One-day ex vivo culture allows effective gene transfer into human nonobesediabetic severe combined immune-deficient repopulating cells using high-titer vesicular stomatitis virus G protein pseudotyped retrovirus

Retrovirus-mediated gene transfer into long-lived human pluripotent hematop oietic stem cells (HSCs) is a widely sought but elusive goal. A major probl em is the quiescent nature of most HSCs, with the perceived requirement for ex vivo prestimulation in cytokines to induce stem cell cycling and allow stable gene integration. However, ex vivo culture may impair stem cell func tion, and could explain the disappointing clinical results in many current gene transfer trials, To address this possibility, we examined the ex vivo survival of nonobese diabetic/severe combined immune-deficient (NOD/SCID) r epopulating cells (SRCs) over 3 days. After 1 day of culture, the SRC numbe r and proliferation declined twofold, and was further reduced by day 3; sel f-renewal was only detectable in noncultured cells. To determine if the per iod of ex vivo culture could be shortened, we used a vesicular stomatitis v irus G protein (VSV-G) pseudotyped retrovirus vector that was concentrated to high titer, The results showed that gene transfer rates were similar wit hout or with 48 hours prestimulation. Thus, the use of high-titer VSV-G pse udotyped retrovirus may minimize the loss of HSCs during culture, because e fficient gene transfer can be obtained without the need for extended ex viv o culture. (C) 1999 by The American Society of Hematology.