Chemically defined and xeno-free media enables the derivation of human extended pluripotent stem cell lines from discarded blastocysts with a high efficiency.

Human extended pluripotent stem cells (hEPS) had been reported to be derived from discarded blastocysts, whereas the derivation method of hEPS cells was extremely complex in this protocol with feeder and animal serum conditions, which also limited the safety and homogeneity of hEPS. Here, we report an optimized, highly efficient protocol by utilizing chemically defined and xeno-free media for the derivation of human extended pluripotent stem cell lines from discarded blastocysts. With this method, we successfully isolated hEPS cell lines from discarded blastocysts with an efficiency of 46%. Chemically defined and xeno-free media simplified the process of hEPS cell isolation with a higher survival rate of cell aggregation passaging from outgrowth. To our knowledge, this is the first report of hEPS cells being efficiently derived from discarded blastocysts under chemically defined and xeno-free conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13578-025-01410-0.