the growth factors are not added for several days, growth factor withdrawal will induce differentiation in vitro. In this case, immunohistochemical analyses show the presence of marker antigens for oligodendrocytes (e.g., 04) and astrocytes (e.g., GFAP) in addition to markers for neural precursor cells (e.g., nestin). 04- and GFAP-positive cells may account for 20-40% and 30-70% of the cell population, respectively. In addition, cells expressing neuronal antigens may be found in these cultures.

The generation of neuronal and glial precursor cells in form of neural spheres is also initiated after a 4 to 8-day-period in N3FL medium. To enrich neural cells, cells grown in N3FL medium are harvested mechanically, triturated to a single cell suspension and further propagated in uncoated cell culture dishes in serum-free medium which may contain the growth factors bFGF and EGF. Within a few days, the cells will form cellular aggregates (neural spheres) which are primarily composed of nestin-positive neural precursor cells. The neural spheres can be further propagated as a suspension culture. In contrast, differentiated neural cells and non-neural cells tend to adhere to the surface of the cell culture dishes.

As soon as small spheres have formed, they are removed from the culture and transferred into new uncoated cell culture dishes. Within a few (5-7) days, the spheres may be used for transplantation purposes. In this case the neural precursors within the spheres differentiate into mature neural cells which innervate the host brain. Small undifferentiated neural spheres may be frozen in liquid nitrogen in serum-free freezing medium and thawed and differentiated at a later time point.

The invention also relates to differentiated neural cells in a sphere composition that may be suitable for transplantation. These cells may be obtained through in vitro differentiation of the ES cell-derived precursor cells with neuronal or glial properties described herein. To induce in vitro differentiation, growth factors are withdrawn and spheres composed of undifferentiated neural precursor cells are plated, e.g., in cell culture dishes coated with polyornithine and fibronectin. In these conditions the spheres rapidly adhere to the surface of the cell culture dish and, in addition to nestin-positive neural precursors, yield neurons, astrocytes and oligodendrocytes. Adhering neurons can be further differentiated to express a variety of neuronal markers, e.g., MAP2, beta-III-tubulin, synapsin, cholinacetyltransferase, tyrosin hydroxylase, GABA, glutamate, serotonin, peripherin and calbindin. Maturation and survival of the differentiated neurons can be enhanced by addition of neurotrophins, e.g., BDNF or neurotrophin 3 (NT-3).

In serum-free medium containing growth factors such as, for example, bFGF and EGF, the ES cell-derived neural spheres may be maintained in suspension culture for several weeks. At that stage, the spheres may grow to a large size easily detectable with the naked eye. In these conditions an increasing level of cell differentiation may be observed within the neural spheres. Thus, these spheres can be used to transplant ES cell-derived neurons even at an advanced stage of differentiation. This is not possible using differentiated ES cell-derived neurons in monolayer culture as harvesting of these cells will invariably lead to major damage of the neuronal processes and destruction of the neuronal cell bodies.

During the last couple of years numerous factors have been identified which influence the differentiation of neuronal cell populations. These factors may, for example, lead to polarization within neural tissue. For example, it was shown that the product of the gene Sonic hedgehog induces a ventral phenotype in neural tissue (Ericson et al., Cell 81:747-756, 1995). It is to be expected that such factors also influence the differentiation of neural cells generated artificially from ES cells. For the specialist it is obvious that the application of such factors will permit the generation of neurons and glial cells with specific phenotypes. For example, induction of a ventral mesencephalic phenotype may yield cells suitable for transplantation into Parkinson patients. In cultured fragments of neural tissue it has already been shown that Sonic hedgehog can induce dopaminergic ventral mesencephalic neurons (Wang et al., Nature Med. 1:1184-1188, 1995).

For the generation of glial precursor cells from ES cell-derived neural spheres, the spheres are propagated in suspension in growth factor-containing serum-free medium until they start adhering to the uncoated surface of the cell culture dish. During this phase, bFGF and EGF, individually or in combination, may be used as growth factors. Following adhesion of the spheres, cells with glial morphology migrate from the spheres onto the surface of the cell culture dishes (so-called touch-down cells). The precise development of this cell population is not understood. Presumably, increasing cell differentiation within the spheres leads to formation of glial precursors which exhibit enhanced adhesion and migration behavior. Spheres producing touch-down cells may be used as generators for glial precursors. To that end, spheres generating glial precursors are cultured only for short periods of time (<1 day) in uncoated cell culture dishes. As soon as glial cells have adhered, the spheres are mechanically detached from the dish and transferred into another dish. This will yield ring-shaped monolayers of glial precursors which can be further proliferated in the presence of growth factors, e.g., bFGF and EGF (applied individually or in combination).

`Touch-down cells` generated in this manner express the neural antigen A2B5 (Eisenbarth et al., Proc. Natl. Acad. Sci. USA 76:4913-4917, 1979) and, upon growth factor withdrawal, differentiate into astrocytes and oligodendrocytes. Using immunohistochemical methods, marker antigens for oligodendrocytes (e.g., 04) and astrocytes (e.g., GFAP) can be detected in these differentiated cells. Undifferentiated touch-down cells may be frozen in liquid nitrogen in serum-free freezing medium without loosing their proliferation and differentiation potential. Glial precursor cells generated in this manner may also be used for transplantation in the nervous system.

The differentiation of the ES cell-derived glial precursors can be influenced by addition of single factors. Addition of CNTF (ciliary neurotrophic factor) shortly before und during growth factor withdrawal will promote astrocytic differentiation. Addition of the thyroid hormone T3 during this stage will result in enhanced differentiation of oligodendrocytes. Addition of serum-containing media during or after growth factor treatment results in a strong increase in the number of astrocytic cells in these cultures.

For the specialist it is obvious that the frozen neural precursor cells may, after thawing, also be used for transplantation. Cells frozen at earlier stages of the in vitro differentiation process may be thawed in a standard manner and further propagated and passaged until a homogenous population of bi- and multipolar cells is obtained.

The methods described herein may further be combined with established cell separation cell sorting procedures. For example, neural subpopulations may be separated at defined time points using fluorescent-activated cell sorting (FACS), immunopanning, or similar methods. A detained sorting and subclassification may permit the generation of replacement cells (including genetically modified replacement cells) tailored to the individual patients needs. Since both ES cells and the ES cell-derived neural precursor cells described herein can be frozen and thawed without loosing their properties, it is possible to establish cell banks, including autologous cell banks.

The methodology described herein permits the generation of neural precursor cells, e.g. neuronal, astrocytic and oligodendroglial cells, in a purity and in quantities required, e.g., for the repair of defects in the nervous system. Cells generated with the methodology described herein contain, for example, only few or no primitive embryonic and non-neural cells. The purity of the neural precursor cells described herein far exceeds the purity of approximately 85% which was previously described by Okabe et al. (Mech. Dev. 59:89-102, 1996). The methodology described herein permits the generation of neural precursor cells in a purity of up to 100%. In addition, the methodology described herein permits the generation of large numbers of neural precursor cells without depending on brain tissue. The neural precursor cells described herein may be obtained from ES cells of various species, e.g., mouse, rat, hamster, birds, fish, swine, cattle, primate or humans. Both, established ES cell lines and ES cells derived from embryos may be used. In addition, the ES cells may be derived from proliferated oocytes. The oocytes may be enucleated and implanted with a cell nucleus derived, for example, from differentiated tissue, permitting the generation of autologous oocytes and ES cells. ES cells or ES-like cells may also be obtained from embryonic germ cells. The ES cells may be genetically modified with standard procedures. For example, a defective gene may be replaced by its `normal` counterpart using homologous recombination. In addition, genes may be deleted using standard methods. These procedures have been extensively used in mice and are, therefore, state of the art.

The rapid proliferation of ES cells and their amenability to genetic modification permits the generation of large numbers of genetically modified neuronal and glial precursor cells. Combined with the extensive migration potential of neuronal and glial precursor cells, this permits the population of large areas of the nervous system with genetically modified precursor cells which may substitute missing factors or secrete polypeptides designed for neuroprotection or other applications. Genetic modification of the cells may also be used to remove genes encoding surface antigens which are involved in transplant rejection. Such a strategy may permit a broad clinical application of ES cell-derived precursor cells without the need for immunosuppression.

The neural precursor cells described herein may also by used as therapeutic medical tools for the treatment of neural defects. A typical example for the application of the neural precursor cells described herein is the reconstitution of lost or functionally impaired neurons by transplanted neural precursor cells.

In order to replace lost neurons and to improve neurological deficits associated with this loss, neural spheres described herein may, for example, be grown for 4-7 days in suspension and then implanted into brain regions exhibiting neuronal loss. Six weeks following transplantation, differentiated neurons innervating the host brain may be observed. Donor-derived axons extending into the host brain tissue may, for example, be detected with antibodies to donor-specific neural antigens. The neuronal reconstitution also leads to functional improvement. This may be demonstrated in behavioral tests in ibotenic acid-lesioned rats before and after transplantation. To that end, large numbers of striatal neurons are destroyed by stereotaxic injection of the neurotoxin ibotenic acid. The resulting defect shows similarities to Huntingtons disease. This experimental model is, therefore, frequently used as an animal model of this disease. After unilateral ibotenic acid lesion, the operated animals exhibit a functional asymmetry and a quantifiable abnormal rotation behavior which may be induced by certain drugs, e.g., amphetamine. A transplant rich in neuronal cells may normalize the rotation behavior. This normalization appears to depend particularly on the number of GABAergic neurons within the transplant. The ibotenic acid lesion model and the functional evaluation of neural transplants by analysis of the rotation behavior have been described extensively (Bjorklund et al., in: Functional Neural Transplantation, Seiten 157-195, Raven Press, New York, 1994). Transplantation of the neural spheres described herein into the brain of ibotenic acid-lesioned rats results in significant postoperative improvement of the abnormal rotation behavior induced by the ibotenic acid lesion. Transplants of, e.g., neural cells into the human nervous system are already being performed in patients (Olanow et al., TINS 19:102-109, 1996).

The glial precursor cells described herein, obtained either from neural spheres or monolayer cultures, may also be used as a tool for the therapy of neural defects. A typical example for the application of the glial precursor cells described herein is the remyelination of demyelinated brain regions by cell transplantation. Since the glial precursor cells described herein exhibit a pronounced migratory potential, they may be used for the treatment of demyelinating diseases encompassing large areas of the CNS. In this case a localized injection of the cells may suffice to populate and remyelinate large areas of the CNS. A typical example of a disease which might be treated in this manner is multiple sclerosis (MS), a disease of unknown cause which is typically associated with multiple foci of demyelination in different regions of the CNS. Transplantation of the neural precursors described herein may be used to remyelinate such defects. In this case the pronounced migration potential of the neural precursor cells may be exploited to target and repair numerous areas of demyelination from single or few implant sites.

In order to myelinate myelin-deficient brain regions, cells derived from monolayer cultures or from ES cell-derived neural spheres may be propagated in growth factor-containing media, harvested mechanically and triturated to a single cell suspension. Transplantation of such cell suspensions into myelin-deficient brain regions typically yields donor-derived astrocytes and oligodendrocytes which are easily detectable by approximately three weeks post transplantation. The transplanted cells typically enwrap host axons with myelin sheaths. Using immunohistochemical methods, myelin proteins such as myelin basic protein (MBP) and proteolipid protein (PLP) can be detected in these myelin sheaths.

The ES cell-derived neural precursors described herein may also be used for the in vitro generation of polypeptides for clinical and commercial applications.

 

Claim 1 of 16 Claims

1. A method for the generation of an isolated, non-tumorigenic cell composition consisting essentially of embryonic stem cell-derived neural precursor cells, and neuronal cells derived from the embryonic stem cell-derived neural precursor cells comprising: (a) culturing mouse or human embryonic stem cells that are not human genetically modified embryonic stem cells to produce neural precursor cells; (b) culturing the neural precursor cells from (a) in a first growth factor-containing serum-free medium, the first medium comprising basic fibroblast growth factor (bFGF); (c) culturing the cells from (b) in a second growth factor-containing serum-free medium, the second medium comprising bFGF and epidermal growth factor (EGF); and (d) culturing the cells from (c) in a third growth factor-containing serum-free medium, the third medium comprising bFGF and platelet-derived growth factor (PDGF), to obtain the cell composition consisting essentially of embryonic stem cell-derived neural precursor cells, and neuronal cells derived from the embryonic stem cell-derived neural precursor cells.
 

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