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Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells
Akiko Tanuma-Takahashi1,2, Momoko Inoue1,2, Kazuhiro Kajiwara1,2
1Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan.
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.
Insights
Down syndrome (trisomy 21) impairs epidermal stem cell function. Rescued cells with normal chromosome number restore keratinocyte differentiation and enable 3D skin formation, offering therapeutic potential.
Area of Science:
- Stem Cell Biology
- Developmental Biology
- Genetics
Background:
- Down syndrome (trisomy 21) is linked to cellular and systemic abnormalities, including skin issues.
- Limited research exists on trisomy 21's impact on epidermal stem cells and progenitor dysfunction.
- This study investigates keratinocyte differences between Down syndrome and normal cells using induced pluripotent stem cells.
Purpose of the Study:
- To compare keratinocytic characteristics between Down syndrome and euploid cells.
- To assess the differentiation potential of trisomy 21-induced pluripotent stem cells (T21-iPSCs) versus autonomous rescued disomy 21-iPSCs (D21-iPSCs).
- To evaluate the capacity for epidermal stratification and 3D skin formation.
Main Methods:
- Keratinocytic differentiation of T21-iPSCs and D21-iPSCs.
- Culture optimization using Rho kinase inhibitor and mouse feeder cells for extended keratinocyte lifespan.
- Dispase-based purification for cell sheet formation.
- 3D air-liquid interface culture to assess stratification.
Main Results:
- Disomy 21-iPSCs, unlike T21-iPSCs, successfully differentiated into epidermal cells expressing keratinocytic markers (KRT14, TP63).
- Keratinocyte lifespan was extended to over 34 population doublings (160 days) using optimized culture conditions.
- Disomy iPSC-derived keratinocytes formed epidermal sheets and generated 3D skin constructs with dermal fibroblasts.
Conclusions:
- Autonomous trisomy-rescued iPSCs can restore keratinocyte differentiation and function.
- These cells exhibit stratification potential for manufacturing 3D skin.
- This suggests a pathway for restoring epidermal integrity in Down syndrome-related dermatological conditions.
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