Preincubation with Endothelial Cell Monolayers Increases Gene Transfer Efficiency into Human Bone Marrow CD34 + CD38 - Progenitor Cells

Abstract

Retroviral gene transfer studies targeting bone marrow CD34⁺CD38^- stem cells have been disappointing because of the rarity of these cells, their G₀ cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34⁺CD38^- stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34⁺CD38^- population to enter the G₁ or G₂/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34⁺CD38^- population into cell cycle but did not maintain cells with the CD34⁺CD38^- phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34⁺CD38^- cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34⁺CD38^- cells (11.4 ± 5.6 and 10.9 ± 5.2%, respectively) than in either steady state bone marrow CD34⁺CD38^- cells (0.6 ± 1.7 and 0.2 ± 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34⁺CD38^- cells (1.4 ± 3.5 and 0.0%, respectively; p < 0.01 and p< 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34⁺CD38^- cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.