Cerqueira Mariana T, Frias Ana M, Reis Rui L, Marques Alexandra P
3B's Research Group-Biomaterials, Biodegradable and Biomimetics, Avepark-Parque de Ciência e Tecnologia, Zona Industrial da Gandra, Guimarães, Portugal.
ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Portugal.
Methods Mol Biol. 2019;1879:101-110. doi: 10.1007/7651_2018_146.
Epidermal stem cells (EpSCs) isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular EpSCs multipotent fraction and their subsequent separation from the remaining primary human keratinocytes (hKC) culture. Those include rapid adherence of freshly isolated hKC to collagen type IV through the β1-integrin ligand and Rho-associated protein kinase inhibitor (Rocki) Y-27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f/CD71 expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f/CD71 sub-populations. The step-by-step methodology herein described indulges the boosting and consecutive purification and separation of interfollicular epidermal stem cells, from human keratinocytes cultures.
表皮干细胞（EpSCs）的分离仍面临困难，主要是因为仍需要明确的方法和标志物。本文提出的方法整合了一系列策略，以实现毛囊间EpSCs多能部分的富集，并随后将其与其余原代人角质形成细胞（hKC）培养物分离。这些策略包括通过β1整合素配体使新鲜分离的hKC快速黏附于IV型胶原，并向培养物中添加Rho相关蛋白激酶抑制剂（Rocki）Y-27632，随后进行免疫磁珠分离，以基于CD49f/CD71的联合表达获得细胞群体。流式细胞术是辅助方法，用于分析处理对早期表皮标志物角蛋白19/5/14表达率的影响，以及与CD49f/CD71亚群的相关性。本文所述的逐步方法有助于从人角质形成细胞培养物中促进并连续纯化和分离毛囊间表皮干细胞。
