A feeder-free culture system supports long-term expansion and germline competence of bovine formative embryonic stem cells†.

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Abstract
Bovine embryonic stem cells (bESCs) are a cornerstone for next-generation applications such as cell-cultured meat, large-animal gene editing, and embryo-stem cell breeding systems. Although primed bESCs and bovine expanded potential stem cells have recently been established, their routine maintenance commonly relies on mouse embryonic fibroblast or bovine fetal fibroblast feeder layers, which are complex, labor-intensive, poorly standardized, and incompatible with scalable manufacturing. Here, we report a simplified feeder-free culture platform for bESCs in which a commercially available basal medium is combined with Growth Factor Reduced Matrigel to generate a three-dimensional, niche-mimetic substrate. Using this approach, we successfully derived a novel feeder-free bESCs (FF-bESCs) that can be robustly expanded for more than 60 passages without ROCK inhibitors, while maintaining a normal karyotype, high proliferation rates, and stable expression of core pluripotency markers. Functional assays confirmed that FF-bESCs possess bona fide pluripotency, as evidenced by their ability to form embryoid bodies in vitro and generate teratomas containing derivatives of all three germ layers in vivo. Transcriptomic profiling further revealed that FF-bESCs align with a formative pluripotent state. Notably, these cells can be directed to differentiate into primordial germ cell-like cells. Our feeder-free culture system provides a scalable, reproducible foundation for advancing bESC-based technologies in bovine precision genome editing and germ cell differentiation for embryo-stem cell breeding systems.
Funding Information
  • National Key Research and Development Program of China (2022YFD1302202)
  • China Agricultural University Basic Scientific Research Fund Program (2022TC114)
  • Major Science and Technology Project of Xinjiang Autonomous Region (2023A02011)

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