Expression of GLVR-1 and GLVR-2 on human CD34+CD38- Bone marrow cells following co-culture with porcine microvascular endothelial cells (PMVEC) and liquid culture with PMVEC-CM

Retroviral based gene therapy using hematopoietic stem cells (HSC) is limited by the low level of gene transduction into primitive CD34+CD38- cells. Inefficient retroviral transduction of CD34+CD38- cells is caused in part by the non-cycling status of CD34+CD38- cells and the very low levels of amphotropic receptor expression in these cells. We have previously demonstrated that co-culture of bone marrow CD34+CD38- cells on PMVEC monolayers and liquid culture with 10% Conditioned media from PMVECs induces >70% of these cells into G1 or G2/S/M phase after 72 hours. Since the PMVEC co-culture system and liquid culture with PMVECConditioned media support a high level of cell cycling in primitive CD34+CD38- cells. we decided to examine the expression of GLVR-1 and GLVR-2 on CD34+CD38- cells following expansion in both of these systems. Purified bone marrow CD34+ cells were cultured with PMVEC monolayers plus GMCSF t IL-3 + IL-6 + SCF + Flt-3 ligand for 7 days and compared to liquid culture plus 10% PMVEC-Conditioned media plus the same cytokines for 7 days. After 7 days, the expanded hematopoietic cells were harvested and sorted by FACS for CD34+CD38- and CD34+CD38+ subsets. CD34+CD38- cells were defined as <1% of all CD34+ cells remaining after expansion. Total cellular RNA was extracted from these cells and analyzed by reverse transcriptase PCR for amphoR (GLVR-2) mRNA and GLVR-1 mRNA levels. The level of GLVR-1 and GLVR-2 mRNA was normalized to the level of Beta-actin mRNA in each sample and compared to the level of GLVR-1 and GLVR-2 mRNA in KGla ceils. Consistent with previous reports, we observed that day 0 (non-expanded) CD34+CD38- cells express low levels of GLVR-1 and GLVR-2 mRNA (2.0% and 7.5% relative to Betaactin). Following 7 days of PMVEC co-culture, expression of GLVR-1 and GLVR-2 remained detectable at low levels (1.8% and 1.6%, respectively). Following 7 days of liquid culture with PMVEC-CM, expression of GLVR-1 and GLVR-2 was also detectable at low levels (4.3% and 2.0%, respectively). These data suggest that the expression of GLVR-1 and GLVR-2 mRNA is low on steady state bone marrow CD34+CD38- cells and that expansion of these primitive HPCs on PMVEC monolayers or in liquid culture with PMVEC-CM does not alter the expression of these receptors. However, since both PMVEC-coculture and liquid culture with PMVEC-CM induce >70% of the CD34+CD38- subset into cell cycle, we are currently testing the efficiency of retroviral-mediated gene transfer into CD34+CD38- cells expanded in these systems.