| Hepatocytes, which are the main performer of liver physiological function, are widely used in basicresearch, drug development, as well as in clinical applications for patients with some liver diseases.However, the shortage of applicable hepatocytes severely restricts the research progress related withhepatocyte, so as to hinder its process of transformation and application in the clinical. Therefore, toexplore the way how to get a large number of pure, healthy and qualified hepatocytes is of great importance.Due to the isolated primary hepatocytes are relatively difficult to culture and donor livers are limited, so ithas been focusing on inducing hepatocytes from other type cells which are easy to culture. So far,remarkable progresses have been made in research on this aspect, but due to some inevitable limitations,the initial cells or induction strategies have their own advantages and disadvantages. Among all of theinitial cells, human umbilical cord mesenchymal stem cells (hUC-MSCs), which not only has the propertiesof stem cell, but also has the advantage of rich source, easy isolation, high proliferation rate, and has alower immunologenicity and hepatogenic capacities, is the ideal source of hepatocytes. In this study, usingthe strategy of direct transdifferentiation of differentiated cells to hepatocyte-like cells for reference, weemployed lentiviral-mediated gene transfer technology to delivery the liver-enriched transcription factorsGATA4and HNF4α into hUC-MSCs and induce them to directionally differentiate into hepatocyte-likecells, and some good results have been obtained.In this study, we achieved hUC-MSCs from umbilical cord stroma by using the method of tissue blockadherent culture. At the culture of3~4d, cells with triangular or spindle-shaped spread out from the tissuesgap and removed the tissues at the time of7d. Another7d of culture, the cells clustered to about90%confluence and then passaged. After passaging, cells attached to the tissue within4h, rapidly grew after2d,showing a typical fibroblast-like morphology. The majority of cells were spindle-shaped or flat star-shapedwith a wide cytoplasm within stress fiber patterns inside, oval nuclei containing large and obvious nucleoli.When the cells reached full convergence of basic, it showed a growth of parallel or whirlpool. These cellswere CD44positive by immunofluorescence staining.In this study, we chose the above-mentioned hUC-MSCs as the original cells of induced hepatocytes.Recombination lentiviral vectors expressing GATA4or HNF4α were prepared by cotransfection of each transfer plasmid and package plasmids into HEK293T cells via calcium phosphate transfection method, andthen hUC-MSCs passaged5~7times were infected by the combination of the two concentrated lentiviruses.In about two weeks after infection, the cell clones with a typical epithelial morphology and GFP expressingwere observed. These induced cells have the morphological characteristics of hepatocyte-like cells, whichshowed a small triangular epithelium with a larger nucleus containing1~3nucleolus and most of which hadcytoplasmic processes. RT-PCR detection of hepatic marker genes expression, it showed both of nativehUC-MSCs and induced cells expressed CK19, EpCAM, CK18, ALB, G6P, GLUL and MET, notably,compared with native hUC-MSCs, induced cells exhibited that the TAT expression turn on but IL6expression turn off; Furthemore, the induced cells have the capacity of ICG uptake and release.In conclusion, we achieved hUC-MSCs from the umbilical cord stroma and then they were induced todirectionally differentiate into hepatocyte-like cells by overexpression of GATA4and HNF4α. |
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