A simple and standardized method supports efficient derivation of clinical-grade human embryonic stem cells under feeder- and xeno-free conditions

Abstract Background Human embryonic stem cells (hESCs), as naturally pluripotent stem cells, constitute a pivotal cell source for cell replacement therapies. Yet, the generation of clinically compliant hESC lines under feeder-free and xeno-free conditions remains inefficient. Moreover, the derivation of hESCs from clinically surplus and discarded low-quality embryos using the standardized, translation-ready culture systems has not been reported. Methods By optimizing culture conditions and inner cell mass (ICM) isolation method, we developed a method that significantly improves the derivation efficiency of hESC lines from clinically surplus and discarded frozen-thawed embryos under feeder- and xeno-free conditions. The derivation protocol is operationally simple and easily standardized, with the reagents commercially available and of GMP-grade. Results Using this protocol, we successfully established 16 hESC lines. Among blastocysts with morphologically distinct ICMs of grades A and B, the derivation efficiency achieved approximately 60%, with all three grade A ICMs yielding viable hESC lines (100% derivation efficiency for grade A). Notably, for embryos with poorly developed ICMs (grade C), the derivation efficiency of hESC lines approached 30%, showing the protocol’s robustness across varying ICM quality. Adhering to GMP standards, we derived two clinical-grade hESC lines, which were demonstrated biological safety, sustained pluripotency, and the capacity for three-germ-layer differentiation. Conclusions Our study offers a robust, standardized, and simple method for deriving clinical-grade hESCs. Efficient derivation, propagation and banking of hESC lines from frozen-thawed embryos would offer a valuable cell source for advancing regenerative medicine, disease modeling, and drug development.