Center for Regenerative Medicine and Skeletal Development, Department of Reconstructive Sciences, University of Connecticut Health, Farmington, Connecticut, USA.
Department of Oral Health and Diagnostic Sciences, School of Dental Medicine, University of Connecticut Health, Farmington, Connecticut, USA.
Cell Reprogram. 2021 Feb;23(1):1-13. doi: 10.1089/cell.2020.0046. Epub 2020 Dec 29.
Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.
从人类胚胎干细胞(hESC)和人类诱导多能干细胞(hiPSC)分化角质形成细胞已成为伤口愈合研究的重要工具,并且在无法获得患者细胞的情况下,也可用于研究皮肤疾病。已经发表了几种使用 hiPSC 集落片段或类胚体的角质形成细胞分化方案,其中一些需要长时间分化或延长使用试剂混合物。在这项研究中,我们提出了一种简化的方法,可在不到 4 周的时间内,从单个 hiPSC 与分化因子、维甲酸和骨形态发生蛋白 4(BMP4)高效产生大量均匀分化的角质形成细胞。单个 hiPSC 的低接种密度可导致分化过程中细胞死亡最小的角质形成细胞培养物,角蛋白 14 阳性细胞的均匀性高达 96%,且角质形成细胞成熟标志物的百分比低,与早期传代的原代角质形成细胞相当。hiPSC 衍生的角质形成细胞保持增殖状态,可长时间维持,并可在高钙条件下以与原代人角质形成细胞相同的方式进行终末分化。此外,hiPSC 衍生的成纤维细胞和角质形成细胞的共培养始终形成器官型 3D 皮肤等效物。因此,通过该方法产生的角质形成细胞是下游应用的可行细胞来源。