Ectopic expression of factor VIII in MSCs and hepatocytes derived from rDNA targeted hESCs.
Ectopic expression of factor VIII in MSCs and hepatocytes derived from rDNA targeted hESCs.
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rDNA 靶向 hESC 衍生的 MSC 和肝细胞中因子 VIII 的异位表达。
DOI：
10.1016/j.cca.2018.08.007
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发表时间：
2018
影响因子：
5
通讯作者：
Liang Desheng
中科院分区：
文献类型：
--
作者：
Sun Qianru;Liu Xionghao;Wu Yong;Niu Wenbin;Long Panpan;Liu Jing;Lei Ming;Hu Youjin;Wu Lingqian;Li Zhuo;Liang Desheng
血友病A是一种由FVIII基因缺陷引起的X连锁隐性出血性疾病，可导致自发性关节出血或危及生命的出血。目前，以细胞为基础的基因治疗通过可移植细胞和整合基因表达载体的体内转导为HA的治疗提供了一种有吸引力的方法。在本研究中，我们通过非病毒靶向载体pHrn将B结构域缺失的FVIII基因表达盒靶向人胚胎干细胞(HESCs)的核糖体DNA(RDNA)。靶向克隆的hESCs在体外和体内均可扩增并保留分化为三层胚层的主要多潜能特性。重要的是，在确定的诱导条件下，靶向的hESCs可以分化为功能性间充质干细胞(MSCs)和肝细胞，相关的特异性细胞标志物和功能检测证实。成瘤实验表明，这些细胞在未来的应用中相对安全。基因表达分析表明，外源性FVIIImRNA和FVIII2蛋白均存在于分化的MSCs和肝细胞中。这些结果表明，通过针对hESCs rDNA位点的基因打靶，可以为HA治疗提供可移植的遗传修饰细胞的持久细胞来源。
Hemophilia A is an X-linked recessive bleeding disorder caused byFVIIIgene deficiency, which may result in spontaneous joint hemorrhages or life-threatening bleeding. Currently, cell-based gene therapyviaex vivo transduction of transplantable cells with integrating gene-expressing vectors offers an attractive treatment for HA. In present study, we targeted an expression cassette of B-domain-deletedFVIIIinto the ribosomal DNA (rDNA) locus of human embryonic stem cells (hESCs) by transfection with a nonviral targeting plasmid pHrn. The targeted hESCs clone could be expanded and retained the main pluripotent properties of differentiation into three germ layers bothin vitroandin vivo. Importantly, under defined induction conditions, the targeted hESCs could differentiated into functional mesenchymal stem cells (MSCs) and hepatocytes, as validated by relevant specific cell markers and functional examination. Tumorgenesis assay demonstrated that these cells are relatively safe for future applications. Analysis on gene expression revealed that exogenousFVIIImRNA andFVIIIproteins were both present in differentiated MSCs and hepatocytes. These results indicated that through gene targeting at hESCs rDNA locus a persistent cell source of transplantable genetic-modified cells can be accomplished for HA therapy.