Ex vivo expansion of autologous bone marrow CD34(+) cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons

Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to re sult in a diminished proliferative capacity or acquisition of a homing defe ct that might compromise marrow repopulation. Our group has demonstrated th at human HSC expanded ex vivo in the presence of porcine microvascular endo thelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, ou r laboratory has established a myeloablative, fractionated total body irrad iation conditioning protocol for autologous marrow transplantation in baboo ns. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 4 3, and 59 days postirradiation, respectively. Immunomagnetically selected C D34(+) marrow cells from two baboons were placed in PMVEC coculture with ex ogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granu locyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobbleston e area-forming cells (CAFC) over input. Both baboons became transfusion ind ependent by day 23 posttransplant and achieved absolute neutrophil count (A NC) >500/mu L by day 25 +/- 1 and platelets >20,000/mu L by day 29 +/- 2, T his hematopoietic recovery was delayed in comparison to two animals that re ceived either a graft consisting of freshly isolated, unexpanded CD34(+) ce lls or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergo ne cycling, and that the population of cells expressing a CD34(+) CD38(-) p henotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell div ision during ex vivo expansion in the presence of endothelial cells to prov ide a graft capable of rescuing a myeloablated autologous host. (C) 1999 by The American Society of Hematology.