Extracellular Matrix Maintains Protein Levels Of OCT4 And SOX2 In Human Embryonic Stem Cells

Derived from the inner cell mass of human preimplantation blastocysts,human embryonic stem cells(hESCs)possess the features of indefinite self-renewal in vitro and pluripotency conferring the capability on hESCs to differentiate into all somatic cell types as well as germ cells,providing powerful resources for application in regenerative medicine and pharmaceutical development.In the past decades,various methods have been developed to support the long-term hESC culture in vitro,relying on combined utilization of multiple growth components such as media containing various growth factors and small molecules.Among them,extracellular matrices(ECMs)play functionally central roles in hESC attachment and self-renewal maintenance.However,the underlying mechanisms remain largely unknown.In the current study,we found that hESC detachment in clumps upon collagenase IV(COL4)treatment led to the protein amount decrease on SOX2 and OCT4 quickly and drastically,which could be rescued by EGTA treatment along with COL4 digestion or calcium chloride supplement in the cell culture medium prior to COL4 treatment.Using small molecules targeting protein degradation pathways,we demonstrate that the above protein level attenuation is induced by protein degradation occurring through both lysosome and calpain pathways.Mechanistically,our present study unravels that Rac1/COFILIN cascade may be responsible for signal transduction from ECM to nuclei with the aim to maintain the protein homeostasis of SOX2 and OCT4.Collectively,this study reveals a previously unknown role of ECM in fine-tuning the protein level of SOX2 and OCT4 through actin cytoskeleton system,and gives novel solutions to optimize the method of COL4-mediated hESC detachment in clumps,providing new clues for the understanding of regulatory mechanisms elicited by ECM,and the development of better hESC culture systems.