title: 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.
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.
authors: Division of Pediatric Hematology and Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
authors: V I
authors: VI
authors: Rebel
authors: M
authors: M
authors: Tanaka
authors: J S
authors: JS
authors: Lee
authors: S
authors: S
authors: Hartnett
authors: M
authors: M
authors: Pulsipher
authors: D G
authors: DG
authors: Nathan
authors: R C
authors: RC
authors: Mulligan
authors: C A
authors: CA
authors: Sieff
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured / Q000637:transplantation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008562:Membrane Glycoproteins; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000502:physiology*; D013997:Time Factors; D014183:Transplantation, Heterologous; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000502:physiology*
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins