miRNA-122 promotes mouse embryonic stem cell-derived hepatic precursor cells to differentiate into hepatocytes |
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Authors: | DENG Xiao-geng QIU Rong-lin WU Yao-hao LI Zhi-xi ZENG Le-xiang ZHANG Jie ZHOU Jia-jia TANG Jing DENG Jie-min |
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Affiliation: | Department of Pediatric Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China. |
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Abstract: | AIM:To investigate whether miRNA-122 (miR-122) promotes the differentiation of mouse embryonic stem cell (ESC)-derived hepatic precursor cells (HPCs) into hepatocytes. METHODS:Mouse ESCs were initially induced to differentiate into HPCs by stimulating with fibroblast growth factor 4 (FGF-4), sodium butyrate and dexamethasone (Dex) sequentially. Then a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP was constructed by Gateway technology and transfected into the mouse ESC-derived HPCs 9 d after induction, so as to gain the cells with stable high expression of miR-122. The morphological changes of transfected cells were observed under inverted phase-contrast microscope. The liver-specific gene expression levels were detected by real-time RT-PCR. The liver-specific protein expression levels were also detected by immunofluorescence method. The liver functions were assessed by indocyanine green (ICG) uptake experiment, glycogen staining and urea synthesis function test. RESULTS:The mouse ESCs were successfully induced into HPCs by stimulating with FGF-4, sodium butyrate and Dex sequentially. At 6 d after transfection of miR-122, the morphology of the cells was closer to the mature hepatocytes. The mRNA levels of liver-specific genes such as albumin (ALB), transthyretion, α1-antitrypsin, glucose-6-phosphatase, cytokeration 8, cholesterol 7α-hydroxylase and cytochrome P450 3A4 were up-regulated. The expression levels of liver-specific proteins such as ALB and cytokeratin 18 were increased, while alpha-fetoprotein was decreased. The results of ICG uptake experiment, glycogen staining and urea synthesis function test indicated that the hepatocyte functions were strengthened as compared with control group. CONCLUSION:The combination of FGF-4, sodium butyrate and Dex successfully induces the mouse ESCs into HPCs. Over-expression of miR-122 effectively promotes the differentiation and maturation of mouse HPCs. |
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Keywords: | Embryonic stem cells Hepatocytes miRNA-122 |
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