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J Anat. Oct 2006; 209(4): 469–480.
PMCID: PMC2100356

Cartilage, SOX9 and Notch signals in chondrogenesis

Abstract

Cartilage repair is an ongoing medical challenge. Tissue engineered solutions to this problem rely on the availability of appropriately differentiated cells in sufficient numbers. This review discusses the potential of primary human articular chondrocytes and mesenchymal stem cells to fulfil this role. Chondrocytes have been transduced with a retrovirus containing the transcription factor SOX9, which permits a greatly improved response of the cells to three-dimensional culture systems, growth factor stimulation and hypoxic culture conditions. Human mesenchymal stem cells have been differentiated into chondrocytes using well-established methods, and the Notch signalling pathway has been studied in detail to establish its role during this process. Both approaches offer insights into these in vitro systems that are invaluable to understanding and designing future cartilage regeneration strategies.

Keywords: cartilage, chondrocyte, chondrogenesis, mesenchymal stem cell, Notch, osteoarthritis, SOX9

Introduction

Diseases of articular joints present a major medical, social and economic burden on society and this will inevitably increase as the proportion of the elderly in the population increases (Woolf & Pfleger, 2003). The impact is particularly serious to those affected and the pain, suffering and loss of personal mobility and independence that comes with joint diseases are currently mitigated by drug treatments for pain relief and the eventual orthopaedic replacements of the joint. The replacement of damaged and diseased joints, such as hips and knees, with plastic and metal articulations is extremely successful (Jones et al. 2005), but the area where limitations remain is in younger patients, where the lifetime of the patient is likely to be well beyond the lifetime of the prosthesis. There is therefore great interest in the development of biologically based repairs, to reconstruct the damaged tissues in joints using living cells that will integrate with the patient's tissues and yield a functional joint, which will ‘outlive’ the patient. The ultimate challenge of repairing and regenerating joints ravaged by osteoarthritis is massive and should not be underestimated; it is also unlikely to be achieved quickly, or in one step. However, progress towards that goal is being made by tackling less severe clinical problems, such as focal defects in articular cartilage that arise as a result of joint trauma and sports injury. Here the problem is limited to repairing a small area of articular cartilage; it therefore does not involve repairing other tissues and it is also being carried out in an otherwise healthy joint. The use of autologous chondrocytes as a cell source for cartilage repair procedures has been in use for over 10 years and follow-up studies suggest that the treatment can provide real benefit (Peterson et al. 2002). However, it is recognized that improvements still need to be made to make the procedure more reliable, more successful and to extend its application to larger defects. Such improvements will lead to the ability to treat more complex problems and eventually to tackling osteoarthritis (OA). A key issue in any of these procedures is what source of cells might be used to promote a biological repair. We have investigated two quite different sources of cells, primary articular chondrocytes and adult bone marrow stem cells. The aim has been to understand better some of the issues involved in establishing the chondrocyte phenotype and driving the efficient production of a cartilage matrix.

Articular chondrocytes

Chondrocyte phenotype

The study of chondrocytes has for a long time had concern with how to retain its differentiated phenotype during cell expansion in culture, when the cells typically switch from a matrix-forming, collagen type II-expressing phenotype, to a non-matrix-forming collagen type I-expressing phenotype (Benya et al. 1977). Many culture systems have been able partially to prevent the change in phenotype by preserving the rounded shape of the chondrocytes in culture (Glowacki et al. 1983; Guo et al. 1989) and cell rounding has been shown to help restore the chondrocyte phenotype following monolayer expansion (Benya & Shaffer, 1982; Liu et al. 1998). The regulation of phenotype by chondrocytes grown as monolayers, or in three-dimensional (3D) culture, has been described as de-differentiation and re-differentiation, but this ignores the fact that the change in the pattern of chondrocyte gene expression is caused by the removal of the cell from its matrix environment, which completely alters the range of signals the cell is receiving and initiates processes such as cell division, which are clearly suppressed in chondrocytes in cartilage. The fact that early in culture the changes in gene expression can be reversed by returning the cell to an environment that mimics cartilage, such as agar or alginate gels, shows that these changes are initially just modulations of the chondrocyte phenotype, rather than switches in differentiation state. There is a need for much more precise molecular methods of detecting and characterizing the epigenetic changes that accompany cell differentiation and distinguishing them from the modulation of phenotype caused by changes in the physical and biochemical signals to which the cell responds. It appears that with the prolonged culture of chondrocytes, both types of change are occurring, as it is clear that the initial gene expression changes progress rapidly in culture (Murdoch et al. 2003) before there is much cell division and at this stage the cells retain the ability to recover a matrix-forming phenotype. However, after 5–7 passages in culture, the capacity to recover the matrix-forming phenotype is severely diminished (Zaucke et al. 2001). This suggests that progressive epigenetic changes occur over several cell cycles and these prevent a ‘chondrocyte response’ when the cells are placed in a 3D environment, which is chondrogenic for early-passage cells.

Retroviral SOX9 transduction of human articular chondrocytes

Characterization of the gene expression changes that occur when primary chondrocytes are cultured on monolayer has revealed several major features. For example, expression of the transcription factor SOX9 is reduced as the cells are expanded and this coincides with the progressive decline in the response of the cells to chondrogenic environment (Stokes et al. 2001; Hardingham et al. 2002) and this was true in both normal and OA chondrocytes (Tew et al. 2005). The SOX9 protein is an important regulator of the chondrocyte phenotype and controls the expression of the genes COL2A1 (Lefebvre et al. 1997), COL9A1 (Zhang et al. 2003), COL11A2 (Bridgewater et al. 1998), aggrecan (Sekiya et al. 2000) and cartilage link protein (Kou & Ikegawa, 2004), which all encode important cartilage extracellular matrix (ECM) proteins. SOX9 binds to the promoter elements of these genes and forms transactivating complexes with other proteins such as SOX5/SOX6 (Lefebvre et al. 1998), CREB/p300 (Tsuda et al. 2003) and c-Maf (Huang et al. 2002). To determine whether higher levels of SOX9 expression would alone be sufficient to help regain a chondrocyte matrix-forming phenotype, we have retrovirally transduced human articular chondrocytes with SOX9.

Retroviral transduction requires dividing cells, and a high transduction efficiency (80–90%) was possible with human articular chondrocytes (Li et al. 2004) by culturing them in a medium that promotes rapid proliferation (Barbero et al. 2003). TFP medium contains transforming growth factor β1 (TGFβ1), fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor BB (PDGF-BB), with 10% fetal bovine serum (FBS). In the presence of these three growth factors the chondrocytes divide more rapidly, they become spindle-shaped and demonstrate dramatically reduced contact inhibition. Barbero et al. (2003) also reported that after culture in TFP the chondrocytes showed increased multipotency with some cells able to differentiate into osteoblasts and adipocytes as well as regaining a chondrocyte phenotype. Once transduced the cells expressed 10-fold higher levels of SOX9 and this level remained high as the cells were further passaged. All subculture following transduction was conducted in a standard growth medium, first to avoid the high cost of the growth factors as the cells expand and, secondly, so that the effect of the SOX9 gene on the chondrocytes was not complicated by any altered plasticity of the cells in TFP. As a consequence of SOX9 transduction, monolayer chondrocyte COL2A1 mRNA levels were raised slightly, but still remained at a low level. However, the presence of SOX9 was able to potentiate the expression of COL2A1 and other matrix genes when the cells were placed in alginate bead culture for 14 days (Li et al. 2004). This demonstrated that the cells had an increased potential to respond to chondrogenic signals after they were transduced with SOX9. This result led us to investigate whether the higher SOX9 levels were able to improve the formation of a cartilaginous ECM in the type of cell aggregate system commonly used in chondrogenic differentiation of mesenchymal stem cells (Johnstone et al. 1998).

Matrix-forming phenotype of SOX9-transduced chondrocytes

The chondrocytes transduced with the SOX9 retrovirus showed a much stronger matrix-forming response to 3D chondrogenic cell aggregate cultures (500 000 cells per aggregate). This response was further enhanced by the addition of serum and by including anabolic factors such as insulin-like growth factor 1 (IGF1), TGFβ3 and bone morphogenetic protein-2 (BMP-2). Under such conditions the SOX9-transduced cultures were able to produce a uniform cartilaginous ECM throughout the cell aggregate, which included high levels of collagen type II and higher levels of glycosaminoglycan deposition (Tew et al. 2005). Cell aggregates formed from control-transduced chondrocytes were able to make some chondrogenic response, but the deposition of glycosaminoglycan and collagen type II was far lower than in the SOX9-transduced cultures. It is important to note that the response of OA-derived chondrocytes was no different from chondrocytes derived from healthy joints. This suggests that the pathology of donor cartilage was not a hindrance to chondrocyte use in tissue repair procedures.

In parallel experiments dermal fibroblasts did not respond to chondrogenic culture conditions after transduction with SOX9. Therefore, the response to SOX9 suggested that the chondrocytes, even following extensive expansion in monolayer culture, retained properties that enabled their response to chondrogenic signals. A recent study describing the application of adenoviruses containing SOX9, SOX5 and SOX6 cDNAs reported that dermal fibroblasts became responsive to chondrogenic cell aggregate cultures when transfected with all three SOX genes (Ikeda et al. 2004). The same study also reported that these three transcription factors could increase COL2A1 mRNA levels in non-chondrocytic immortalized cell lines, although in only one cell line (HuH-7), which expressed moderate levels of endogenous SOX5 and SOX6 already, was the up-regulation substantial. SOX9 was not found to induce the expression of SOX5 and SOX6 in articular chondrocytes and their ability to respond to transduction with SOX9 alone may therefore be aided by the existing levels of expression of SOX5 and SOX6.

Enhancement of cartilage matrix deposition with culture in lowered oxygen

Being avascular, chondrocytes in articular cartilage exist in an environment of low oxygen tension in vivo (Silver, 1975). It is reported that in lowered oxygen conditions there is increased COL2A1 gene expression when chondrocytes are grown in 3D culture systems and that this involves increases in endogenous SOX9 expression (Domm et al. 2002; Murphy & Polak, 2004). In our experiments, SOX9 transduced articular chondrocytes in the presence of IGF1 and TGFβ3, formed even larger cell aggregates, when cultured under lowered oxygen (5%) conditions. After 14 days of culture SOX9-transduced cell aggregates had a 24% greater wet weight. This appeared to reflect increased proliferation as the DNA content was increased by 15% whereas glycosaminoglycan content was unchanged (Fig. 1A). The level of expression of COL1A1 and COL2A1 were unchanged in lower oxygen (Fig. 1B), but from increased immunostaining there appeared to be a preferential increase in deposition of type II collagen, but no change in type I collagen. The basis of this selective effect requires more investigation as it implies some translational control that favours collagen type II. There were also some morphological differences. In low oxygen toward the centre of the SOX9 cell aggregates there was more intense pericellular safranin-O staining (Fig. 1C). This raised the possibility that low oxygen contributed to regional variations in glycosaminoglycan density reminiscent of the territorial and interterritorial matrices found within articular cartilage and so potentially the production of a more functional ECM. It was interesting that control cultures were highly responsive to lowered oxygen, with increased wet weight, DNA and glycosaminoglycan content after 14 days in culture and COL2A1 gene expression was also raised. However, in spite of this increased response of the control cell aggregate to lowered oxygen, they still fell short of the matrix assembly achieved by SOX9-transduced cells.

Fig. 1
Cartilage extracellular matrix deposition can be stimulated in late-passage human articular chondrocytes by SOX9 transduction and hypoxic culture conditions. Fourteen-day cell aggregate cultures formed from articular chondrocyte at passage 10 were transduced ...

Absence of hypertrophy in cultured OA chondrocytes

An important factor to assess in an in vitro engineered cartilage is evidence of chondrocyte terminal differentiation. Chondrocyte hypertrophy and ECM calcification would be an undesirable characteristic of any articular cartilage repair tissue. However, we have found only low gene expression of type X collagen and no immunodetectable protein in cell aggregates formed from SOX9-transduced chondrocytes (anti-deer antler type X collagen mouse monoclonal antibody was a kind gift from Dr Gary Gibson, Detroit, USA). Therefore, there was no evidence of hypertrophy, or terminal differentiation in the passaged articular chondrocytes even when they were derived from the residual cartilage on an OA joint (Brew et al. 2004; Tew et al. 2005).

The fall in SOX9 expression in isolated chondrocytes would appear to be a critical factor in the loss of matrix-forming phenotype in monolayer culture. The progressive decrease in SOX9 expression in successive passage in culture may also contribute to the subsequent and more gradual loss of an ability to regain easily a matrix-forming phenotype when placed in a chondrogenic environment. We have demonstrated that increasing SOX9 expression in human OA chondrocytes was sufficient to regain responses to all types of chondrogenic stimuli, including 3D culture, anabolic growth factors and low oxygen tension. There is therefore great interest in identifying signals that can promote endogenous SOX9 expression in cultured chondrocytes and thereby prime them for matrix assembly.

In the context of tissue engineering in general, the chondrocyte forms an example of a differentiated cell with a phenotype that is highly dependent on interactions with the ECM that surrounds it and this property of cell–matrix interactions is likely to make an important contribution to the phenotype of many other differentiated cells. The profound effect the cell environment has on cell phenotype needs to be considered in any in vitro manipulation of cells for tissue engineering, where specific differentiated function is being sought. Much has been learnt from early embryo development of the signals that direct cell differentiation and tissue morphogenesis, but in seeking to recapitulate these events in vitro it appears advisable also to provide the appropriate cell environment with matrix signals and physical cues that may be necessary to complement the factors driving differentiation.

Human Bone Marrow Mesenchymal Stem Cells (hMSCs)

Background

Over the past 10 years there has been an interest in the application of MSCs for clinical therapies in regenerative medicine, including the production of cartilage from chondrocytes. The presence of MSCs within mature adult animals was recognized 40 years ago with the pioneering work of Friedenstein and co-workers (Friedenstein et al. 1966, 1968). MSCs from bone marrow were shown to differentiate into the three well-defined mesenchymal cell lineages of adipocytes, osteocytes and chondrocytes driven by defined culture conditions (Pittenger et al. 1999). In spite of the many advances in the understanding of MSC biology, the standard technique for MSC isolation from bone marrow (Mackay et al. 1998; Pittenger et al. 1999; Solchaga et al. 2005) is still based on the early work (Friedenstein et al. 1970).

Cell surface markers have been used to characterize MSCs, using monoclonal antibodies raised against SH2, an epitope present on CD105 (endoglin) (Haynesworth et al. 1992; Barry et al. 1999) and SH3 and SH4, which are epitopes present on CD73 (ALCAM) (Haynesworth et al. 1992; Barry et al. 2001a). The multilineage potential of MSCs has been characterized following isolation by FACS using a monoclonal antibody raised against the STRO-1 antigen (Simmons & Torok-Storb, 1991; Gronthos et al. 1994) and the low-affinity nerve growth factor receptor (Quirici et al. 2002). In the defining work of Pittenger et al. (1999) MSCs were characterized as being CD29+, CD44+, CD71+, CD90+, CD106+, CD120+ and CD124+. This technique of cell sorting is limited in that the expression of cell surface molecules differs according to the length of time and conditions of culture and the methods used to prepare the samples (Devine, 2002; Jackson et al. 2002). In practice, FACS and MACS has been used in negative selection protocols for the removal of HSCs positive for the CD14, CD34 and CD45 cell surface markers, leaving behind an MSC-enriched population (Baddoo et al. 2003). MSC populations have been enriched by flow cytometry (Zohar et al. 1997). MSCs were characterized by low forward angle scatter (smaller cell size), low side angle scatter (low cytoplasmic granularity) and low protein content. When plated, the isolated MSC population developed a high proliferative capacity and multipotent potential towards osteogenic, adipogenic, chondrogenic and smooth muscle cell lineages. A protocol for MSC isolation from bone marrow has also been described based upon the differences in size between the MSC and haematopoeitic stem cell (HSC) populations (Hung et al. 2002).

The in vitro culture conditions reported for the chondrogenic differentiation of MSCs are well documented and yet largely empirical (Heng et al. 2004; Indrawattana et al. 2004). MSCs have been cultured at high density with a high-glucose, serum-free culture medium supplemented with dexamethasone and pro-chondrogenic factors such as TGF-βs and bone morphogenic proteins (BMPs) (Johnstone et al. 1998; Mackay et al. 1998; Yoo et al. 1998; Pittenger et al. 1999; Barry et al. 2001b). In order to develop and optimize in vitro chondrogenesis, a more detailed understanding of the molecular signalling pathways involved is required.

Chondrogenesis of hMSCs

We have investigated some of the early signalling events in the chondrogenic differentiation of human bone marrow-derived stem cells. Cells were cultured in serum-free media containing a nutrient supplement and the pro-chondrogenic factors dexamethasone and TGF-β3 (Yoo et al. 1998; Johnstone et al. 1998; Mackay et al. 1998; Pittenger et al. 1999; Barry et al. 2001b). Contrary to earlier published reports, these initial experiments with hMSCs failed to result in any detectible chondrogenesis. However, others had noted that the isolation and expansion of the MSCs in media supplemented with FGF-2 increased chondrogenic potential (Digirolamo et al. 1999; Mastrogiacomo et al. 2001; Tsutsumi et al. 2001; Solchaga et al. 2005) and this proved to be necessary to gain reliable chondrogenesis with the hMSCs we used.

Culture of hMSCs with FGF-2 caused differences in cell morphology. Control cells were flatter, polygonal and spread-out compared with the FGF-2-treated MSCs, which were smaller with a spindle-like, fibroblastic morphology (Solchaga et al. 2005). Furthermore, flow cytometry showed a one-third reduction in cell size in the FGF-2-treated hMSCs. At low cell density these morphologically distinct MSC populations were evident in control cultures comparable with other reports (Johnstone et al. 1998; Satomura et al. 1998; Digirolamo et al. 1999). The smaller, fibroblastic MSCs have been defined as type I cells and the large, flat hMSCs as type II cells (Mets & Verdonk, 1981). The type I MSC population had a faster growth kinetic than the type II population and could also be derived from the type II hMSCs. In the present study the type II-like hMSCs predominated in non-FGF-2-treated cultures and the type I cells in FGF-2-treated hMSC cultures.

FGF-2-treated MSCs showed increased proliferation (Mastrogiacomo et al. 2001), which together with other evidence suggested that FGF-2 selectively expanded a subpopulation of cells, rather than being an inductive agent for the whole population. This was supported by the detection of increased telomere length after FGF-2 expansion (Bianchi et al. 2003) and given that no telomerase activity was detected during culture it was concluded that the presence of FGF-2 had selected a subpopulation of MSCs that intrinsically had longer telomeres. There are conflicting reports on the necessity of FGF-2 during the expansion stages of the hMSCs for chondrogenesis. Solchaga et al. (2005) reflected on this point as arising from differences in either the composition of the basal media or lot-to-lot variability of the FCS source. Whatever the reasoning behind this interlaboratory variability, it is apparent that further standardization and characterization of tissue culture protocols and reagents is necessary for the development of clinical cartilage tissue engineering using MSCs.

SOX gene expression during chondrogenesis

In assays of chondrogenic differentiation in 14-day cell aggregate cultures the FGF-2-treated hMSCs already contained higher expression of SOX9 and SOX5 prior to the assay. The expression of these genes and that of the other transcription factor SOX6 increased throughout culture and was higher than in non-FGF-2-treated hMSCs. Gene expression of COL2A1 and COLXA1 also increased throughout the culture and there was increased glycosaminoglycan accumulation and histological evidence of collagen type II and collagen type X protein deposition. Cellular proliferation showed a main increase in DNA during the first 7 days of culture, prior to the major weight gain, which clearly thus occurred through deposition of cartilage-specific ECM proteins and proteoglycans and the retention of water (Hardingham & Fosang, 1992; Mackay et al. 1998). The accumulation of a glycosaminoglycan-rich ECM and up-regulation of the chondrocyte-specific ECM genes COL2A1, COLXA1 and aggrecan mainly occurred from day 7 onwards. The thousand-fold changes in COL2A1 and COLXA1 gene expression were considerably greater than the changes in aggrecan expression, which consistently showed only small changes. Increased expression of these genes, as well as that of minor cartilage proteins involved in regulating the macromolecular assemblies within the ECM, such as cartilage oligomeric protein (COMP), decorin and fibromodulin, have been shown to increase from day 3 and 8 of chondrogenic pellet culture (Barry et al. 2001b). Immunolocalization data have also shown the initial deposition of aggrecan, link protein, COMP, decorin, biglycan, KS and chondroitin-4-sulphate from between day 3 and 5 of chondrogenic pellet culture (Yoo et al. 1998; Barry et al. 2001b). Radiolabelled sulphate incorporation, a measure of glycosaminoglycan biosynthesis, was also reported to be greatest from day 5 onwards (Yoo et al. 1998). Together, these results indicate that signalling events are likely during the first few days of the culture prior to the deposition and organization of a cartilage ECM.

Expression of collagen type I and type X

Although collagen type II is a characteristic gene product of chondrocytes as it forms the major structural framework of cartilage tissue, it is COL1A1 that is expressed in undifferentiated hMSCs (Martin et al. 2001) and there was no significant down-regulation of it during chondrogenesis, when there is a dramatic increase in collagen type II deposition (Barry et al. 2001b). There were also similarly high levels of expression of the COL2A1 gene throughout the 14 days of chondrogenic culture. However, immunohistochemistry at day 14 showed collagen type I protein only towards the surface layers of the cell aggregate and the central chondrogenic region was strongly stained for collagen type II.

A consistent feature of chondrogenesis of hMSCs is the up-regulation of COLXA1 gene expression in parallel with the increase in COL2A1. Immunohistochemical analysis of hMSC chondrogenic cell aggregates showed that the collagen type X protein is localized to areas that also contain collagen type II and that some of the cells in these areas appear enlarged, suggesting that they are undergoing hypertrophy. The collagen type X protein is classically known to be produced during chondrocyte hypertrophy in the growth plate of long bones and in that context its expression only rises when the production of collagen type II is decreasing and indeed it is used to identify terminally differentiated hypertrophic chondrocytes (Schmid & Linsenmayer, 1985; Ornitz & Marie, 2002; Shen, 2005). The concurrent up-regulation of COLXA1 with COL2A1 is thus under different regulatory control than in growth plate and shows that expression of collagen type X can occur in chondrogenic cells that are actively assembling matrix, rather than only as a portent of vascular invasion and matrix destruction. In order for hMSCs to be used for the repair of articular cartilage defects, it is important that the consequences of the expression of collagen type X on cartilage function are more completely understood.

Notch signalling during hMSC chondrogenesis

The cell–cell contacts that occur during 3D cell aggregate culture mimic the mesenchymal condensation that initiates chondrogenesis during development and provides an ideal environment for Notch signalling. Previous studies have shown that the Notch pathway is active during the early stages of chondrogenesis and during the transition of prehypertrophic to hypertrophic chondrocytes in murine and chick limb development (Crowe et al. 1999; Watanabe et al. 2003). Similarly, immunolocalization studies have revealed the presence of Notch receptors and ligands in both murine and bovine articular cartilage (Hayes et al. 2003; Dowthwaite et al. 2004). Previous studies had shown the Notch-2 receptor to be broadly distributed throughout articular cartilage and the epiphyseal growth plate, being expressed on proliferating, prehypertrophic and hypertrophic chondrocytes (Crowe et al. 1999; Hayes et al. 2003). The exact purpose for such a widespread distribution is unclear, although it has been speculated that Notch-2 may modulate the activities of Notch-1 and Notch-3 (Shimizu et al. 2002), thereby influencing the development and growth of cartilage and also the homeostasis of the mature tissue (Hayes et al. 2003). During murine development Notch-3 is specifically localized to the deep layers of articular cartilage and to hypertrophic chondrocytes within the epiphyseal growth plate (Hayes et al. 2003). A previous report stated that Notch-3 could halt the proliferation of A7r5-N3IC cells (a stably transfected vascular smooth muscle cell line) at subconfluent cell densities, whilst promoting cell cycle progression upon confluence (Campos et al. 2002). It was suggested that Notch-3 was involved in inhibiting proliferation of terminally differentiated chondrocytes within the cartilage tissue where no cell–cell contacts are formed (Hayes et al. 2003). Notch-1 was localized to cells within the surface zone of articular cartilage in bovine and murine tissue (Hayes et al. 2003; Dowthwaite et al. 2004). Particular attention has been paid to this cell population because it contains a pool of chondroprogenitor cells that are able to differentiate into connective tissue cell lineages, including chondrocytes, myocytes, tenocytes and osteoblasts (Dowthwaite et al. 2004).

In a recent study we investigated the gene expression of the Notch receptors (Notches 1–4) and the Notch ligands (Jagged-1, Jagged-2, DLL-1, DLL-3 and DLL-4) (Oldershaw et al. 2005). During chondrogenesis in high-density 3D cell aggregate culture there was a general down-regulation of Notch receptors. Notch-2 message levels were significantly reduced within 24 h, but the reduction in Notch-3 expression was slower and it quickly became the most highly expressed Notch receptor. This may relate to it having a particular role in reducing the proliferative capacity of the hMSCs simultaneous with them undergoing chondrogenic differentiation (Campos et al. 2002; Hayes et al. 2003). At day 0, Notch-1 was transiently up-regulated, as was a known downstream gene HES-1 (Jarriault et al. 1998). During murine skeletal development Notch-1 is strongly localized within the condensing mesenchyme during the early stages of chondrogenesis (Watanabe et al. 2003). A larger second wave of Notch signalling was evident by a transient increase in Jagged-1 gene expression and the concurrent up-regulation of HEY-1, peaking at day 2 of pellet culture. Jagged-1 can itself be a downstream target of Notch signalling (Luo et al. 1997; Ascano et al. 2003; Ross & Kadesch, 2004), and its up-regulation provided additional evidence that the Notch pathway was active very early in chondrogenic cell aggregate.

Role of Jagged-1 in chondrogenesis

Mutations in the Jagged-1 gene result in haploinsufficiency of Jagged-1 at the cell surface and are responsible for Alagille syndrome (AGS), an autosomal dominant disorder that causes multi-organ defects in the eyes, kidneys, heart, liver, face and skeleton (Alagille et al. 1975; Joutel & Tournier-Lasserve, 1998; Loomes et al. 1999; Crosnier et al. 2000; Kamath et al. 2002; Krantz, 2002). Skeletal abnormalities include ‘butterfly vertebrae’, craniofacial abnormalities and a growth retardation phenotype (Kamath et al. 2002; Alagille et al. 1975). This implies that Jagged-1-mediated Notch signalling is involved in the chondrogenic steps of both intramembranous and endochondral ossification. Embryonic mice that were homozygous null for the Jagged-1 gene failed to develop as embryos owing to vascular abnormalities whilst heterozygous Jagged-1 null mice displayed ocular defects, similar to those in AGS (Xue et al. 1999). However, mice that were doubly heterozygous for the Jagged-1 null allele and a Notch-2 hypomorphic allele exhibited other AGS-like phenotypes, including heart, eye, liver and kidney abnormalities as well as growth retardation. It has therefore been proposed that Notch-2 is a genetic modifier of Jagged-1 signalling and this may explain the broad spectrum of phenotypes and clinical severity associated with AGS (McCright et al. 2002) and may relate to the wide distribution of Notch-2 during cartilage development (Crowe et al. 1999; Hayes et al. 2003).

To understand more about the impact of Jagged-1-mediated Notch signalling on chondrogenesis we investigated the effects of over-expressing Jagged-1 in hMSCs (Oldershaw et al. 2005). The transduction of hMSCs with adenoviral Jagged-1 increased Jagged-1 expression and activated Hey-1 expression, confirming that it triggered Notch signalling. When Jagged-1-transduced hMSCs were assessed in chondrogenic cell-aggregate culture the results showed a complete inhibition of chondrogenesis, whereas chondrogenesis occurred normally in vector control transduced hMSCs. There were reduced wet weights of the cell aggregates, low glycosaminoglycan depostion and collagen type II histological staining and suppressed activation of cartilage matrix protein genes. It has been well documented that the ectopic expression of the Notch ligands Serrate and Delta during dorsal/ventral compartmentalization and wing formation in the Drosophila melanogaster dampens the Notch signal (Speicher et al. 1994; Couso et al. 1995; Diaz-Benjumea & Cohen, 1995; Thomas et al. 1995; Doherty et al. 1996) and a model of ligand-mediated inhibition of Notch signalling has been described (LaVoie & Selkoe, 2003). Notch ligands can be processed by the same proteolytic machinery as the Notch receptor (Qi et al. 1999; Lieber et al. 2002). Therefore, ADAM-17/TACE-mediated cleavage of the ligand's ectodomain may diminish the pool of cell surface ligand available to activate Notch receptors and thereby reduce Notch signalling (LaVoie & Selkoe, 2003). Furthermore, it has been proposed that the Jagged-1 cytoplasmic fragment (CTF) could inhibit Notch signal transduction by actively competing with the S2/NEXT cleavage product for the presenilin-dependent γ-secretase complex (LaVoie & Selkoe, 2003).

In the Jagged-1 transduced hMSCs there was sustained Jagged-1 and HEY-1 gene expression throughout the 14 days of chondrogenic culture, indicating that the Notch pathway was continuously active during this time course. It has been shown that prolonged Notch signalling maintained the stem-cell-like phenotype in neural progenitors (Nye et al. 1994; Ohtsuka et al. 1999; Handler et al. 2000; Solecki et al. 2001; Kageyama et al. 2005), myocyte progenitors (Nye et al. 1994; Shawber et al. 1996; Kuroda et al. 1999) and preadipocytes (Ross et al. 2004). Within bone marrow (the compartment from which the hMSCs were derived), Jagged-1-expressing osteoblasts promote the expansion and maintenance of the haematopoietic stem cells (Calvi et al. 2003).

The results of our present study suggest that Jagged-1-mediated Notch signalling maintained the progenitor-like status of the transduced hMSCs and thereby suppressed their differentiation. It may also imply that the switch-off of Notch signalling evident at day 3–4 of culture is essential for chondrogenesis to proceed (Fig. 2). The transient Jagged-1-mediated Notch signalling may thus form a cue that initiates chondrogenesis and it may also serve as a cell–cell signal to co-ordinate and synchronize the subsequent differentiation. It may therefore be that the two waves of Notch signalling that we observed during the first 5 days of chondrogenic pellet culture were required to prime the hMSC population for chondrogenesis, creating a check-point mechanism that permits chondrogenic differentiation under the appropriate developmental cues (Nofziger et al. 1999; Martinez Arias et al. 2002). The knowledge of these key cellular events in this differentiation process provides valuable information to devise cartilage regeneration strategies that will enable better control of chondrogenesis and the efficient elaboration of cartilaginous tissue.

Fig. 2
Schematic model of Notch signalling during chondrogenic differentiation of MSCs. Transient Notch signalling was observed during the first 3 days of chondrogenic differentiation by MSCs. Overexpression of the Notch ligand Jagged-1 caused suppression of ...

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