Center for Regenerative Medicine, National Center for Child Health and Development Research Institute, 2-10-1 Okura, Setagaya, Tokyo, 157-8535, Japan.
Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, 105-8471, Japan.
Stem Cell Res Ther. 2021 Aug 25;12(1):476. doi: 10.1186/s13287-021-02448-w.
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.
人类 21 号染色体的额外拷贝会改变细胞表型以及免疫和代谢系统。唐氏综合征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，很少有研究关注三体 21（T21）对表皮干细胞和祖细胞功能障碍的影响。在这里，我们通过分化来自三体诱导多能干细胞（T21-iPSC）和自主挽救的二倍体 21-iPSC（D21-iPSC）的细胞，研究了唐氏综合征和二倍体细胞之间角蛋白细胞特征的差异。
我们采用了角蛋白细胞分化 T21-iPSC 和 D21-iPSC 的方案。为了增殖 T21-和 D21-iPSC 衍生的角蛋白细胞和细胞片形成，在生长速度降低时引入了在鼠饲养细胞上补充 Rho 激酶抑制剂的培养基。在传代之前，进行了具有差异 dispase 反应性的角蛋白细胞群体的选择。进行三维（3D）气液界面以评估 iPSC 衍生的角蛋白细胞分化和形成分层鳞状上皮的能力。
与 T21-iPSC 相比，挽救三体的二倍体 21-iPSC 能够进行表皮分化，并在分化时表达角蛋白细胞标志物，如 KRT14 和 TP63。在含有 Rho 激酶抑制剂的培养基中，iPSC 衍生的角蛋白细胞在鼠饲养细胞上的寿命可以成功延长，超过 160 天的 34 个以上的群体倍增。基于 dispase 的二倍体 iPSC 衍生的角蛋白细胞的纯化有助于表皮片形成。具有延长寿命的挽救三体的二倍体 21-iPSC 衍生的角蛋白细胞与真皮成纤维细胞成分结合生成 3D 皮肤。
源自自主挽救的 iPSC 的角蛋白细胞具有分层的能力，可用于制造具有角蛋白细胞功能恢复的 3D 皮肤。
