Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures [RNA-seq]

Purpose: Comparison of genes expression pattern in normal human epidermal keratinocytes (NHEKs) which were cultured at several specific temperatures or with rapamycin. Methods: Basically, NHEKs were cultured with irradiated 3T3J2 feeder cells in cFAD medium (Pr Green's method) for 1 week. NHEKs were cultured at 32, 35, 36, 37 and 38 degrees C from beginning to end of cell culture, respectively. Furthermore, 100 nM rapamycin-added cFAD medium and ed at 37 degrees C. 7days after cell cuture, NHEKs were harvested for RNAseq analysis. Results: The culture condition at 32 degrees C and 37 degrees C with rapamycin maintained human epidermal keratinocyte stem cells. Conclusions: mTORC1 inhibition via temperature changes favors ex vivo maintenance of human epidermal keratinocyte stem cells. mRNA profiles in human epidermal keratinocytes which were cultured at some specific temperatures or with 100 nM rapamycin.

研究目的：比较在不同特定温度下培养，或使用雷帕霉素（rapamycin）处理的正常人表皮角质形成细胞（normal human epidermal keratinocytes, NHEKs）的基因表达模式。实验方法：基础培养方案为将NHEKs与辐照处理的3T3J2饲养细胞共同培养于cFAD培养基（采用Pr Green法配制）中，培养周期为1周。实验设计如下：其一，分别将细胞置于32、35、36、37、38℃的环境中完成全程培养；其二，使用添加100nM雷帕霉素的cFAD培养基，并于37℃下培养。细胞培养7天后，收集NHEKs用于RNA测序（RNAseq）分析。实验结果：32℃培养条件以及添加雷帕霉素的37℃培养条件，可维持人表皮角质形成细胞的干细胞特性。研究结论：通过温度调控或抑制mTORC1信号通路，可助力人表皮角质形成细胞干细胞的体外维持。本数据集包含在特定温度下或经100nM雷帕霉素处理的人表皮角质形成细胞的mRNA表达谱。