Early Stages Of Chondrogenesis Research Articles

Influence of DNA-methylation at multiple stages of limb chondrogenesis

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models.

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

Neuromedin B is a new player in cartilage formation

Purpose: In view of the importance of the TGFβ pathway in cartilage homeostasis and its deregulation in rheumatic diseases, we aimed at identifying genes modulating by TGFβ3 in the early phases of mesenchymal stromal cells (MSC) differentiation into chondrocytes and deciphering their role in maintaining cartilage phenotype. Methods: Transcriptomic analysis was performed on human bone marrow MSC, induced to differentiate into chondrocytes using micropellet culture with/without TGFβ3 and stopped at day 0.5/1/2/3 as compared to d0. Expression of chondrocyte markers and novel genes was quantified by RT-qPCR and western-blotting. Gain- and loss-of-function experiments were performed using recombinant neuromedin B (NMB) and siRNA transfection on MSC induced to chondrogenesis and in mature OA chondrocytes. Results: Transcriptomic data analysis identified genes significantly modulated in TGFβ3-induced micropellets. We selected NMB as one of the most highly up-regulated gene and as a secreted factor. Expression was increased by a 40-fold factor at d1, stabilized at d2-d3 and returned to basal expression at d21 of chondrogenesis. Enhanced expression of NMB was specific to chondrogenesis. NMB basal expression in MSC positively and significantly correlated with SOX9, COL2B, aggrecan and COL10 expression at d21. Down-regulation of NMB expression in MSCs resulted in partial inhibition of chondrogenesis while addition of rNMB did not impact differentiation. Expression of NMB and NMBR was also detected in chondrocytes. Addition of rNMB did not impact chondrogenic (COL2B, ACAN), hypertrophic (MMP13, AP) and fibrotic (COL1, COL3) markers but NMB inhibition induced these markers. Finally, we showed that NMB expression is associated with intracellular Ca2+ influx and calcineurin activity required for Sox9-induced chondrogenesis. Conclusions: NMB induction in the early stages of chondrogenesis is required for MSC differentiation. NMB expression needs to be down-regulated in late stages of chondrogenesis and finely regulated in mature chondrocytes to maintain their phenotype. High expression of NMB in MSC might be a predictive marker of chondrogenic potential.

The effect of the combination of TGF-β1 and BMP2 with high-density pellet cell culture during chondrogenic differentiation of pluripotent stem cells.

Introduction: The osteoarthritis is a serious threat for well-developed and ageing countries. Present techniques of treatment of damaged cartilage are not sufficient. Hence, new strategies should be developed. One of the potential sources for the regeneration of cartilage is pluripotent stem cells (PSC). Aim: The development of an efficient protocol of chondrogenic differentiation using PSC. Material and methods: The human embryonic stem cell line (BG01V) was used in this study. The chondrogenic differentiation was performed using high-density pellet culture in the presence of TGF-β1 (10 ng/ml) and BMP2 (100 ng/ml). After 21 days gene expression analysis of markers related to chondrogenesis was done. Additionally, the histological staining was performed to detect the deposition of proteoglycans and collagens in differentiated pellet culture. Results: Obtained pellets exhibited decreased expression of pluripotent markers. The upregulation of mesodermal marker and type II collagen was observed in differentiated pellets in the presence of applied growth factors. The histological analysis revealed mild deposition of proteoglycans and collagens. Conclusion: The presented approach enables to obtain chondrogenic pellets in their early stages of chondrogenesis.

MicroRNA-218 promotes early chondrogenesis of mesenchymal stem cells and inhibits later chondrocyte maturation

BackgroundMicroRNAs (miRNAs) reportedly participate in the mesenchymal stem cell (MSC) chondrogenic differentiation regulation. We objected to examine how miR-218 regulate chondrogenic differentiation of synovium-derived MSCs (SDSCs) and the maturation of RCJ3.1C5.1 chondrocytes. SDSCs were sourced from the knee joint synovium of New Zealand white rabbits, and their multilineage differentiation potentials were examined. The level of miR-218 was measured during SDSC chondrogenic differentiation, together with determination of SDSCs chondrogenic markers and RCJ3.1C5.1 chondrocytes maturation markers expression level after transfection of miR-218 mimics/inhibitor.ResultsmiR-218 expression was notably upregulated in early chondrogenesis but mostly ceased during the maturation phases of chondrogenic differentiation in SDSCs. The transfection of miR-218 mimics notably enhanced SDSCs chondrocytes differentiation, as evidenced by augmented expressions of chondrogenic markers (SOX9, COL2A1, ACAN, GAG, and COMP) in terms of mRNA and protein level, and the inhibition of miR-218 yielded opposite resutls. Additionally, miR-218 overexpression substantially suppressed the expression of osteogenic markers (ALP, BSP, COL1A1, OCN and OPN) during the early stage of chondrogenesis while increasing that of chondrogenic markers (SOX9, COL2A1, ACAN, GAG and COMP). However, miR-218 mimics notably suppressed maturation markers (CMP, COL10A1, MMP-13 and VEGF) expression in RCJ3.1C5.18 chondrocytes, and the miR-218 inhibitor promoted the expression of these maturation markers. We proposed miR-218 plays a regulatory role on 15-hydroxyprostaglandin dehydrogenase (HPGD), which plays a key role in chondrogenic differentiation, and this finding indicates that miR-218 directly regulates HPGD expression in SDSCs.ConclusionOur study suggests that miR-218 contributes to early chondrogenesis while suppressing later chondrocyte maturation. The miR-218-HPGD pathway offers us a perspective into how SDSCs differentiate into chondrogenic cells.

TGFβi is involved in the chondrogenic differentiation of mesenchymal stem cells and is dysregulated in osteoarthritis

Transforming growth factor-β (TGFβ) is a major regulator of cartilage homeostasis and its deregulation has been associated with osteoarthritis (OA). Deregulation of the TGFβ pathway in mesenchymal stem cells (MSCs) has been proposed to be at the onset of OA. Using a secretome analysis, we identified a member of the TGFβ family, TGFβ-induced protein (TGFβi or βIGH3), expressed in MSCs and we investigated its function and regulation during OA. Cartilage, bone, synovium, infrapatellar fat pad and bone marrow-MSCs were isolated from patients with OA or healthy subjects. Chondrogenesis of BM-MSCs was induced by TGFβ3 in micropellet culture. Expression of TGFβi was quantified by RT-qPCR, ELISA or immunohistochemistry. Role of TGFβi was investigated in gain and loss of function experiments in BM-MSCs and chondrocytes. TGFβi was up-regulated in early stages of chondrogenesis and its knock-down in BM-MSCs resulted in the down-regulation of mature and hypertrophic chondrocyte markers. It likely occurred through the modulation of adhesion molecules including integrin (ITG)β1, ITGβ5 and N-cadherin. We also showed that TGFβi was upregulated invitro in a model of OA chondrocytes, and its silencing enhanced the hypertrophic marker type X collagen. In addition, TGFβi was up-regulated in bone and cartilage from OA patients while its expression was reduced in BM-MSCs. Similar findings were observed in a murine model of OA. Our results revealed a dual role of TGFβi during chondrogenesis and pointed its deregulation in OA joint tissues. Modulating TGFβi in BM-MSCs might be of interest in cartilage regenerative medicine.

Hyaluronan microenvironment enhances cartilage regeneration of human adipose-derived stem cells in a chondral defect model

Hyaluronan (HA) is an important extracellular matrix component in the early stage of chondrogenesis. This study aimed to investigate the application of an HA microenvironment for human adipose-derived stem cells (hADSCs)-based articular cartilage regeneration. HA-enriched fibrin (HA/Fibrin) hydrogels were synthesized and characterized for use as HA microenvironments. The cell viability and chondrogenic gene expression of hADSCs cultured in HA/Fibrin (HA/Fibrin/hADSC) and Fibrin (Fibrin/hADSC) hydrogels were tested in vitro. A chondral defect created in osteochondral core explants ex vivo was used to test chondral defect regeneration by HA/Fibrin/hADSC or Fibrin/hADSC hydrogels. The results showed that HA/Fibrin hydrogels exhibited an increased swelling ratio and matrix stiffness and a smoother surface with more interconnected pores than in Fibrin hydrogels. The viability of hADSCs in HA/Fibrin/hADSC hydrogels was not altered, but they exhibited higher chondrogenic gene expression than those in Fibrin/hADSC hydrogels. For chondral defect regeneration, the HA/Fibrin/hADSC hydrogels exhibited the most cartilaginous tissue neo-formation, chondral integration and sGAG content in the surrounding tissue. This study demonstrated that an HA microenvironment enhances hADSC-mediated cartilage regeneration in chondral defects and thus may be used for ADSC-based articular cartilage tissue engineering.

Randomized double-blind study of a single intra-articular injection of allogeneic mesenchymal precursor cells + hyaluronan compared to hyaluronan alone to explore clinical and structural outcomes in patients post anterior cruciate ligament reconstruction

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Specific synovial joint progenitor cell populations uniquely contribute to articular cartilage growth and repair

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Co-delivery of Cbfa-1-targeting siRNA and SOX9 protein using PLGA nanoparticles to induce chondrogenesis of human mesenchymal stem cells

During stem cell differentiation, various cellular responses occur that are mediated by transcription factors and proteins. This study evaluated the abilities of SOX9, a crucial protein during the early stage of chondrogenesis, and siRNA targeting Cbfa-1, a transcription factor that promotes osteogenesis, to stimulate chondrogenesis. Non-toxic poly-(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. Coomassie blue staining and circular dichroism revealed that the loaded SOX9 protein maintained its stability and bioactivity. These NPs easily entered human mesenchymal stem cells (hMSCs) in vitro and caused them to differentiate into chondrocytes. Markers that are typically expressed in mature chondrocytes were examined. These markers were highly expressed at the mRNA and protein levels in hMSCs treated with PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. By contrast, these cells did not express osteogenesis-related markers. hMSCs were injected into mice following internalization of PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. When the injection site was excised, markers of chondrogenesis were found to be highly expressed at the mRNA and protein levels, similar to the in vitro results. When hMSCs internalized these NPs and were then cultured in vitro or injected into mice, chondrogenesis-related extracellular matrix components were highly expressed.

Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics

BackgroundOsteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA. New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. MethodsTo discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. ResultsExpression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. ConclusionThe results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. General significanceThese cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents.

Cell proliferation is promoted by compressive stress during early stage of chondrogenic differentiation of rat BMSCs

The presence of an appropriate number of viable cells is prerequisite for successive differentiation during chondrogenesis. Chondrogenic differentiation has been reported to be influenced by mechanical stimuli. This research aimed to study the effects of cyclic compressive stress on cell viability of rat bone marrow-derived MSCs (BMSCs) during chondrogenesis as well as its underlying mechanisms. The results showed that dynamic compression increased cell quantity and viability remarkably in the early stage of chondrogenesis, during which the expression of Ihh, Cyclin D1, CDK4, and Col2α1 were enhanced significantly. Possible signal pathways implicated in the process were explored in our study. MEK/ERK and p38 MAPK were not found to function in this process while BMP signaling seemed to play an important role in the mechanotransduction during chondrogenic proliferation. In conclusion, dynamic compressive stress could enhance cell viability during chondrogenesis, which might be achieved by activating BMP signaling.

FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis

One of the earliest events during chondrogenesis is the formation of condensations, a necessary pre-requisite for subsequent differentiation of a chondrogenic phenotype. Members of the Fibronectin Lecucine Rich Transmembrane (FLRT) proteins have been shown to be involved in cell sorting and neurite outgrowth. Additionally, FLRT2 is highly expressed at putative sites of chondrogenic differentiation during craniofacial development. In this study, we demonstrate that FLRT2 plays a role in mediating cell proliferation and cell-cell interactions during early chondrogenesis. Clones of stable transfectants of a murine chondroprogenitor cell line, ATDC5, were established in which FLRT2 was knocked down or overexpressed. Cells in which FLRT2 was knocked down proliferated at a slower rate compared to control wild-type ATDC5 cells or those containing a non-coding shRNA. In addition, FLRT2 knockdown cells formed numerous lectin peanut agglutinin (PNA) stained aggregates and exhibited higher expression of the cell adhesion molecule, N-cadherin. In an in vitro wound healing assay, fewer FLRT2 knockdown cells appeared to migrate into the defect. Surprisingly, the FLRT2 knockdown cells demonstrated increased formation of Alcian blue-stainable extracellular matrix, suggesting that their reduced aggregate formation did not inhibit subsequent chondrogenic differentiation. The opposite trends were observed in ATDC5 clones that overexpressed FLRT2. Specifically, FLRT overexpressing cells proliferated faster, formed fewer PNA-positive aggregates, accumulated increased Alcian blue-positive matrix, and migrated faster to close a wound. Collectively, our findings provide evidence for a role of FLRT2 in enhancing cell proliferation and reducing intercellular adhesion during the early stages of chondrogenesis.

Evolutionarily Conserved, Growth Plate Zone-Specific Regulation of the Matrilin-1 Promoter: L-Sox5/Sox6 and Nfi Factors Bound near TATA Finely Tune Activation by Sox9

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate.

Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.

Transglutaminase 2 regulates early chondrogenesis and glycosaminoglycan synthesis

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-β activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.

MED and PSACH COMP mutations affect chondrogenesis in chicken limb bud micromass cultures

Mutations in cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We studied the effects of over-expression of wild type and mutant COMP on early stages of chondrogenesis in chicken limb bud micromass cultures. Cells were transduced with RCAS virus harboring wild type or mutant (C328R, PSACH; T585R, MED) COMP cDNAs and cultured for 3, 4, and 5 days. The effect of COMP constructs on chondrogenesis was assessed by analyzing mRNA and protein expression of several COMP binding partners. Cell viability was assayed, and evaluation of apoptosis was performed by monitoring caspase 3 processing. Over-expression of COMP, and especially expression of COMP mutants, had a profound affect on the expression of syndecan 3 and tenascin C, early markers of chondrogenesis. Over-expression of COMP did not affect levels of type II collagen or matrilin-3; however, there were increases in type IX collagen expression and sulfated proteoglycan synthesis, particularly at day 5 of harvest. In contrast to cells over-expressing COMP, cells with mutant COMP showed reduction in type IX collagen expression and increased matrilin 3 expression. Finally, reduction in cell viability, and increased activity of caspase 3, at days 4 and 5, were observed in cultures expressing either wild type or mutant COMP. MED, and PSACH mutations, despite displaying phenotypic differences, demonstrated only subtle differences in their cellular viability and mRNA and protein expression of components of the extracellular matrix, including those that interact with COMP. These results suggest that COMP mutations, by disrupting normal interactions between COMP and its binding partners, significantly affect chondrogenesis.

Tensile Loading Modulates Bone Marrow Stromal Cell Differentiation and the Development of Engineered Fibrocartilage Constructs

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

Activin A expression regulates multipotency of mesenchymal progenitor cells

IntroductionBone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); however, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency.MethodsFor this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources.ResultsThe multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs.ConclusionsThis comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.

TGF-beta3 inhibits chondrogenesis by suppressing precartilage condensation through stimulation of N-cadherin shedding and reduction of cRREB-1 expression.

Transforming growth factor-beta (TGF-beta) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-beta3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-beta3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-beta3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-beta3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.