MicroRNAs Profile Identification And Their Roles On Hepatic Differentiation Of Mesenchymal Stem Cells

【BACKGROUND】End-stage liver disease represents a worldwide health problem. The incidence of endstage liver disease is still increasing all around the world. During a one-year period,30-50%of patients with decompensated cirrhosis were died. Currently, livertransplantation provides the only effective therapy for such patients. However, it isaffected by lack of donor organs, risk of rejection, and high costs.Researches demonstrated that adult stem cell including hematopoietic stem cells andmesenchymal stem cell can break through the limitation of their germ layer of origin anddifferentiate into hepatocyte-like cells both in vitro and in vivo. So, adult stem celltransplantation can be a potential treatment for end stage liver disease. But the mechanismof hepatic differentiation of stem cells is still unknown. The clinical application of adultstem cells for cytotherapy was inhibited by the limitation of traditional induction methods such as long induction period, complex induction methods and low differentiationefficiency.MicroRNAs comprise a group of non-coding small RNAs (17-25nt) that are involvedin post-transcriptional regulation and have been identified in various plants and animals.Their spatiotemporal expression patterns make them play an important role in cell fatedetermination. Recently, studies found that using specific microRNA group can convertsomatic cells into another cell type. microRNA play important role in liver development,but the specific microRNA profile and their role on hepatic differentiation of adult stemcell is still unknown.【AIMS】The aim of the study is to identify the microRNAs profile and clarify their rolesduring hepatic differentiation of mesenchymal stem cells. First, establish a stable cellmodel of hepatic differentiation from MSC. Then using this cell model, to screen the keymicroRNAs involved in the hepatic differentiation of MSC through microarray and so on.Finally, to clarify the structure role of the key microRNAs.【METHODS】1. Purified hematopoietic stem cells were isolated from peripheral-blood usingimmunomagnetic beads. Purified mesenchymal stem cells were isolated from humanumbilical cord lining. Cell morphology, flow cytometry, and osteogenic differentiationability were done for stem cell identification. After MSC was successfully isolated, hepaticdifferentiation of MSC was induced using Hepatogenic Differentiation Kit. Then hepaticgene expression in mRNA and protein level, PAS staining, urea assay and LDL uptakeassay were done to confirm that induced MSC posses the characterization of hepatocyte.2. Seven time points of cellular total RNA including undifferentiated MSC andhepatic differentiated for2days,6days,10days,14days,22days and26days werecollected for microRNA microarray analysis. The expression of altered expressedmicroRNAs was confirmed by qRT-PCR. To confirm that the altered microRNAs werehepatic differentiation specific, dynamic microRNA profiles of hepatic differentiatedMSCs were also compared with the microRNA profile of osteogenic differentiation of MSCs, normal hepatocyte cell line and hepatoma carcinoma cell.3. MSC were infected with lentivirus packaged with siRNAs for miR-1246,miR-1290, miR-148a, miR-30a, miR-424and miR-542-5p respectively for6days wereinduced for hepatic differentiation with a Hepatogenic Differentiation Kit. Using miR-122mimic as control, MSC were transfected with single miR-mimic or mimics mix. Thenhepatic gene expression in mRNA and protein level, PAS staining, urea assay and LDLuptake assay were done to confirm that microRNA mediated induced hepatocyte fromMSCs posses the characterization of hepatocyte. Finally, transplantated iHep into acuteliver injured mice to confirm that microRNA-mediated hepatocyte from MSCs can alsoposses the hepatic function in vivo.【RUSULTS】1. adult stem cells isolation and hepatic differentiationFlow cytometry analysis confirmed that about99%cells isolated from peripheralblood using MACS was CD34positive cells, and displayed a small round non-adherencesignature. After culture and expansion of CD34positive cells with four different culturemedium, SFM34medium with IL3, SCF, and GM-SCF showed a best efficient expansion.However, after expansion, CD34positive cells changed into CD34negative cells.Isolated MSCs from human umbilical cord lining displayed a typical fibroblast-likeappearance as MSCs. Flow cytometry analysis confirmed the MSC signature of MSCs atP3with a high expression of CD105.The contamination of the culture with hematopoieticcells and endothelial cells was excluded by the absence of CD34and CD31. Moreover,MSCs can differentiate into osteoblast-like cells, thus demonstrating their multipotentdifferentiated potential as MSCs.QRT-PCR results demonstrated that the expression of HNF4α, ALB and CK18wasincreased after1week of induction and reached the highest level after26days.Immunofluorescence results showed MSC can express AFP, ALB and CK18after2weeksof induction. PAS staining demonstrated that MSCs submitted to the hepaticdifferentiation protocol were able to specifically store glycogen compared withundifferentiated MSCs after induction for2weeks. After induction for2weeks, all hepatic differentiated MSCs could uptake LDL. Moreover, The urea production ability of MSCsincreased significantly after induction by hepatic differentiation medium and reached thehighest level at day22. These results indicated that MSCs differentiated into cells withsignificant hepatic gene expression and hepatic functions.2. microRNA profile identificationAt seven time points (2days,6days,10days,14days,22days and26days) duringthe hepatic differentiation of MSCs, cellular total RNA was collected, including fromundifferentiated MSCs, for microRNA microarray analysis. A total of1205human and144human viral microRNAs were analyzed. The normalized data demonstrated that61microRNAs displayed consistent changes and were altered at least2-fold between MSCsand hepatic differentiated MSCs. Based on fold changes and expression level, sevenover-expressed microRNAs that were altered at least four fold, and six over-expressedmicroRNAs that were highly expressed, Six under-expressed microRNAs that werealtered at least four fold, and ten under-expressed microRNAs that were highlyexpressed were selected for further qRT-PCR analyses. The qRT-PCR results demonstratedthat the expression patterns of miR-542-5p, miR-148a, miR-1290, miR-424, miR-30a andmiR-1246were consistent with the microarray results. With the exception of miR-3646,all of the under-expressed microRNAs according to microarray were also decreased whenanalyzed by qRT-PCR. However, only miR-146a displayed consistent changes.6over-expressed and8under-expressed microRNAs in hepatic differentiated MSCselected by microarray and qRT-PCR was analyzed in osteogenic differentiated MSC atday6and day14. The qRT-PCR results showed several microRNAs which wasover-expressed in hepatic differentiation was under-expressed in osteogenic differentiationand several microRNAs which was under-expressed in hepatic differentiation wasover-expressed in osteogenic differentiation. The expression of these microRNAs in L02,which is a normal human hepatocyte cell line, and in HepG2, which is a hepatocellularcarcinoma cell line, cultured with hepatic differentiation medium also showed that theexpression of over-expressed(or under-expressed) microRNAs in both L02and HepG2was even lower(or higher) than in MSCs. These results indicate that microRNA profile of hepatic differentiation was different from that of osteogenic differentiation and hepatocyteand hepatocellular carcinoma cells.3. the role of microRNAs on hepatic differentiation of MSCAfter HGF-induced for6days, the ALB mRNA expression of MSC and MSCinfected with miRNA-control-Lev can up-regulated. Whereas, MSC infected withmiR-1246-RNAi-Lev, miR-1290-RNAi-Lev, miR-148a-RNAi-Lev, miR-30a-RNAi-Lev,miR-424-RNAi-Lev, miR-542-5p-RNAi-Lev cannot over-express albumin. Moreover,after HGF-induced12days, MSC successfully infected with miRNA-control-Lev (withgreen fluorescence) can uptake Dil-Ac-LDL(with red fluorescence), however, MSCsuccessfully infected with miR-1246-RNAi-Lev, miR-1290-RNAi-Lev,miR-148a-RNAi-Lev, miR-30a-RNAi-Lev, miR-424-RNAi-Lev, miR-542-5p-RNAi-Levcannot uptake Dil-Ac-LDL, and only several hMSCs without miRNA-RNAi-Levinfection can uptake Dil-Ac-LDL. Additionally, miR-1246-RNAi-Lev,miR-1290-RNAi-Lev, miR-148a-RNAi-Lev, miR-30a-RNAi-Lev, miR-424-RNAi-Lev,and miR-542-5p-RNAi-Lev can also down regulate ALB and G6P expression in HepG2cell.Single miRNA mimic transfection can promote ectopic overexpression themicroRNA, but cannot promote MSC express hepatocyte marker gene ALB. SevenmicroRNA mimics combination not only can increase seven microRNA ectopicexpressions at the same time, but also can make MSC over express early, middle and latehepatic gene, promote the morphology of MSC convert from fibroblast-like morphologyto epithelial morphology, posses hepatic function, and rescues CCL4-induced liver injurymice.【CONCLUTION】We successfully isolated purified hematopoietic stem cells from peripheral-blood andpurified mesenchymal stem cells from human umbilical cord lining. we identified thedynamic microRNA profiles of hepatic differentiated human umbilical cord lining-derivedmesenchymal stem cells through microRNA microarray and qRT-PCR. We found thatdown-regulation of any one of the six hepatic differentiation-specific microRNAs can inhibit HGF-induced hepatic differentiation. Although overexpression of any one of thesix microRNAs alone or liver-enriched miR-122cannot initiate hepatic differentiation,ectopic overexpression of seven microRNAs together can stimulate MSC conversion intofunctionally mature induced hepatocytes. The current study provides informative data onchanges in microRNAs during the differentiation of MSCs into hepatocytes and indicatethat microRNAs have the capability of directly converting MSCs to a hepatocytephenotype in vitro.