Differentiation Of Rat Adipose Tissue-derived Mesenchymal Stem Cells Into Hepatocyte-like Cells

So far, there has no ideal therapeutic measure to deal with terminal liver diseases and some of heritage liver diseases, and cell therapy and bioartificial liver which aim to supply functional liver cells are bold and useful attempts. But liver cells are limited in clinical application as they cannot be auto-obtained, the number of homogeneity liver cells and the preserving time are limited. So there is an urgent need to search for alternatives to hepatocytes. And the finding of multi-differentiation ability of adult stem cells brings a ray of hope to people. Of various kinds of adult stem cells, bone-marrow mesenchymal stem cells(BMSCs) and umbilical cord blood-derived mesenchymal stem cells(UCBMSCs)are frequently reported for their hepatocyte differentiation ability. Since Zuk et al. found the multi-differentiation ability of adipose-derived mesenchymal stem cells(ADMSCs)in 2001, ADMSCs have become the centre of medical attention soon because of their unique advantages such as their abundant source, easy auto-obtainment with little trauma. It has been found in some researches that ADMSCs can be differentiated into adipose cells, cartilage cells, bone cells, myocardial cells, nerve cells,endothelial cells in suitable conditions, but there have been much fewer reports on the hepatic lineage differentiation. Our present research on the hepatocyte differentiation of ADMSCs may provide a lot of novel and promising cell resources for both cell therapy for liver diseases and bioartificial liver.Our research attempted to investigate the hepatocyte differentiation capability of ADMSCs by different induction methods in vitro and in vivo. Firstly, Rat adipose tissue was harvested ,stem cells were islated using the method of collagenase enzyme digestion, then the cells were amplified, sub-cultivated, and we identified stem cells derived from adipose tissue by detecting cell surface marker expression using immunofluorescence and by evaluating their multi-directional differentiation ability by inducing them into adipose cells, osteoblast and neuroglial cells.Secondly, hepatocyte growth factor (HGF) and fibroblast growth factor-4(FGF-4) were used to induce the ADMSCs into hepatic lineage. The morphology change was observed by inverted microscope. 14 and 21 days after the induction, albumin, as a hepatic special marker, was detected by immunofluorescence. Cell culture media were collected at the indicated times after the induction, and urea nitrogen was detected by urea nitrogen detection kit. In this way, the hepatocyte differentiation ability of ADMSCs in vitro by providing suitable condition was studied.Then the ADMSCs were obtained as described above. Stem cells isolated from the rat adipose tissue were amplified and sub-cultivated. The second passage ADMSCs were labeled by PKH26 in vitro. The partial hepatectomy models were made. And the labeled ADMSCs were autotransplanted by portal vein injection. Two weeks later, the livers were cut off and frozen sections were made. The labeled cells in the liver were observed by fluorescence microscope and their albumin expression was detected by immunofluorescence.The results showed that cells derived from adipose tissue expressed CD29 strongly, but they did not express CD31 and CD45, they can be induced into adipose cells, osteoblast and neuroglial cells by using different inducing method. These results confirm that the cells we derived from adipose tissue is stem cells which have the multi-directional differentiation ability. The hepatocyte-like morphology was observed and albumin was detected by immunofluorescence 14 days after the induction. Urea nitrogen detection showed the differentiated ADMSCs had the function of urea production in a time-dependent manner. ADMSCs could be obtained from the adipose tissue and easily amplified in vitro; the labeling rate of PKH26 on ADMSCs was about 95%. There were sporadic PKH26-labeled cells among hepatocytes in the liver tissue section. And most of the cells expressed albumin.In summary, by our experimental process, a kind of stem cell with multi-directional differentiation ability can be obtained from the rat adipose tissue which is called adipose tissue-derived mesenchymal stem cel(lADMSC). In vitro, ADMSCs can be induced into hepatic lineage which has some of hepatocytes'features in morphology, phenotype and function. In vivo, they can also be induced into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.The research showed that ADMSCs can be induced into hepatic lineage in vitro and in vivo in optimal environment. It provide, to a certain degree, an experimental proof for the applications of ADMSCs in cell-based therapy for liver disease, bioartificial liver and liver tissue engineering.