Next-generation sequencing to characterize the molecular basis for the differentiation of ES cells into testicular somatic cell-like cells and germ cell-like cells

Taking advantage of the tesco-cfp transgenic mouse line in which CFP is exclusively expressed in Sertoli cells under the control of a testis-specific enhancer (tesco), we established an embryonic stem cell from the mouse line (i.e. tcESC). We also created tcESC constitutively expressing mouse Sf1 transgene (tc;Sf1ESC). When we induced the differentiation of the two ESC lines into testicular somatic cell-like cells (TesLCs), tc;Sf1ESCs efficiently developed seminiferous tubule-like structures with the appearance of CFP-positive Setoli cell-like cells (SCLCs) in the tubules. On the other hand, epiblast-like cells (EpiLCs) were generated from ESCs having a germ cell-specific Prdm1-gfp transgene. Co-culture of tc;Sf1TesLCs with Prdm1-gfp EpiLCs resulted in self-organised aggregates, or testicular organoids. In the organoid, the EpiLCs differentiated into GFP-positive PGCLCs or gonocyte-like cells that were enclosed within a seminiferous tubule-like structure composed of CFP-positive SCLCs. Overall design: SCLCs and PGCLCs were isolated by flow cytometry from day 12 of TesLC culture and day 6 of testicular organoids, respectively. Then RNA was extracted from these cells, as well as tcESCs and tc;Sf1ESCs. All samples were in biologically duplicate. Hight-throughput sequencing was performed on Illimina HiSeq4000 platform.