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

中文翻译：

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自主三体性拯救细胞中角质形成细胞表型的恢复

人类 21 号染色体的额外拷贝可以改变细胞表型以及免疫和代谢系统。唐氏综合症与许多与健康相关的问题和与年龄相关的疾病有关，包括皮肤异常。然而，很少有研究关注 21 三体 (T21) 对表皮干细胞和祖细胞功能障碍的影响。在这里，我们通过从 21 三体诱导的多能干细胞 (T21-iPSC) 和自主拯救的二体 21-iPSC (D21-iPSC) 分化细胞来研究唐氏综合征和整倍体细胞之间角化细胞特征的差异。我们采用了 T21-iPSC 和 D21-iPSC 的角化细胞分化方案。对于 T21-和 D21-iPSC 衍生的角质形成细胞的增殖和细胞片形成，随着生长速率的降低，在小鼠饲养细胞上加入了补充有 Rho 激酶抑制剂的培养基。在传代之前，选择具有差异分散酶反应性的角质形成细胞群。进行三维 (3D) 气液界面以评估 iPSC 衍生的角质形成细胞分化和形成复层鳞状上皮的能力。与三体性 21-iPSCs 相比，三体性拯救的二体性 21-iPSCs 能够进行表皮分化并在分化时表达角化细胞标志物，如 KRT14 和 TP63。iPSC 衍生的角质形成细胞的寿命可以在含有 Rho 激酶抑制剂的培养基中的小鼠饲养细胞上成功延长，在 160 天的时间内达到 34 次以上的群体倍增。二体 iPSC 衍生角质形成细胞的基于分散酶的纯化有助于表皮层的形成。三体性拯救的二体性 21-iPSC 衍生角质形成细胞具有延长的寿命，可与真皮成纤维细胞成分结合生成 3D 皮肤。来自自主三体性拯救的 iPSC 的角质形成细胞具有分层制造 3D 皮肤的能力，并恢复角质形成细胞的功能。