A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34⁺ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures

Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34⁺CD38^- cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34⁺CD38^- cells contain the self-renewing, long-term culture- initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34⁺CD38^- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34⁺ cells and the CD34⁺CD38^- subset before and after ex vivo expansion on PMVECs. Purified human CD34⁺ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony- stimulating factor (GMCSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34⁺ population and the CD34⁺CD38^- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34⁺CD38^- subset were found to be in G₁, and 51% were in G₂/S/M phase of the cell cycle. More remarkably, 53% of the CD34⁺CD38^- subset were in G₁, and 17% were in G₂/S/M phase after 7 days of PMVECs coculture. In contrast, only 22% of the CD34⁺CD38^- subset remaining after 7 days of stroma-free culture were in G₁, and 6% were in G₂/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expansion of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34⁺ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L- selectin) increased after ex vivo expansion. In contrast, CD34⁺ cells expanded on stroma-free cultures showed lower and more variable expression of CD62L and CD15s. These findings demonstrate that the primitive CD34⁺CD38^- subset of marrow progenitor cells can be induced to enter cell cycle and can be significantly expanded ex vivo on a hematopoietic supportive microenvironment (PMVECs) while preserving the expression of cell adhesion molecules that may be important in stem cell homing and engraftment.