Ex vivo expansion of hematopoietic stem cells using adenovirus

造血干细胞(HSC)的体外扩增技术因其在多种临床应用中的潜能而备受关注。这样的技术已经在一定程度上通过各种细胞因子的组合或连续灌流培养而实现。然而，还需要更多的改进，特别是在原始造血祖细胞的扩增方面。我们在这里提出了一种新的分子方法，可能有可能补偿目前的扩张。我们设计了一种腺病毒载体，在纯化的人CD34+外周血(PB)细胞上瞬时表达人表皮生长因子受体(EGFR)，并试图用EGF体外扩增这些细胞。由于我们发现CD34+PB细胞暴露于细胞因子诱导了腺病毒内化受体的表面表达，并使这些细胞对腺病毒感染具有容许性，因此我们在细胞因子存在的情况下将携带EGFR基因的腺病毒载体感染这些细胞。双色流式细胞仪分析显示，60.3%+/-22.4%的CD34+细胞表达腺病毒载体介导的EGFR。长期培养启动细胞实验表明，腺病毒载体可以转导更多的原始祖细胞。随后，我们尝试在含有EGF的悬浮培养中扩增这些细胞5天。甲基纤维素克隆实验显示，EGF在5d内诱导集落形成单元池增殖5.0-+/-2.4倍。高效的腺病毒基因导入未成熟造血细胞的简便方法被证明是很有前景的，这项技术可能适用于一种旨在体外扩增造血祖细胞的新策略。

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.