Setthawong Piyathip, Phakdeedindan Praopilas, Tiptanavattana Narong, Rungarunlert Sasitorn, Techakumphu Mongkol, Tharasanit Theerawat
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
Biochemistry Unit, Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
Theriogenology. 2019 Mar 15;127:32-40. doi: 10.1016/j.theriogenology.2018.12.033. Epub 2018 Dec 21.
Induced pluripotent stem cells (iPSCs) are generated by reprogramming of somatic cells using four transcription factors: OCT4, SOX2, KLF-4, and c-MYC (OSKM). However, reprogramming efficiency of iPSCs is currently poor. In this study, we used the Sertoli line as a novel cell source for somatic cell reprogramming. Neonatal testes were collected from 1-week-old piglets. The testes were digested by a two-step enzymatic method to isolate Sertoli cells. The latter were transfected with retroviral vectors expressing OSKM. The Sertoli iPSC-like colonies were subjected to morphological analysis, alkaline phosphatase staining, RT-PCR, G-banding karyotyping, in vitro differentiation, and in vivo differentiation. Primary Sertoli cells had polygon-shaped morphology and manifested phagocytic activity as determined by a fluorescent bead assay. Sertoli cells also expressed the anti-Müllerian hormone protein in the cytoplasm. According to RT-PCR results, these cells expressed Sertoli cell markers (FSHR, KRT18, and GATA6) and endogenous transcription factors genes (KLF4 and c-MYC). A total of 240 colonies (0.3% efficiency) were detected by day 7 after viral transduction of 72500 cells. The Sertoli iPSC-like colonies contained small cells with a high nucleus-to-cytoplasm ratio. These colonies tested positive for alkaline phosphatase staining, expressed endogenous pluripotency genes, and had a normal karyotype. All these cell lines could form in vitro three-dimensional aggregates that represented three germ layers of embryonic-like cells. A total of two cell lines used for in vivo differentiation produced high-efficiency teratoma. In conclusion, Sertoli cells can efficiently serve as a novel cell source for iPSC reprogramming.
诱导多能干细胞（iPSC）是通过使用四种转录因子：OCT4、SOX2、KLF-4和c-MYC（OSKM）对体细胞进行重编程而产生的。然而，目前iPSC的重编程效率较低。在本研究中，我们使用支持细胞系作为体细胞重编程的新型细胞来源。从1周龄仔猪收集新生睾丸。通过两步酶法消化睾丸以分离支持细胞。将后者用表达OSKM的逆转录病毒载体转染。对支持细胞来源的类iPSC集落进行形态学分析、碱性磷酸酶染色、逆转录聚合酶链反应（RT-PCR）、G带核型分析、体外分化和体内分化。原代支持细胞具有多边形形态，通过荧光珠试验确定其具有吞噬活性。支持细胞在细胞质中也表达抗苗勒管激素蛋白。根据RT-PCR结果，这些细胞表达支持细胞标记物（促卵泡激素受体、细胞角蛋白18和GATA6）和内源性转录因子基因（KLF4和c-MYC）。在对72500个细胞进行病毒转导后第7天，共检测到240个集落（效率为0.3%）。支持细胞来源的类iPSC集落包含核质比高的小细胞。这些集落碱性磷酸酶染色呈阳性，表达内源性多能性基因，并且具有正常核型。所有这些细胞系都能在体外形成代表胚胎样细胞三个胚层的三维聚集体。总共两个用于体内分化的细胞系产生了高效畸胎瘤。总之，支持细胞可以有效地作为iPSC重编程的新型细胞来源。
