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
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
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DOI：
10.1182/blood.v93.7.2217.407a01_2217_2224
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发表时间：
1999-04-01
期刊：
影响因子：
20.3
通讯作者：
Sieff, CA
中科院分区：
文献类型：
--
作者：
Rebel, VI;Tanaka, M;Sieff, CA
逆转录病毒介导的基因转移到长寿的人多能造血干细胞（hsc）是一个广泛寻求但难以实现的目标。一个主要的问题是大多数造血干细胞的静态特性，需要体外预刺激细胞因子来诱导干细胞循环并允许稳定的基因整合。然而，体外培养可能会损害干细胞功能，并且可以解释目前许多基因转移试验中令人失望的临床结果。为了解决这一可能性，我们检查了非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）再生细胞（src）的体外存活超过3天。培养1 d后，SRC数量和增殖下降2倍，第3天进一步降低；自我更新仅在未培养的细胞中检测到。为了确定是否可以缩短离体培养时间，我们使用水疱性口炎病毒G蛋白（VSV-G）假型逆转录病毒载体浓缩到高滴度，结果表明，在没有或有48小时预刺激的情况下，基因转移率相似。因此，使用高滴度VSV-G伪型逆转录病毒可以最大限度地减少造血干细胞在培养过程中的损失，因为无需延长离体培养即可获得有效的基因转移。(C) 1999年由美国血液病学会出版。
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. (C) 1999 by The American Society of Hematology.