Transplantation of Mesenchymal Stem Cells into Experimental Injured Mice Liver

  This study was designed to investigate the hepatogenic potential of bone marrow derived mesenchymal stem cells (BM-MSCs) and to evaluate the ability of differentiated hepatocytes to repair surgically induced liver damaged mice. In the first experimental work; The mesenchymal stem cells were isolated from the bone marrow of the thigh bones of 4-8 weeks old male mice based on the ability of adherence to plastic surfaces. The reactivity of isolated mesenchymal stem cells was tested immunocytochemically for MCSs markers. Results showed a positive reactivity towards the MSC markers (CD 105 and CD 90), and a negative reactivity towards the hematopoietic stem cells markers (CD 34 and CD45). Differentiation of BM-MSCs to hepatocytes was induced by adding different growth factors including hepatocyte
growth factor (HGF), fibroblast growth factor-4 (FGF4), oncostatin-M and dexamethasone to Minimal Essential Medium (MEM tissue culture medium) supplemented with 10% of fetal bovine serum. Results revealed that the differentiation medium used was very efficient in directing the BM-MSCs to the hepatocytes, which immunocytochemically showed positive reactivity to specific hepatocyte markers including albumin (ALB) and alpha-fetoprotien (AFP) as well as to cytochrome oxidase p450 (CYP3A4).  The quantitative Elisa assay of CYP3A4 exhibited a significant increase in enzyme levels during the three weeks of differentiation. The level was significantly higher after 21 days of differentiation (65.27ng/ml) in comparison with the first and second weeks of differentiation (16.73 and 33.00 ng/ml) respectively.
       In the second experimental work; the ability of the differentiated hepatocytes to be used as a cellular therapy was studied after induction of subtotal hepatectomy surgical operation in mice which were subjected to a severe injury after removing of most of the liver parts of. Mice were divided into four groups as follows: Group A contained healthy mice served as control group.  Group B contained induced liver damage mice. Group C contained mice with induced liver damage and transplanted with 0.7ml of 5×10 5  of differentiated hepatocytes by intrasplenic injection, Group D contained mice with induced liver damage and transplanted with 0. 7 ml  of 5×105  of BM-MSCs by intrasplenic injection.After 10 days of transplantation, results of the liver/ body weight ratio of each mice group showed significant differences as compared to the control group (0.26 g). Mice transplanted with hepatocytes and BM-MSCs showed a significant increase in liver/ body weight ratio (0.20 and 0.19) g respectively in comparison with liver damaged mice (0.17g). Serum Alanine Amino Transferase (ALT),  Aspartate Amino Transferase (AST) and Alkaline Phosphatase (ALP) showed a significant increase hepatectomized mice (403, 226  and 36.21 IU/L) respectively in comparison with the healthy control (218, 125 and 16 IU/L) respectively Mice transplanted with differentiated hepatocytes showed a significant improvement in liver function enzymes (ALT, AST and ALP), (263, 146 and 17 IU/L) respectively in comparison with mice transplanted with bone marrow mesenchymal stem cells (280, 183 and 22 IU/L) respectively.  Histological sections of liver in mice with damaged liver showed a severe injury in liver tissue characterized by inflammatory and cellular infiltration as well as cytoplasmic vacuolation and degeneration of hepatocytes. Liver sections of mice treated with hepatocytes revealed regeneration of the damaged liver represented by healing of cellular infiltration and reduction of tissue damage. liver sections of mice treated with BM-MSCs showed slight regeneration with a fewer lesions. In conclusion, differentiated hepatocytes were successfully able to repair the liver damage resulting from surgical liver injury in addition to the improvement of serum level of liver function enzymes which were more effective than bone marrow mesenchymal stem cells used for the same goal.