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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2217-2224
One-Day Ex Vivo Culture Allows Effective Gene Transfer Into Human
Nonobese Diabetic/Severe Combined Immune-Deficient Repopulating Cells
Using High-Titer Vesicular Stomatitis Virus G Protein Pseudotyped
Retrovirus
Vivienne I. Rebel,
Mayumi Tanaka,
Jeng-Shin Lee,
Sheila Hartnett,
Michael Pulsipher,
David G. Nathan,
Richard C. Mulligan, and
Colin A. Sieff
From the Division of Pediatric Hematology and Oncology, Dana- Farber
Cancer Institute, Boston; and the Departments of Pediatrics and
Genetics, Harvard Medical School and Howard Hughes Medical Institute,
Children's Hospital, Boston MA.
Retrovirus-mediated gene transfer into long-lived human pluripotent
hematopoietic stem cells (HSCs) is a widely sought but elusive goal. A
major problem 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 function, 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) repopulating cells
(SRCs) over 3 days. After 1 day of culture, the SRC number and
proliferation declined twofold, and was further reduced by day 3;
self-renewal was only detectable in noncultured cells. To determine if
the period of ex vivo culture could be shortened, we used a vesicular
stomatitis virus G protein (VSV-G) pseudotyped retrovirus vector that
was concentrated to high titer. The results showed that gene transfer
rates were similar without or with 48 hours prestimulation. Thus, the
use of high-titer VSV-G pseudotyped retrovirus may minimize the loss of
HSCs during culture, because efficient gene transfer can be obtained
without the need for extended ex vivo culture.
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