Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

Akiko Tanuma-Takahashi; Momoko Inoue; Kazuhiro Kajiwara; Ryo Takagi; Ayumi Yamaguchi; Osamu Samura; Hidenori Akutsu; Haruhiko Sago; Tohru Kiyono; Aikou Okamoto; Akihiro Umezawa

doi:10.1186/s13287-021-02448-w

Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

Stem Cell Res Ther. 2021 Aug 25;12(1):476. doi: 10.1186/s13287-021-02448-w.

Authors

Akiko Tanuma-Takahashi^{1

2}, Momoko Inoue^{1

2}, Kazuhiro Kajiwara^{1

2}, Ryo Takagi^{1

3}, Ayumi Yamaguchi¹, Osamu Samura², Hidenori Akutsu¹, Haruhiko Sago⁴, Tohru Kiyono⁵, Aikou Okamoto², Akihiro Umezawa⁶

Affiliations

¹ Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan.
² Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, 105-8471, Japan.
³ Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, 162-8666, Japan.
⁴ Center for Maternal-Fetal, Neonatal and Reproductive Medicine, National Center for Child Health and Development, Tokyo, 157-8535, Japan.
⁵ Project for Prevention of HPV-related Cancer, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Chiba, 277-8577, Japan.
⁶ Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan. umezawa@1985.jukuin.keio.ac.jp.

Abstract

Background: An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs).

Methods: Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium.

Results: Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component.

Conclusions: Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

Keywords: 3D skin; Induced pluripotent stem cells; Keratinocytes; Trisomy 21; Trisomy rescue.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation
Induced Pluripotent Stem Cells*
Keratinocytes
Mice
Phenotype
Trisomy* / genetics