Hypo-osmolarity promotes naive pluripotency by reshaping cytoskeleton and increasing chromatin accessibility

[Display omitted] •Hypo-osmolarity facilitates the spontaneous transition of primed EpiSCs to naive ESCs, independent of exogenous transcription factors expression.•Hypo-osmolarity activates the PI3K-AKT-SP1 pathway, reshapes cytoskeleton, and increases chromatin accessibility in EpiSCs.•Under hypo-osmotic condition, naive ESC delays its transition to formative and primed states.•Mouse embryonic fibroblasts are effectively converted to naive pluripotency by hypo-osmolarity during the late stage of reprogramming. Cell fate determination and transition are of paramount importance in biology and medicine. Naive pluripotency could be achieved by reprogramming differentiated cells. However, the mechanism is less clear. Osmolarity is an essential physical factor that acts on living cells, especially for pluripotent cells, but its significance in cell fate transition remains unexplored. To investigate the role of osmolarity in cell fate transition and its underlying mechanism. Flow cytometry, quantitative real-time PCR, teratoma and chimeric mice assays were performed to assess reprogramming efficiency and characterize iPSCs. TEM, immunofluorescence staining, western blot, chemical treatment and genetic modification were utilized to evaluate cell morphology, signaling pathways, cytoskeleton and nuclear structure. Multiomic sequencings were applied to unveil the transcriptome, histone markers and chromatin accessibility of EpiSCs in hypo-osmotic condition. In hypo-osmotic condition, the reprogramming efficiency of hypo-osmotic EpiSCs increased over 60-fold than that of iso-osmotic cells (1100 vs 18 colonies per 3 × 105 cells), whereas no colony formed in hyper-osmotic cells. As expected, the converted cells displayed naive pluripotency. The hypo-osmotic EpiSCs exhibited larger cell size, nuclear area and less heterochromatin; ATAC-seq and ChIP-seq confirmed the increased accessibility of naive pluripotent gene loci with more H3K27ac. Mechanistically, hypo-osmolarity activated PI3K-AKT-SP1 signaling in EpiSCs, which reshaped cytoskeleton and nucleoskeleton, resulting in genome reorganization and pluripotent gene expression. In contrast, hypo-osmolarity delayed the ESCs’ exit from naive pluripotency. Moreover, in MEFs reprograming, hypo-osmolarity promoted the conversion to naive pluripotency. Hypo-osmolarity promotes cell fate transition by remodeling cytoskeleton, nucleoskeleton and genome via PI3K-AKT-SP1 pathway.