| Objective:Previous study of our subject group have proved that normal rat liver homogenate supernatants could induce HUCMSCs differentiate into i-Heps(Hepatocyte-like cells) with partial function of hepatocytes. Taking into account the characteristics of stem cells that they could "home" to the injured target organ with blood circulation after transplantation, we hypothesized that fibrotic liver tissue microenvironment may be more conducive to the differentiation of stem cells into hepatocytes. In this study, we intend to use rat fibrotic liver homogenate supernatants to simulate fibrotic liver tissue microenvironment in vitro,to investigate the differentiation potential of HUCMSCs into hepatocytes by evaluating changes of cell morphology, expression of hepatocyte biomarkers and formation of hepatocyte-specific functions. Furthermore, a comparative study of the differentiation efficiency of HUCMSCs into hepatocytes under fibrotic liver tissue microenvironment and normal liver tissue microenvironment was carried out, so as that under fibrotic rats blood microenvironment and normal rats blood microenvironment.This paper was aimed to provide theoretical and experimental basis for stem cell transplantation in treatment of hepatic fibrosis.Methods:1. Differentiation of human umbilical cord mesenchymal stem cells into hepatocytes under rat fibrotic liver tissue microenvironmentLiver fibrosis was induced in the SD rats by repeated intraperitoneal injections of thioacetamide at a dose of 200 mg/kg body weight twice a week for 4 weeks, fibrotic liver tissues were used to prepare liver homogenate supernatants. Passage 3 HUCMSCs were used and divided into control group(cells were cultured in DMEM/F12 with 10% fetal bovine serum) and fibrotic liver homogenate supernatants group(cells were cultured in DMEM/F12 with 10% fetal bovine serum and 50g/L fibrotic liver homogenate supernatants),cells were cultured for 7 days. The morphological changes of the cells were recorded; the protein level of CK18, AFP and CYP3A4 was evaluated using Western Blot. Glycogen storing ability of the cells was assessed by PAS staining. Furthermore, the synthesis of albumin and urea of the cells was measured.2. A comparative study of the differentiation efficiency of human umbilical cord mesenchymal stem cells into hepatocytes under fibrotic liver tissue microenvironment and normal liver tissue microenvironmentPassage 3 HUCMSCs were used and divided into control group(cells were cultured in DMEM/F12 with 10% fetal bovine serum),fibrotic liver homogenate supernatants group(cells were cultured in DMEM/F12 with 10% fetal bovine serum and 50g/L fibrotic liver homogenate supernatants), normal liver homogenate supernatants group(cells were cultured in DMEM/F12 with 10% fetal bovine serum and 100g/L normal liver homogenate supernatants). The morphological changes of the cells in each group were recorded; the protein level of CK18,AFP,CYP3A4,CYP2E1,CYP2D6 and TPH2 was evaluated using Western Blot. Furthermore, the synthesis of albumin of the cells was measured.3. Differentiation of human umbilical cord mesenchymal stem cells into hepatocytes under blood microenvironment of rats with hepatic fibrosisSerum samples of liver fibrotic rats and normal rats were collected. Passage 3 HUCMSCs were used and divided into three groups:liver fibrotic rats serum group, cells were cultured in DMEM/F12 with 10% fetal bovine serum and 5ml/L serum from rats with hepatic fibrosis; normal rats serum group, cells were cultured in DMEM/F12 with 10% fetal bovine serum and 5ml/L serum from normal rats; control group, cells were cultured in DMEM/F12 with 10% fetal bovine serum. Cells were cultured for 7 days. The morphological changes of the cells were recorded; The expression of AFP and CK18 were detected by immune fluorescence; The protein level of ALB, TPH2 and CYP3A4 was evaluated using Western Blot; The synthesis of urea of the cells was measured.Results:1. In the first part of our study, under the effect of fibrotic liver homogenate supernatants, the stem cells of fusiform shape began to lose their sharp edges and progressively shrunk, and then they changed into i-Heps with the morphous of round and irregular shape. Compared with the control group, the resulted i-Heps in fibrotic liver homogenate supernatants group exhibited a panel of human hepatocyte biomarkers including CK18,AFP,CYP3A4 and hepatocyte-specific functions glycogen storage. The concentrations of BUN and ALB in supernatant of the control group were (0.43±0.07)mmol/L and(8.08±0.41)?g/mL while that of the experimental group were (2.52±0.20)mmol/L and(41.48±4.11)?g/mL, the differences between two groups reached statistical significance (p<0.01;p<0.01).2. In the second part of our study, after a seven-day inducement, the stem cells in liver homogenate supernatants groups lost their fusiform shape and changed into i-Heps with the morphous of round shape. Compared with HUCMSCs in control group, the resulted i-Heps in liver homogenate supernatants groups exhibited human hepatocyte biomarkers CK18 and AFP. The cells in control group could express a little amount of CYP2E1 while cells in two liver homogenate supernatants groups could express CYP3A4, CYP2E1, CYP2D6, TPH2. Compared with control group, the expression level of CYP2E1 in liver homogenate supernatants groups increased significantly (p<0.01),however, the relative level of CYP3A4, CYP2E1, CYP2D6, TPH2 in two liver homogenate supernatants groups didn't reach statistical significance (p>0.05). At the same time, compared with control group, the concentration of albumin in liver homogenate supernatants groups markedly increased (p<0.01) while that of two liver homogenate groups had no significant difference (p>0.05).3. In the third part of our study, no morphological changes of cells in any group occurred in 7 days.After inducement, the cells in liver fibrotic rats serum group could express human hepatocyte biomarkers AFP,CK18 and hepatocyte-specific-function proteins ALB, TPH2 and CYP3A4 while cells in normal rats serum group and control group didn't. The concentration of BUN of normal rats serum group was (0.40±0.04)mmol/L, control group was (0.38±0.04)mmol/L, while that of liver fibrotic rats serum group was (0.74±0.07)mmol/L. The differences between normal rats serum group and control group did not reach statistical significance (p>0.05) while that between liver fibrotic rats serum group and normal rats serum group or liver fibrotic rats serum group and control group reached statistical significance (p<0.05).Conclusion:1. In rat fibrotic liver tissue microenvironment, HUCMSCs could differentiate into i-Heps with hepatocyte biomarkers and hepatocyte-specific functions such as glycogen storage, urea production and albumin secretion. i-Heps could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.2. Fibrotic liver tissue microenvironment and normal liver tissue microenvironment both could induce HUCMSCs differentiate into i-Heps. To achieve the same effect, compared with normal liver tissue, fibrotic liver tissue required lower concentration, suggesting that fibrotic liver tissue microenvironment may be more conducive to stem cell differentiation.3. Under the influence of serum from rats with hepatic fibrosis, HUCMSCs began to differentiate along a direction to hepatocyte lineage and to possess some features of hepatocytes, suggesting that the differentiation process of HUCMSCs into i-Heps has begun just after transplantation. |
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