Matrix Stiffness Regulates Myocardial Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Through Piezo1

At present,myocardial infarction?MI?is one of the most important causes of sudden cardiac death?SCD?worldwide.Once cardiomyocytes?CMs?are damaged,irreversible loss will occur due to adult cardiomyocytes without almost regenerative capacity.Stem cells have strong self-renewal and multidirectional differentiation potential,which brings new hope for the treatment of myocardial injury,especially umbilical cord mesenchymal stem cells?hUC-MSCs?,which have the advantages of turning waste into treasure,non-invasive extraction,and no ethical controversy,etc.Therefore,we chose hUC-MSCs as research objects.Life can't live without cells,while cells can't survive without extracellular matrix.Each organ or tissue in the body has its own unique characteristics of stiffness,which means that the cells in the body are under conditions of different extracellular matrix stiffness.As a mechanical property,substrate stiffness can affect a range of biological behavior,including cell shape,migration,proliferation and differentiation.Biological materials or scaffolds prepared artificially,which can imitate cell microenvironment,are used to cell culture and research,guiding stem cell fate of differentiation.Nowadays,directing stem cell differentiation is one of the main focuses in stem cell research.Piezo is a mechanically sensitive non-selective cation channel with pressure-activated properties,which senses changes in the mechanical force of the cell membrane and reacts rapidly.It has been found that mechanical stimulation such as matrix stiffness can activate Piezo1 to regulate stem cells differentiation.It is known that the stiffness of normal mature myocardial tissue and fibrotic myocardial tissue are 10-20kPa and 35-70kPa,respectively.In this thesis,we intended to use biological materials that mimicking the matrix stiffness of healthy heart tissue or fibrotic myocardium,to observe its role and mechanism in regulating the differentiation of hUC-MSCs into cardiomyocytes,and to propose new ideas of finding alternative cells for damaged cardiomyocytes.Primary hUC-MSCs were obtained by the method of tissue block and surface markers of hUC-MSCs were identified that expressed CD44,CD90 and CD105without expressing hematopoietic cell markers CD34 and CD45,which were in accordance with the minimum standard for the identification of MSCs the International constituted by Cell Therapy Association?ISCT?.This study prepared polyacrylamide?PAAM?gels with different stiffness?13-16kPa and 62-68kPa?according to previous research work.The morphological changes of hUC-MSCs were observed when hUC-MSCs were cultured on different stiffness matrices.At 13-16kPa,the cell morphology appeared as a short column,which was similar to cardiomyocytes.At 62-68kPa,the cell morphology showed polygonal shape,which was similar osteoblasts.The expression of early myocardial markers Nkx2.5,GATA4,Mesp1 and the mature myocardial markers cTnT,cTnI,?-actin was detected by qPCR and Western Blot.The results showed that at 13-16kPa,the expression of early myocardial and mature myocardial markers were increasing on day 7th,indicating that the matrix stiffness of 13-16kPa tended to induce the differentiation of hUC-MSCs into myocardium.However,on the substrate of 62-68kPa,the expression of both Nkx2.5 and?-actin were higher than the control on day 7th,indicating that the matrix stiffness of 62-68kPa was not obvious for the differentiation of hUC-MSCs into the myocardium.The expression of Piezo changed during the process of matrix stiffness-induced differentiation of hUC-MSCs into myocardial direction.The study found that on the first day,the expression of Piezo1 increased with the stiffness gradient of 1-10kPa,13-16kPa,35-38kPa and 62-68kPa,although the expression Piezo2 changed,this change was irregular.Therefore,Piezo1 was selected as the next research object.Subsequent studies found that the expression of Piezo1 tends to be stable on the soft substrate over the time,but the sensitivity of Piezo1 on the stiff substrate lasted longer.At the same time,the expression of integrin?1 and Ca²⁺was also found to change on different matrix stiffness significantly.The expression of integrin?1 and Ca²⁺on stiff substrate was higher than on soft substrate.In conclusion,hUC-MSCs showed a distinct tendency to differentiate into the myocardium on a matrix stiffness of 13-16kPa,while the tendency of the matrix at62-68kPa regulating myocardial differentiation of hUC-MSCs was not obvious.In this induced differentiation process,Piezo1 can sense the mechanical stimulation of matrix stiffness,possibly through interaction with integrin?1 and regulation of Ca²⁺concentration.