Decades of work have identified the signaling pathways that regulate the differentiation of chondrocytes during bone formation, from their initial induction from mesenchymal progenitor cells to their ...terminal maturation into hypertrophic chondrocytes. Here, we review how multiple signaling molecules, mechanical signals and morphological cell features are integrated to activate a set of key transcription factors that determine and regulate the genetic program that induces chondrogenesis and chondrocyte differentiation. Moreover, we describe recent findings regarding the roles of several signaling pathways in modulating the proliferation and maturation of chondrocytes in the growth plate, which is the 'engine' of bone elongation.
The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and ...heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.
It has been discussed whether reduced glutathione (GSH) could promote the chondrogenic differentiation ability of human umbilical cord mesenchymal stem cells (hUC-MSCs). hUC-MSCs were isolated from ...human umbilical cord and their specificity was identified, then induced into cartilage-like cells in chondrogenic induction medium with transforming growth factor beta 1 (TGF- beta 1), especially with GSH. The morphological change before and after induction was observed through inverted phase contrast microscope, Type II collagen (COL2-A1) and glycosaminoglycan (GAG) were analyzed qualitatively by Toluidine blue and immunofluorescence technique, respectively, the contents of COL2-A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2-A1 were assayed by real-time fluorescence quantitative PCR. After continuously cultured for 21 days with GSH, Toluidine blue staining and immunofluorescence reaction were all positive in basic induction medium group (group B), basic induction medium +0.5% dimethylsulfoxide (DMSO) group (group BD) and basic induction medium +0.5% DMSO +500 mu M GSH group (group BDG). Moreover, compared with group B and group BD, the contents of COL2-A1 and GAG in group BDG relatively increased and the mRNA expression level of COL2-A1 and GAG also comparatively increased (P < 0.05) and both had a significant statistical significance (P < 0.05). So GSH might promote the induction of hUC-MSCs to differentiate into cartilage-like cells.
Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its ...proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically.
N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and ...organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.
Summary Objective Metalloproteinases (MMPs) are key regulators of osteoarthritis (OA) and collagen degradation and have been shown to participate in endochondral ossification. The aim of this study ...was to determine whether microRNA-320 (miR-320) regulates the expression of MMP-13 in chondrogenesis and inflammation. Experimental design miR-320 expression was assessed in vitro , in the ATDC5 cell model of chondrogenesis and in interleukin-1β (IL-1β)-treated primary mouse chondrocytes (PMCs), and in vivo , in normal and OA human cartilage by in situ hybridization. ATDC5 and PMCs were transfected with miR-320 or its antisense inhibitor (anti-miR-320), respectively. The roles of activated MAP kinases (MAPK) and NF-κB were evaluated by using specific inhibitors. Direct interaction between miR-320 and its putative binding site in the 3′-untranslated region (3′-UTR) of Mmp-13 mRNA was confirmed by the luciferase reporter assay. Results miR-320 expression was elevated in chondrogenic and hypertrophic ATDC5, while significantly reduced in OA cartilage compared with normal cartilage. Stimulation with IL-1β led to a significant reduction in miR-320 expression in PMCs. Upregulation of MMP-13 expression was correlated with downregulation of miR-320 expression in both PMCs and ATDC5. Overexpression of miR-320 suppressed the activity of a reporter construct containing the 3′-UTR and inhibited MMP-13 expression in both ATDC5 and IL-1β-treated PMCs, while treatment with anti-miR-320 enhanced MMP-13 expression. NF-κB and MAPK activation downregulated miR-320 expression. Conclusion Cartilage development and homeostasis are influenced by miR-320, which directly targets MMP-13 and regulates chondrogenesis and the IL-1β-stimulated catabolic effect in mouse chondrocytes.
Replacement of degenerated cartilage with cell‐based cartilage products may offer a long‐term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell‐based ...FDA‐approved cartilage products; yet human marrow‐derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF‐β1), growth/differentiation factor 5 (GDF‐5), and bone morphogenetic protein 2 (BMP‐2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF‐β1, 100 ng/mL GDF‐5, or 100 ng/mL BMP‐2 supplementation during three‐dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF‐β1, GDF‐5, and BMP‐2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage‐specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF‐β1, GDF‐5, and BMP‐2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self‐assembling method to generate robust, scaffold‐free neocartilage constructs using expanded hACs and hMSCs. Stem Cells 2015;33:762–773
Bony fusion caused by pathological new bone formation manifests the clinical feature of ankylosing spondylitis (AS). However, the underlying mechanism remains elusive. Here we discovered spontaneous ...kyphosis, arthritis and bony fusion in mature CD4-Cre;Ptpn11
mice, which present the pathophysiological features of AS. A population of CD4-Cre-expressing proliferating chondrocytes was SHP2 deficient, which could differentiate into pre-hypertrophic and hypertrophic chondrocytes. Functionally, SHP2 deficiency in chondrocytes impeded the fusion of epiphyseal plate and promoted chondrogenesis in joint cavity and enthesis. Mechanistically, aberrant chondrocytes promoted ectopic new bone formation through BMP6/pSmad1/5 signaling. It is worth emphasizing that such pathological thickness of growth plates was evident in adolescent humans with enthesitis-related arthritis, which could progress to AS in adulthood. Targeting dysfunctional chondrogenesis with Smo inhibitor sonidegib significantly alleviated the AS-like bone disease in mice. These findings suggest that blockade of chondrogenesis by sonidegib would be a drug repurposing strategy for AS treatment.
Abstract Electrospinning has recently gained much interest due to its ability to form scaffolds that mimic the nanofibrous nature of the extracellular matrix, such as the size and depth-dependent ...alignment of collagen fibers within hyaline cartilage. While much progress has been made in developing bulk, isotropic hydrogels for tissue engineering and understanding how the microenvironment of such scaffolds affects cell response, these effects have not been extensively studied in a nanofibrous system. Here, we show that the mechanics (through intrafiber crosslink density) and adhesivity (through RGD density) of electrospun hyaluronic acid (HA) fibers significantly affect human mesenchymal stem cell (hMSC) interactions and gene expression. Specifically, hMSC spreading, proliferation, and focal adhesion formation were dependent on RGD density, but not on the range of fiber mechanics investigated. Moreover, traction-mediated fiber displacements generally increased with more adhesive fibers. The expression of chondrogenic markers, unlike trends in cell spreading and cytoskeletal organization, was influenced by both fiber mechanics and adhesivity, in which softer fibers and lower RGD densities generally enhanced chondrogenesis. This work not only reveals concurrent effects of mechanics and adhesivity in a fibrous context, but also highlights fibrous HA hydrogels as a promising scaffold for future cartilage repair strategies.
Mesenchymal stem cells are known to exert immunomodulatory effects in inflammatory diseases. Immuneregulatory cells lead to progressive joint destruction in rheumatoid arthritis (RA). Proinflammatory ...cytokines, such as tumour necrosis factor α (TNF‐α) and interleukins (ILs) are the main players. Here, we studied progenitor cells from RA cartilage (RA‐CPCs) that are positive for IL‐17 receptors to determinate the effects of inflammation on their chondrogenic potenial. IL‐17A/F reduced the chondrogenic potential of these cells via the upregulation of RUNX2 protein and enhanced IL‐6 protein and MMP3 mRNA levels. Blocking antibodies against IL‐17 positively influenced their repair potential. Furthermore, treating the RA‐CPCs with the anti‐human IL‐17 antibody secukinumab or the anti‐TNF‐α antibody adalimumab reduced the proinflammatory IL‐6 protein level and positively influenced the secretion of anti‐inflammatory IL‐10 protein. Additionally, adalimumab and secukinumab in particular reduced RUNX2 protein to promote chondrogenesis. The amelioration of inflammation, particularly via IL‐17 antagonism, might be a new therapeutic approach for enhancing intrinsic cartilage repair mechanisms in RA patients.
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