Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.     

Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study

Mesenchymal stem cells (MSC) have the potential to differentiate into distinct mesenchymal tissues including cartilage, which suggest these cells as an attractive cell source for cartilage tissue engineering approaches. Our objective was to study the effects of TGF-beta1, hyaluronic acid and synovial fluid on chondrogenic differentiation of equine MSC. For that, bone marrow was aspirated from the tibia of one 18-month-old horse (Haflinger) and MSC were isolated using percoll-density centrifugation. To promote chondrogenesis, MSC were centrifuged to form a micromass and were cultured in a medium containing 10 ng/ml TGF-beta1 or 0.1mg/ml hyaluronic acid (Hylartil, Ostenil) or either 5%, 10% or 50% autologous synovial fluid as the chondrogenesis inducing factor. Differentiation along the chondrogenic lineage was documented by type II collagen and proteoglycan expression. MSC induced by TGF-beta1 alone showed the highest proteoglycan expression. Combining TGF-beta1 with hyaluronic acid could not increase the proteoglycan expression. Cultures stimulated by autologous synovial fluid (independent of concentration) and hyaluronic acid demonstrated a pronounced, but lower proteoglycan expression than cultures stimulated by TGF-beta1. The expression of cartilage-specific type II collagen was high and about the same in all stimulated cultures. In summary, hyaluronic acid and autologous synovial fluid induces chondrogenesis of equine mesenchymal stem cells, which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chondrogenic differentiation.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF-beta 1 and BMP-2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF-beta 1 and BMP-2 on rat periosteum-derived cells during in vitro chondrogenesis in a three-dimensional aggregate culture. RT-PCR analyses for gene expression of cartilage-specific matrix proteins revealed that treatment with BMP-2 alone and combined treatment with TGF-beta 1 and BMP-2 induced time-dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP-2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF-beta 1 and BMP-2 showed no such expression. Treatment with TGF-beta 1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP-2-treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti-type II and type X collagen antibodies. In contrast, at 14 days, TGF-beta 1 + BMP-2-treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti-type X collagen antibody. Expression of BMPR-IA, TGF-beta RI, and TGF-beta RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP-2, TGF-beta 1 and TGF-beta 1 + BMP-2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP-2 induces neochondrogenesis of rat periosteum-derived cells, and that TGF-beta 1 modulates the terminal differentiation in BMP-2 induced chondrogenesis.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

Validation of a high-throughput microtissue fabrication process for 3D assembly of tissue engineered cartilage constructs

Described here is a simple, high-throughput process to fabricate pellets with regular size and shape and the assembly of pre-cultured pellets in a controlled manner into specifically designed 3D plotted porous scaffolds. Culture of cartilage pellets is a well-established process for inducing re-differentiation in expanded chondrocytes. Commonly adopted pellet culture methods using conical tubes are inconvenient, time-consuming and space-intensive. We compared the conventional 15-mL tube pellet culture method with 96-well plate-based methods, examining two different well geometries (round- and v-bottom plates). The high-throughput production method was then used to demonstrate guided placement of pellets within a scaffold of defined pore size and geometry for the 3D assembly of tissue engineered cartilage constructs. While minor differences were observed in tissue quality and size, the chondrogenic re-differentiation capacity of human chondrocytes, as assessed by GAG/DNA, collagen type I and II immunohistochemistry and collagen type I, II and aggrecan mRNA expression, was maintained in the 96-well plate format and pellets of regular size and spheroidal shape were produced. This allowed for simple production of large numbers of reproducible tissue spheroids. Furthermore, the pellet-assembly method successfully allowed fluorescently labelled pellets to be individually visualised in 3D. During subsequent culture of 3D assembled tissue engineered constructs in vitro, pellets fused to form a coherent tissue, promoting chondrogenic differentiation and GAG accumulation.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Calcification or dedifferentiation: Requirement to lock mesenchymal stem cells in a desired differentiation stage

A current challenge in mesenchymal stem cell (MSC)‐based cartilage repair is to solve donor and tissue‐dependent variability of MSC cultures and to prevent chondrogenic cells from terminal differentiation like in the growth plate. The aim of this study was to select the best source for MSC which could promise stable cartilage formation in the absence of hypertrophy and ectopic in vivo mineralization. We hypothesized that MSC from synovium are superior to bone marrow‐ and adipose tissue‐derived MSC since they are derived from a joint tissue. MSC were characterized by flow cytometry. MSC pellets were cultured under chondrogenic conditions and differentiation was evaluated by histology, gene expression analysis, and determination of alkaline phosphatase activity (ALP). After chondrogenic induction, pellets were transplanted subcutaneously into SCID mice. MSC from bone marrow, adipose tissue, and synovium revealed similar COL2A1/COL10A1 mRNA levels after chondrogenic induction and were positive for collagen‐type‐X. Bone marrow‐derived and adipose tissue‐derived MSC showed significantly higher ALP activity than MSC from synovium. Low ALP‐activity before transplantation of pellets correlated with marginal calcification of explants. Surprisingly, non‐mineralizing transplants specifically lost their collagen‐type II, but not collagen‐type I deposition in vivo, or were fully degraded. In conclusion, the lower donor‐dependent ALP activation and reduced mineralization of synovium‐derived heterotopic transplants did not lead to stable ectopic cartilage as known from articular chondrocytes, but correlated with fibrous dedifferentation or complete degeneration of MSC pellets. This emphasizes that beside appropriate induction of differentiation, locking of MSC in the desired differentiation state is a major challenge for MSC‐based repair strategies. J. Cell. Physiol. 219: 219–226, 2009. © 2008 Wiley‐Liss, Inc.     

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.This work was supported by grants R92-001-1 and R92-001-2 from the Veterans General Hospital, Taipei, and grant NSC-92-2314-B-075-022 from the National Science Council, Taiwan.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-β1, TGF-β1 + LiCl, TGF-β1 + SB216763, TGF-β3, TGF-β3 + LiCl, and TGF-β3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, β-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-β in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-β3 appeared much better than TGF-β1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since β-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-β3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.     

Chondrogenic differentiation of human mesenchymal stem cells: a comparison between micromass and pellet culture systems

High-density cell culture is pivotal for the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Two high-density cell culture systems, micromass and pellet culture, have been used to induce chondrogenic differentiation of hMSCs. In micromass culture, the induced-cartilage tissues were larger, more homogenous and enriched in cartilage-specific collagen II but the fibrocartilage-like feature, collagen I, and hypertrophic chondrocyte feature, collagen X, were markedly decreased compared to those in pellet culture. Furthermore, real time RT-PCR analysis demonstrated that collagen II and aggrecan mRNA were up-regulated while collagen X and collagen I mRNA were down-regulated in micromass culture. Thus, the micromass culture system is a promising tool for in vitro chondrogenic studies.     

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P < 0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P < 0.05) and that of type I collagen significantly decreased (P < 0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Regulation of MHC class II expression and antigen processing in murine and human mesenchymal stromal cells by IFN-gamma, TGF-beta, and cell density

Mesenchymal stromal cells (MSC) possess immunosuppressive properties, yet when treated with IFN-gamma they acquire APC functions. To gain insight into MSC immune plasticity, we explored signaling pathways induced by IFN-gamma required for MHC class II (MHC II)-dependent Ag presentation. IFN-gamma-induced MHC II expression in mouse MSC was enhanced by high cell density or serum deprivation and suppressed by TGF-beta. This process was regulated by the activity of the type IV CIITA promoter independently of STAT1 activation and the induction of the IFN regulatory factor 1-dependent B7H1/PD-L1 encoding gene. The absence of direct correlation with the cell cycle suggested that cellular connectivity modulates IFN-gamma responsiveness for MHC II expression in mouse MSC. TGF-beta signaling in mouse MSC involved ALK5 and ALK1 TGF-beta RI, leading to the phosphorylation of Smad2/Smad3 and Smad1/Smad5/Smad8. An opposite effect was observed in human MSC where IFN-gamma-induced MHC II expression occurred at the highest levels in low-density cultures; however, TGF-beta reduced IFN-gamma-induced MHC II expression and its signaling was similar as in mouse MSC. This suggests that the IFN-gamma-induced APC features of MSC can be modulated by TGF-beta, serum factors, and cell density in vitro, although not in the same way in mouse and human MSC, via their convergent effects on CIITA expression.