While much work on bone marrow stem cells can be accomplished in vitro, the ultimate test of such cells must involve studies of engraftment in vivo. For this purpose it is essential to utilize animal models, and the mouse has been most widely exploited for this purpose. There are, however, significant differences between rodents and large animals with respect to studies of bone marrow transplantation. In particular, the preparative regimen by which chimerism is routinely achieved in mice is far too toxic for use in human beings. In this laboratory, partially inbred miniature swine have been developed as a large animal preclinical model for studies of transplantation biology. These animals are very similar to man in parameters related to bone marrow transplantation, and they therefore provide a unique large animal model for such studies: They are the only large animals in which transplantation across defined major histocompatibility barriers can be reproducibly performed. Previous studies from this laboratory have established conditions under which bone marrow can be transplanted between MHC matched animals successfully, leading to longterm survival. The main goal of this proposal is therefore to derive conditions for long-term cultures of normal and retrovirally transduced bone marrow stem cells and to examine in vivo reconstitution of MHC matched animals with these cultured cells. Specifically, we shall: 1) Establish culture conditions capable of maintaining and propagating porcine bone marrow stem cells; 2) Assess the potential of such cells to reconstitute lethally irradiated miniature swine; and 3) Utilize these conditions for reconstitution of animals with autologous bone marrow transduced with allogeneic Class II genes. Because of the similarity between these animals and human beings it is hoped that studies of cultured stem cells and the requirements for their engraftment in this model will be clinically applicable.
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