Osmolar modulation drives reversible cell cycle exit and human pluripotent cell differentiation via NF-?? and WNT signaling
Terminally differentiated cells are regarded as the most stable and common cell state in adult organisms as they reside in growth arrest and carry out their cellular function. Improving our understanding of the mechanisms involved in promoting cell cycle exit would facilitate our ability to manipulate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, we demonstrated that a hyperosmolar environment enforced a protective p53-independent quiescent state in dedifferentiated and pluripotent stem cells (PSCs)-derived models of human hepatocytes and endothelial cells, depicting the endodermal and mesodermal lineages. Prolonged culture in hyperosmolar conditions stimulated transcriptional and functional cell maturation. Interestingly, hyperosmolar conditions did not only trigger cellular maturation but were also necessary to maintain this maturated state, as switching back to serum osmolarity caused the loss of maturation markers and the gain of proliferative markers. Transcriptome analysis revealed activation of NF-?? and repression of WNT signaling as the two main pathways downstream of osmolarity-regulated growth arrest and cell maturation, respectively. In pluripotent cell differentiation, blocking NF-?? signaling under hyperosmolar conditions disrupted cell cycle arrest and cell maturation. In contrast, WNT activation reduced transcriptional maturation without effects on the cell cycle. These studies revealed that increased osmolarity serves as a biochemical signal to promote long-term growth arrest, transcriptional changes, and maturation into different lineages, serving as a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely. Overall design: HUH7.5 cells and iPSC-derived HLCs were differentiated under hyperosmolar conditions