# Ascorbic acid enhances in vitro primordial germ cell-like cell differentiation from mouse ESCs

## TL;DR

Ascorbic acid improves the efficiency of generating primordial germ cell-like cells from mouse embryonic stem cells in the lab.

## Contribution

A new efficient protocol for PGCLC differentiation using ascorbic acid and epigenetic modulation is established.

## Key findings

- Ascorbic acid and transferrin supplementation significantly increased DPPA3-mCherry-positive PGCLC formation.
- Combining AA, transferrin, BMP4, and BMP8B further enhanced PGCLC specification.
- The protocol enables reproducible derivation of PGCLCs and embryonic germ cells.

## 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). In vitro 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 remains 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) using CRISPR–Cas9-mediated knock-in. We implemented an embryoid body (EB)-based differentiation strategy under a non-adherent, defined culture condition, which systematically examined factors influencing PGCLC specification. PGCLC specification was assessed temporarily by flow cytometer-based quantification of DPPA3-mCherry expressing cells. Given the established role of ten–eleven translocation 1 (TET1)-mediated epigenetic regulation in PGC development, we evaluated that ascorbic acid (AA), a known activator of TET1, acts as a potent enhancer of PGCLC induction from ESCs.

Flow cytometric analysis revealed a substantial enrichment of DPPA3-mCherry-positive PGCLC upon AA and transferrin supplementation compared to standard EB medium. The PGCLC specification was further enhanced by combined supplementation with BMP4 and BMP8B in AA- and transferrin-supplemented basal EB medium. The DPPA3-mCherry PGCLC further characterize for the 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 mouse embryonic stem cells (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.