Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

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

doi:10.1186/s13287-021-02448-w

Abstract

BackgroundAn 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).MethodsOur 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.ResultsTrisomy-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.ConclusionsKeratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

Highlights

An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems
Rescued disomy 21 Induced pluripotent stem cells (iPSCs), but not trisomy 21 iPSCs, are likely to differentiate into keratinocytes We employed a standard protocol for differentiation of T21iPSCs and D21-iPSCs into keratinocytes (Fig. 1a)
The differentiated cells derived from T21- and D21-iPSCs, i.e., keratinocyte progenitor cells, resembled normal human epidermal keratinocytes (NHEK) at passage 2 (Fig. 1b)

Summary

Introduction

An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Keratodermatoses and abnormality of elastic fibers contribute to dermatological disorders such as anetoderma and keratosis pilaris in Down syndrome patients [4,5,6] Genetic signatures of these dermatological diseases have been reported using keratinocytes derived from patient-specific iPSCs [7]. Generation of disease-specific induced pluripotent stem cells (iPSC) allows an alternative paradigm for modeling human genetic disease in vitro [9, 10]. Directed differentiation from these iPSC theoretically enables researchers to derive any given diseaseaffected human cell-type [11]. Disease-specific iPSCs, serve as a powerful tool for investigating the mechanisms of Down syndrome [9, 12, 13]

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