Coculture of human cd34'cd38' cells with porcine microvascular endothelial cells (PMVEC) or pmvecconditioned media enhances retroviral mediated gene transfer

Retrovirai-mediated gene transfer into hematopoietic progenitor cells using Moloney-murine leukemia vims (Mo-MLV)-based vectors has proved difficult due to the inability to transduce nondividing ceils. We have previously shown that PMVECs and PMVEC-Conditioned media (PMVEC-CM) support the proliferation and expansion of human CD34+CD38~ hematopoietic progenitor cells. In this study, we examined the efficiency of retroviral gene transfer into CD34 CD38" cells expanded on PMVEC monolayers and in stroma-free liquid suspension cultures containing cytokines plus PMVEC-CM. Purified CD34' bone marrow cells were cultured directly on PMVEC monolayers in the presence of optimal concentrations of GMCSF, IL-3.IL-6, SCF, and Flt-3 ligand for 7 days or in stroma-free cultures with 10% PMVEC-CM plus cytokines for 5 days. Both culture systems induced >70% of the CD34 CD38' cells to enter d or 02/S/M phase of the cell cycle. Harvested hematopoietic cells were then transduced using high-speed centrifugation with retroviral supernatant (spinoculation x 3 days, Mo-MLV-alkaline phosphatase vector) from cither PA317 or PO13 packaging cell lines. After 3 days, CD34 CD38" and CD34 CD38cells were FACS sorted , stained for rv-alkaline phosphatase activity and assayed in methylcellulose colony forming assays to assess for long term gene expression. CD34'CD38" cells expanded on PMVEC monolayers were highly transduced with vectors from both PG13 and PA317 (43% and 15% of the cells stained positively for rv-alkaline phosphatase, respectively) and CD34 CD38' cells expanded in liquid culture with 10% PMVEC-CM were also highly transduced with both PG13 and PA317 (28% and 33%). In contrast, unstimulated CD34'CD38' showed markedly lower transduction efficiency with both PG13 and PA317 (1% and 3%, respectively). Greater than 95% of the colony-forming progenitor cells (CFU-GEMM and CFU-GM) derived from sorted transduced CD34 CD38" cells cultured on PMVECs or in stroma-free cultures containing PMVEC-CM expressed rv-alkaline phosphatase activity. These results demonstrate that ex-vivo expansion using PMVEC monolayers or PMVEC-CM offers an efficient and attractive means of introducing therapeutic genes into CD34 hematopoietic stem and progenitor cells. CD34 hematopoietic cells transduced with this high level of efficiency have both experimental and clinical applications in metabolic and hematopoietic disorders.