Novel feeder-cell-free 3C system promotes the transition from primed to formative pluripotency, self-renewal, and germline differentiation in rabbit embryonic stem cells in vitro

作者：Xiao Chen, Zhihui Liu, Qing Li, Rahman Ullah, Yuan Gao, Xiyue Wang, Lanjun Liu, Fuliang Du · 发表于：Frontiers in Cell and Developmental Biology · 年份：2026 · DOI：10.3389/fcell.2026.1841436 · 研究领域：Pluripotent Stem Cells Research、Reproductive Biology and Fertility、Neurogenesis and neuroplasticity mechanisms

Introduction: Most rabbit embryonic stem (rbES) cell lines established in embryonic fibroblast feeder layer systems predominantly exhibit pluripotency features restricted to a "primed" state, which hinders their directed differentiation into reproductive germ cells and limits their capacity for germline transmission. Methods: We developed a novel 3C system containing IWP2, DZNep, and TSA in iFLY medium supplemented with basic fibroblast growth factor, human leukemia inhibitory factor, and Noggin. Pluripotency maintenance and germline differentiation were validated by gene expression analysis and induction with BMP4, stem cell factor, and epidermal growth factor. Results: The 3C system efficiently maintained the pluripotency of rbES cells with stable expression of core pluripotent genes. These cells showed upregulated expression of partial naïve markers and expressed formative pluripotent markers. Moreover, 3C cultured rbES cells were successfully differentiated into primordial germ cell like cells (PGCLCs). Discussion: The 3C culture system confers rbES cells with naïve pluripotency and self renewal ability, as well as strong potential for directed germ cell differentiation. This system provides a reliable stem cell platform for genetic modification and breeding in livestock.