Discovery, localization, harvest, and propagation of an FGF2...

Details Discovery, localization, harvest, and propagation of an FGF2... Discovery, localization, harvest, and propagation of an FGF2...

1999-03-18

2004-11-02

Falk, Anne-Marie (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S325000, C435S366000, C435S368000, C435S383000, C435S384000, C435S455000, C436S513000

Reexamination Certificate

active

06812027

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to neuronal progenitor cells which have been identified in both tissue cultures and histological sections of the adult human brain. The present invention provides methods for the localization, characterization, harvest, and propagation of neuronal progenitor cells derived from adult humans.
BACKGROUND OF THE INVENTION
The damaged adult mammalian brain is incapable of significant structural self-repair. Terminally differentiated neurons are incapable of mitosis, and compensatory neuronal production has not been observed in any mammalian models of structural brain damage (Korr, 1980; Sturrock, 1982). Although varying degrees of recovery from injury are possible, this is largely because of synaptic and functional plasticity rather than the frank regeneration of neural tissues. The lack of structural plasticity of the adult brain is partly because of its inability to generate new neurons, a limitation that has severely hindered the development of therapies for neurological injury or degeneration. Indeed, the inability to replace or regenerate damaged or dead cells continues to plague neuroscientists, neurologists, and neurosurgeons who are interested in treating the injured brain. During the last several years, however, a considerable body of evidence has evolved that suggests a marked degree of cellular plasticity in the adult as well as in the developing CNS. In particular, recent work on neural progenitor cells, derived from both embryos and adults, has suggested strategies for directed neuronal regeneration and structural brain repair. These include the use of neural stem cells which are the multipotential progenitors of neurons and glia that are capable of self-renewal (Davis, 1994; Gritti, 1996; Kilpatrick, 1993; Morshead, 1994; Stemple, 1992; Goldman, 1996; Weiss, 1996a).
In the adult human brain, both neuronal and oligodendroglial precursors have been identified as well, and methods for their harvest and enrichment have been established. Neural precursors have several characteristics that make them ideal vectors for brain repair. They may be expanded in tissue culture, providing a renewable supply of material for transplantation. Moreover, progenitors are ideal for genetic manipulation and may be engineered to express exogenous genes for neurotransmitters, neurotrophic factors, and metabolic enzymes (reviewed in Goldman 1998; Pincus 1998; and Goldman & Luskin 1998).
In embryonic neurogenesis, the proliferation of neuronal precursors takes place at the surface of the central canal lining the neural tube (Jacobson, 1991). The central canal ultimately forms the ventricular system of the adult. This neurogenic layer is referred to as the ventricular/subventricular zone in development, and the ependymal/subependymal zone (SZ) in adults (Boulder Committee, (1970). In development, mitogenesis in the ventricular/subventricular zone is followed by the migration of newly generated neurons and glia along radial guide fibers into the brain parenchyma, including that of the cortical plate (LaVail, 1971; Rakic, 1971; Rakic, 1974; Sidman, 1973).
A variety of signals, including both humoral and contact-mediated factors, have been described which influence the proliferation, differentiation, and survival of stem cells and their progeny. Work on model systems derived from the peripheral nervous system has suggested that the neurotrophins (Anderson, 1986; DiCicco-Bloom, 1993; Murphy, 1991; Sieber-Blum, 1991), neurotransmitters (Pincus, 1990), and traditional growth factors (DiCicco-Bloom, 1988; Murphy, 1994; Shah, 1994) may all influence the development of precursors in vitro. In the CNS, soluble growth factors, particularly basic fibroblast growth factor (FGF-2) regulate neuronal precursor proliferation (DeHamer, 1994; Deloulme, 1991; Drago, 1991; Gensburger, 1987; Gritti, 1996; Kilpatrick, 1995; Kitchens, 1994; Murphy, 1990; Palmer, 1995; Qian, 1997; Ray, 1994; Ray, 1993; Vescovi, 1993). In mixed cell cultures derived from rat embryonic cerebrum, the addition of FGF-2 stimulated the proliferation of neuronal precursors (Gensburger, 1987). Similarly, FGF-2 stimulated the proliferation of a multipotential neural progenitor in fetal mice, which gave rise to neurons and astrocytes (Kilpatrick, 1995). Embryonic rat hippocampal, spinal cord, and olfactory neuron progenitors all have been shown to proliferate in the presence of FGF-2 (DeHamer, 1994; Deloulme, 1991; Ray, 1994; Ray, 1993). Not surprisingly, FGF-2 may also regulate precursor division in concert with other factors; this has been demonstrated in the coordinate regulation of neuronal precursor division by insulin-like growth factor I and FGF-2 (Drago, 1991), as well as oligodendrocyte precursor division by FGF-2 and platelet-derived growth factor (McKinnon, 1993; Wolswijk, 1992).
Where FGF2 had been shown to promote the division of neuronal precursor cells and, hence, the specific generation of neurons, epidermal growth factor (EGF) has also been shown to influence the proliferation of uncommitted neural precursors (Kitchens, 1994; Lu, 1996; Ray, 1994; Reynolds, 1992b; Reynolds, 1992a; Santa-Olalla, 1995; Weiss, 1996b). In dissociated cultures of embryonic mouse striata grown in suspension without culture substrata, EGF induced the proliferation of progenitor cells and the formation of floating “neurospheres” of cells, which expressed nestin (Reynolds, 1992a). Nestin is an intermediate filament protein expressed not only by CNS stem cells (Dahlstrand, 1992; Lendahl, 1990a), but also by young neurons reactive astrocytes, and radial glia. When these neurospheres were dissociated and plated onto poly-L-ornithine-coated plates, &ggr;-aminobutyric acid- and substance P-expressing neurons and glial fibrillary acidic protein-expressing astrocytes were generated (Ahmed, 1995). Similar effects were reported in adult striatal cultures (Reynolds, 1992b). In this culture preparation, the actions of EGF were mimicked by its membrane-bound homolog, transforming growth factor &agr;, but not by nerve growth factor, FGF-2, platelet-derived growth factor, or transforming growth factor &bgr;. A similar action of EGF on precursor cells derived from embryonic and adult rat spinal cord has also been reported (Ray, 1994; Weiss, 1996).
Although it is now possible to isolate and cultivate populations of neural precursors in vitro, the ability to direct specific neuronal phenotypes has remained elusive. In the EGF-generated sphere model, multipotent progenitors differentiated into neurons, which expressed &ggr;-aminobutyric acid and substance P, as well as astrocytes and oligodendrocytes (Reynolds, 1992b; Reynolds, 1992a; Vescovi, 1993; Weiss, 1996b). Other neuronal phenotypes were rare, and their directed differentiation into defined transmitter phenotypes has not yet been demonstrated. In this regard, Raff et al. (Raff, 1988; Raff, 1983) suggested that growth factors control the development of a bipotential glial progenitor. Sequential exposure to specific combinations of platelet-derived growth factor, ciliary neurotrophic factor, and neurotrophin 3 can direct clonal expansion of the oligodendrocyte/Type 2 astrocyte (02A) progenitor cell in vitro, and drive an intrinsic clock that times oligodendrocyte development (Barres, 1994; Lillien, 1988; Raff, 1988; Temple, 1985). Nonetheless, a similarly directed differentiation of multipotent stem cells along specific neuronal lines has not yet been clearly demonstrated.
The persistence of neuronal precursors in the adult mammalian brain may permit the design of novel and effective strategies for central nervous system repair. However, although methods for the characterization and propagation of progenitors derived from adult rodents have been described, no such methods have allowed the high-yield harvest of purified native progenitors. Furthermore, no methods have been reported for obtaining or propagating such progenitor cells from adult human brain tissue.
SUMMARY OF THE INVENTION
The present invention provides human neural or neuronal progenitor cells isolated

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