MBD3 Knockdown Promotes the Differentiation of Bovine iPSCs into Primordial Germ Cell-like Cells Associated with Altered Bivalent Modification of TGF-β-Related Genes

Abstract

Highlights What are the main findings? MBD3 knockdown during bovine iPSC reprogramming significantly enhances the differentiation efficiency of primordial germ cell-like cells (PGCLCs) under a 2D monolayer culture system. MBD3-knockdown iPSCs exhibit elevated bivalent histone modifications (H3K4me3/H3K27me3) at TGF-β-related gene loci and upregulated DPPA4/DNMT3L expression, which primes enhanced NODAL signaling to drive PGCLC formation. What are the implications of the main findings? MBD3 functions as an epigenetic gatekeeper that restricts germline competence during reprogramming, and its depletion predisposes iPSCs toward a PGCLC fate. These findings provide a mechanistic basis for optimizing bovine iPSC-derived PGCLC protocols through targeted epigenetic modulation, with potential applications in livestock breeding and germline conservation. Abstract Methyl-CpG binding domain protein 3 (MBD3) is a member of the nucleosome remodeling and deacetylase (NuRD) corepressor protein complex, which plays a pivotal role in embryonic development, pluripotent stem cell (PSC) differentiation, and induced pluripotent stem cell (iPSC) reprogramming. However, whether MBD3 is also involved in iPSC differentiation into primordial germ cell-like cells (PGCLCs) is unclear. In this study, bovine iPSCs generated by MBD3 knockdown (MBD3 KD) during reprogramming were subjected to PGCLC differentiation using a monolayer cell culture method. Our results revealed that MBD3 KD enhanced the ability of bovine iPSCs to differentiate into PGCLCs. MBD3-KD bovine iPSC-derived PGCLCs exhibited the characteristics of in vivo primordial germ cells (PGCs) in a migratory state. MBD3 KD increased NODAL signal transduction during the induction of PGCLCs and was associated with increased expression of PRDM1 and SOX15. Moreover, MBD3 KD increased the bivalent modification of transforming growth factor beta (TGF-β)-related genes concomitant with the upregulation of DPPA4 and DNMT3L expression in bovine iPSCs, and this epigenetic change was associated with enhanced NODAL signaling. Our results indicate that MBD3 KD during reprogramming favors the differentiation of bovine iPSCs into PGCLCs, further highlighting the impact of MBD3 on histone modifications during germ cell development.