Chondrogenic differentiation of adult mesenchymal stem cells and embryonic cells in collagen scaffolds

Many cell types and cellular microenvironments have been explored for articular cartilage tissue engineering. We compared the potential of bone marrow-derived mesenchymal stem cells (MSCs) and P19 embryonic carcinoma cells (ECCs), a pluripotent derivative of embryonic stem cells (ESCs), for cartilage histogenesis in porous collagen scaffolds in vitro. We found that while both MSCs and ECCs express α-smooth muscle actin (α-SMA), only MSCs exhibit condensation and contraction necessary for cartilage histogenesis. Furthermore, histology confirmed that only MSCs exhibited sulfated glycosaminoglycans and collagen type II formation after 14 days in culture. We conclude that MSCs appear to be superior over ECCs for cartilage regeneration under particular culture conditions. The α-SMA-expressing ECCs may not have contracted due to the absence of actin unit polymerization or the absence of myosin molecules. Our observations may explain the absence of a contractile scar in fetal wound healing.     

Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels

Human embryonic stem cells (hESCs) have the potential to self-renew and generate multiple cell types, producing critical building blocks for tissue engineering and regenerative medicine applications. Here, we describe the efficient derivation and chondrogenic differentiation of mesenchymal-like cells from hESCs. These cells exhibit mesenchymal stem cell (MSC) surface markers, including CD29, CD44, CD105, and platelet-derived growth factor receptor-alpha. Under appropriate growth conditions, the hESC-derived cells proliferated without phenotypic changes and maintained MSC surface markers. The chondrogenic capacity of the cells was studied in pellet culture and after encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with exogenous extracellular proteins or arginineglycine- aspartate (RGD)-modified PEGDA hydrogels. The hESC-derived cells exhibited growth factor- dependent matrix production in pellet culture but did not produce tissue characteristic of cartilage morphology. In PEGDA hydrogels containing exogenous hyaluronic acid or type I collagen, no significant cell growth or matrix production was observed. In contrast, when these cells were encapsulated in RGDmodified poly(ethylene glycol)hydrogels, neocartilage with basophilic extracellular matrix deposition was observed within 3 weeks of culture, producing cartilage-specific gene up-regulation and extracellular matrix production. Our results indicate that precursor cells characteristic of a MSC population can be cultured from differentiating hESCs through embryoid bodies, thus holding great promise for a potentially unlimited source of cells for cartilage tissue engineering.     

Chondrogenic differentiation of human mesenchymal stem cells in three-dimensional alginate gels

We characterized the temporal changes in chondrogenic genes and developed a staging scheme for in vitro chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in three-dimensional (3D) alginate gels. A time-dependent accumulation of glycosaminoglycans, aggrecan, and type II collagen was observed in chondrogenic but not in basal constructs over 24 days. qRT-PCR demonstrated a largely characteristic temporal pattern of chondrogenic markers and provided a basis for staging the cellular phenotype into four stages. Stage I (days 0-6) was defined by collagen types I and VI, Sox 4, and BMP-2 showing peak expression levels. In stage II (days 6-12), gene expression for cartilage oligomeric matrix protein, HAPLN1, collagen type XI, and Sox 9 reached peak levels, while gene expression of matrilin 3, Ihh, Homeobox 7, chondroadherin, and WNT 11 peaked at stage III (days 12-18). Finally, cells in stage IV (days 18-24) attained peak levels of aggrecan; collagen IX, II, and X; osteocalcin; fibromodulin; PTHrP; and alkaline phosphatase. Gene profiles at stages III and IV were analogous to those in juvenile articular and adult nucleus pulposus chondrocytes. Gene ontology analyses also demonstrated a specific expression pattern of several putative novel marker genes. These data provide comprehensive insights on chondrogenesis of hMSCs in 3D gels. The derivation of this staging scheme may aid in defining maximally responsive time points for mechanobiological modulation of constructs to produce optimally engineered tissues.     

骨髓间充质干细胞向软骨细胞的分化

背景：以骨髓间充质干细胞构建组织工程气管尚缺乏理想的特异性表面标志物,对其鉴定主要依赖细胞形态学、细胞表型及诱导分化的功能进行分析。 目的：体外分离培养、鉴定兔骨髓间充质干细胞,观察在特定条件下向气管软骨细胞分化的潜能。 方法：无菌环境取兔骨髓,经全骨髓贴壁筛选法分离培养细胞至第2代,流式细胞术鉴定第1、第2代细胞表面抗原CD44、CD45的表达。无菌环境取气管,经酶消化法分离培养气管软骨细胞,甲苯胺蓝染色鉴定软骨细胞蛋白聚糖的合成。在使用转化生长因子β1的基础上,将骨髓间充质干细胞与气管软骨细胞通过Transwell小室非接触式共培养,倒置显微镜观察细胞形态,甲苯胺蓝染色鉴定蛋白聚糖的合成,荧光实时定量PCR鉴定Ⅱ型胶原和蛋白聚糖 mRNA的表达。 结果与结论：分离、培养的细胞呈长梭形、不规则形聚集生长,传代后细胞生长速度明显增快,呈鱼群状聚集生长。第1代有96.97%的细胞表达CD44、13.72%的细胞表达CD45,第2代有99.11%的细胞表达CD44、8.54%的细胞表达CD45。气管软骨细胞甲苯胺蓝染色阳性。在诱导后,骨髓间充质干细胞形态逐渐由长梭形变为三角形或不规则形,表达软骨细胞特异性Ⅱ型胶原和蛋白聚糖 mRNA基因,甲苯胺蓝染色示阳性。结果表明全骨髓贴壁筛选法可成功分离培养骨髓间充质干细胞,第2代纯度较高,且在特定诱导条件下具有分化为气管软骨细胞的潜能。     

Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces

Human mesenchymal stem cells (MSCs) were cultured on polystyrene surfaces modified with photoreactive azidophenyl-derivatives of three different chargeable polymers, poly(acrylic acid) (PAAc), polyallylamine (PAAm), and poly(ethylene glycol) (PEG). The MSCs adhered and spread both on a PAAm-modified surface and on PAAc-modified and polystyrene (control) surfaces. However, the cells adhered more easily to the PAAm-modified surface. The MSCs did not attach to the PEG-modified surface and aggregated to form pellets immediately after cell seeding. The cells proliferated on the PAAc-, PAAm-modified and control surfaces with culture time, formed a monolayer, and aggregated to form pellets. The cells in the pellets that formed on the PAAm- and PEG-modified surfaces after 2 weeks culture had a round morphology and the extracellular matrices were positively stained by safranin O and toluidine blue, while those that formed on the PAAc-modified and control surfaces had a spindle, fibroblast-like morphology and were not positively stained by safranin O and toluidine blue. The pellets that formed on the PAAm- and PEG-modified surfaces contained significantly higher levels of sulfated glycosaminoglycans than did those that formed on the PAAc-modified and control surfaces. Type II collagen and cartilage proteoglycan were immunohistologically detected in the pellets that formed on PAAm- and PEG-modified surfaces, but not those that formed on the PAAc-modified and control surfaces. The MSCs cultured on the PAAm- and PEG-modified surfaces expressed a high level of cartilaginous genes encoding type II collagen and aggrecan, while the MSCs cultured on the PAAc-modified and control surfaces did not express these genes. These results suggest that the PAAm-modified surface supported cell adhesion and proliferation and also promoted chondrogenic differentiation of the MSCs. The PAAc-modified and polystyrene surfaces supported cell adhesion and proliferation, but not chondrogenic differentiation. The PEG-modified surfaces did not support cell adhesion, but did promote chondrogenic differentiation. The adhesion, proliferation, and differentiation of the MSCs could be controlled by surface chemistry.     

胎盘来源间充质干细胞的体外诱导成软骨分化

背景：胎盘间充质干细胞已被证实有较强的增殖能力,且能向成骨细胞、成神经细胞、类肝样细胞等诱导分化,但对其成软骨细胞诱导分化的研究不多。 目的：在体外诱导人胎盘间充质干细胞成软骨细胞分化。 方法：体外培养人胎盘间充质干细胞并鉴定。取第3代胎盘间充质干细胞,调整细胞悬液浓度为1.6×1010 L-1,在24孔板中央分别滴5,10,15 μL细胞悬液,培养2 h(目的是形成微团);然后加入间充质干细胞培养基或软骨诱导培养基培养14 d,阿利新蓝染色,进行大体观察及倒置显微镜观察。 结果与结论：应用间充质干细胞培养基培养的对照组,细胞增殖形成大量贴壁细胞,贴壁细胞具有典型的间充质细胞形态;应用软骨诱导培养基培养后,细胞只保持微团,不继续增殖形成贴壁细胞,微团基部无贴壁细胞,随着接种细胞数量的增加,微团直径增大。说明人胎盘来源间充质干细胞具有向软骨细胞分化的能力,可作为组织工程、细胞治疗等应用的种子细胞来源。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

生长分化因子5诱导脂肪干细胞复合Ⅰ型胶原支架成软骨细胞的分化

背景：前期实验中发现生长分化因子5可以诱导脂肪干细胞向软骨细胞分化,但在复合Ⅰ型胶原支架的体外培养条件下其向软骨细胞分化的能力尚未见研究报道。 目的：探讨生长分化因子5诱导脂肪干细胞复合Ⅰ型胶原支架向软骨细胞分化的能力。 方法：从兔脂肪组织中分离培养脂肪干细胞,使用倒置相差显微镜观察细胞形态,使用免疫荧光对其表型进行鉴定。在复合Ⅰ型胶原支架条件下,加入外源性生长分化因子5对脂肪干细胞向软骨细胞进行诱导,在诱导14 d时,采用苏木精-伊红染色和扫描电镜对诱导的细胞进行形态学观察。在诱导7,14和21 d时,采用反转录PCR方法检测诱导的细胞的Ⅱ型胶原和蛋白多糖mRNA表达情况。 结果与结论：原代脂肪干细胞贴壁生长,呈梭形、多角形分布,细胞表面抗原CD44、CD49d 阳性,CD106 阴性。生长分化因子5诱导的脂肪干细胞与Ⅰ型胶原支架黏附良好,增殖能力旺盛,细胞表面存在着大量的细胞外基质分泌,Ⅱ型胶原和蛋白聚糖mRNA表达水平明显增加。说明生长分化因子5能够成功诱导复合Ⅰ型胶原支架的脂肪干细胞成软骨细胞分化。     

基底膜基质/壳聚糖诱导骨髓间充质干细胞成软骨分化

背景：课题组前期研究发现基底膜基质能够定向诱导骨髓间充质干细胞向软骨方向分化,但其力学性能与实际应用有较大差距,还需要进一步研究。 目的：制备兼具适宜力学性能和优异生物相容性的壳聚糖/基底膜基质水凝胶支架用于软骨修复。 方法：以京尼平为交联剂,将壳聚糖溶液与基底膜基质按2︰1,1︰1,1︰3比例混合制成水凝胶,接种大鼠骨髓间充质干细胞,培养14 d。经材料力学测试、细胞增殖、活细胞染色、酶联免疫吸附测试以及阿尔新蓝染色等方法评价材料诱导细胞成软骨分化能力。 结果与结论：在基底膜基质内添加壳聚糖后,材料力学性能从0.48 kPa上升到1.78 kPa。标志性蛋白分泌结果显示,纯壳聚糖组早期诱导成软骨活性高于其他组,但后期诱导能力减弱,而含基底膜基质各组在后期能够保持较好的诱导活性,其中壳聚糖/基底膜基质=1︰1组材料具有一定的力学强度,且Ⅱ型胶原和Ⅹ型胶原的表达量在14 d较其他组高。结果表明实验制备的壳聚糖/基底膜基质水凝胶具有良好的力学性能,并能够促进骨髓间充质干细胞向软骨方向分化,可用于软骨组织工程研究。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair

For the development of new therapeutical cell-based strategies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. We present a systematic and detailed study on the performance and biological impact of a simple and efficient labelling protocol for human mesenchymal stem cells (hMSCs). Commercially available very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake via endocytosis was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabelled cells, VSOP-labelling did neither influence the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect hMSCs in undergoing adipogenic, osteogenic or chondrogenic differentiation, as demonstrated histologically and by gene expression analyses. The efficiency of the labelling protocol was assessed with high-resolution MR imaging at 11.7T. VSOP-labelled hMSCs were visualised in a collagen type I hydrogel, which is in clinical use for matrix-based articular cartilage repair. The presence of VSOP-labelled hMSCs was indicated by distinct hypointense spots in the MR images, as a result of iron specific loss of signal intensity. In summary, this labelling technique has great potential to visualise hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.     

Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, interest in methods focused on biological repair has increased. Several tissue engineering approaches using different cell types and hydrogels/scaffolds have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering attention has focused on the use of mesenchymal stem cells (MSCs). Additionally, while rigid scaffolds have been demonstrated to allow MSC differentiation to the chondrocyte-like cells of the IVD, hydrogels are being increasingly studied as they allow minimally invasive implantation without extensive damage to the IVD. Here, we have studied the temperature-sensitive hydrogel chitosan-glycerophosphate (C/Gp), seeded with human MSCs and cultured for 4 weeks in standard medium. We have analysed the gene and protein expression profile of the MSCs and compared it to that of both nucleus pulposus (NP) cells and articular chondrocytes cultured in C/Gp. Gene expression analysis for chondrocytic-cell marker genes demonstrated differentiation of MSCs to a phenotype which showed similarities to both articular chondrocytes and NP cells. Conventional PCR demonstrated a lack of expression of osteogenic marker genes and the hypertrophic marker gene type X collagen. MSCs also secreted both proteoglycans and collagens in a ratio, which more closely resembled that of NP cells than articular chondrocytes. These results therefore suggest that MSC-seeded C/Gp gels could be used clinically for the regeneration of the degenerate human IVD.     

Chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells induced by growth differentiation factor 5

BACKGROUND: Growth differentiation factor 5 is a member of the transforming growth factor superfamily and one of the earliest markers of joint development. Growth differentiation factor 5 has an important role in cartilage repair. OBJECTIVE: To explore the action mechanism of growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Rabbit bone marrow mesenchymal stem cells were isolated and cultured. CCK-8 assay was used to detect the effect of different mass concentrations of growth differentiation factor 5 on the proliferation activity of bone marrow mesenchymal stem cells. RT-PCR was utilized to detect the expression of genes related to chondrogenic differentiation of bone marrow mesenchymal stem cells induced by different mass concentrations of growth differentiation factor 5. To further investigate the action mechanism of growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells, we added inhibitor XAV-939 and activator Laduviglusib of Wnt/β-catenin signaling pathways to induce cell culture for 14 days. RT-PCR and western blot assay were performed to detect the expression of cartilage-related genes and Wnt/β-catenin signaling pathway proteins. RESULTS AND CONCLUSION: (1) CCK-8 results showed no significant effect of growth differentiation factor 5 on the proliferation of bone marrow mesenchymal stem cells. (2) Growth differentiation factor 5 promoted the expression of cartilage-related genes type II collagen, aggrecan and Sox9, among which growth differentiation factor 5 induced a significant upregulation of cartilage-related genes in the 50 ng/mL group. (3) Addition of Laduviglusib, an activator of Wnt/β-catenin signaling pathway, upregulated Sox9, β-catenin and type II collagen expression (P < 0.05). Addition of XAV939, an inhibitor of Wnt/β-catenin signaling pathway, down-regulated Sox9, β-catenin and type II collagen expression (P < 0.05). (4) Taken together, growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells may be associated with the activation of the Wnt/β-catenin signaling pathway. © 2024, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells induced by acellular cartilage sheets

Acellular cartilage sheets (ACSs) have been used as scaffolds for engineering cartilage with mature chondrocytes. In this study we investigated whether ACSs possess a chondrogenic induction activity that may benefit cartilage engineering with multipotent stem cells. Bone marrow-derived mesenchymal stem cells (BMSCs) isolated from newborn pigs were expanded in vitro and seeded on ACSs that were then stacked layer-by-layer to form BMSC-ACS constructs. Cells seeded on polyglycolic acid/polylactic acid (PGA/PLA) scaffolds served as a control. After 4 weeks of culture with or without additional chondrogenic factors, constructs were subcutaneously implanted into nude mice for another 4 weeks. Cartilage-like tissues were formed after 4 weeks of culture. However, formation of cartilage with a typical lacunar structure was only observed in induced groups. RT-PCR showed that aggrecan, COMP, type II collagen and Sox9 were expressed in all groups except the non-induced BMSC-PGA/PLA group. At 4 weeks post-implantation, cartilage formation was achieved in the induced BMSC-ACS group and partial cartilage formation was achieved in the non-induced BMSC-ACS group, confirmed by safranin O staining, toluidine blue staining and type II collagen immunostaining. In addition, enzyme-linked immunosorbent assay demonstrated the presence of transforming growth factor-β1, insulin-like growth factor-1 and bone morphogenic protein-2 in ACSs. These results indicate that ACSs possess a chondrogenic induction activity that promotes BMSC differentiation.     

不同生长因子诱导骨髓间充质干细胞向软骨细胞表型转化的机制

背景：关节软骨损伤后,自身修复能力十分有限,以往医学手段无法对其进行再生修复,利用干细胞治疗软骨损伤彻底扭转了这一局面。 目的：探讨不同生长因子诱导骨髓间充质干细胞向软骨细胞表型的转化机制。 方法：取第5代大鼠骨髓间充质干细胞,利用不同的生长因子及生长因子组合对大鼠骨髓间充质干细胞进行诱导,分别为TGF-β1组、TGF-β1+IGF-1组、BMP-2+IGF-1组、TGF-β1+BMP-2组、TGF-β1+IGF-1+BMP-2组、空白对照组。诱导后21 d进行阿尔新蓝染色和茜素红染色,RT-PCR检测Ⅱ型胶原mRNA表达。 结果与结论：阿新蓝染色观察可见细胞胞浆与间质存在异染情况,蛋白多糖呈绿色表达,经茜素红染色未见橘红色钙结节。初步推断,骨髓间充质干细胞已分化形成软骨细胞,不表达骨细胞表型。空白对照组Ⅱ型胶原mRNA表达呈阴性。与TGF-β1组比较,BMP-2+IGF-1组Ⅱ型胶原mRNA表达显著偏低,TGF-β1+BMP-2组和TGF-β1+IGF-1+BMP-2组Ⅱ型胶原mRNA表达显著偏高(P < 0.05);且TGF-β1+IGF-1+BMP-2组Ⅱ型胶原mRNA表达显著高于其他各组(P < 0.05)。结果表明,转化生长因子β1具备单独诱导骨髓间充质干细胞向软骨分化的能力,采用胰岛素样生长因子1、骨形态发生蛋白2、转化生长因子β1在诱导骨髓间充质干细胞向软骨分化时具有协同作用,可以发挥出最大的诱导软骨分化效应。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type I alpha 2-deficient mouse

Fetal mesenchymal stem cell (fetal MSC) therapy has potential to treat genetic diseases with early onset, including those affecting the kidney and urinary tract. A collagen type I alpha 2-deficient mouse has a deletion in the alpha2 chain of the procollagen type I gene, resulting in the synthesis of abnormal alpha1(I)(3) homotrimers, which replace normal alpha 1(I)2 alpha 2(I)1 heterotrimers and a glomerulopathy. We first confirmed that col1 alpha 2-deficient homozygous mice show abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSCs from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4-12 week-old col1 alpha 2-deficient mice. Using bioluminescence imaging, in situ hybridization and immunohistochemistry in transplanted col1 alpha 2-deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells. Real-time RT-PCR demonstrated that this effect was seen at an engraftment level of 1% of total cells in the kidney, albeit higher in glomeruli. We conclude that intrauterine transplantation of human fetal MSCs improves renal glomerulopathy in a collagen type I-deficient mouse model. These data support the feasibility of prenatal treatment for hereditary renal diseases.     

Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer

Poly(N-isopropylacrylamide) (PNIPAAm) is known to be thermally responsive material and has a lower critical solution temperature (LCST, 32 degrees C) at which a macromolecular transition from a hydrophilic to a hydrophobic structure occurs. Chitosan is a useful natural polymeric biomaterial due to its biocompatibility and biodegradable properties. It has good characteristics for cell attachment, proliferation and viability. The aim of this study was to assess the ability to differentiate from mesenchymal stem cells (MSCs) to chondrocytes and mass formation using a newly developed injectable material, a thermosensitive (water-soluble chitosan-g-PNIPAAm) gel, and evaluate cartilage formation in vivo after injecting a cell-thermosensitive gel complex. The MSCs were cultured in the chitosan-PNIPAAm in vitro. Fluorescence-activated cell sort analysis, viability test, collagen type I, II, X formation and the aggrecan levels were examined. These cultured cells can be easily recovered from a copolymer gel by simply lowering the temperature. An animal study was performed to assess cartilage formation in the submucosal layer of the bladder of rabbits. The cartilage formation could be detected. This can be used to treat vesicoureteral reflux or reflux esophagitis by the effective mass effect. This is a simple method (sol-gel technique in LCST), and good cartilage formation occurs in the bladder tissue.     

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.