Extended culture of 2D gastruloids to model human mesoderm development

  • Nat Methods. 2025 Jun;22(6):1355-1365. doi: 10.1038/s41592-025-02669-4.
Bohan Chen  1 ,  Hina Khan  #  2 ,  Zhiyuan Yu  #  1 ,  LiAng Yao  #  2 ,  Emily Freeburne  2 ,  Kyoung Jo  2 ,  Craig Johnson  2 ,  Idse Heemskerk  3  4  5  6  7
Affiliations
  • 1. Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA.
  • 2. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
  • 3. Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
  • 4. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
  • 5. Center for Cell Plasticity and Organ Design, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
  • 6. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. [email protected].
  • 7. Department of Physics, University of Michigan, Ann Arbor, MI, USA. [email protected].
  • # Contributed equally.
PMID: 40335707 DOI: 10.1038/s41592-025-02669-4
Abstract

Micropatterned human pluripotent stem cells treated with BMP4 (two-dimensional (2D) gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high-throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have been studied only up to about 2 days owing to a loss of organization beyond this time with earlier protocols. Here we report an extended 2D gastruloid model to up to 10 days. We discovered a phase of highly reproducible morphogenesis between 2 and 4 days during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral plate mesoderm-like cells on the colony border and paraxial mesoderm-like cells further inside the colony. Single-cell transcriptomics showed strong similarity of these cells to mesoderm in human and nonhuman primate embryos. Our results illustrate that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.

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