Microrna Expression Of Human Adipose Stem Cells During Chondrogenic Differentiation And Studies Of Chondrogenic Induction Of Cell Carrier Complex

Part ⅠStudy of Human Adipose Stem Cellsin Vitroand Induction Culture of CellCarrier ComplexObjective:To study in vitro methods of human adipose-derived stem cells and chondrogenic induction of cell carrier complex.Methods:Three samples of adipose tissues were obtained from donors who underwent elective liposuction or other abdominal surgery.Human adipose-derived mesenchymal stem cells(hADSCs) were harvested from adipose tissue by isolated and digested, and experienced primary culture and serial subcultivation; To analyze the growth of hADSCs and draw growth curve through MTT; Collection ofhADSCs at passage 3, the cell surface markersCD29, CD34, CD45, CD90andCD105, were detected using a flowcytometry; hADSCs cultured in the adipogenic induction medium were identified through oil redOstaining. hADSCs cultured in the osteogenic medium, and the expression of bonesialoprotein(BSP),osteopontin(OPN) and osteonectin(ON)were confirmed by luciferase reporter assays. hADSCs cultured in the chondrogenic medium were determined by toluidine bluestaining, and the expression of collagen type II was detected by immunohistochemical examination. hADSCs at passage 3mixed withdifferent concentrations ofcollagenprotein intothe cellcarrier complexgel, which was determined by toluidine bluestaining and the expression of collagen type Ⅱ was detected by immunohistochemical examinationafter14d.Results:hADSCs with high activity were harvested by isolation and culture, the proliferation curve of the cells likes S-shaped. The cells were positive forCD29, CD90 and CD 105, and negative for CD34 and CD45. The morphology of hADSCs cultured in adipogenic induction medium were round, and there were transparent lipid dropletsin these cells, lipid droplets stained by oil red Owere bright red. hADSCs afterosteogenic inductionwereirregular in shape,luciferase reporter assays confirmed the expressionof bonesialoprotein, osteopontin and osteonectin. Morphology of hADSCs followed by chondrogenic differentiation becamecobblestone, extracellular matrix stained by toluidineblue were blue, immunohistochemicalstainingof collagen type Ⅱ was positive.Alcian bluestaining ofcellcarrier complex was blue, immunohistochemistry examination showed the expression of collagen type Ⅱ in the mixed collagen group was strongerthan in the single collagengroup.Conclusions:Ahigh degree homologyhADSCs with multip lediff-erentiation potential were successfully isolated. The vectors of mixed collagenproteinduringhADSCs differentiateinto chondrocytes, may be used asan ideal scaffold for repairingarticularcartilage defects.Part ⅡMicroRNA expression profiles of human adipose-derived stem cells during chondrogenic differentiationObjectives:To detect the miRNA expression profile during the chondrogenic differentiation of hADSCs and identify the potential mechanism through which miRNAs may affect the process of chondrogenesis.Methods:(1) Three samples of adipose tissues were obtained from donors who underwent elective liposuction or other abdominal surgery.Human adipose-derived mesenchymal stem cells(hADSCs) were harvested from adipose tissue by isolated and digested; (2) hADSCs surface markers(CD29, CD44, CD49andCD45) were detected using a flowcytometry hADSCs.(3)The expression of chondrogenic related protein after hADSCs chondrogenic induction were detected using immunohistochemistry. (4) The miRNA expression profiles were then obtained through a miRNA array,20 miRNAs that were differentially expressed by at least two-fold, and these miRNAs included twelve upregulated miRNAs and eight downregulated miRNAs. The northern blot analysis further confirmed the miRNA expression levels. (5) Putative targets of the miRNAs were predicted using the algorithms TargetScan (www.targetscan.org), MiRanda (www. microrna.org), and miRBase Targets (microrna.sanger.ac.uk). The predicted targets were detected using Western blot analysis and validated through a luciferase reporter assay. (6) we conducted functional analysis to detect the role of miR-196a and miR-490-5p in the chondrogenic differentiation of hADSCs, which were transfected with miR-490-5p lentivirus, ELISA assay used for quantitative measurement of chondrogenic differentiation markers (Col2A1, Col10A1 and aggrecan). (7) hADSCs following the induction of chondrogenic differentiation transfected with BMPR2 siRNA lentivirus, BMPR2 protein expression was determined by Western blot analysis. ELISAassay for quantitative measurement of chondrogenic differentiation markers (Col2A1, Col10A1 and aggrecan).Results:(1) hADSC cells at passage 3, which included small, round individual cells or several cells clumped together suspended in the medium. The cells were positive forCD29, CD44 and CD49, and negative forCD45. (2) The morphology of the cells in the chondrogenic induction medium was altered and consisted of polygon-shaped cells, which are typical of chondrocyte morphology.The level of Col II by immunohistochemical examinationg was significantly higher, showed that hADSCs subjected to chondrogenic differentiation. (3) the miRNAs expression levels of hADSCs in all 3 sets of hADSCs before and after the induction of chondrogenic differentiation were detected using miRNA microaray chips. The miRNAs that exhibited at least two-fold differential expression in the hADSCs before and after the induction of chondrogenic differentiation, and include twelve upregulatedmiRNAs (miR-196a, miR-143, miR-383, miR-193b, let-7i, miR-26a, miR-539, miR-199a-3p, miR-337-5p, miR-146a-5p, miR-646, and miR-381) and eight downregulatedmiRNAs (miR-490-5p, miR-1307, miR-125b, miR-96-3p, miR-302-3p, miR-23a-3p, miR-590, and miR-510). (4) A northern blot analysis was conducted to detect the expression levels of eightrepresentativedifferentially expressed miRNAsidentified using the microarray, including four upregulatedmiRNAs (miR-196a, miR-193b, miR-383 and miR-143) and four downregulatedmiRNAs (miR-490-5p, miR-1307, miR-125b and miR-590). The northern blot results showed thatmiR-196a was overexpressed, and miR-490-5p was significantly downregulated in all three samples. (5) we performed functional analysis to detect the role of the two miRNAs (miR-490-5p and miR-196a) in the chondrogenic differentiation of hADSCs.However, we only found miR-490-5p, but not miR-196a (data not shown), showed obviously effect on the chondrogenic differentiation.However,whenthe cellswere transfected with miR-490-5p lentivirus on day 12,the cell morphology was reversed onday18 compared with the control lentivirus group.we used ELISA assay for quantitative measurement of chondrogenic differentiation markers (Col2A1, Col10A1 and aggrecan) and they were gradually upregulated on days 12,15, and 18; however, the levels of whichwere downregulated in the cells transfected with miR-490-5p lentivirus. These results demonstrated that miR-490-5p can inhibit thechondrogenic differentiation of hADSCs. (6) Bioinformatics searches identified SOX-2 and BMPR2 as putative targets of miR-490-5p, which are related to chondrogenic differentiation.Awestern blot analysis was then performed to examine the protein expression of the predicted targets SOX-2 and BMPR2. The results showed that the expression levels of SOX-2 and BMPR2 in the chondro-differentiated hADSC were upregulated, and the transfection of the cells with miR-490-5p lentivirus resulted in the downregulation of BMPR2 after hADSC differentiation, whereas the expression of SOX-2 was not obviously affected. Furthermore, luciferase reporter assays confirmed that miR-490-5p suppressed the BMPR2 3’UTR luciferase activity by 40%, and the luciferase activity was completely rescued with mutated BMPR2 3’UTR. Therefore, we confirmed that BMPR2 is a direct target of miR-490-5p. (7) BMPR2 siRNAlentivirus was transfected into hADSCs cellsand resulted significant knockdown of BMPR2 protein expression in a time-dependent manner following the induction of chondrogenic differentiation, and the immunohistochemical examination demonstrated that the expression level of Col Ⅱ was downregulated. We conducted ELISA analysis and found that the levels of Col2A1, Col10A1 and aggrecan were downregulated on days 12,15, and 18 in the cells transfected with BMPR2 sirna lentivirus.Conclusions:In this study, we identified a set of miRNAs that may play key roles in the regulation of the chondrogenic differentiation of hADSCs. The results may provide a basis for further investigation of the molecular mechanism of miRNAs in hADSCchondrogenesis.