Park Su Cheol, Zeng Changjun, Tschudy-Seney Benjamin, Nguyen Ngoc Tue, Eun Jong Ryeol, Zhang Yanling, Ramsamooj Rajendra, Zhang Yanghong, Zhao Min, Theise Neil D, Zhou Huaijun, Zern Mark A, Duan Yuyou
1 Department of Internal Medicine, University of California Davis Medical Center , Sacramento, California.
2 Institute for Regenerative Cures, University of California Davis Medical Center , Sacramento, California.
Stem Cells Dev. 2015 Jul 1;24(13):1506-14. doi: 10.1089/scd.2015.0022. Epub 2015 May 12.
A large number of cancer stem cells (CSCs) have been isolated and identified; however, none has been cultured in an unlimited manner in vitro without losing tumorigenicity and multipotency. In this study, we successfully clonogenically cultured a newly identified CD34+ liver CSC (LCSC) on feeder cells up to 22 passages (to date) without losing CSC property. Cloned CD34+ LCSC formed a round packed morphology and it could also be cryopreserved and recultured. Stem cell markers, CD34, CD117, and SOX2; normal liver stem cell markers, alpha fetoprotein, CK19, CK18, and OV6; putative CSC markers, CD44, CD133, EpCAM, and CD90; as well as CD31 were expressed in cloned CD34+ LCSC. SOX2 was the major factor in maintaining this LCSC before colonization, and interestingly, OCT4, SOX2, NAONG, Klf4, c-Myc, and Lin28 were upregulated in association with symmetric self-renewal for colony growth of CD34+ LCSC on feeder cells. Gene expression patterns of in vitro differentiation were consistent with our in vivo finding; furthermore, the tumorigenicity of cloned CD34+ LCSC was not different from uncloned CD34+ LCSC sorted from parental PLC. These results show that our cloned CD34+ LCSC maintained CSC property, including self-renewal, bipotency, and tumorigenicity after long-term culture, demonstrating that this LCSC can be cultured in an unlimited manner in vitro. Thus, establishing pure population of CSCs isolated from the patients will provide an opportunity to explore the mechanisms of tumorigenesis and cancer development, and to identify unique biomarkers presenting potential indicators of drug efficacy against CSCs for establishment of a novel strategy for cancer therapy.
大量癌症干细胞（CSCs）已被分离和鉴定；然而，尚无一种能在体外以无限方式培养而不丧失致瘤性和多能性。在本研究中，我们成功地在饲养细胞上对新鉴定的CD34+肝CSC（LCSC）进行了克隆培养，传代至22代（迄今为止），且未丧失CSC特性。克隆的CD34+ LCSC形成圆形聚集形态，还可进行冷冻保存和再培养。干细胞标志物CD34、CD117和SOX2；正常肝干细胞标志物甲胎蛋白、CK19、CK18和OV6；假定的CSC标志物CD44、CD133、EpCAM和CD90；以及CD31在克隆的CD34+ LCSC中均有表达。SOX2是在定植前维持这种LCSC的主要因素，有趣的是，OCT4、SOX2、NANOG、Klf4、c-Myc和Lin28在饲养细胞上与CD34+ LCSC集落生长的对称自我更新相关而上调。体外分化的基因表达模式与我们的体内发现一致；此外，克隆的CD34+ LCSC的致瘤性与从未克隆的亲代PLC中分选的CD34+ LCSC并无差异。这些结果表明，我们克隆的CD34+ LCSC在长期培养后仍保持CSC特性，包括自我更新、双能性和致瘤性，证明这种LCSC可在体外以无限方式培养。因此，建立从患者中分离出的CSC纯群体将为探索肿瘤发生和癌症发展机制提供机会，并鉴定独特的生物标志物，这些生物标志物是针对CSC的潜在药物疗效指标，从而建立一种新的癌症治疗策略。
