Generation of Pig iPS Cells: A Model for Cell Therapy
Generation of Pig iPS Cells: A Model for Cell Therapy
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DOI：
10.1007/s12265-010-9233-3
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发表时间：
2011-04-01
影响因子：
3.4
通讯作者：
Izpisua Belmonte, Juan Carlos
中科院分区：
文献类型：
--
作者：
Montserrat, Nuria;Garreta Bahima, Elena;Izpisua Belmonte, Juan Carlos
猪体细胞重编程为诱导多能干细胞提供了再生医学领域的巨大进步，因为猪代表了新兴细胞疗法临床前测试的理想大型动物模型。然而，当前的猪诱导多能干细胞（piPSCs）需要使用耗时且费力的逆转录病毒或慢病毒转导方法，以便在饲养细胞存在的情况下异位表达多能性相关转录因子Oct4， Sox2， Klf4和c-Myc。在这里，我们描述了一种简单的方法，通过在明胶包被板中单次转染表达Oct4， Sox2, Klf4， c-Myc和绿色荧光蛋白（GFP）报告基因的cag驱动的多顺反子质粒来产生piPSC，有或没有饲养细胞。在我们的系统中，与连续三次逆转录病毒转导相似的多顺反子结构相比，在明胶涂层上从成年猪耳成纤维细胞中衍生出pipsc的效率和重编程率更高。我们的piPSCs表达经典的胚胎干细胞标记，表现出稳定的核型并形成畸胎瘤。此外，我们还开发了一种简单的方法，从pipsc中产生体外自发跳动的心肌细胞样细胞。总的来说，我们的初步结果为大规模生产无xeno和无整合的iPSC奠定了基础，并为iPSC技术在大型动物环境中的临床前应用提供了有力的工具。
Reprogramming of pig somatic cells to induced pluripotent stem cells provides a tremendous advance in the field of regenerative medicine since the pig represents an ideal large animal model for the preclinical testing of emerging cell therapies. However, the current generation of pig-induced pluripotent stem cells (piPSCs) require the use of time-consuming and laborious retroviral or lentiviral transduction approaches, in order to ectopically express the pluripotency-associated transcription factors Oct4, Sox2, Klf4 and c-Myc, in the presence of feeder cells. Here, we describe a simple method to produce piPSC with a single transfection of a CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc and a green fluorescent protein (GFP) reporter gene, in gelatine-coated plates, with or without feeder cells. In our system, the derivation of piPSCs from adult pig ear fibroblasts on a gelatine coating showed a higher efficiency and rate of reprogramming when compared with three consecutive retroviral transductions of a similar polycistronic construct. Our piPSCs expressed the classical embryonic stem cell markers, exhibit a stable karyotype and formed teratomas. Moreover, we also developed a simple method to generate in vitro spontaneous beating cardiomiocyte-like cells from piPSCs. Overall, our preliminary results set the bases for the massive production of xeno-free and integration-free piPSCs and provide a powerful tool for the preclinical application of iPSC technology in a large animal setting.