Bone marrow repopulation by human marrow stem cells following long-term expansion culture on a porcine endothelial cell line

Short-term exposure of murine hematopoietic stem cell (HSC) populations to cytokines may result in the acquisition of a defect in the ability of these cells to engraft. We utilized a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous SCF, IL-3, IL-6, and GM-CSF to expand human CD34⁺Thy-1⁺Lin marrow cells. Expansion of total cells, CD34⁺ cells, CFU-GM, BFU-E, and cobblestone area-forming cells (CAFC) were evaluated in comparison to a stromal cell-free liquid culture system to which cytokine combinations including SCF, IL-3, IL-6, GM-CSF, G-CSF, and LIF were added. CD34⁺Thy-1⁺Lin^- cells were detectable in both culture systems for up to three weeks. These cells were re-isolated and their ability to engraft allogeneic human fetal bone fragments implanted into C.B-17 seid/seid mice (SCID-hu bone) was evaluated. CD34Thy1⁺Lin^- cells expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage progeny present eight weeks post engraftment. In contrast, grafts composed of similar numbers of CD34⁺Thy-1⁺Lin^- cells expanded in stroma-free cultures did not lead to multilineage repopulation. Proliferation analysis utilizing the membrane dye PKH26 revealed that by one week of culture the majority (>80%) of the cells in the PMVEC cocultures expressing the primitive CD34⁺CD38 phenotype had undergone cell division. Virtually none (<1%) of the cells expanded in suspension culture remained negative for CD38. To test the possibility that the expanded cell population responsible for marrow repopulation might express an altered phenotype, the entire expanded cell product from each culture system was injected into SCID-hu bones after two to four weeks of culture. Again, cells expanded in PMVEC coculture were consistently capable of multilineage marrow engraftment whereas the expanded cell product of the stroma-free cultures did not repopulate SCID-hu bone.These data suggest that the ex vivo expansion of hematopoietic stem cells in the presence of cytokines alone results in an engraftment defect which can be overcome by coculture with PMVEC.