Momiji Embryonic Stem Cell System Enabling Live Imaging of Random X-Chromosome Inactivation In Vitro During Embryonic Development

Manami Koshiguchi; Nao Yonezawa; Yu Hatano; Kazuo Yamagata; Shin Kobayashi

doi:10.1007/7651_2025_681

Momiji Embryonic Stem Cell System Enabling Live Imaging of Random X-Chromosome Inactivation In Vitro During Embryonic Development

Methods Mol Biol. 2025 Nov 12. doi: 10.1007/7651_2025_681. Online ahead of print.

Authors

Manami Koshiguchi^{1

2}, Nao Yonezawa³, Yu Hatano^{3

4}, Kazuo Yamagata³, Shin Kobayashi⁵

Affiliations

¹ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.
² RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, Japan.
³ Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan.
⁴ Department of Biomedical Sciences, School of Biological and Environmental Sciences, Kwansei Gakuin University, Nishinomiya, Hyogo, Japan.
⁵ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan. kobayashi.shin@aist.go.jp.

PMID: 41217624
DOI: 10.1007/7651_2025_681

Abstract

In eutherian mammals, X-chromosome inactivation (XCI) equalizes X-linked gene dosage between XX female mammals and XY male mammals. Random XCI (rXCI) begins shortly after implantation, but its direct observation in vivo is difficult. In vitro differentiation of female embryonic stem cells (ESCs) provides a useful model to study rXCI, although conventional XX ESCs often lose one X chromosome during culture and/or differentiation, hindering accurate analysis. We developed the Momiji ESC (version 2) system, in which each X chromosome carries distinct fluorescent reporters and drug-resistance markers. Drug selection before differentiation prevents X-chromosome loss, enabling faithful rXCI modeling and long-term single-cell live imaging. This protocol uses spinning-disk confocal microscopy with Z-stack acquisition, rapid multi-tile imaging, and low-phototoxicity time-lapse observation to monitor rXCI onset and progression for up to 7 days. The Momiji ESC system offers a robust platform for visualizing and analyzing XCI dynamics in vitro.

Keywords: Cell fate tracing; Embryonic stem cell differentiation; Epigenetics; Fluorescent reporter; Live-cell imaging; Momiji ESC; Single-cell tracing; Spinning-disk confocal microscope; X-chromosome inactivation (XCI).