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, Colin A. Sieff

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2217-2224
Number of pages8
JournalBlood
Volume93
Issue number7
StatePublished - Apr 1 1999
Externally publishedYes

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Gene transfer
Retroviridae
Stem cells
Hematopoietic Stem Cells
Genes
Cell culture
Stem Cells
Pluripotent Stem Cells
Cell Culture Techniques
Cell Count
Cell Proliferation
vesicular stomatitis virus G protein
Cytokines
Survival

ASJC Scopus subject areas

  • Hematology

Cite this

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. / Rebel, Vivienne I.; Tanaka, Mayumi; Lee, Jeng Shin; Hartnett, Sheila; Pulsipher, Michael; Nathan, David G.; Mulligan, Richard C.; Sieff, Colin A.

In: Blood, Vol. 93, No. 7, 01.04.1999, p. 2217-2224.

Research output: Contribution to journalArticle

Rebel, Vivienne I. ; Tanaka, Mayumi ; Lee, Jeng Shin ; Hartnett, Sheila ; Pulsipher, Michael ; Nathan, David G. ; Mulligan, Richard C. ; Sieff, Colin A. / 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. In: Blood. 1999 ; Vol. 93, No. 7. pp. 2217-2224.
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