Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation.
Productive hepatitis C virus infection of stem cell-derived hepatocytes reveals a critical transition to viral permissiveness during differentiation.
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DOI：
10.1371/journal.ppat.1002617
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Tang H
中科院分区：
文献类型：
--
作者：
Wu X;Robotham JM;Lee E;Dalton S;Kneteman NM;Gilbert DM;Tang H
从患者活检中分离出的原代人肝细胞代表了丙型肝炎病毒（HCV）感染在生理上最相关的细胞培养模型，但这些原代细胞不易获得，表现出个体差异，并且在很大程度上难以进行基因操作。从多能干细胞分化而来的肝细胞样细胞提供了一种有吸引力的替代方案，因为它们不仅克服了这些缺点，而且还可以为研究和细胞治疗提供无限的非癌细胞来源。尽管有这样的前景，但分化的人类肝细胞样细胞 (DHH) 是否允许 HCV 感染尚未得到探索。在这里，我们报告了一种基于源自人类胚胎 (hESC) 和诱导多能干细胞 (iPSC) 的 DHH 的新型感染模型。在无饲养层条件下在化学成分确定的培养基中产生的 DHH 受到细胞培养物中衍生的 HCV (HCVcc) 和患者衍生病毒 (HCVser) 的感染。多能干细胞和定形内胚层不允许HCV感染，而肝祖细胞持续感染并将感染性颗粒分泌到培养基中。对感染的允许程度与肝脏特异性 microRNA-122 的诱导和影响 HCV 复制的细胞因子的调节相关。针对干细胞中必需细胞辅助因子的 RNA 干扰导致分化后产生 HCV 抗性肝细胞样细胞。用 HCV 患者血清直接感染培养细胞、研究病毒许可的特定阶段以及产生具有所需表型的转基因细胞的能力对于宿主-病原体相互作用和细胞治疗具有广泛的意义。用于感染丙型肝炎病毒（HCV）的生理相关细胞培养模型很少，并且来自患者血清的病毒感染效率低下。源自多能干细胞的分化人肝细胞样细胞表现出肝功能，但尚未用于 HCV 感染研究。在这里，我们报告了一种基于这些肝细胞样细胞的新型感染模型。干细胞和定形内胚层成功抵抗了 HCV 感染，而源自干细胞的肝祖细胞则被人类和细胞培养物衍生的 HCV 有效感染。我们确定了从抗性到易感性的转变点，并通过比较基因分析，确定了与易感性相关的宿主因素。人类胚胎干细胞的基因改造加上肝分化，产生了能够抵抗丙型肝炎病毒感染的肝细胞样细胞。我们的研究建立了一种新的用于HCV感染的非癌性和可再生细胞培养系统，允许在体外直接感染细胞，确定肝细胞分化过程中HCV易感性的明确转变，并证明在体外产生抗病毒人肝细胞样细胞的可行性。
Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy. Physiologically relevant cell-culture models for infection with hepatitis C virus (HCV) are scarce, and infection by viruses derived from patient serum has been inefficient. Differentiated human hepatocyte-like cells derived from pluripotent stem cells demonstrate hepatic functions but have not been explored for HCV infection studies. Here we report a novel infection model based on these hepatocyte-like cells. Stem cells and definitive endoderm successfully resisted HCV infection, whereas hepatic progenitor cells derived from the stem cells were productively infected by both human- and cell-culture-derived HCV. We determined the point of transition from resistance to susceptibility and, by comparative gene profiling, identified the host factors that were correlated with susceptibility. Genetic modification of human embryonic stem cells, coupled with hepatic differentiation, generated hepatocyte-like cells that were resistant to HCV infection. Our study establishes a new noncancerous and renewable cell-culture system for HCV infection, permits direct infection of cells by patient sera in vitro, identifies a defined transition to HCV susceptibility during hepatocyte differentiation, and demonstrates the feasibility of generating virus-resistant human hepatocyte-like cells in vitro.
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影响因子：
14.9
作者：
Kenworthy R;Lambert D;Yang F;Wang N;Chen Z;Zhu H;Zhu F;Liu C;Li K;Tang H
通讯作者：
Tang H
影响因子：
46.9
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影响因子：
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作者：
Kuritzkes DR
通讯作者：
Kuritzkes DR
影响因子：
56.9
作者：
Jopling, CL;Yi, MK;Sarnow, P
通讯作者：
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影响因子：
32.4
作者：
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通讯作者：
Luban, J