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 r apid proliferation and expansion of human CD34(+)CD38(-) cells that are cap able of multilineage engraftment within the bone marrow of a secondary host . CD34(+)CD38(-) cells contain the self-renewing, long-term culture-initiat ing cells (LTC-IC) that are ideal targets for retroviral gene transfer expe riments. Previous experiments attempting retroviral infection of CD34(+)CD3 8(-) cells have failed partly because these cells do not enter cell cycle i n response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34(+) cel ls and the CD34(+)CD38(-) subset before and after ex vivo expansion on PMVE Cs. Purified human CD34(+) cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-sti mulating factor (GMCSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF ) + Flt-3 ligand. The total CD34(+) population and the CD34(+)CD38(-) subse t 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) pro genitor cells. After 7 days of coculture with PMVECs, 44% of the CD34(+)CD3 8(+) subset were found to be in G(1), and 51% were in G(2)/S/M phase of the cell cycle. More remarkably, 53% of the CD34(+)CD38(-) subset were in G(1) , and 17% were in G(2)/S/M phase after 7 days of PMVEC coculture. In contra st, only 22% of the CD34(+)CD38(-) subset remaining after 7 days of stroma- free culture were in G(1), and 6% were in G(2)/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (s ialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was mai ntained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, an d CD62L (L selectin) increased after ex vivo expansion. In contrast, CD34() cells expanded on stroma-free cultures showed lower and more variable exp ression of CD62L and CD15s. These findings demonstrate that the primitive C D34(+)CD38(-) subset of marrow progenitor cells can be induced to enter cel l cycle and can be significantly expanded ex vivo on a hematopoietic suppor tive microenvironment (PMVECs) while preserving the expression of cell adhe sion molecules that may be important in stem cell homing and engraftment. ( C) 1999 International Society for Experimental Hematology. Published by Els evier Science Inc.