Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures [RNA-seq]. 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. Overall design: mRNA profiles in human epidermal keratinocytes which were cultured at some specific temperatures or with 100 nM rapamycin.

数据集目的：比较经不同特定温度培养或经雷帕霉素（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：将NHEKs与辐照处理的3T3J2饲养细胞共同培养于cFAD培养基（Pr Green法）中，培养周期为1周。分别设置全程培养温度为32℃、35℃、36℃、37℃及38℃的实验组；另设置添加100 nM雷帕霉素的cFAD培养基组，于37℃条件下培养。细胞培养7天后，收集NHEKs进行RNA测序（RNAseq）分析。实验结果：32℃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：通过温度变化实现的mTORC1抑制，有利于人表皮角质形成细胞干细胞的体外维持。整体实验设计：对经特定温度培养或经100 nM雷帕霉素处理的人表皮角质形成细胞进行mRNA表达谱分析。