Maintenance of human hematopoietic stem cells during ex vivo progenitor cell expansion on a porcine endothelial cell line

We utilized a porcine microvascular endothelia! cell (PMVEC) coculture system to evaluate the m vitro expansion of human marrow cells expressing the CD34 and Thy-1 antigens but lacking the lineage-associated markers CD2, CD14, CD15, CD16, CD19, and glycophorin (Ihr). CD34Thyin' cells were isolated by fluorescence-activated cell sorting (FACS) and cultured on PMVEC monolayers in the presence of IL-3, IL-6, and GM-CSF ±kit ligand and their phenotype and biological activity followed over three weeks. CD34 cells, as well as CFU-GM and CFU-MK, were expanded over this culture period. To assess stem cell activity in the cocultures, cobblestone area-forming cells (CAFC) and cells capable of in vivo marrow repopulation were assayed. The total number of CAFC assayable on a murine stromal cell line was maintained or increased over the culture period. The expanded cells from the PMVEC cocultures retained the ablility to repopulate fragments of human fetal bone implanted into C.B-17 seid/seid mice (SCID-hu bones) with multilineage CDI9 B-lymphoid and CD33 myeloid as well as CD34⁺ progeny after eight weeks. Cells in the PMVEC cocultures expressing the original CD34Thylin' phenotype were re-isolated by FACS over the three-week period and compared to the cells used to initiate the expansion cultures. These cells exhibited no loss in CAFC frequency and were capable of multilineage and CD34 cell engraftment in SCID-hu bone assays. These data contrasted with those obtained in an adherent cell-free culture system. While CD34Thylin" cells and CAFC were maintained for similar lengths of time in the absence of endothelial cells, the expanded cells exhibited a diminished capacity to repopulate SCID-hu bone. These data indicate that the ex vivo expansion of CD34Thylin' cells in PMVEC coculture generates grafts with increased progenitor eel! content as well as stem cells capable of competitive marrow repopulation.