Low temperature and mTOR inhibition favor stem cell maintenance in human keratinocyte cultures

Abstract

Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single‐cell analysis that long‐term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications. image Cultured human epidermal keratinocyte stem cells respond to a small drop in temperature through thermoTRP channels connected to mTORC1 signaling. mTORC1 inhibition favors the maintenance of a stem cell phenotype in human keratinocyte cultures. A small variation in temperature impacts human keratinocyte stem cell behavior in culture. Temperature regulates mTORC1 signaling through calcium‐permissive thermoTRP channels in human keratinocytes. Lower temperature and mTORC1 inhibition similarly impact on gene expression in cultured human keratinocytes. Continuous inhibition of mTORC1 inhibits keratinocyte stem cell differentiation and favors the maintenance of stemness.