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Establishment of human gastrulating stem cells with the capacity of stable differentiation into multiple gastrulating cell types

Cell Research volume 35, pages 719–734 (2025)Cite this article

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Abstract

Pluripotent stem cells (PSCs) have been derived from various species, but most culture systems stabilize only a single PSC type. By contrast, epiblast cells in vivo exist along a continuum and interact dynamically with both embryonic and extraembryonic cells, interactions missing in standard PSC cultures. This absence limits the self-organizing potential of PSCs and leads to disorganized tissue formation in teratomas. To address this, we developed a unified culture system that supports the stable differentiation of epiblast-like cells into multiple key human gastrulating cell types, collectively called human gastrulating stem cells (hGaSCs). hGaSCs, composed of endoderm-like, mesoderm-like, ectoderm-like, amnion ectoderm-like, and primordial germ cell-like cells, maintain a stable balance during long-term culture. In 3D culture, hGaSCs self-assemble into gastruloid-like structures (hGaSC-gastruloids) that model aspects of a Carnegie Stage 7 human embryo, including gastrulation and germ layer specification. Using hGaSC-gastruloids, we modeled the effects of valproic acid (VPA) on human gastrulation and uncovered molecular pathways underlying VPA-induced malformations. When transplanted into the seminiferous tubules, hGaSCs formed embryo-like structures, progressing through fetal tissue and organ development, unlike the disorganized growth seen in teratomas. In conclusion, hGaSCs provide a versatile platform to study human gastrulation, early organogenesis, developmental defects, and drug teratogenicity, with promising applications in tissue and organ generation from cultured stem cells.

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Fig. 1: Establishment of stable hGaSCs.
Fig. 2: Structure, morphology, and transcriptional characterization of hGaSC-gastruloids.
Fig. 3: Morphological and transcriptional characterization of t-Gastruloids formation from hGaSCs transplanted testes.
Fig. 4: Single-cell visualization of the ordered gastrulation pattern of hGaSCs in the testis.

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Acknowledgements

We thank Dangsheng Li and Yan Liu (Nanjing Medical University) for their valuable discussions and comments on our manuscript. This work was supported by the National Key R&D Program grant 2021YFC2700302 (Y.Y.), the National Natural Science Foundation of China grant 82122025 (Y.Y.), and 82221005 (J.S.), the National Key R&D Program grant 2021YFC2700200 (Y.C.), and Jiangsu Province Excellent Postdoctoral Program 2023ZB725 (M.H.).

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Author notes

  1. These authors contributed equally: Mingqian Huang, Mengqi Chen, Gege Yuan, Yiqiang Cui.

Authors and Affiliations

  1. State Key Laboratory of Reproductive Medicine and Offspring Health, Department of Histology and Embryology, Nanjing Medical University, Nanjing, Jiangsu, China

    Mingqian Huang, Mengqi Chen, Gege Yuan, Yiqiang Cui, Bin Shen, Zhaode Liu, Bohang Zhang, Junqing Chen, Dingdong Chen, Shuangshuang Qiu, Yichun Zhang, Li Liu, Yunfei Zhu, Hao Zhang & Yan Yuan

  2. The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University, Nanjing, Jiangsu, China

    Li Liu

  3. State Key Laboratory of Reproductive Medicine and Offspring Health, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu, China

    Lianju Qin & Jiayin Liu

  4. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jun Wu

  5. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jun Wu

  6. Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Jun Wu

  7. State Key Laboratory of Reproductive Medicine and Offspring Health, Women’s Hospital of Nanjing Medical University, Nanjing Women and Children’s Healthcare Hospital, Nanjing Medical University, Nanjing, Jiangsu, China

    Jiahao Sha

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  1. Mingqian Huang
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Contributions

Conceptualization: J.S., Y.Y.; methodology: M.H., H.Z., G.Y., M.C., Y.C., S.Q.; investigation: M.H, H.Z., G.Y., M.C., Z.L., D.C., B.S., L.Q., J.L., L.L., J.C., Y.F.Z.; visualization: M.H., H.Z., G.Y., M.C., B.Z., Y.Y., Y.C.Z.; funding acquisition: J.S., Y.Y., Y.C., M.H.; project administration: J.S., Y.Y., J.W.; supervision: J.S., Y.Y.; writing — original draft: J.S., Y.Y., M.H., H.Z., G.Y., M.C.; writing — review & editing: J.S., J.W., Y.Y.

Corresponding authors

Correspondence to Hao Zhang, Jun Wu, Yan Yuan or Jiahao Sha.

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Huang, M., Chen, M., Yuan, G. et al. Establishment of human gastrulating stem cells with the capacity of stable differentiation into multiple gastrulating cell types. Cell Res 35, 719–734 (2025). https://doi.org/10.1038/s41422-025-01146-z

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