Piezo1-ERK1/2-YAP Signaling Cascade Regulates The Proliferation Of Urine-derived Stem Cells On Collagen Gels

ObjectiveUrine derived stem cells(USCs)are an attractive cell source for cell therapy and tissue engineering,but the strategies for promoting the proliferation of USCs in vitro and their related molecular mechanisms are currently facing challenges in the research and application of USCs.important question.The cellular microenvironment is an important factor affecting cell proliferation,such as extracellular matrix stiffness,and soluble signals composed of growth factors and cytokines,which play a key regulatory role in stem cell identity maintenance,self-renewal,and differentiation.When cultured in vitro,stem cells are exposed to an altered extracellular environment,so it is critical to create an in vitro environment in which cells can survive.Natural collagen exhibits specific peptide sequences that can be recognized by cell surface receptors,allowing cell adhesion and spreading,and is well reconstituted in many in vivo environments.It has been reported that the non-selective calcium channel Piezo1 can respond to changes in the external environment and regulate the nuclear expression of Yes-Associated Protein(YAP),thereby participating in cell proliferation.However,the effect of Piezo1 and YAP on the proliferation of Urine derived stem cells(USCs)is still unclear.Therefore,in this study,we cultured USCs into collagen-contained dishes(COL)to study their proliferation,and cultured USCs in collagen non-contained dish(NON)as a control,and the role of Piezo1-ERK1/2-YAP signaling pathway in the proliferation of USCs was investigated.Methods and Contents1.The surface markers of USCs were identified by fluorescence identification.2.The activity and proliferation ability of P4 and P8 USCs were detected by MTT and cell scratch assay.3.The proliferation ability of USCs under different culture environments was studied by EDU-488 cell proliferation kit.4.The expression of YAP and Piezo1 in USCs under different culture environments was studied by immunofluorescence staining experiments.5.The expressions of YAP,Piezo1,ERK1/2,P-ERK1/2 and LATS1 in USCs in different culture environments was detected by western blotting.6.The YAP blocker Verteporfin was used to interfere with the proliferation of USCs to studied whether YAP was involved in regulating the proliferation of USCs.7.The calcium ion activity in USCs under different culture conditions was detected by using the calcium ion fluorescent probe Fluo-4AM.8.Using Piezo1 inhibitor(Gs MTx4)and activator(Yoda1)to interfere with the proliferation of USCs,to verify the cascade regulation relationship between Piezo1 and YAP in regulating the proliferation of USCs.Results1.USCs do not express hematopoietic stem cell surface markers CD31 and CD45;express MSCs surface markers CD44 and CD133,express ESC surface markers SSEA4 and pericyte surface markers CD146,PDGFRB and NG2.2.The results of MTT cell viability assay and cell scratch assay showed that the proliferation ability of USCs decreased significantly after passage to 8 passages.3.USCs cultured on collagen-contained dish have better proliferative ability.4.VP intervention affects the nucleocytoplasmic shuttling of YAP in USCs and then affects the proliferation of USCs,indicating that YAP is involved in regulating the proliferation of USCs.5.Piezo1 is involved in the proliferation of USCs.6.Piezo1 inhibitor Gs MTx4 can down-regulate the expression of calcium ion and ERK1/2 signal after inhibiting the expression of Piezo1,regulate the expression of YAP into the nucleus,and then down-regulate the proliferation of USCs.7.Piezo1 inducer Yoda1 activates Piezo1,and by up-regulating the expression of calcium ion and ERK1/2 signal,it regulates the increased expression of YAP into the nucleus and then up-regulates the proliferation of USCs.ConclusionsUSCs had good proliferation ability in the collagen gel microenvironment,and their proliferation regulation depended on the regulation of Piezo1 channel.Piezo1 acts on the upstream of YAP in USCs,and can induce the opening of Piezo1 channels in USCs in the collagen culture environment,and the increase of calcium influx activates ERK1/2signaling to regulate the expression of YAP into the nucleus,thereby regulating the proliferation of USCs.Therefore,Piezo-ERK1/ 2-YAP is an important signaling cascade pathway involved in regulating the proliferation of USCs on collagen gels.USCs are ideal seed cells for bladder regeneration and repair,and the study of their proliferation mechanism will provide a theoretical basis for the construction of tissue-engineered bladder reconstruction.