In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Objective

Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)–based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration.

Methods

A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp‐hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage‐forming ability.

Results

Coculture of hPi‐GFP chondrocytes with Kp‐hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor β superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin‐like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp‐hMSCs acquired neocartilage‐forming potential within the collagen scaffold.

Conclusion

These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell‐based transplantation for future clinical use.

     

Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation

Objective

Development of stem cell therapies for regenerating the nucleus pulposus (NP) are hindered by the lack of specific markers by which to distinguish NP cells from articular chondrocytes (ACs). The purpose of this study was to define the phenotype profile of human NP cells using gene expression profiling and to assess whether the identified markers could distinguish mesenchymal stem cell (MSC) differentiation to a correct NP cell phenotype.

Methods

Affymetrix MicroArray analyses were conducted on human NP cells and ACs, and differential expression levels for several positive (NP) and negative (AC) marker genes were validated by real‐time quantitative polymerase chain reaction (PCR) analysis. Novel marker gene and protein expression was also assessed in human bone marrow–derived MSCs (BM‐MSCs) and adipose tissue–derived MSCs (AD‐MSCs) following differentiation in type I collagen gels.

Results

Analysis identified 12 NP‐positive and 36‐negative (AC) marker genes that were differentially expressed ≥20‐fold, and for a subset of them (NP‐positive genes PAX1, FOXF1, HBB, CA12, and OVOS2; AC‐positive genes GDF10, CYTL1, IBSP, and FBLN1), differential expression was confirmed by real‐time quantitative PCR. Differentiated BM‐MSCs and AD‐MSCs demonstrated significant increases in the novel NP markers PAX1 and FOXF1. AD‐MSCs lacked expression of the AC markers IBSP and FBLN1, whereas BM‐MSCs lacked expression of the AC marker IBSP but expressed FBLN1.

Conclusion

This study is the first to use gene expression profiling to identify the human NP cell phenotype. Importantly, these markers can be used to determine the in vitro differentiation of MSCs to an NP‐like, rather than an AC‐like, phenotype. Interestingly, these results suggest that AD‐MSCs may be a more appropriate cell type than BM‐MSCs for use in engineering intervertebral disc tissue.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Human articular chondrocytes secrete parathyroid hormone–related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis

Objective

The use of bone marrow–derived mesenchymal stem cells (MSCs) has shown promise in cell‐based cartilage regeneration. A yet‐unsolved problem, however, is the unwanted up‐regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs.

Methods

MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice.

Results

The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC‐conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose‐dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone–related protein (PTHrP) throughout the culture period, whereas PTHrP was down‐regulated in favor of IHH up‐regulation in control MSCs after 2–3 weeks of chondrogenesis. The main inhibitory effects seen with HAC‐conditioned medium were reproducible by PTHrP supplementation of unconditioned medium.

Conclusion

HAC‐derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

     

Gremlin 1, Frizzled‐related protein,and Dkk‐1 are key regulators of human articular cartilage homeostasis

Objective

The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation.

Methods

Whole‐genome gene‐expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA.

Results

Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled‐related protein (FRP), and Dkk‐1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long‐bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single‐nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population‐based cohorts.

Conclusion

Taken together, our study identified Gremlin 1, FRP, and Dkk‐1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

     

Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

Objective

To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS‐4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic “unsilencing.”

Methods

Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS‐4 was analyzed with methylation‐sensitive restriction enzymes, followed by polymerase chain reaction amplification.

Results

Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late‐stage OA cartilage were immunopositive. The overall percentage of nonmethylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP‐13, 81% versus 47% for MMP‐9, 57% versus 30% for MMP‐3, and 48% versus 0% for ADAMTS‐4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at −110 for MMP‐13, −36 for MMP‐9, −635 for MMP‐3, and −753 for ADAMTS‐4.

Conclusion

This study provides the first evidence that altered synthesis of cartilage‐degrading enzymes by late‐stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.

     

Higher chondrogenic potential of fibrous synovium– and adipose synovium–derived cells compared with subcutaneous fat–derived cells: Distinguishing properties of mesenchymal stem cells in humans

Objective

Mesenchymal stem cells from synovium have a greater proliferation and chondrogenic potential than do those from bone marrow, periosteum, fat, and muscle. This study was undertaken to compare fibrous synovium and adipose synovium (components of the synovium with subsynovium) to determine which is a more suitable source for mesenchymal stem cells, especially for cartilage regeneration, and to examine the features of adipose synovium–derived cells, fibrous synovium–derived cells, and subcutaneous fat–derived cells to determine their similarities.

Methods

Human fibrous synovium, adipose synovium, and subcutaneous fat were harvested from 4 young donors and 4 elderly donors. After digestion, the nucleated cells were plated at a density considered proper to expand at a maximum rate without colony‐to‐colony contact. The surface epitopes, proliferative capacity, cloning efficiency, and chondrogenic, osteogenic, and adipogenic differentiation potentials of the cells were compared.

Results

Fibrous synovium– and adipose synovium–derived cells were higher in STRO‐1 and CD106 and lower in CD10 compared with subcutaneous fat–derived cells. Cells derived from fibrous and adipose synovium had higher proliferative potential and colony‐forming efficiency compared with subcutaneous fat–derived cells, both in mixed‐population and in single‐cell–derived cultures. In chondrogenic assays, pellets from fibrous synovium– and adipose synovium–derived cells produced more cartilage matrix than did cell pellets from subcutaneous fat. Osteogenic ability was also higher in fibrous synovium– and adipose synovium–derived cells, whereas adipogenic potential was nearly indistinguishable among the 3 populations. Differentiation potential of the cells was similar between young and elderly donors.

Conclusion

Cells derived from the fibrous synovium and from the adipose synovium demonstrate comparable chondrogenic potential. Adipose synovium–derived cells are more similar to fibrous synovium–derived cells than to subcutaneous fat–derived cells.

     

Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling

Objective

We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow–derived mesenchymal stem cells (MSCs), with consideration of sex‐dependent differences in cartilage repair.

Methods

Bone marrow was obtained from the iliac crest of young men. Density‐gradient centrifugation–separated human MSCs proliferated as a monolayer in serum‐containing medium. After confluence was achieved, aggregates were created and cultured in a serum‐free differentiation medium. We added different concentrations of 17β‐estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane‐impermeable E2–bovine serum albumin (E2‐BSA), ICI 182.780 alone, G‐1 (an agonist of G protein–coupled receptor 30 [GPR‐30]), and G15 (a GPR‐30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP‐13) expression.

Results

The existence of intracellular and membrane‐associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2‐BSA in a dose‐dependent manner. Furthermore, E2 enhanced type X collagen and MMP‐13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR‐30 agonists alone (G‐1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G‐1. Furthermore, the specific GPR‐30 antagonist G15 reversed the GPR‐30–mediated inhibition of chondrogenesis and up‐regulation of hypertrophic gene expression.

Conclusion

The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR‐30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling.

     

Reduction of CpG‐induced arthritis by suppressive oligodeoxynucleotides

Objective

Bacterial DNA contains immunostimulatory CpG motifs that cause inflammation when injected into the knee joints of normal mice. We examined whether synthetic oligodeoxynucleotides (ODN) that suppress CpG‐induced immune responses prevent CpG‐induced arthritis.

Methods

CpG, suppressive, and/or control ODN were injected into the knees of BALB/c mice. Joint swelling and inflammation were evaluated by physical measurement, by histologic analysis of joint tissue, and by magnetic resonance imaging.

Results

Immunostimulatory CpG DNA induced local arthritis, characterized by swelling of the knee joints, the presence of inflammatory cell infiltrates, the perivascular accumulation of mononuclear cells, and hyperplasia of the synovial lining. Administering suppressive (but not control) ODN reduced the manifestations and severity of arthritis up to 80%.

Conclusion

Suppressive ODN may be useful for the prevention or treatment of arthritis induced by bacterial DNA.