Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin

The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have imp eded studies to optimize the retroviral transduction of stem cells from adu lt sources. To address this problem, first a cytokine combination was defin ed that would both maximize the kinetics of adult BM CD34(+)CD38(-) cell mi togenesis and minimize the period of prestimulation required for the transd uction of these cells by a MSCV-GFP/neo(r) virus in tissue culture dishes i n the absence of fibronectin. Three days of stimulation with flt3-ligand, S teel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and s ufficient to obtain 83% +/- 2% GFP(+) CD34(+)CD38(-) cells, 75% +/- 10% G41 8-resistant clonogenic progenitors, and 50% +/- 20% transduced long-term cu lture-initiating cells as recovered 48 hours after a single exposure to vir us. Moreover, this was accompanied by a several-fold increase in viral rece ptor (pit-1) messenger RNA transcripts in the target cells. Using this pres timulation protocol, repeated daily exposure to new virus (3x) did not alte r the proportion of transduced cells over that obtained with a single expos ure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-beta(2) M(-/-)) mice were also efficiently transduced (10%-20% GFP(+) human lymphoi d and myeloid cells present 6-8 weeks after transplant) using a g-day prest imulation and infection protocol. A clinically useful efficiency of retrovi rus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activati on into cycle and concomitant increased expression of virus receptor transc ripts before virus exposure, (Blood, 2000;96:2432-2439) (C) 2000 by The Ame rican Society of Hematology.