Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes
Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes
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DOI：
10.1002/ijc.20126
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发表时间：
2004-06-20
影响因子：
6.4
通讯作者：
Yang, WK
中科院分区：
文献类型：
--
作者：
Hung, SC;Yang, DM;Yang, WK
来源于骨髓的hMSC可用作研究细胞谱系分化和组织重塑的物种特异性细胞培养系统。然而，由于复制性衰老，hMSCs通常具有较短的体外寿命。因此，我们使用高剂量的逆转录病毒载体LXSN-16 E6 E7转染衰老供体的hMSCs，获得了表达HPV 16 E6/E7 mRNA的活跃增殖的细胞系，命名为KP-hMSCs。而亲本hMSC在30个PD后停止生长，KP-hMSC可以繁殖超过100个PD。培养程序，以避免选择压力和拥挤的细胞生长，KP-hMSCs没有表现出肿瘤转化的迹象，通过软琼脂锚定非依赖性生长和NOD-SCID小鼠致瘤性测定。KP-hMSCs的31种CD标记物的细胞荧光谱与亲本原代hMSCs相似，除了一些形态学变化和最初非常小的CD 34（dim）CD 38（+）CD 50（+）细胞群的扩增。在特定的体外刺激条件下，KP-hMSCs可以响应并沿间充质（骨、脂肪和软骨）和非间充质（神经元）细胞谱系沿着分化。本研究结果表明，HPV 16 E6/E7基因转导的hMSCs可以获得永生化，并通过精心的培养程序保持其不发生肿瘤转化，从而为干细胞研究和临床应用提供了有用的材料。(C)2004 Wiley-Liss，Inc.
hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16 E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50(+) cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application. (C) 2004 Wiley-Liss, Inc.