Soluble stromal factors enhance gene transfer in primitive human hematopoietic cells in mobilized peripheral blood

Abstract

Hematopoietic stem cells present mobilized peripheral blood (MPB) are attractive targets for retroviral gene therapy. Previously, we have found that in contrast to primitive hematopoietic cells in umbilical cord blood, primitive cells in G-CSF MPB are not efficiently transduced. Based on earlier studies demonstrating a positive effect of the fetal liver stromal cell line AFT024 on the maintenance of primitive hematopoietic cells ex vivo, we hypothesized that AFT024 may enhance the level of gene transfer (GT) into these cells. To test this hypothesis, CD34+ cells from G-CSF MBP were cultured in the presence or absence of irradiated AFT024 cells using trans-well (non-contact) cultures for 4 days with either G-CSF, SCF and TPO (GST; 100 ng/mL) or Flt3-L, SCF, IL-7 and TPO (FS7T; 10-20 ng/mL), followed by infection on two consecutive days with a GALV-pseudotyped MFG-EGFP retroviral vector on fibronectin fragment FN CH-296 (Retronectin®, Takara Shuzo). Cultured and transduced cells were evaluated for the level of expansion as well as for the level of GT in CFC and LTC-IC assays. AFT024 enhanced the expansion of CFC and LTC-IC (1.5 to 2-fold). The level of GT in CFC (ranging from 1 to 26% in BFU-E and CFU-GM; n=10) was higher in the groups pre-stimulated with GST as compared to FS7T. In contrast, the level of GT in LTC-IC (ranging from 1.5 to 47%) was higher with FS7T (n=6), but only in the presence of AFT024. Finally, the overall recovery of transduced LTC-IC in the presence of AFT024 with FS7T was 6-fold higher as compared to our previously used strategy using GST in the absence of stroma (p<0.001). These data demonstrate that the recovery of transduced primitive hematopoietic cells in G-CSF MPB can be enhanced using low doses of early acting cytokines and a soluble factor produced by AFT024.