ObjectiveTo investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts. MethodsLiver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks;fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10%FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded;the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured. ResultsIn experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43±0.07) mmol/L and (8.08±0.41) μg/mL in the control group and were (2.52±0.20) mmol/L and (41.48±4.11) μg/mL in the experimental group, showing significant differences (t=24.160, P=0.000;t=19.810, P=0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. ConclusionHUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.
Objective To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes and its mechanisms. Methods Eighteen male adult Sprague Dawley rats [weighing, (200±20) g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor (EGF), hepatocyte growth factor (HGF), oncostatin M (OSM), and basic fibroblastic growth factor (bFGF). Passage 3 HUCMSCs were divided into 3 groups: cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from rats with hepatic fibrosis (group A), in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from normal rats (group B), and in DMEM/F12 containing 10% fetal bovine serum (group C). The morphological changes of the cells were observed. The expressions of α-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunofluorescence. The protein levels of albumin (ALB), tryptophan 2, 3-dioxygenase (TPH2), and CYP3A4 and MAPK/ERK signal pathway protein (P-ERK) were detected using Western blot. The content of blood urea nitrogen (BUN) was measured by diacetyl m onoxime method. Results HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were (21.42±0.32) pg/mL and (17.57±0.31) pg/mL respectively, showing significant difference (t=14.989, P=0.000); the concentrations of OSM were (129.96±0.65) pg/mL and (98.44±1.32) pg/mL respectively, showing significant difference (t=37.172, P=0.000); the concentrations of HGF were below the detection limit and (1.03±0.12) ng/ mL respectively; and the concentrations of bFGF were lower than the detection limit in both groups. No morphological changes of cells were observed in both groups at 7 days, and there was no significant difference between groups. At 7 days after culture, the cells in group A could express human hepatocyte biomarkers of AFP, CK18 and hepatocyte-specific-function proteins of ALB, TPH2, and CYP3 A4 while cells in groups B and C did not. Western blot showed that cells in each group could express P-ERK protein. The relative level of P-ERK protein in group A was significantly higher than that in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The BUN concentration of group A [(0.74±0.07) mmol/ L] was significantly higher than that of groups B [(0.40±0.04) mmol/ L] and C [(0.38±0.04) mmol/L] (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). Conclusion Under the condition of hepatic fibrosis, the level of HGF will increase while EGF and OSM will decrease. The formed blood microenvironment will activate MAPK/ERK signal pathway in HUCMSCs, induce them differentiate into hepatocytes.