Generation of porcine induced-pluripotent stem cells from Sertoli cells

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.