Effect Of Mimic Liver Tissue Microenvironment On Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Hepatocyte-like Cells

Objective：To investigate the differentiation ability of human umbilicalcord mesenchymal stem cells (hUCMSCs) to differentiate into hepatocyte-likecells on morphology, the expression of hepatocyte markers alpha fetoprotein(AFP), cytokeratin-18(CK-18) and liver metabolism enzyme tryptophan2,3-dioxygenase (TPH2), rat liver tissue homogenate supernatant (LHS) was usedin the present study to mimic liver tissue microenvironment. This studyprovided a therapeutic strategy of stem cell for end-stage liver disease.Methods：1Isolation, and identification of hUCMSCsUmbilical cord was obtained from puerpera delivering full-term infants insterile environment and stored in0.9%NaCl sterile solution at4℃.Theumbilical cord was rinsed with sterile phosphate buffered saline to remove theresidual blood and cut into3-4cm sections. Incise the umbilical cord alongtheir long axis to expose the blood vessels and pull the umbilical cord vein andthe two arteries away. The umbilical cord matrix (wharton’s jelly) was scrapedoff and cut into0.5-1mm~3pellets. The pellets were cultured in DMEM/F12supplemented with10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin at37℃,5%CO2and saturating humidity. The pelletswere aspirated off and the cells were passaged when they were80%–90%confluent. The hUCMSCs at passage3were trypsinized and resuspended inphosphate buffered saline and the flow cytometry analysis was conducted toidentify phenotype of hUCMSCs. The hUCMSCs at passage3in thelogarithmic phase were trypsinized, resuspended and plated in a24-well plateat a density of2×10~4/well. The cultures were induced by osteogenicdifferentiate medium and adipogenic differentiate medium respectively. After 14days of induction, Von Kossa staining was conducted to evaluate calciumdeposition and Oil Red O staining was conducted to evaluate lipid droplets.2Hepatogenic differentiation of hUCMSCs induced by LHSSprague-Dawley (SD) rats were anesthetized by2%pentobarbitalsodium (3mL/kg, intraperitoneal injection), fixed and disinfection. Rats wereperformed a midline laparotomy. The distal end of the hepatic portal vein wasperformed ligature and the proximal end of the hepatic portal vein wascannulated and perfused with0.9%NaCl sterile solution (ice-cold) for30min.The liver was removed from the rat to an ice bath after perfusion and then theliver capsule and ligaments were dissected. The liver tissues (150mg) werehomogenized on ice in DMEM/F12(1mL). After homogenization, thehomogenate was centrifuged at15000×g for30min at4℃. The suspernatantswere combined, carefully filtered with0.22μm filters and stored as aliquots at-70℃.The hUCMSCs at passage3in the logarithmic phase were trypsinized,resuspended and plated in35mm plastic dishes at a density of1×105/dish withDMEM/F12medium contains10%FBS. When the cells grew to80%confluence, the hepatogenic differentiation groups were cultured in LHS toinduce hUCMSCs to hepatogenic differentiate. This study was divided into6groups: negative control group (LHS), positive control group (QSG-7701cells), standard control group (hUCMSCs) and hepatogenic differentiationgroups (hUCMSCs were induced by LHS for3,5,7days respectively). Theinvert microscope was used to observe the morphology of cells in each group.Total RNA was isolated by TRIzol Reagent. RT-PCR was conducted toanalyze the mRNA of hepatocyte markers AFP, CK-18and of livermetabolism enzyme TPH2.The hUCMSCs at passage3in the logarithmic phase were trypsinized,resuspended and plated in a T-25plastic flask at a density of5×105/flask withDMEM/F12medium containing10%FBS. The procedure of induction andgroups of experiment vide supra. The total protein of cells in each group wasisolated by Extraction Reagent. Western blot was conducted to detect the expression of hepatocyte markers AFP, CK-18and of liver metabolismenzyme TPH2.3Statistical AnalysisAll experiments were repeated three times independently. Results aregiven as the mean±SD. Statistical analysis of data were performed byOne-Way ANOVA followed by Dunnett t test with SPSS13.0. P＜0.01wasconsidered statistical significance.Results：1Isolation and identification of hUCMSCsThe fibroblast-like cells began to outgrow from the umbilical cord pelletsat the10th-14th day of the primary culture and reached80%-90%confluenceas a whirlpool or radiation after7-10days later. Flow cytometry revealed thatthe cells high expressed MSC signature CD29, CD44, CD105, CD73andCD90, but lack expression of hematopoietic cells marker CD34, CD45andendothelial cells specific marker CD31, as well as of HLA class II (HLA-DR).Under the influence of osteogenic differentiation medium, the appearance ofhUCMSCs changed from spindle shape into irregular shape with increasingtime of induction, meanwhile the nodules of calcium mineralization wasformed and showed by Von Kossa stain. Under the influence of adipogenicdifferentiation medium, The spindle shape of hUCMSCs flattened andbroadened with increasing time of induction and the lipid droplets was formedin cytoplasm and positively stained by Oil Red O. These results showed thathUCMSCs share the signature and differentiation ability with MSCs.2Hepatogenic differentiation of hUCMSCs induced by LHSUnder the induction of the LHS, the hUCMSCs of fusiform began to losetheir sharp edges and progressively shrunk at the3rd day of induction. Thetriangle and polygon shape cells appeared at the5th day and almost allchanged into irregular or oval shape hepatocyte-like cells at the7th day ofinduction. The cells of standard control group were still fibroblast-like, thoughincreased in density.The expression of several hepatocyte specific markers were investigated on both mRNA and protein levels. RT-PCR and Western blot analysis showedthat, compared with standard control group, the level of AFP and CK-18increased more significantly in each hepatogenic differentiation group (P＜0.01). The level of AFP gradually increased and reached the peak at the5thday of induction. Meanwhile, the level of CK-18was significantlyup-regulated with a time-dependent manner.The expression of mRNA and protein of liver metabolism enzyme wereinvestigated by RT-PCR and Western blot. Compared with standard controlgroup, the level of TPH2increased more significantly in each hepatogenicdifferentiation group (P＜0.01) with a time-dependent manner.Conclusions：1Human umbilical cord mesenchymal stem cells characterized asmesenchymal stem cells could be isolated from umbilical cord matrix(Wharton’s Jelly) by using explant method in vitro.2Human umbilical cord mesenchymal stem cells induced by liver tissuehomogenates supernatant are able to differentiate into hepatocyte-like cellswith expression of hepatocyte markers in vitro.3The high expression of liver metabolism enzyme in hepatocyte-like cellssuggested that the cells obtained hepatocyte function.