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Background <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月1日

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  • Qiong Zhang,
  • Huicheng Chen,
  • Jintao Cun,
  • Qianqian Liu,
  • Hao Wang,
  • Luxi Jiang,
  • Shubo Hu,
  • Hongkun Wang,
  • Jingxin Wang,
  • Jian Xu,
  • Jinyue Shi,
  • Fangyuan Sun,
  • Xianlang Xiong,
  • Zhen Liang,
  • Yuansong Yu,
  • Yuanyuan Du

摘要

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.