Ex vivo expansion of hematopoietic stem cells using adenovirus

使用腺病毒体外扩增造血干细胞

基本信息

批准号：
09671091
负责人：
CHIBA Shigeru
金额：
$ 2.24万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Ex vivo expansion of hematopoietic stem cell (HSC) is an attractive technology for its potency of a variety of clinical applications. Such a technology has been achieved to some extent with combinations of various cytokines or continuous perfusion cultures. However, much more improvement is required especially for expansion of primitive hematopoietic progenitors. We propose here a novel molecular approach that might have the potential to compensate the current expansion. We designed an adenovirus vector to transiently express human epidermal growth factor receptor (EGFR), which is known to transduce only a mitogenic, but not a differentiation signal to mouse bone marrow cells on human purified CD34+ peripheral blood (PB) cells, and tried to expand these cells with EGF ex vivo. Because we found that exposure of CD34+ PB cells to cytokines induced surface expression of adenovirus-intemalization receptor and rendered these cells permissive to adenovirus infection, we infected these cells with the adenovirus vector carrying EGFR gene in the presence of cytokines. Two-color flow cytometric analysis demonstrated that 60.3% +/- 22.4% of CD34+ cells expressed the adenovirus-mediated EGFR.Moreover, long-term culture-initiating cell assay showed that adenovirus vector could transduce more primitive progenitors. Subsequently, we tried to expand these cells in suspension culture with EGF for 5 days. Methylcellulose clonal assay showed that EGF induced 5.0- +/- 2.4-fold proliferation of the colony-forming unit pool during 5 days of expansion. The simple procedure of efficient adenovirus gene delivery to immature hematopoietic cells proved promising, and this technique was potentially applicable for a novel strategy aiming at ex vivo expansion of hematopoietic progenitors.

造血干细胞（HSC）的离体扩增因其在多种临床应用中的功效而成为一项有吸引力的技术。这种技术已经在一定程度上通过多种细胞因子的组合或连续灌注培养来实现。然而，还需要更多的改进，特别是对于原始造血祖细胞的扩增。我们在这里提出了一种新颖的分子方法，它可能有潜力补偿当前的扩张。我们设计了一种腺病毒载体来瞬时表达人表皮生长因子受体（EGFR），已知该受体仅在人纯化的 CD34+ 外周血（PB）细胞上向小鼠骨髓细胞转导促有丝分裂信号，而不是分化信号，并尝试用 EGF 离体扩增这些细胞。因为我们发现CD34+PB细胞暴露于细胞因子会诱导腺病毒内化受体的表面表达并使这些细胞允许腺病毒感染，所以我们在细胞因子存在下用携带EGFR基因的腺病毒载体感染这些细胞。双色流式细胞术分析表明，60.3%+/-22.4%的CD34+细胞表达腺病毒介导的EGFR。此外，长期培养起始细胞分析表明，腺病毒载体可以转导更多的原始祖细胞。随后，我们尝试用 EGF 悬浮培养这些细胞 5 天。甲基纤维素克隆测定表明，EGF 在 5 天的扩增过程中诱导集落形成单位库增殖 5.0-+/-2.4 倍。将腺病毒基因有效递送至未成熟造血细胞的简单程序被证明是有前途的，并且该技术可能适用于旨在体外扩增造血祖细胞的新策略。