The Interactions Between BMSCs And Hepatocytes/Hepatic Stellate Cells Under Different Substrate Elastic Modulus

Bone marrow mesenchymal stem cells(BMSCs)-hepatocytes and BMSCs-hepatic stellate cells(HSCs)were co-cultured on Polyvinyl alcohol(PVA)hydrogel substrates which have been prepared at different elastic modulus(4.5 ± 0.47 kPa?19 ± 3.51 kPa?37 ±2.09 kPa),using a non-contact cell co-culture model.Then,qPCR technique and flow cytometry were applied to research the effects of mechanical microenvironment and co-culture interactions on the differentiation of BMSCs,as well as the function and phenotype alteration of intrahepatic cells,for the purpose to understand the cell behaves during the stem cell therapy of hepatic fibrosis more detailly.The results show that the BMSCs,hepatocytes and HSCs could grow well on all these PVA hydrogel substrates with three different elastic modules,the expression of ?-Smooth muscle actin(?-SMA)and collagen type I(Col1a1)in both of BMSCs and HSCs which were cultured separately showed an increasing trend with the increase of substrate elastic modulus,indicating that the mechanical microenvironment will affect the differentiation of BMSCs and the activation of HSCs.In the same time,mechanical properties of substrates had no significant effect on the expression of albumin(Alb)of hepatocytes during 48 h.And then,the Alb expression increases with the increase of elastic modulus at 72 h in hepatocytes.The interaction between BMSCs and HSCs,BMSCs on those three substrates elastic modulus will promote the expression of ?-SMA,Col1a1,and reduce the expression of PPAR? in HSCs cells,which will promote the activation of HSCs,and the promotion effect will be more obvious on the substrate of 37 kPa.HSCs could also affect the differentiation of BMSCs through paracrine effects.HSCs promoted the expression of Alb in BMSCs on all three substrates and inhibited the expression of ?-SMA and Col1a1 in BMSCs on substrate of 37 kPa,however,the effect on soft substrates were not obvious.Therefore,co-culture with HSCs may have the potential to inhibit the differentiation of BMSCs into myofibroblasts and promote their differentiation into hepatocyte-like cells.It maybe has positive influences during the stem cells therapy of hepatic fibrosis.And as described,those effects correlated with substrates elastic modulus at certain degree.In the interactions between BMSCs and hepatocytes,BMSCs can promote the proliferation of hepatocytes,and the promotion effect is more obvious on the substrate of 4.5 kPa.Hepatocytes can also affect the differentiation of BMSCs through paracrine effect,Hepatocytes inhibited the expression of ?-SMA and promoted the expression of Alb in BMSCs too.i.e.,Hepatocytes would inhibit the differentiation of BMSCs into myofibroblasts as well as HSCs.The results show that the differentiation of BMSCs could be affected by the mechanical microenvironment,the co-culture of hepatocytes/hepatic stellate cells,and BMSCs also affect the function of intrahepatic cells correspondingly.This study is conducive to the follow-up study of stem cell therapy of liver fibrosis,provide clues to understand how to reversal of liver fibrosis or to avoid aggravating fibrosis.