Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP⁺) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP⁺ I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP⁺ and AP⁻ I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP⁺ I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP⁺ I-MEFs. Together with sestrin 1 upregulation, we found that AP⁺ I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP⁺ I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP⁺ phenotype and achieve a quiescent state characterized by a new transcriptional network.