Directed in vitro differentiation of marrow stromal cells into neural cell progenitors

#Human marrow stromal cells (hMSCs) are multipotential adult stem cells that contribute to the regeneration of tissues such as bone, cartilage, fat, and muscle (1997, Friedenstein, et al., Exp. Hematol. 4(5):267-274; 1997, Prockop, D J, Science, 276(5309):71-74; 1999, Pittenger, et al., Science, 284(5411):143-147; 1998, Ferrari et al., Science, 279(5356): 1528-1530).

#The recent discovery of stem cell populations in the central nervous system (CNS) has generated intense interest, since the brain has long been regarded as incapable of regeneration (Reynolds and Weiss, 1992, Science 255:1707-1710; Richards et al., 1992, Proc. Natl. Acad. Sci. USA 89:8591-8595; Morshead et al., 1994, Neuron 13:1071-1082). Neural stem cells (NSCs) are capable of undergoing expansion and differentiating into neurons, astrocytes and oligodendrocytes in vitro (Reynolds and Weiss, 1992, Science 255:1707-1710; Johansson et al., 1999, Cell 96:25-34; Gage et al., 1995, Annu. Rev. Neurosci. 18:159-192; Vescovi et al., 1993, Neuron 11:951-966). NSCs back transplanted into the adult rodent brain survive and differentiate into neurons and glia, raising the possibility of therapeutic potential (Lundberg et al., 1997, Exp. Neurol. 145:342-360; Lundberg et al., 1996, Brain Res. 737:295-300; Renfranz et al., 1991, Cell 66:713-729; Flax et al., 1998, Nature Biotech. 16:1033-1039; Gage et al., 1995, Proc. Natl. Acad. Sci. USA 92:11879-11883; Svendsen et al., 1997, Exp. Neurol. 148:135-146). However, the inaccessibility of NSC sources deep in the brain severely limits clinical utility. The recent report demonstrating that NSCs can generate hematopoietic cells in vivo suggests that hematopoietic stem cell populations may be less restricted than previously thought (Bjornson, 1999, Science 283:534-537).

#Recent data suggest that MSCs can also be induced to differentiate into neural cells in vivo. It has been found that hMSCs integrate and migrate along the known pathway for the migration of neural stem cells after being infused into rat brain (Azizi, et al., 1998, PNAS, 95(7):3908-3913). Other data demonstrate that mouse MSCs (mMSCs) labeled with BrdU migrate to both forebrain and cerebellum without disruption of normal brain structure when injected into the lateral ventricle of a neonatal mouse (Kopen, et al., 1999, PNAS, 96(19):10711-10716). Some of the mMSCs differentiated into cells that had astrocyte morphology and expressed the astrocyte-specific protein glial fibrillary acid protein (GFAP). Further, some of the mMSCs appeared in the olfactory bulb and the internal granular layer of the cerebellum, both of which are neuron-rich regions. Finally, the Kopen study also demonstrated that some BrdU-labeled mMSCs found in the reticular formation of the brain stem were positive for neurofilament.

#The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiment(s) which are presently preferred. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

#FIG. 1, comprising FIGS. 1A-1D, is a quartet of images depicting induction of neural morphology in hMSCs. FIG. 1A is an image depicting untreated hMSCs. FIGS. 1B, 1C, and 1D depict hMSCs treated with 0.5 millimolar isobutylmethylxanthine (IBMX) and 1 millimolar dibutyryl cyclic AMP (dbcAMP) for 6 days. Arrows represent differentiated neuron-like cells while undifferentiated hMSCs are indicated by an arrow head. 200× Magnification.

#FIG. 2 is a graph representing cellular proliferation and neural differentiation in hMSCs treated with 0.5 millimolar IBMX and 1 millimolar dbcAMP. The data points represent mean ± standard deviation of results of experiments performed in triplicate. Cells were scored as hMSCs with neural morphology by the presence of a refractile cell body, multipolar processes, and growth-cone-like structures.

#The invention herein described demonstrates another way to differentiate MSCs to cells with many characteristics of early neurons and glia. The method of differentiation involves increasing levels of intracellular cyclic AMP by treating the MSCs with dibutyryl cyclic AMP (dbcAMP) and isobutylmethylxanthine (IBMX).

#The invention comprises and utilizes the discovery that increasing cyclic AMP levels in marrow stromal cells mediates differentiation of the cells into neural cells expressing a variety of early-expressed neuron-specific markers (e.g. neuron-specific enolase (NSE), vimentin, MAP1B, and the like). The cells exhibit other neuron-like phenotypic characteristics such as, but not limited to, spherical and retractile cell bodies exhibiting typical neural perikaryal appearance, cell bodies extending long processes terminating in growth cones and filopodia typical of neurons. Thus, the methods disclosed herein induce marrow stromal cell differentiation into immature neural cells. Such methods are crucial in the development of cell-based therapeutics for treatment of central nervous system (CNS) disorders, diseases or conditions. Indeed, prior to the present invention, the lack of clarity with respect to neural differentiation has severely impeded the development of CNS therapeutics.

#The invention includes a method of inducing an isolated marrow stromal cell to differentiate into an isolated neural cell. Embodiments of the method of the invention are described in the Examples section herein. Generally, bone marrow cells are isolated from a donor, stromal cells are obtained therefrom, and the stromal cells are subsequently cultured in vitro on culture plates using standard cell culture techniques, e.g., as described in the materials and methods section of the Examples. Preferably, the donor is a human, however, the invention is intended to encompass a mammalian donor and should not be limited to the specific donors disclosed herein.