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

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

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. afukuda@tokai.ac.jp.
  • 6 The Institute of Medical Sciences, Tokai University, Isehara, Japan. afukuda@tokai.ac.jp.
  • 7 Micro/Nano Technology Center, Tokai University, Hiratsuka, Kanagawa, Japan. afukuda@tokai.ac.jp.
  • 8 Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan. afukuda@tokai.ac.jp.
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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