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The Ex Vivo Expansion of Feline Marrow Cells Leads to Increased Numbers of BFU-E and CFU-GM but a Loss of Reconstituting Ability

Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington and Pacific Northwest Research Foundation, Seattle, Washington, USA

Key Words. Ex vivo expansion • Feline hematopoietic stem cells • Enrichment of HSC

Dr. Janis L. Abkowitz, Division of Hematology, University of Washington, Box 357710, Seattle, Washington 98195-7710, USA.

Some studies in mice suggest that hematopoietic stem cells can be maintained and possibly expanded ex vivo. As there is a paucity of data from larger animals, we have studied hematologic reconstitution following autologous marrow transplantation in cats. Transplantation of very low density marrow cells (<1.050 g/ml), termed "1050 cells," at 2 x 10⁵ cells/kg leads to rapid hematopoietic recovery (granulocytes >200/µl by day 20 ± 2 and platelets >50 x 10³/µl by day 21 ± 3). Recovery rates are comparable when 1-2 x 10⁷ nucleated marrow cells/kg are infused, suggesting that reconstituting cells are enriched 50- to 100-fold in the 1050 cell preparation. To explore if the numbers of reconstituting cells could be expanded ex vivo, 1050 cells were cultured in the presence of 5 ng/ml recombinant human interleukin 1ß, 10 ng/ml recombinant canine (rc)G-CSF, 2 U/ml rHu erythropoietin, and 5 ng/ml rc stem cell factor. Maximum numbers of BFU-E and colony-forming units-granulocyte/macrophage (CFU-GM) were generated at day 6. However, when 10⁶ 1050 cells/kg (5x that needed for hematologic recovery) were cultured for six days and all resulting cells infused into irradiated donor animals, two of nine (22%) engrafted. Even when flt3 ligand (100 ng/ml) was added to cultures, only two of five animals (40%) engrafted (p = NS versus studies without flt3 ligand). These data confirm that BFU-E and CFU-GM provide inaccurate estimates of reconstituting cells and demonstrate that the number or function of feline reconstituting cells is impaired by in vitro culture with cytokines.

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