Visualization Of Human Adipose-Derived Stem Cells Into Chondrocytes And Roles Of Wnt/β-catenin Signaling In Chondrogenic Differentiation

Objectives:Mesenchymal stem cells (MSCs) are thought to be multipotent cells which candifferentiate into cells of multiple tissue lineages. Maintenance of chondroid cellsphenotype from MSCs is crucial for tissue homeostasis, but cells will dedifferentiatein vitro via long-term cultured. Wnt/β-catenin signaling pathway regulatesproliferation, differentiation, maturity and apoptosis of MSCs. There is very littleinsight into the mechanisms of action of this signaling during chondrogenesis inhuman adipose-derived stem cells (hADSCs). In this study, we tested the hypothesisthat Wnt/β-catenin signaling pathway regulated in vitro chondrogenesis in hADSCs.Simultaneously, we aimed to probe the intrinsic mechanism of dedifferentiation ofchondroid cells in the late stage of differentiation, in order to provide a feasiblesolution for preventing chondroid cells dedifferentiation.Methods:1. The hADSCs were isolated from human subcutaneous fatty tissue by digestion ofcollagenaseⅠand adhesive screening method. Expressions of cell surface antigensand cell cycle were detected by flow cytometry. The hADSCs were cultured inadipogenic medium, osteogenic medium and chondrogenic medium, respectively.After induction, the cells were observed by oil red O staining, alkaline phosphatasestaining, Von Kossa staining and toluidine blue staining, respectively.2. We observed the change in morphological and biomechanical properties betweenchondroid cells and normal chondrocytes through atomic force microscope (AFM),and we took the lead in detecting plasma protein parameter in molecular scale. Weutilized Flow Cytometry and Laser Confocal Scanning Microscopy (LCSM) toanalyze integrin β1expression and F-actin distribution during chondrogenic differentiation of hADSCs.3. Using DKK-1or Wnt-1to shut down or activate Wnt/β-catenin signaling. Duringthe process of chondrogenic differentiation, the expression of Wnt/β-catenin pathwayprotein was examined by Western blot, the fluorescence intensity of β-catenin in cellnuclei was detected by confocal laser scanning microscope, expression of genescharacterized chondrogenesis was determined by realtime RT-PCR.Results:1. The hADSCs expressed CD44, CD29, CD1and CD105, but did not express CD34,CD35and HLA-DR.85.12%of cells was in G0/G1phase. After adipogenic induction,the cells were positive for oil red O staining, and the cells were positive for alkalinephosphatase staining and Von Kossa staining after osteogenic induction and positivefor toluidine blue staining after chondrogenic induction.2. Gene expression of chondrocyte-specific markers collagen type II, SOX-9andaggrecan increased during chondrogenesis of hADSCs, peaking at day12, and thendecreased. hADSCs (ADS) presented an irregular long spindle shape.12-daydifferentiation cells (12DD) and normal chondrocytes (NC) presented irregular smalltriangle or polygon.21-day differentiation cells (21DD) had been transformed tospindle shape with prominent structure. Rq and Ra of12DD had the maximum valuesin the cells of each differentiation group, but they were still less than those of NC, andthe difference had statistical significance.3. In chondrogenic differentiation process, adhesion force was increased graduallyand reached the maximum at12DD then descended gradually as differentiationcontinued. Nevertheless, adhesion force of12DD was still lower than that of NC.Young’s modulus of12DD was about2-fold higher than ADS, and equivalented toNC (P>0.05). The maximum value was at21DD as (3.518±0.381) kPa.4. The fluorescence intensity of ADS and21DD were all relatively weak with smoothand thin filamentous shape and deranged distribution. F-actin of NC and21DD couldbe clearly seen as bundles of fibers orient along the cell long axis. They hadhomogeneous distribution. They presented the degree of increase. From nucleus tomembrane, they had dense distribution. 5. Our outcome showed that NC had the highest fluorescence intensity of integrinβ1.With the chondrogenic differentiation of ADSCs, the fluorescence intensity ofintegrinβ1was enhanced gradually and reached to the peak at12DD, but the intensityof12DD was still weaker than NC. That of21DD was obviously weaker than12DDand NC. Flow cytometry was adopted for the quantification of integrinβ1of fourgroups (ADS,12DD,21DD, NC). Integrinβ1content of NC was the highest, up to90.53%, next was12DD,75.36%and the contents of21DD and ADS were only43.02%and39.84%, respectively.6. Protein expression analysis of several key components of Wnt/β-catenin pathway in21-day chondrogenic cultures showed that Wnt1, β-catenin, GSK3β sharply increasedat day12, peaking at day18, and then declined. A Wnt/β-catenin target gene, TCF1,was also closely followed the expression of Wnt1. These results were consistent withendonuclear β-catenin fluorescence intensity. Simultaneously, gene expression ofchondrocyte-specific markers collagen type II, SOX-9and aggrecan increased duringchondrogenesis of hADSCs, peaking at day12, and then decreased. Whereas when aWnt inhibitor was added to cultures as inhibition days0-21that COL II, SOX-9andaggrecan elevated continuously. In contrast, as Wnt1added to cultures causedcontinuous sustained Wnt/β-catenin signaling over21days, and resulted in the geneexpression of chondrocyte-specific markers were strongly suppressed. As added wnt1to cultures just on days18-21showed a rapidly decline in chondrocyte-specificmarkers expression after day18.Conclusion:1. We used the adhesive screening method which reported before and successfullyisolated MSCs from human adipose tissue. We followed the procedures to inducechondroid cell differentiation, and RT-PCR results suggest that the induced chondroidfrom hADSCs possess the expression signature of normal chondrocytes.2. There was no obvious morphological change could be observed under an invertedmicroscope when the dedifferentiation occurred. But the diversity between12DD and21DD could be found by AFM and realtime RT-PCR. Our study revealed a novelviewpoint that the amount and distribution of integrin β1might probably play a critical role in mediating chondroid cells maturity and dedifferentiation. And itsuggested that integrin β1might be a new marker and possible therapeutic target forphenotypic maintenance in chondroid cells.3. Wnt/β-catenin signaling must be switched off for chondrogenesis to proceed, whichis beneficial to mature and maintain phenotype of chondroid cells.