Mairim Alexandra Solis

Gene regulatory networks establish cell identity during development by regulating transcription factor binding at cis-regulatory elements, many of which require chromatin remodelers for accessibility. Although these key principles of gene regulation in mammalian development have emerged, the contribution of chromatin remodellers to early human embryogenesis remains unclear. Here, we show that the SWI/SNF ATPases SMARCA2 and SMARCA4 are essential for establishing gene regulatory programs and cell identity in human pre-implantation development by facilitating enhancer accessibility. Using both human blastoids and human blastocysts, we find that loss of SMARCA2/4 disrupts blastoid epiblast identity, enhances trophectoderm initiation, and impairs inner cell mass formation and developmental progression. Integrative single-cell chromatin accessibility and transcriptome profiling in blastoids reveals that SMARCA2/4 safeguard naïve epiblast and trophectoderm programs by maintaining enhancer and transcription factor binding motif accessibility. These findings identify SWI/SNF chromatin remodellers as critical regulators of embryonic lineage specification during human pre-implantation development.

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. Naive hPSCs efficiently differentiate towards embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm, and 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 confound analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. We successfully expanded for up to 25 passages 8 different naive hPSCs lines across 5 laboratories. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating eliminated fibroblast contamination. Naive hPSCs cultured on serum exhibited more efficient germ layer specification, retained trophectoderm potential and high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins such as vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective and therefore widely applicable 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 facilitate the investigation of mechanistic insights into developmental and disease modelling.