Feeder-free culture of naive human PSCs preserving embryonic, extraembryonic and blastoid potential

Conventional human pluripotent stem cells (hPSCs) are widely used to study early embryonic development, generate somatic cells, and model diseases, with differentiation potential aligned to a post-implantation epiblast identity. In the past decade, naive hPSCs, representing a pre-implantation stage, have been derived, exhibiting broader differentiation potential, including embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm. Naive hPSCs can also self-organize into blastocyst-like structures called blastoids. However, their culture typically relies on mouse embryonic fibroblasts (MEFs), which are variable, resource-intensive, and can contaminate analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating minimised fibroblast contamination. Naive hPSCs cultured on serum exhibited faster exit from pluripotency, more efficient germ layer specification, and retained trophectoderm potential with high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins like vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective alternative for naive hPSC culture, maintaining developmental potential, reducing DNA mutations, and eliminating MEF-related confounding factors. We believe serum coating will expand the use of naive hPSCs to large-scale studies and mechanistic insights of developmental and disease modelling. Overall design: RNA-seq profiling of naive human embryonic stem cells cultured on serum (fetal bovine serum) coating and after differentiation of these cells in various conditions (N2B27, E8, mTeSR, XAV and FAX) for up to 8 days on Geltrex.