Three-dimensional suspension induction of porcine EPSCs into PGCs

IF 2.5 2区农林科学 Q3 REPRODUCTIVE BIOLOGY

Theriogenology Pub Date : 2025-10-20 DOI:10.1016/j.theriogenology.2025.117718

Liming Yuan , Xiaolong Wu , Xiaoxiao Fu , Zuo Lei, Hongzhao Shi, Wenjie Xu, Yaya Zhao, Jiannan Li, Xueting Li, Lexi Gao, Na Li, Jinlian Hua

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Abstract

Primordial germ cells (PGCs), the precursors of germ cells, originate from the post-implantation epiblast under the regulation of signals from other germ layers. Elucidating the origin and differentiation mechanisms of PGCs is essential for understanding embryonic development and germ cell formation. This study delineated the cell fate characteristics and key molecular expression patterns during the differentiation of porcine extended pluripotent stem cells (pEPSCs) into epiblast-like cells (EpiLCs) and subsequently into PGCs, while also revealing the phenomenon and regulatory mechanisms of spontaneous PGC specification during this process. pEPSCs were first induced into EpiLCs using N2B27 medium with Activin A and bFGF, then further differentiated via 3D suspension culture. Analysis of key markers revealed a complex cell fate transition. During EpiLC induction, the core marker OTX2 was highly expressed, alongside upregulation of other lineage markers. Notably, even without exogenous BMP4, the PGC marker PRDM1 was detectable at 36 h, indicating spontaneous PGC specification. In subsequent embryoid bodies (EBs), PRDM1 expression increased alongside pluripotency gene reactivation, confirming advanced PGC differentiation. This work establishes that pEPSCs can differentiate into PGCs via an “EpiLCs→EB” pathway and demonstrates spontaneous PGC specification during the EpiLC stage. These findings provide a foundation for understanding the regulatory mechanisms of PGC differentiation in vitro and for optimizing PGCs induction systems.

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三维悬浮诱导猪EPSCs转化成PGCs

原始生殖细胞（PGCs）是生殖细胞的前体，在胚胎着床后外胚层的信号调控下形成。阐明PGCs的起源和分化机制对理解胚胎发育和生殖细胞形成至关重要。本研究揭示了猪延伸多能干细胞（pEPSCs）向上皮样细胞（epiblast样细胞，EpiLCs）和向PGCs分化过程中的细胞命运特征和关键分子表达模式，揭示了这一过程中PGC自发规范的现象和调控机制。首先用N2B27培养基和激活素A和bFGF将pEPSCs诱导成EpiLCs，然后通过三维悬浮培养进一步分化。关键标记的分析揭示了一个复杂的细胞命运转变。在EpiLC诱导过程中，核心标记OTX2高表达，同时其他谱系标记上调。值得注意的是，即使没有外源性BMP4， PGC标记物PRDM1在36 h时也可以检测到，这表明PGC是自发形成的。在随后的胚状体（EBs）中，PRDM1的表达随着多能性基因的再激活而增加，证实了PGC的晚期分化。本研究证实了pEPSCs可以通过“EpiLC→EB”途径分化为PGCs，并证明了在EpiLC阶段自发的PGC规范。这些发现为进一步了解PGC体外分化调控机制和优化PGC诱导系统奠定了基础。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Theriogenology 农林科学-生殖生物学

CiteScore

5.50

自引率

14.30%

发文量

387

审稿时长

72 days

期刊介绍： Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.