Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs

Abstract

The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.

Germline stem cells in human

“…87,95 In general, hPGCs/hPGCLCs express a range of types of key genes, including (1) pluripotency markers: NANOG, POU5F1, ALPL, KLF4, LIN28, KIT, NANOS3, SSEA-1, SSEA-4, DPPA3 (also called as STELLA), and ZFP42 (also known as REX1), 17,20,21,[161][162][163][164][165][166][167] (2) cell-surface makers: CD38, EPCAM, ITGA6 (INTEGRIN alpha 6), ITGB3, FGFR3, KIT, and ALPL, 20,21,167,168 (3) germline markers: SOX17, BLIMP1 (also known as PRDM1), TFAP2C, PRDM14, DDX4 (also known as VASA), DAZL, and TCL1A, 20,21,167,[169][170][171][172] (4) amnion-related genes: CDX2 and GATA3, 24 (5) mesoderm markers: EOMES, NODAL, SP5, and T, 20,21 (6) transcription factors: SOX17, BLIMP1, SOX15, GATA4, PRDM14, SALL4, and UTF1, 20,21,[172][173][174][175] and ( 7) epigenetic regulation factors: DNA demethylation dioxygenases (TET1, TET2, and TET3), protein arginine methyltransferase 5(PRMT5), and DND microRNAmediated repression inhibitor 1(DND1). 20,167,176,177 The types and expression patterns of these marker genes reflect corresponding states of hPGC development and cell identity. For example, expression levels of pluripotency genes gradually decrease in hPGCs from embryos of 4-19 weeks, 167 indicating a slow loss of pluripotency.…”

Early Gonadal Development and Sex Determination in Mammal

“…On the other hand, the same gene expression patterns were observed for human PGCLCs and PGCs [ 165 ], cementing the feasibility of researching PGCs formation in vitro. Several genes important in PGCs formation and maintenance, such as TFAP2C [ 193 , 194 ], SSEA1 [ 195 ], DND1 [ 196 ], and SOX15 [ 197 ], were identified by employing sequencing technology, cell biology techniques, and genome editing technology on PGCLCs. However, there were still many challenges ahead; for instance, human PGCLCs derived in vitro could not meiosis completely during the embryonic stage [ 198 ].…”