ORIGINAL RESEARCH article
Front. Cell Dev. Biol.
Sec. Stem Cell Research
Ascorbic Acid enhances in vitro Primordial germ cell-like cell differentiation from mouse ESCs
Umesh Kumar
Nithyapriya Kumar
P Chandra Shekar
Kumarasamy Thangaraj
CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India
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Abstract
Primordial germ cells (PGCs), the precursors of sperm and oocytes, are specified from a subset of epiblast cells during the post-implantation stage of mammalian embryonic development. Over the past decade, primordial germ cell-like cells (PGCLCs) have been successfully generated in vitro from mouse and human embryonic stem cells (ESCs). Invitro PGCLC differentiation provides a powerful system to study germ cell specification and epigenetic reprograming. Notably, mouse PGCLCs can further mature into functional sperm following transplantation into neonatal testes.. Despite these advancements, in vitro PGCLC differentiation remain inefficient, highlighting gaps in our understanding of PGC specification. To identify strategies for improving differentiation efficiency, we generated a Dppa3-mCherry PGC reporter mouse ESC line (TDM11) by CRISPR-Cas9 mediated knock-in. We employed an Embryonic body (EBs) based differentiation strategy under non adherent, defined culture condition systematically examine factors influencing PGCLC specification. PGCLC specification was assessed temporarily by flow cytometer-based quantification of DPPA3-mCherry expressing cells. Given the established role of Tet1 mediated epigenetic regulation play an implicating role in PGC development, we evaluated that ascorbic acid (AA), a known activator of Tet1, acts as a potent enhancer of PGCLC induction from ESCs. Flowcytometric analysis revealed a significant enrichment of DPPA3-mCherry positive PGCLC upon AA and Transferrin supplementation compared to standard EBs medium. The PGCLC specification was further enhanced by combined supplementation with BMP4 and BMP8B in AA Transferrin supplemented basal EBs medium. The DDPA3-mCherry PGCLC further characterize for expression of other PGC specific genes and successful derivation of embryonic germ cells. Building upon this finding, we established a highly efficient and reproducible protocol for in vitro PGCLC differentiation from mESCs by modulating epigenetic regulation through AA. This system provides a valuable platform for dissecting the molecular mechanism and epigenetic reprograming during early germ cell development and potential therapeutic applications.
Summary
Keywords
BMP signaling, DPPA3 (Stella), epigenetic reprogramming, Germ cell specification, In vitro germ cell differentiation, mESCs, PGCLCs, Tet-mediated DNA demethylation
Received
28 November 2025
Accepted
29 January 2026
Copyright
© 2026 Kumar, Kumar, Chandra Shekar and Thangaraj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: P Chandra Shekar; Kumarasamy Thangaraj
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