Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing

  • Stem Cell Res Ther. 2025 Jul 18;16(1):389. doi: 10.1186/s13287-025-04501-4.
Nami Motosugi  1 ,  Keita Hasegawa  2 ,  Natsumi Kurosaki  1 ,  Erika Kawaguchi  1 ,  Kenji Izumi  1  3 ,  Yumi Iida  4 ,  Misaki Higashiseto  4 ,  Keiko Yokoyama  4 ,  Ayumi Sasaki  4 ,  Kazuhiko Nakabayashi  2 ,  Atsushi Fukuda  5  6  7  8
Affiliations
  • 1. Department of Molecular Life Sciences, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
  • 2. Department of Maternal-Fetal Biology, National Center for Child Health and Development, Tokyo, Japan.
  • 3. Department of Obstetrics and Gynecology, Tokai University School of Medicine, Isehara, Japan.
  • 4. Department of Life Science Support, Research Innovation Center, University Hospitals Sector, Tokai University, Isehara, Kanagawa, Japan.
  • 5. Department of Molecular Life Sciences, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan. [email protected].
  • 6. The Institute of Medical Sciences, Tokai University, Isehara, Japan. [email protected].
  • 7. Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, Japan. [email protected].
  • 8. Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan. [email protected].
PMID: 40682144 DOI: 10.1186/s13287-025-04501-4
Abstract

The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST, its efficiency remains low. Here, we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.

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