Stem cell research & therapy · 2025
A simple and standardized method supports efficient derivation of clinical-grade human embryonic stem cells under feeder- and xeno-free conditions.
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Zhang Qiong, Chen Huicheng, Cun Jintao, Liu Qianqian, Wang Hao, Jiang Luxi, Hu Shubo, Wang Hongkun, Wang Jingxin, Xu Jian, Shi Jinyue, Sun Fangyuan, Xiong Xianlang, Liang Zhen, Yu Yuansong, Du Yuanyuan
Аннотация
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.
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.