A stem cell is a cell with extensive proliferation potential that can differentiate into several cell types. The quintessential stem cell is the embryonal stem (ES) cell which has unlimited self renewal and multipotent differentiation potential. Tissue specific stem cells have less self-renewal ability and are not multipotent. A number of recent studies suggest that tissue specific stem cells may have the ability to generate cells of tissues from unrelated organs. Whether this unexpected plasticity constitutes "transdifferentiation" or whether a small population of multipotent stem cells persists in post-natal tissues is not known The finding that stem cells exist in post-natal tissues with previously unknown proliferation and differentiation potential opens up the possibility of using autologous stem cells to treat a host of degenerative, traumatic or congenital diseases. Aside from the immunological advantage of using autologous stem cells, tissue derived stem cells are also not encumbered by ethical considerations. We identified cells in human (h), murine (m) and rat (r), post-natal bone marrow (BM) that have, at the single cell level, multipotent differentiation and extensive proliferation potential. which we named Multipotent Adult Progenitor Cell or MAPC. (A) hMAPC, mMAPC and rMAPC differentiate in vitro mesodermal cell types, cells with neuroectodermal and with endodermal features. (B) Using retroviral marking and evaluation of the integrant insertion site, we have shown for hMAPC that multi-lineage differentiation is derived from single cells. (C) hMAPC, mMAPC and rMA.PC express active telomerase and can undergo 90+ cell doublings without telomere shortening, suggesting that they do not senesce. (D) hMAPC. mMAPC and rMAPC express oct-4, Rex-i mRNA and a fraction of cultured hMAPC stain with the Ab, SSEA-4. (E) We have evidence that MAPC engraft in vivo and persist for 2+ months even though single cell origin of the engrafted cells that differentiate in multiple tissue specific cells has not yet been proven. (F) Similar cells with extensive proliferative and multilineage differentiation potential can be found in murine brain and muscle. Because MAPC containing cultures are still heterogeneous, we plan to further characterize the phenotype and genotype of MAPC. We will also test the hypothesis that MAPC may exist in other organs but that the phenotype of MAPC derived from different organs will be similar. This will be accomplished in the following 3 aims: SA1: To define the cell-surface phenotypic characteristics and expressed gene profile of MAPC. As has been shown for other stem cells, we believe that MAPC can be selected from fresh tissue to high purity based on cell surface or functional (e.g. quiescence) characteristics. The expressed gene profile of MAPC will allow us to discriminate between MAPC and tissue committed stem cells, such as MSC. We also have preliminary evidence that MAPC may exist in tissues other than BM and plan to further characterize MAPC from other organs in mouse. SA2: To further define the growth requirements of MAPC. We will test if autocrine factors are necessary for optimal MAPC growth in vitro, and whether presence of more committed progenitors negatively impacts on culture of MAPC to homogeneity. SA3: To define the in vivo differentiation capacity of mMAPC. Final proof that MAPC are true stem cells will require that we demonstrate long-term engraftment and differentiation into mesodermal and non-mesodermal cell types at the clonal level in vivo. We will test whether MAPC differentiate in vivo to tissue specific cells and whether this is enhanced when organs are damaged.