Ex vivo expanded human CD34⁺ progenitor cells retain their primitive cell adhesion molecule profile when co-cultured with porcine microvascular endothelial cells and exposed to cytokines

Various ex vivo culture systems are being utilized to expand CD34⁺ progenitor cells for bone marrow transplantation and gene therapy protocols. However, it is evident that exposure to cytokines alone may result in a defect in engraftment capacity. Previously, we have demonstrated that porcine microvascular endothelial cells (PMVECs) and cytokines support a rapid proliferation and expansion of CD34⁺CD38 progenitor cells. These cells have no engraftment defect and are capable of homing and multilineage engrafbnent. In this study, we compared the cell adhesion molecule profile on purified CD34⁺ bone marrow cells before and after ex vivo expansion on PMVECs. Purified CD34' bone marrow cells were co-cultured with PMVEC for 7 days in the presence of optimal concentrations of GM-CSF+IL-3+IL-6+FLT3-ligand. CD34⁺ andCD34'CD38 cell populations increased 10.5 and 152-fold, respectively with absolute increases in the number of CFU-GM (22.7 fold), CFU-Mix (10.0 fold) and BFU-E (4.6 fold) progenitor cells. PMVEC co-culture had no significant effect on LFA-I (CD1 la), LFA-3 (CD58), Mac-1 (GDI Ib), CD44, ICAM (CD54), E-selectin (CD62E) or P selectin (CD62P) expression on expanded CD34⁺ cells. VLA-4 (CD49d) and L-selectin (CD62) were detected on a greater number of ex vivo expanded CD34⁺ cells and expressed at a higher density when compared to untreated CD34' cells. Both CD43 and sLex were expressed at high levels on >87% of untreated CD34⁺ cells. In some cultures, following ex vivo expansion we detected two CD34⁺ cell populations, CD34⁺ "sLex l'i' CD43 "h (46 %) and CD34~i 'LexJ"'CD43J" (54 %). The decreased expression of sLex and CD43 post ex vivo culture directly correlated with the early appearance of more differentiated CD34⁺ cells in culture (36% CD34⁺ Ii|M CD 15 w ). We speculate that the CD34⁺M lLex'l"CD43'lw subset is likely to contain the more mature, committed progenitor cells, whereas the CD34' 1 ksLex CD43'"'h subset contains the less differentiated, Lin progenitor cells. These studies demonstrate that CD34⁺ CD38'bone marrow cells can be significantly expanded ex vivo on PMVECs while preserving the expression of multiple cell adhesion molecules which have been suggested to be important in stem cell homing and bone marrow engraftment. Thus, the defect in engrafbnent capacity of CD34⁺ cells expanded/maintained in stroma-frce culture systems may be due solely to the inadequate preservation of pivotal adhesion molecules, which we have shown can be overcome by ex vivo expansion on endothelial cells (PMVECs).