CULTURE AND CHARACTERIZATION OF HEMATOPOIETIC PROGENITOR CELLS FROM MINIATURE SWINE

Miniature swine are being used as a large animal model in which cultur ed and retrovirus-transduced hematopoietic stem cells (HSC) can be tes ted in a reproducible manner for their long-term in vivo repopulating ability. As part of these studies, long-term bone marrow culture (LTBM C) and progenitor colony assay systems were developed and used to char acterize the in vitro growth potential and in vivo frequency of hemato poietic progenitors in this species. We found that LTBMCs initiated wi th a single marrow inoculum produced myeloid colony progenitors contin uously for at least 7 weeks. The sites of myelopoietic activity in the se cultures were uniquely restricted to isolated, morphologically dive rse germinal centers rather than more disperse cobblestone patches. We also used the progenitor assay to screen several human and murine rec ombinant cytokines for cross-reactivity to swine bone marrow cells, in cluding interleukin-3 (IL-3), IL-6, IL-11, granulocyte and granulocyte -macrophage colony-stimulating factors (G-CSF and GM-CSF), c-kit ligan d (also called mast cell growth factor [MGF]), and erythropoietin (Epo ). With the exception of human and murine IL-3, each of the cytokines tested induced swine progenitor colony formation to varying degrees, w ith some combinations leading to the formation of primitive multilinea ge and high proliferative potential colonies. Finally, in an attempt t o characterize alternative sources of HSC from swine, we compared the progenitor content of adult and juvenile swine bone marrow and fetal l iver. The fetal liver samples were found to be highly enriched for bot h primitive and mature progenitors, while analysis of postnatal marrow samples revealed an approximately two-fold decline in overall progeni tor frequency between the ages of 10 and 20 weeks. Taken together, the se studies demonstrate the development and use of in vitro culture met hods for characterizing hematopoietic elements from miniature swine an d suggest a hierarchy of progenitor cell content in various hematopoie tic tissues from this large animal model.