The Role Of Different Substrate Elasticity On Regulating Rapid Differentiation Of Heparg Cells Into Hepatocyte-like Cells

Background & Aims: acute liver failure(ALF)is a clinical syndromes of multi-organ failure with 80% mortality rates,which is induced by massive hepatocellular necrosis.Liver transplantation is the most effective treatment at present,while it still can't be widely applied to clinic as scarcity of donor liver,immunological rejection,expensive cost.Only in the USA,about 20% patients with ALF were died without donor liver,resulting that studuing biological artificial liver to prolong the survival of patients with ALF and improve their living conditions had brought hot attention.However,it is difficult to access a large number of available hepatocytes for biological artificial liver everytime.HepaRG cell,was first developed in 2002,is a new kind of liver progenitor cell Line,HepaRG cell can be induced to differentiate into functionalhepatocyte-like cells and bile duct cells under dimethyl sulfoxide(DMSO)and suitable for biological artificial liver(BAL),but DMSO also inhibits specially hepatic function of differentiated cells such as expression of ALB and cytochrome P450,elimination of ammonia and cell proliferation.In recent years,it is found that the extracellular matrix(ECM)can regulate cell biology behavior such as cell migration,proliferation,differentiation.According to this,the differentiation of HepaRG cells were induced by substrate elasticity gradient ECM system in our study,to preliminary investigate the potential of substrate elasticity on regulating rapid differentiation of HepaRG cells into hepatocyte-like cells and further supply new hepatocyte for BAL.Methods: 1.The 4 kinds of substrate elasticity(4s group,8s group,16 s group,Glass group)and ALB-GFP-reporter system were made,HepaRG cells were differentiated under the induction of four kinds of substrates with different elasticity modulus.Detected albumin(ALB)expression and observed morphological changes of cells were used to verify the differentiated effects of 4 groups respectively.2.Alamar bule was performed to detect the effect of substrate elasticity on the cell proliferation.3.Immunofluorescent staining was performed to detect yes-associated protein(YAP)expression of cells,primarily exploring the reason resulting in HepaRG cells differentiation.Results: 1.ALB-GFP-reporter system and Image J software showedthat: at the 4th hour,the relative expressions of ALB inside the HepaRG cells between 4s group and 8s group,16 s group or Glass group were not statistically significant differences(t=0.791,1.389,2.481,P>0.05);at the4 th day,the expressions of ALB among the 4 groups were statistically significant differences(F=82.60,P<0.05),the relative expression of 4s group was higher than 16 s group,Glass group,with statistically significant differences(t=12.41,12.52,P<0.05),there was not statistically significant difference between 4s group and 8s group(t=2.603,P>0.05);at the 7th day,there were statistically significant differences among the 4 groups(F=27.02,P<0.05),the relative expression of 4s group was statistically different from 8s group,16 s group,Glass group(t=3.266,6.725,8.005,P<0.05).2.Inverted microscope showed that an undifferentiated elongated cell morphology was observed initially which was able to actively divide when they reached confluency within 4 days;At the seventh day two morphologically different cell types appear,one forms clusters of granular epithelial cells closely resembling typical adult primary hepatocytes with one or two nuclei,while the second surrounding the former is flattenned and retains a clear cytoplasm resembling bile canaliculus-like structures,the hepatocyte-like cellsr represent 50%-55%.3.The results of alamar blue: at the 4th,the cell number of 4s group was not significantly different from 8s group,16 s group,Glass group(t=1.550,0.518,3.106,P>0.05);at the 7th day,thecell number of 4s group was also not significantly different from 8s group,16 s group,Glass group(t=0.025,1.408,3.889,P>0.05).4.The results of Immunofluorescence: YAP on the softest substrate were positive mainly in the cytoplasm,the YAP on the middle hardness substrate were positive in the both cytoplasm and nucleus,the YAP on the hardest substrate were positive mainly in nucleus.Conclusions: 1.Soft substrate can regulate rapid differentiation of HepaRG cells into hepatocyte-like-cells.2.Cells proliferation were not inhibited by soft substrate,and HepaRG cells potentially provide appropriate hepatocytes for BAL.3.YAP plays an important role on HepaRG cells rapid differentiation into hepatocyte-like-cells under oft substrate.