Matrix Stiffness Regulates Neural Differentiation Of Umbilical Cord Mesenchymal Stem Cells Through BMPR

Matrix stiffness has important effects on many functions and behaviors such as cell migration,proliferation,and differentiation.At present,Umbilical cord mesenchymal stem cells?UC-MSCs?are the ideal cells currently used for cell therapy due to their strong self-renewal and multidirectional differentiation.Different matrix stiffness can induce mesenchymal stem cells?MSCs?to different directions,especially MSCs induced to the nerve on the soft matrix.However,in the process of differentiation,the molecular mechanism which cells sense matrix stiffness and how to convert mechanical signal stimulation into biological signals is not yet clear.This study continued the previous work to prepare a matrix with different stiffness,using UC-MSCs as the research object,detecting the role and molecular mechanism of bone morphogenetic protein receptor?BMPR?on the matrix stiffness induce neural differentiation of UC-MSCs through qPCR and Western Blot.According to previous research work,polyacrylamide?PAAM?was prepared by polymerizing cross-linking of acrylamide and bisacrylamide,which successfully achieved matrix stiffness of 1-10 kPa,35-38 kPa,and 62-68 kPa,respectively.The UC-MSCs were cultured on different stiffness matrices and the morphological changes of UC-MSCs were observed under microscope.Previous studies showed that cells cultured at 35-38 kPa exhibited needle-shaped and myogenic differentiation.At 62-68kPa,cell morphology showed polygonal shape and differentiated to bone.In this study,UC-MSCs were cultured on a substrate with a stiffness of 1-10 kPa.The cell morphology was elliptical,which was similar to that of nerve cells.The results of qPCR and Western blot showed that the expressions of Nestin and?III tubulin in UC-MSCs were significantly increased on the 1-10 kPa,indicating that the 1-10 kPa can induce UC-MSCs to differentiate into neurons.In order to explore the role of BMPR in this differentiation process,the BMPR expression level of UC-MSCs on the matrix stiffness was measured.The results showed that UC-MSCs highly expressed BMPR on a substrate with a stiffness of 1-10kPa.After the addition of BMPR inhibitor?LDN-193189?,the expression of the nerve markers Nestin and?III tubulin in UC-MSCs was significantly reduced at 1-10 kPa,and BMPR was considered to be involved in this differentiation process.In order to investigate the molecular mechanism underlying BMPR,Western blot was used to detect downstream Smads of BMPR and AKT,GSK-3?and FAK downstream of integrins.The results showed that the phosphorylation of Smad protein was reduced at1-10 kPa after addition of BMPR inhibitors.The expression levels of AKT,GSK-3?and FAK were also significantly suppressed.It is thought that downstream of BMPR may play a role through the Smad pathway and cross-talk occurs downstream of integrin.In addition,the calcium ion fluorescent probe was loaded into the UC-MSCs and it was found that when the BMPR inhibitor was added,the fluorescence intensity of Ca²⁺produced by the intracellular fluorescent probe was also reduced.Therefore,it is considered that calcium ion-mediated signal transduction may be possible.It also participates in the process of neural differentiation of UC-MSCs.In summary,in this study,different stiffness matrices were successfully prepared,and UC-MSCs were found to be able to differentiate into neural directions on a substrate with a stiffness of 1-10 kPa.During this differentiation process,BMPR plays an important role in the differentiation process,possibly through the regulation of Smad proteins,or cross-talk with integrins.The calcium ion-mediated signaling pathway may also be involved in this regulation process,which is of great significance for further revealing the complex molecular mechanism that matrix stiffness induces neural differentiation of hUC-MSCs.