RESUMO
Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency1. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >1010-fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells2,3, differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine.
Assuntos
Reprogramação Celular , Epigênese Genética , Células Germinativas , Técnicas In Vitro , Feminino , Humanos , Masculino , Âmnio/citologia , Proteínas Morfogenéticas Ósseas/metabolismo , Reprogramação Celular/genética , Metilação de DNA/genética , Células Germinativas/metabolismo , Células Germinativas/citologia , Sistema de Sinalização das MAP Quinases , Mitose/genética , Oxigenases de Função Mista/deficiência , Oogênese/genética , Oogônios/citologia , Oogônios/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas/genética , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Rim , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Rim/metabolismo , Rim/embriologia , Rim/crescimento & desenvolvimento , Camundongos , Macaca fascicularis , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/genética , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.