School of Arts, Sciences and Humanities, University of Sao Paulo (USP), Sao Paulo, Brazil.
Department of Biochemistry, Federal University of Sao Paulo (UNIFESP), Sao Paulo, Brazil.
J Cell Biochem. 2019 Aug;120(8):13141-13155. doi: 10.1002/jcb.28589. Epub 2019 Mar 19.
Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.
表皮分化是一个复杂的过程，角质形成细胞在大约 15 到 30 天内经历形态和生化变化。异常角质形成细胞分化涉及几种皮肤病的病理生理学。在这种情况下，间充质干细胞（MSC）作为一种研究正常和病理条件下皮肤生物学的有前途的方法出现。在此，我们研究了脐带 MSC 向角质形成细胞的分化。MSC 在 Dulbecco 改良 Eagle 培养基（DMEM）（增殖培养基）中培养，在特征鉴定后，通过在含有表皮生长因子（EGF）和氯化钙离子的无血清角质形成细胞培养基（KSFM）中培养细胞来诱导分化。培养在 DMEM 中的细胞用作对照。根据细胞形态、p63、兜甲蛋白和细胞角蛋白（KRT）KRT5、KRT10 和 KRT14 的表达、定量聚合酶链反应、Western blot 分析或免疫荧光，以及表皮激肽酶活性的检测，从第 1 天到第 23 天评估培养物。在含有 EGF 和 1.8 mM 氯化钙的无血清角质形成细胞培养基中培养的细胞中，第 1 天可见 KRT5 和 KRT14 的表达，随后第 7 天可见 p63 的表达。KRT10 的表达在第 7 天被检测到，而兜甲蛋白在这之后才被观察到。与对照组相比，第 11 天 KSFM 培养的细胞中的激肽酶（KLK）活性更高。这些数据表明 MSC 分化为角质形成细胞与人类表皮中的情况相似。KLK 活性检测似乎是监测 MSC 向角质形成细胞系分化的一种很好的方法，为更好地理解皮肤生物学提供了有用的工具。
