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）的体内扩张是一种有吸引力的技术，其效力各种临床应用。通过各种细胞因子或连续灌注培养物的组合，已经实现了这种技术。但是，对于扩大原始造血祖细胞的扩展需要更多的改进。我们在这里提出了一种新型的分子方法，该方法可能具有补偿当前扩张的潜力。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），该受体仅转导促丝分裂，但不转导向小鼠骨髓细胞的分化信号在人类纯化的CD34+外周血（PB）上，并尝试通过EGF EX VIVO扩展这些细胞。因为我们发现CD34+ PB细胞暴露于细胞因子诱导腺病毒 - 甘粉化受体的表面表达，并使这些细胞允许在腺病毒感染中允许的细胞，因此我们用腺病毒载体在细胞因子的存在下用腺病毒载体携带EGFR基因感染了这些细胞。两色流式细胞仪分析表明，60.3％+/- 22.4％的CD34 +细胞表达了腺病毒介导的EGFR。此外，长期培养的发射细胞测定法显示腺病毒载体可以传递更原始的祖细胞。随后，我们试图用EGF在悬浮培养中扩展这些细胞5天。甲基纤维素克隆分析表明，EGF在膨胀5天内诱导了菌落形成单位池的5.0- +/- 2.4倍增殖。事实证明，有效的腺病毒基因递送到未成熟的造血细胞的简单程序被证明是有希望的，并且该技术可能适用于一种新型策略，旨在实时扩展造血祖细胞。