Pluripotent stem cells have been detected in multiple tissues in the adult mammal, participating in normal replacement and repair, while undergoing self-renewal (Hay, 1966, Regeneration, Holt, Rinehart and Winston, New York; McKay, 1999, Nature Med. 5:261-262; Lemiscka, 1999, Ann. N.Y. Acad. Sci. 872:274-288; Owens and Friedenstein, 1988, Ciba Foundation Syp. 136, Chichester, U.K. pp. 42-60; Prockop, 1997, Science 276:71-74; Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991, J. Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad. Sci. USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology 97:561-570; Majumdar et al., 1998, J. Cell Physiol. 176:57-66; Pittenger et al., 1999, Science 284:143-147). A subclass of bone marrow stem cells is one prototype, capable of differentiating into osteogenic, chondrogenic, adipogenic and other mesenchymal lineages in vitro (Owens and Friedenstein, 20 1988, Ciba Foundation Symp. 136, Chichester, U.K. pp. 42-60; Prockop, 1997, Science 276; 71-74; Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991, J. Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad. Sci. USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology 97:561-570; Majumdar et al., 1998, J. Cell. Physiol. 176:57-66; Pittenger et al., 1999, Science 284:143-147). These pluripotent cells have been termed marrow stromal cells (MSCs), and have been used clinically to treat osteogenesis imperfecta (Horwitz et al., 1999, Nature Med. 5:309-313).
The 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). The recent report demonstrating that NSCs can generate hematopoietic cells in vivo suggests that stem cell populations may be less restricted than previously thought (Bjornson, 1999, Science 283:534-537).                Adult MSC cells are both self-renewing and multipotential (Owens and Friedenstein, 1988, Ciba Foundation Symp. 136, Chichester, U.K. pp. 42-60; Prockop, 1997, Science 276; 71-74; Ferrari et al., 1998, Science 279:1528-1530; Caplan,1991, J. Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad. Sci. USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology 97:561-570; Majumdar et al., 1998, J. Cell. Physiol. 176:57-66; Pittenger et al., 1999, Science 284:143-147; Sanchez Ramos et al. Exp. Neurol. 2000 164(2) 247-56), thereby fulfilling many of the criteria of a stem cell population.        
Recent studies show that neural stem cells have broad developmental potential, contributing to the development of blood as well as to all germ layers in chimeric embryos (Clarke, D. L. et al., Science (2000) 288: 1660-1663). During early embryonic development of mammalian embryos, developing neural cells share features with developing islet cells. A specific characteristic of neural precursor cells is their expression of the protein nestin (U. Lendhal, L. D. Zimmerman, R. D. McKay Cell: 23, 585 (1990) as described in U.S. Pat. No. 5,338,839. Recently, researchers have found the stem cell marker nestin within developing islet cells (H. Zulewski et al., 2001, Diabetes 50: 521) and others report the differentiation of nestin-positive embryonic stem cells to insulin-secreting structures similar to pancreatic islets in mice (N. Lumelsky et al. supra) and humans (Assady et al., Diabetes 50, August 2001). However, differentiation of insulin-secreting islet-type cells from adult stem cells has not previously been demonstrated. Common mechanisms of control and shared nestin expression point to a close relationship between pancreatic and neural progenitors which give rise to tissues of endodermal and ectodermal embryonic origin respectively.
We have recently demonstrated that MSCs can be induced to differentiate into neuronal cells (D. Woodbury et al., 2000, J. Neur. Sci. Res. 61, 364). Differentiation of MSCs into astrocytes and glial cells (WO 99/43286) has also been demonstrated. These recent studies indicate that rat and human MSCs are capable of differentiating into non-mesenchymal derivatives, suggesting that intrinsic genomic mechanisms of commitment, lineage restriction and cell fate are mutable. Environmental signals apparently can elicit the expression of pluripotentiality that extends well beyond the accepted fate restrictions of cells originating in classical embryonic germ layers.
To define the process of stem cell differentiation and elucidate underlying mechanisms, we have characterized MSCs and developing neurons more extensively, defining expression patterns for representative genes of different lineages and correlating expression with morphologic maturation. Our observations indicate that the “undifferentiated” MSCs express germline, endodermal and ectodermal genes, as well as the expected mesodermal genes. Neuronal differentiation of the MSCs involves complex modulation of these different gene sets, rather than simple on-off switching of neural and non-neural genes. We now describe, for the first time, conditions which permit the growth and expansion of endodermal cells, particularly insulin-producing pancreatic islet cells, differentiated from adult bone marrow stromal cells (MSCs). MSCs constitute a novel source of pancreatic islet cells and represent the only adult cells used for this purpose.
A number of disease states are associated with organs of endodermal lineage which include the liver, stomach, intestine, pancreas, and other endocrine glands. Type 1 and Type 2 diabetes and chronic pancreatitis result from the anatomical and functional loss of insulin-producing beta cells and the ductal and acinar cells, respectively, while uncontrolled proliferation of the ductal cells leads to pancreatic carcinogenesis. The replacement of these cells through regeneration or transplantation could offer lifelong treatment for diabetics and for patients with chronic pancreatitis. However, a major problem in implementing treatment is the lack of sufficient pancreatic/islet cell tissue for transplantation. The present invention officers the potential of generating sizable quantities of insulin-producing cells from adult bone marrow stromal cells.
Gut malignancies and inflammatory bowel diseases are major causes of morbidity and mortality. The cell differentiation techniques disclosed herein may be utilized to gain new insights about initiation, progression and treatment of tumorigenesis and offer new strategies to increase the absorptive function of the intestine.
Liver transplantation is the treatment of choice for many liver diseases. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. Therapeutic liver re-population with bone marrow derived cells holds the hope of overcoming the shortage in donor livers.
Despite the crucial need for obtaining endodermal cells for treatment a number of diseases, disorders, and conditions, associated with tissues of endodermal lineage, no method has previously been available for obtaining large numbers of endodermal cells without encountering the technical and ethical hurdles involved in obtaining adult human or fetal tissue. The present invention overcomes that need, offering the potential of generating sizable quantities of endodermal tissue from adult bone marrow stromal cells.
While previous studies have demonstrated that intrinsic genomic mechanisms of commitment, lineage restriction and mesenchymal cell fate of MSCs are mutable, it was unexpected that these adult cells could be induced to differentiate to cells associated with tissues of endodermal lineage including the liver, stomach, intestine, pancreas, and other endocrine glands.
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