Serum coating enables feeder-free culture of naive human pluripotent stem cells preserving developmental potential
Giada Rossignoli, Michael Oberhuemer, Ida Sophie Brun, Irene Zorzan, Anna Osnato, Anne Wenzel, Emiel van Genderen, Andrea Drusin, Giorgia Panebianco, Nicolò Magri, Moritz Becker, Mairim Alexandra Solis, Chiara Colantuono, Sam Samuël Franciscus Allegonda van Knippenberg, Thi Xuan Ai Pham, Sherif Khodeer, Paolo Grumati, Davide Cacchiarelli, Paolo Martini, Nicolas Rivron, Vincent Pasque, Jan Jakub Żylicz, Martin Leeb, Graziano Martello
Abstract
Abstract Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise in vitro into blastocyst-like structures called blastoids. Naive hPSC maintenance routinely relies on co-culture with mouse embryonic fibroblast (MEFs) as feeder cells, a method prone to variability and analytical confounders. Here, we describe a feeder-free culture system based on serum coating that supports long-term maintenance of naive hPSCs. Across five laboratories, 30 serum batches were evaluated for the expansion of eight naive hPSCs lines for up to 25 passages. Mass spectrometry analysis identified fibronectin and collagens as extracellular matrix proteins consistently present in serum coating. Cells cultured on serum coating displayed growth kinetics, clonogenic capacity, mutation rates, and global gene expression profiles comparable to MEF-based cultures. Importantly, serum-cultured naive hPSCs efficiently underwent germ layer specification, retained trophectoderm competence, and generated blastoids with efficiency similar to MEF-based cultures. Collectively, serum coating provides a scalable, cost-effective, and robust alternative to feeder-based systems, preserving genomic stability and developmental potential while eliminating MEF-associated disadvantages and variability. This platform facilitates large-scale applications of naive hPSCs and enables more reproducible mechanistic studies.