Ex vivo expansion of hematopoietic stem cells using adenovirus

造血干细胞的体外扩增是一项极具吸引力的技术，具有广泛的临床应用前景。这种技术在某种程度上已经通过各种细胞因子的组合或连续灌注培养来实现。然而，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的分子方法，可能有潜力补偿目前的扩张。我们设计了一种腺病毒载体，瞬时表达人表皮生长因子受体（EGFR），这是已知的，只有一个促有丝分裂，但不是一个分化的信号，小鼠骨髓细胞对人纯化的CD 34+外周血（PB）细胞，并试图扩大这些细胞与EGF离体。因为我们发现暴露于细胞因子的CD 34 + PB细胞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，我们用携带EGFR基因的腺病毒载体在细胞因子的存在下感染这些细胞。双色流式细胞仪检测显示60.3% +/- 22.4%的CD 34+细胞表达腺病毒介导的EGFR，长期培养启动细胞实验显示腺病毒载体能扩增更多的原始祖细胞。随后，我们尝试在含有EGF的悬浮培养物中扩增这些细胞5天。甲基纤维素克隆测定表明，EGF诱导5.0 +/- 2.4倍的扩增5天的集落形成单位池。腺病毒基因转染未成熟造血细胞的简单方法被证明是有前途的，这种技术可能适用于一种新的策略，旨在体外扩增造血祖细胞。

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.