Optimized derivation and culture system of human naïve pluripotent stem cells with enhanced DNA methylation status and genomic stability

Yan Bi; Jindian Hu; Tao Wu; Zhaohui Ouyang; Tan Lin; Jiaxing Sun; Xinbao Zhang; Xiaoyu Xu; Hong Wang; Ke Wei; Shaorong Gao; Yixuan Wang

doi:10.1093/procel/pwaf053

Optimized derivation and culture system of human naïve pluripotent stem cells with enhanced DNA methylation status and genomic stability

Protein Cell. 2025 Jun 20:pwaf053. doi: 10.1093/procel/pwaf053. Online ahead of print.

Authors

Yan Bi^{1

2

3}, Jindian Hu^{1

2

3}, Tao Wu^{1

2

3}, Zhaohui Ouyang^{1

2

3}, Tan Lin^{1

3}, Jiaxing Sun^{1

3}, Xinbao Zhang^{1

3}, Xiaoyu Xu^{1

3}, Hong Wang^{1

2

3}, Ke Wei^{2

3}, Shaorong Gao^{1

2

3}, Yixuan Wang^{1

2

3}

Affiliations

¹ Shanghai Key Laboratory of Maternal and Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, School of Life Sciences and Technology, Tongji University, 200092 Shanghai, China.
² Institute for Regenerative Medicine, State Key Laboratory of Cardiology and Medical Innovation Center, Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200120, China.
³ Frontier Science Center for Stem Cell Research, Tongji University, Shanghai 200092, China.

PMID: 40578838
DOI: 10.1093/procel/pwaf053

Abstract

Human naïve pluripotent stem cells (PSCs) hold great promise for embryonic development studies. Existing induction and culture strategies for these cells, heavily dependent on MEK inhibitors, lead to widespread DNA hypomethylation, aberrant imprinting loss, and genomic instability during extended culture. Here, employing high-content analysis alongside a bifluorescence reporter system indicative of human naïve pluripotency, we screened over 1,600 chemicals and identified 7 promising candidates. From these, we developed four optimized media-LAY, LADY, LUDY, and LKPY-that effectively induce and sustain PSCs in the naïve state. Notably, cells reset or cultured in these media, especially in the LAY system, demonstrate improved genome-wide DNA methylation status closely resembling that of pre-implantation counterparts, with partially restored imprinting and significantly enhanced genomic stability. Overall, our study contributes advancements to naïve pluripotency induction and long-term maintenance, providing insights for further applications of naïve PSCs.

Keywords: DNA methylation; bifluorescence reporter system; genomic stability; high content analysis; human naïve culture system.