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Generation of porcine induced-pluripotent stem cells from Sertoli cells

Author:
Setthawong, Piyathip, Phakdeedindan, Praopilas, Tiptanavattana, Narong, Rungarunlert, Sasitorn, Techakumphu, Mongkol, Tharasanit, Theerawat
Source:
Theriogenology 2019 v.127 pp. 32-40
ISSN:
0093-691X
Subject:
GATA transcription factors, Sertoli cells, alkaline phosphatase, anti-Mullerian hormone, cell lines, chromosome banding, cytoplasm, embryonic germ layers, enzymatic treatment, genes, induced pluripotent stem cells, karyotyping, phagocytosis, piglets, retroviral vectors, reverse transcriptase polymerase chain reaction, somatic cells, staining
Abstract:
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.
Agid:
6282863