FGF, TGFb and Wnt crosstalk: embryonic to in vitrocartilage development frommesenchymal stem cellsMairéad A. Cleary1,2, Gerjo J. V. M van Osch1,3*, Pieter A. Brama2, Catharine A. Hellingman3

and Roberto Narcisi11Department of Orthopaedics, Erasmus MC, University Medical Centre, Dr. Molewaterplein 50 3015 GE, Rotterdam, The Netherlands2School of Veterinary Medicine, Veterinary Science Centre, University College Dublin, Belfield, Dublin 4, Ireland3Department of Otorhinolaryngology, Erasmus MC, University Medical Centre, Dr. Molewaterplein 50 3015 GE, Rotterdam,The Netherlands

Abstract

Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promisingtherapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells(MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known fromembryology that members of the fibroblast growth factor (FGF), transforming growth factor-b (TGFb)and wingless-type (Wnt) protein families are involved in controlling different differentiation stagesduring chondrogenesis. Individually, these pathways have been extensively studied but so far attemptsto recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stableand functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe abetter understanding of the simultaneous integration of these factorswill improve howwe relate embryonicchondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define currentknowledge on the crosstalk between the FGF, TGFb andWnt signalling pathways during different stagesof mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCsmight provide insights into how to improve and progress cartilage tissue engineering for the future.Copyright © 2013 John Wiley & Sons, Ltd.

Received 18 September 2012; Revised 30 January 2013; Accepted 23 February 2013

Keywords chondrogenesis; mesenchymal stem cell; cell differentiation; cell signalling; fibroblast growthfactor; transforming growth factor-b; wingless-type protein

1. Background

Articular cartilage has limited intrinsic healing capacity;minor injury leads to progressive damage and degenera-tion. As cartilage consists of only a single cell type and isboth avascular and aneural, it is a good candidate for tissueengineering-based repair strategies. Mesenchymal stemcells (MSCs), present in most adult tissues, including bonemarrow (Osyczka et al., 2002b; Pittenger et al., 1999) andconnective tissue (Adachi et al., 2002; Bosch et al., 2000;De Bari et al., 2001a, 2001b; Gronthos et al., 2001;Nakahara et al., 1991; Noth et al., 2002; Osyczka et al.,

2002a; Vandenabeele et al., 2003; Zuk et al., 2001), repre-sent a population of multipotent progenitor cells capable ofdifferentiation into mesenchymal tissues, such as cartilageand bone (Dominici et al., 2006). MSCs can undergonumerous passages in vitro whilst retaining their mesen-chymal differentiation potential. Thus, they areattractive candidates for cartilage tissue engineering-basedrepair strategies. To date, however, rather than stablehyaline cartilage, MSCs differentiated down the chondrogeniclineage produce terminally differentiated cartilage thatforms bone through endochondral ossification. Such anoutcome is difficult to prevent when it is unclear what theresponsible molecular mechanisms are.

Embryonic cartilage development is primarily investi-gated during the endochondral ossification process of limbdevelopment. Initially, following active cellmigration towardsthe centre of the outgrowing limb bud, a mesenchymalcondensation is formed (Tuan, 2003). This is followed by

*Correspondence to: G. J. V. M. van Osch, Connective Tissue Cellsand Repair Group, Erasmus MC, University Medical CentreRotterdam, Departments of Orthopaedics and Otorhinolaryngology,Dr. Molewaterplein 50, Room Ee1655, 3015 GE Rotterdam, TheNetherlands. E-mail: g.vanosch@erasmusmc.nl

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differentiation of these cells into chondrocytes before aperiod of hypertrophy and finallymineralization (Canceddaet al., 2000; Kronenberg, 2003). Mimicking the differentia-tion process during in vivo embryonic development iswidely accepted to lead to the most desirable outcomesin vitro. Indeed, in vitro chondrogenic differentiation ofMSCs in pellet culture has been shown to take place in asimilar manner (Barry et al., 2001; Hellingman et al.,2010; Pelttari et al., 2006), suggesting thst similar signallingpathways may operate during tissue engineering and limbdevelopment. Accordingly, the molecular regulation ofMSC differentiation, based on knowledge from embryonicbiology studies, has been extensively researched. In fact,current culture conditions for chondrogenic differentiationof MSCs mimic embryonic cartilage development. TGFb isessential in different stages of embryonic chondrogenesisand is commonly used for chondrogenic differentiation ofexpanded chondrocytes and MSCs (Goldring et al., 2006).Similarly, FGF signalling is important for embryonic carti-lage development, and FGF-2 is utilized during expansionof MSCs to improve proliferation and chondrogenic poten-tial (Solchaga et al., 2010). Furthermore, expression pat-terns of Wnt signalling molecules during skeletaldevelopment imply that Wnt signalling is also crucial forchondrogenesis. Consequently, in this review the focus ison the interaction between FGF, TGFb and Wnt signallingduring in vivo and in vitro chondrogenesis. We believe thatby studying these pathways and their interactions in moredepth, and by finding links between embryonic cartilagedevelopment and in vitro chondrogenic differentiation ofMSCs, we will advance our knowledge of chondrogenesisin general and which, more specifically, may lead us to abetter control of in vitro chondrogenic differentiation.

2. FGF–TGFb–Wnt intracellularpathways: a general overview

2.1. FGF signalling

FGF signalling molecules and their cell surface tyrosinekinase receptors (fibroblast growth factor receptors;FGFRs) are involved in the earliest stages of limbdevelopment and in the formation of skeletal elementswithin the developing limb; disruption of FGF signal-ling is linked with chondrodysplasia in humans(Vajo et al., 2000). In vertebrates, 22 members have beenidentified and can be classified as intracrine (FGF-11/12/13/14), paracrine (FGF-1/2/5, FGF-3/4/6, FGF-7/10/22, FGF-8/17/18 and FGF-9/16/20) or endocrine (FGF-19/20/23), depending upon their mechanism of action(Itoh, 2010; Kharitonenkov, 2009).

Four genes, FGFR-1, FGFR-2, FGFR-3 and FGFR-4,encode the FGFRs with alternative splicing in the extracellu-lar domain capable of generatingmultiple receptor isoforms(Zhang et al., 2006). Binding of the receptor triggers activa-tion of several cytoplasmic signal transduction pathways,including Ras/Erk, Akt and the protein kinase C (PKC)

pathways, associated with proliferation and differentiation,cell survival and cell morphology and migration, respec-tively (Dailey et al., 2005; Dorey and Amaya, 2010;Mohammadi et al., 2005; Schlessinger, 2000).

2.2. TGFb signalling

The TGFb superfamily, including TGFb1, TGFb2, TGFb3and bone morphogenic proteins (BMPs), is vitally impor-tant in all stages of chondrogenesis. The importance ofTGFb signalling has been established using several models,identifying roles for family members in proliferation andhypertrophic differentiation of chondrocytes, as well aschondrogenic cell fate determination in mesenchymal cells.Furthermore, this signalling pathway has a critical rolein the maintenance and repair of articular cartilage, anobservation supported by the development of osteoarthritis(OA) in individuals with deregulated TGFb signalling(Wang et al., 2011).

The Smad-dependent TGFb–BMP signalling cascade iswell described and involves two types of receptors (typesI and II) which, upon activation, phosphorylate theirspecific receptor-Smads (R-Smads). These include Smad2and Smad3 for TGFb signalling and Smad1, Smad5 andSmad8 for BMP signalling. Several type I receptors, alsoknown as activin receptor-like kinases (ALKs), have beendescribed (ten Dijke et al., 1994) and it is transductionof TGFb signalling through ALK5, resulting in activationof Smad2/3, which is considered the TGFb superfamilycanonical signalling pathway (Tuli et al., 2003; Zhang,2009). TGFb can also activate the MAPK pathways, withErk, p38 and JNK capable of differentially regulatingTGFb-stimulated chondrogenesis (Mu et al., 2012; Tuliet al., 2003; Zhang, 2009).

Smad turnover is critically regulated by the E3ubiquitin ligases Smurf1 and Smurf2, which are recruitedby the inhibitory Smads (I-Smads: Smad6 and Smad7).The universally expressed Smad7 inhibits all R-Smad sig-nalling, while the more selectively expressed Smad6 prefer-entially inhibits the Smad1/5/8 pathway. Consequently, atmultiple stages Smad7 inhibits chondrocyte differentiation(Iwai et al., 2008).

2.3. Wnt signalling

In all animal species studied, Wnt signalling is involved in amultitude of embryonic events, including chondrogenesis.Indeed, skeletal defects and malformations are associatedwithWnt ligand and receptormutations (Moon et al., 2004).

There are 19 known human Wnt genes (Kawano andKypta, 2003; Miller et al., 2001), the protein products ofwhich exert their effects by signalling through receptor-mediated canonical and non-canonical pathways. Canoni-cal Wnts signal through the frizzled receptors to stabilizecytosolic b-catenin, which then translocates to the nucleuswhere it associates with the LEF/TCF transcription factorfamily. In the absence of Wnt a degradation complex

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phosphorylates b-catenin, marking it for ubiquitination andproteasomal degradation. Consequently, low intracellularlevels of b-catenin are maintained (Dale, 1998; Huelskenand Behrens, 2002; Kawano and Kypta, 2003; Miller et al.,2001; Nusse, 2005).

Non-canonical Wnts induce transcription of targetgenes independent of b-catenin through diverse path-ways, such as the planar cell polarity and Wnt–cGMP/Ca2+ signalling pathway (Semenov et al., 2007; Sethiand Vidal-Puig, 2010). Recently, reports have proposedthat Wnt proteins themselves are neither canonical nornon-canonical. Instead, the signalling pathway activatedby Wnt proteins is determined by specific receptor sets(van Amerongen et al., 2008).

3. FGF–TGFb–Wnt crosstalk: cartilagedevelopment

3.1. Overview

The developing limb constitutes a classic model for thestudy of chondrogenesis. It has been shown (Hellingmanet al., 2010) that in embryonic limb development thereare three sequential stages: mesenchymal condensation(characterized by N-cadherin), chondrogenic differentia-tion (characterized by collagen II) and hypertrophy (char-acterized by collagen X). FGF, TGFb and Wnt signallingare recognized as being important for in vivochondrogenesis and are known to control and influencethese three stages through their cooperative andantagoniztic effects on each other. Clues for controllingin vitro chondrogenesis can be elucidated by studying theprocess of in vivo limb development.

3.2. Proliferation (summarized in Figure 1)

Early limb growth is initiated by the mesoderm (Carringtonand Fallon, 1984; Saunders and Reuss, 1974) and appearsto be under the control of a Wnt–FGF signalling loop,whose members, when expressed ectopically, are capableof inducing limb development (Kawakami et al., 2001). Inlimb initiation, FGF-10, whose expression in the chick isrestricted to the lateral plate mesoderm (LPM), actsupstream of FGF-8 (Martin, 1998; Ohuchi et al., 1995; Xuet al., 1998; Yonei-Tamura et al., 1999). FGF-10 expressionis itself under the influence of Wnt-2b, which is in turncontrolled by FGF-8 (Niswander, 2003). Moreover, FGF-24, as observed in the zebrafish, andWnt-8c are also capableof activating FGF-10 (Fischer et al., 2003).

As limb growth proceeds, the overlying ectodermand the apical ectodermal ridge (AER) in particular main-tain the underlying mesoderm cells in a proliferativeand undifferentiated state (Saunders, 1948; Todt andFallon, 1984).Wnt-3a signalling has been identified as arequirement for AER induction and, within the AER, inducesFGF-8 expression (Kawakami et al., 2001; Kengaku et al.,

1998; Tickle andMunsterberg, 2001). The role of FGF-8 ap-pears to bemaintenance of FGF-10 signalling from the LPM,which is responsible for induction of Wnt-3a expression(Ohuchi et al., 1997; Yonei-Tamura et al., 1999). This roleof FGF-8–Wnt-3a in synergistically promoting proliferationwhile maintaining cells in their undifferentiated state wasmore recently confirmed by ten Berge et al. (2008), whoalso showed that the undifferentiated state is maintainedthrough repression of Sox9 expression. Further confirma-tion of the importance of FGF and Wnt signalling duringearly limb development is that upregulation of Wnt-5a inthe limb mesoderm correlates with increased proliferation,whereas Wnt-5a downregulation correlates with reducedproliferation (Chimal-Monroy et al., 2002). AdditionallyFGF-2, -4 and -8, secreted from the AER, and Wnt-3a arecapable of substituting for the AER (Crossley et al., 1996;Dono and Zeller, 1994; Niswander et al., 1993; Olwin et al.,1994; Savage et al., 1993; Vogel et al., 1996).

TGFb signalling antagonizes these effects of FGF signal-ling. In a recent study (Lorda-Diez et al., 2010), TGFb2and TGFb3 were found to be highly expressed in theAER and, in the presence of TGFb signalling, cell prolifer-ation was seen to be reduced. This was accompaniedby downregulation of Gremlin (Grem), a BMP inhibitor.Normally, by neutralizing BMPs, Gremlin maintainsexpression of FGF-8 in the AER (Merino et al., 1999). Itwould therefore appear that TGFb indirectly preventsproliferation in early limb development through inhibitionof Grem. Other studies have shown BMP-2, also located inthe AER of mouse limb buds, to have opposite effects tothose of FGF-4; BMP-2 decreases proliferation, whileFGF-4 increases proliferation (Hofmann et al., 1996;Martin, 1998; Niswander et al., 1993).

In summary, FGF signalling is pivotal for cell prolifera-tion during early limb development. Together with Wntsignalling, FGF signalling maintains the cells in anundifferentiated state by repressing Sox9 expression inthe AER. TGFb signalling antagonizes the effects of FGFsignalling, but further research is required to clarify theeffects of TGFb signalling in early limb growth.

3.3. Cartilage differentiation and maturation(summarized in Figure 2)

In vivo chondrogenesis occurs in three stages: mesenchymalcondensation (characterized by N-cadherin), chondrogenicdifferentiation (characterized by collagen II) and hypertro-phy (characterized by collagen X).

Mesenchymal condensation, a necessity for initiation ofchondrogenesis, is characterized by the cell adhesionmolecule N-cadherin. Moving towards chondrogenicdifferentiation, N-cadherin expression is downregulated.Increased or prolonged expression of N-cadherin leadsto stabilization of cell–cell adhesion and eventual inhibi-tion of chondrogenesis. b-Catenin is an N-cadherin-related cytoskeleton element, and in limb mesenchymalchondrogenic cultures, misexpression of Wnt-7a was

FGF, TGFb and Wnt crosstalk: embryonic to in vitro cartilage development from MSCs

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Figure 1. Schematic overview of the signals that regulate cell proliferation in the early stages of limb development. FGF signalling iscrucial to maintain cell proliferation and suppresses cell differentiation by repressing SOX9 in the AER. Canonical Wnts are generallyupstream of FGF proteins, but they can also act synergistically with FGFs to inhibit SOX9, maintaining the cells in an undifferentiatedstate. Members of TGFb superfamily counteract the effect of FGF molecules. Only the interactions between FGF, TGFb and Wntproteins are underlined. AER, apical ectodermal ridge; LPM, lateral plate mesoderm. This figure is based on the following literature:Chimal-Monroy et al., 2002; Crossley et al., 1996; Dono and Zeller, 1994; Fischer et al., 2003; Hofmann et al., 1996; Kawakami et al.,2001; Kengaku et al., 1998; Lorda-Diez et al., 2010; Martin, 1998; Merino et al., 1999; Niswander, 2003; Niswander et al., 1993; Ohuchiet al., 1995, 1997; Olwin et al., 1994; Savage et al., 1993; ten Berge et al., 2008; Tickle and Munsterberg, 2001; Vogel et al., 1996; Xu et al.,1998; Yonei-Tamura et al., 1999

Figure 2. Interactions between TGFb and Wnt signalling pathways during cartilage development. During chondrocyte maturation theregulation of Runx2, SOX9 and Twist1 is crucial to determine cell fate. Both Wnt and TGFb pathways are involved in this regulation,acting in a stage-specific manner. Although FGF proteins are known to be important during limb development, to our knowledge thereare no in vivo studies that have investigated their interactions with TGFb or Wnt pathways during cartilage development. This figure isbased on the following literature: Dong et al., 2005; Dong et al., 2006, 2007; Enomoto-Iwamoto et al., 2001; Enomoto et al., 2004;Hinoi et al., 2006; Reinhold and Naski, 2007

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found to prevent chondrogenesis through stabilization ofN-cadherin (Tufan and Tuan, 2001; Tufan et al., 2002).Inhibition of the p38–MAPK pathway resembles the effectof Wnt-7a misexpression in that N-cadherin expression issustained and chondrogenesis inhibited. Thus, Wnt andMAPK signalling are important for initiating chondrogenicdifferentiation (Oh et al., 2000).

TGFb signalling is important both throughout chondrogenesisand for cartilage homeostasis. Consistent with this,Smad3–/– mice and transgenic mice overexpressing adominant-negative form of the TGFb type II receptordevelop skeletal degeneration resembling human OA,indicating an essential role for TGFb in repressing chon-drocyte terminal differentiation (Li et al., 2006; Liuet al., 2002; Serra et al., 1997; Yang et al., 2001).

In immature chondrocytes, TGFb signalling enhancesWnt signalling, leading to inhibition of maturation,whereas in more mature chondrocytes, further matura-tion is inhibited through inhibition of Wnt signalling byTGFb signalling (Dong et al., 2007; Reinhold and Naski,2007). Therefore, at different stages of chondrogenesis,signalling pathways can exert different effects.

Chondrocyte maturation is reliant on an increase inRunx2 levels, accompanied by a decrease in Twist1 levels(Dong et al., 2005, 2006). Runx2 is a known promoterof chondrocyte maturation, with low levels of Runx2required in immature chondrocytes to maintain theundifferentiated state (Enomoto-Iwamoto et al., 2001;Enomoto et al., 2004). Less is known, however, aboutTwist1 and its role in chondrogenesis. Twist1 expressionhas been observed in the LPM prior to formation of thelimb buds (Stoetzel et al., 1995), and is thereafterexpressed in a dynamic manner during limb development(O’Rourke and Tam, 2002). Its primary action appears tobe regulation of mesodermal tissue differentiation,evidenced by retarded development of limb buds inTwist–/– mouse embryos (Chen and Behringer, 1995;Tavares et al., 2001). In contrast to Runx2, immaturechondrocytes express high endogenous levels of Twist1,whereas Twist1 levels in mature chondrocytes areundetectable (Dong et al., 2007; Hinoi et al., 2006).This expression pattern in immature chondrocytes is atleast partly due to TGFb signalling, while in maturechondrocytes, Wnt signalling inhibits Twist1 and promotesRunx2 expression to support chondrocyte maturation(Dong et al., 2007).

Axin, a component of the degradation complex whichmarks b-catenin for proteasomal degradation, modulatesboth Wnt and TGFb signalling pathways. In the presenceof Wnt/b-catenin signalling, a negative feedback loop isestablished whereby Axin2, induced by Wnt/b-cateninsignal, inhibits Wnt/b-catenin signalling (Jho et al., 2002;Leung et al., 2002). In contrast, Axin enhances TGFbsignalling through facilitation of Smad3 phosphorylation andactivation, as well as by enhancing Smad7 phosphorylationand degradation (Furuhashi et al., 2001; Liu et al., 2006).Dao et al. (2010) showed disruption of Axin2 signalling-accelerated chondrocyte maturation, similar to b-cateningain-of-function, indicating a regulatory role for Axin in

differentiating chondrocytes, whereby Axin balances theopposing effects of TGFb andWnt/b-catenin signalling dur-ing chondrogenesis.

The role of FGF proteins and FGFRs during cartilagedevelopment is uncertain and only indirect considerationsare possible concerning their interactions with the Wntand/or TGFb signalling pathways, with several reportssuggesting that stimulation of b-catenin signalling is possiblethrough activation of FGFRs (Goldfarb, 1996; Martin, 1998;Muenke and Schell, 1995). During mesenchymal condensa-tion, FGFR-1 expression is observed in the limb mesenchymeand periphery of the condensation, FGFR-2 expressionwithinthe condensation and FGFR-3 within the core of the conden-sation, but only at the onset of differentiation (Delezoide et al.,1998; Hellingman et al., 2010; Orr-Urtreger et al., 1991; Peterset al., 1992). The role of FGF in the condensing mesenchymeis thought to be induction of Sox9 expression, but absence ofFGF signalling within the mesenchyme shows no effect oncondensation (Colvin et al., 1999, 2001a, 2001b). Interest-ingly, it has been shown that induction of Sox9 by FGF canlead to inhibition of Wnt signalling (Naski and Ornitz, 1998).

As chondrogenesis proceeds, no FGFRs are expressed inhyaline chondrocytes, whereas all FGFRs are present duringhypertrophy (Hellingman et al., 2010). FGFR-1 is expressedin prehypertrophic and hypertrophic chondrocytes; FGFR-2expression is thought to overlap with FGFR-3 expression,which itself is expressed in proliferating chondrocytes(Deng et al., 1996; Peters et al., 1992). This expressionpattern suggests a role for FGFR-1 in maintaining hypertro-phic chondrocytes, with FGFR-3 acting as a master inhibiterof chondrocyte proliferation (Delezoide et al., 1998;Peters et al., 1992; Sahni et al., 1999). Indeed, Ornitz(2005) showed that FGFR-3 disappears in hypertrophicchondrocytes, while FGFR-1 is upregulated in the same cells,supporting a role for FGFR-1 in the regulation of cell survivaland differentiation. FGF-2, -9 and -18 are the ligands mainlyinvolved after mesenchymal condensation; however, onlyknockout of FGF-18 leads to defects in chondrogenesis(Hellingman et al., 2010; Liu et al., 2002; Ohbayashi et al.,2002; Ornitz, 2005). FGF-18–/– mice have a similar pheno-type to FGFR-3–/– mice, thus suggesting that FGF-18 coordi-nates chondrogenesis through FGFR-3.

In summary, regulation of Runx2, Sox9 and Twist iscentral to chondrocyte maturation. Both Wnt and TGFbare central to this regulation, with TGFb signalling workingin a stage-dependent manner. The role of FGF signallingduring chondrogenic differentiation is poorly understood,despite the presence and stage-specific expression patternof FGFRs during chondrogenesis.

4. FGF–TGFb–Wnt crosstalk: in vitroMSC chondrogenesis

4.1. Overview

Similar to developmental chondrogenesis in vivo, in vitrochondrogenic differentiation of MSCs requires the complex

FGF, TGFb and Wnt crosstalk: embryonic to in vitro cartilage development from MSCs

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involvement of growth factors and cell–cell and cell–matrixinteractions. Specific signalling pathways have been identi-fied that regulate differentiation of MSCs to chondrocytes,with the TGFb signalling pathway particularly targeted.Less well characterized are the signals and/or pathwaysthat maintain MSCs in their undifferentiated state. Forclinical and research use, a large number of MSCsis required; however, MSCs lose their differentiationpotential upon continued cell passaging. To overcome thislimitation, FGF-supplemented expansion medium is used.Despite targeting these pathways, however, commonin vitro chondrogenic protocols result in MSC-derivedchondrocytes, which, instead of forming stable articularcartilage, form hypertrophic transient epiphyseal cartilage(Cals et al., 2012; Farrell et al., 2009; Hellingman et al.,2010, 2011; Pelttari et al., 2006; Scotti et al., 2010).

4.2. Proliferation (expansion phase)(summarized in Figure 3)

As already discussed, during limb development FGFs cansubstitute for the AER, with FGF-2, -4 and -8 capableof stimulating proliferation and maintaining theundifferentiated state of cells (Crossley et al., 1996; Vogelet al., 1996). In vitro, FGF-2 is the most common supple-ment used in MSC expansion culture media to sustainMSC self-renewal and therefore maintain the cells in animmature state (Benavente et al., 2003; Tsutsumi et al.,2001). Exposure of MSCs to FGF-2 during expansionimproves cell number and culture time; however, themolecular mechanisms by which this is achieved arepoorly understood.

Sawada et al. (2006) demonstrated that TGFb receptorI and Smad3 expression are increased with time inculture. TGFb signalling has been shown to promotecellular senescence of MSCs through G1 arrest, achievedthrough activation of cyclin D kinase inhibitors (p16, p21and p53). FGF-2, through inhibition of TGFb2 expression,was able to suppress this cellular senescence (Ito et al.,2007). Furthermore, Sacchetti et al. (2007) demonstrateda downregulation of endoglin (CD105), a TGFb co-receptor, following expansion of MSCs in FGF-2 andplatelet-derived growth factor (PDGF)-BB. Therefore, justas with proliferation during in vivo limb development,FGF and TGFb signalling appear to exert opposing effects;FGF signalling promotes proliferation, while TGFb signal-ling inhibits proliferation.

Interestingly, TGFb1 signalling has also been shown tostimulate proliferation (Jian et al., 2006), with MSCtranscriptome analysis revealing TGFb, FGF and PDGF tobe sufficient for serum-free expansion of MSCs. In fact,culture medium consisting of these three factors wasrecently developed for this purpose (Chase et al., 2010).This positive effect of TGFb is dependent upon b-cateninsignalling (Baksh et al., 2007), which itself can alsobe an inhibitor of proliferation. Wnt-5a, through non-canonical signalling, can block proliferation, as canDkk1, an inhibitor of canonical Wnt signalling (Bakshet al., 2007). These opposing effects are probably depen-dent on interaction with FGFs and TGFb signalling, buthow these interactions occur, and the subsequent out-come on chondrogenesis, are not known. Furthermore,the concentration of the signalling molecules appears tobe important. De Boer et al. (2004) reported stimulationof MSC proliferation by Wnt signalling, but this was onlyevident when lower concentrations of Wnt-signallingmolecules were added.

In summary, FGF-2 supplementation is favoured duringexpansion to stimulate proliferation of MSCs while alsomaintaining their undifferentiated state. TGFb signallingcan both inhibit and stimulate proliferation, the latterdepending on activation of b-catenin/CyclinD1. Similarly,canonical Wnt signalling can enhance proliferation, whichis in contrast to non-canonical Wnt signalling, whichreduces proliferation.

4.3. Cartilage differentiation and maturation(summarized in Figure 4)

Prior to chondrogenic induction, MSCs are often firstexpanded in order to obtain sufficient numbers. Asdiscussed, FGF-2 is frequently the growth factor selectedfor this purpose. FGF-2 treatment prior to chondrogenicinduction, however, not only improves MSC proliferationbut also improves the chondrogenic differentiation ofMSCs. Interestingly, Handorf and Li (2011) observed adecrease in OCT3/4 and Nanog levels, suggesting thatFGF-2 reduces the multipotentiality of MSCs, insteadpriming them for the chondrogenic lineage. MSCsexpanded in FGF-2 form larger pellets, due to greater

Figure 3. Schematic overview of the signals that regulate cellproliferation during human MSCs in vitro expansion. In vitro,FGF-2 is the most common growth factor used to stimulate MSCproliferation. As previously shown in vivo, TGFb is mainly knownto inhibit proliferation. However, the TGFb–Smad3 pathway canstimulate proliferation via b-catenin–CyclinD1. Canonical andnon-canonical Wnt signalling exert opposite effects, enhancingor decreasing proliferation respectively. This figure is based onthe following literature: Baksh et al., 2007; De Boer et al.,2004; Ito et al., 2007; Jian et al., 2006; Sacchetti et al., 2007;Sawada et al., 2006

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matrix protein production, with a better developed and,in terms of collagen II and aggrecan, a more homogeneouscartilaginous matrix (Solchaga et al., 2010). Increasingbasal Sox9 protein levels accompany this phenotypeand explain, at least in part, this FGF-2-associatedenhanced chondrogenesis.

The molecular mechanism(s) by which FGF-2 triggersupregulation of Sox9 during expansion, and subsequentenhanced chondrogenesis, is not entirely clear but mayrequire interaction with Wnt signalling pathways. FGF-2is capable of upregulating secreted frizzled-related pro-tein 1 (SFRP1), a Wnt signalling inhibitor, anddownregulating frizzled-7 (FZD7) and Wnt-1-induciblesignalling pathway protein 1 (WISP1), both stimulatorsof Wnt signalling (Solchaga et al., 2005).

Furthermore, MAPK signalling has been implicated inFGF-2 upregulation of Sox9. FGF-2 inhibits MAPK signal-ling through upregulation of two MAPK inhibitors,dual specific protein phosphatase (DUSP) 4 and DUSP6(Solchaga et al., 2005), molecules that inhibit p38 andERK-1/2 (Stanton et al., 2003; Tuli et al., 2003). Thisoccurs independent of TGFb signalling, which in contrastincreases Sox9 levels through stimulation of MAPK signal-ling (Handorf and Li, 2011). Recently, however, it wassuggested that TGFb1 administration during expansion ofMSCs ismore likely to reduce, rather than enhance, Sox9 ex-pression (Handorf and Li, 2011). Alas, the authors did nottest whether TGFb1 administration during expansion hadan effect on chondrogenic potential. Surprisingly, althoughboth FGF-2 and TGFb can signal through theMAPK cascade,information regarding the effect of this interaction onchondrogenesis using MSCs is lacking.

Chondrogenic differentiation of MSCs using the classicpellet culture system proceeds, as in in vivo embryogene-sis, through three stages: condensation, characterized byN-cadherin; differentiation, characterized by collagen II;and terminal differentiation, characterized by collagen X(Barry et al., 2001; Hellingman et al., 2010). Smad2/3and Smad1/5/8 signalling is necessary for induction ofchondrogenesis, with TGFb being the classic growthfactor used for chondrogenic induction. TGFb is capableof initiating and maintaining the chondrogenesis of MSCs(Johnstone et al., 1998) through transient activation ofthe MAPK submembers p38, ERK-1 and JNK (Tuli et al.,2003). Inhibiting MAPK signalling in TGFb1-treatedpellet cultures increases N-cadherin levels, preventingthe transition from precartilage condensation to overtchondrogenic differentiation. Wnt-7a regulates N-cadherinexpression, with increased gene expression observed uponinhibition of MAPK signalling, a finding which correlateswith in vivo data, and which was dependent upon thepresence of TGFb. It is not yet clear, however, how thiscooperation between TGFb and Wnt occurs.

Through a series of cell line experiments, Jian et al.(2006) observed rapid nuclear accumulation of b-cateninin MSCs following TGFb1 treatment, an accumulationwhich was neither the result of a Wnt ligand-dependentprocess nor stabilization of b-catenin levels; it was insteadmediated by Smad3-facilitated b-catenin translocation tothe nucleus. This combination of TGFb and Wnt has beenshown, again in cell lines, to produce pellets that are ofgreater size and have more cartilage matrix productionthan pellets produced from the use of TGFb only (Zhouet al., 2004). Several cell lines also demonstrate the effect

Figure 4. Interactions between FGF, TGFb and Wnt signalling pathways during human MSC chondrogenic induction. TGFb–Smad3 isthe classical intracellular pathway involved in initiating and maintaining chondrogenesis via activation of the transcription factorSOX9. TGFb can also stimulate chondrogenesis through MAPK phosphorylation. This process leads to direct stimulation of SOX9and inhibition of the canonical Wnt pathway that, during chondrogenic differentiation, stimulates the expression of hypertrophicmarkers. In addition to Smad3, members of the TGFb family can signal via Smad1/5/8. This pathway seems to be important in theearly stages of chondrogenesis, but exerts a negative effect if it is maintained throughout chondrogenic induction. Although theuse of FGF proteins during expansion ‘primes’ MSCs for chondrogenic induction by increasing SOX9 levels, exposure of MSCs toFGF proteins during differentiation has a negative impact by promoting hypertrophy through b-catenin signalling. The figure is basedon the following literature: Furuhashi et al., 2001; Jian et al., 2006; Johnstone et al., 1998; Leijten et al., 2012; Tuli et al., 2003;Zhou et al., 2004

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of Axin, a negative regulator in Wnt signalling, as anadapter of Smad3, enhancing the activation of Smad3 inTGFb signalling (Furuhashi et al., 2001). Whether thisregulation exists in human MSCs remains to be clarified.

Once chondrogenesis has begun, signalling throughSmad1/5/8 enhances hypertrophy (Hellingman et al.,2011) and the role of TGFb in promoting hypertrophyhas been extensively studied. Leijten et al. (2012) recentlydemonstrated that chondrogenically differentiating MSCsacquire a gene expression prolife pattern more character-istic of hypertrophic hyaline cartilage than stable articularcartilage. This profiling identified frizzled-related protein(FRZB) and Dkk1, both inhibitors of Wnt signalling, andGrem, a BMP inhibitor, as markers of articular cartilage.Adding all three factors to the culture medium signifi-cantly reduced the expression of collagen X and alkalinephosphatase. Therefore, blocking b-catenin signallingby any of these routes could reduce the production ofhypertrophic cartilage in vitro.

In summary, FGF-2-supplemented expansion medium canenhance subsequent chondrogenesis through upregulation ofSox9 expression, the expression of which is also activated byTGFb/Smad3 signalling, the conventional intracellular path-way involved in initiating and maintaining chondrogenesis.TGFb signalling also stimulates chondrogenesis throughMAPK signalling, promoting Sox9 expressionwhile preventingexpression of hypertrophic markers through inhibition ofb-catenin signalling.

5. Discussion

The therapeutic potential of MSCs has inspired a signifi-cant amount of research over the past decades. Whileour knowledge of the biological processes and molecularmechanisms of these cells in chondrogenesis is alwaysimproving, their successful translation to the clinicremains to be achieved. Instead of stable, functional hyalinecartilage, in vitro chondrogenic differentiation of MSCsproduces a transient hypertrophic cartilage, which whenimplanted in vivo forms bone. Chondrogenesis is an obliga-tory process in endochondral bone formation. It beginswith recruitment of mesenchymal cells into condensations,followed by their differentiation into chondrocytes. Thesechondrocytes become hypertrophic before ultimatelyforming bone. Thus, there appears to be similarity betweenin vivo endochondral ossification and in vitro chondrogenicdifferentiation of MSCs (Barry et al., 2001; Hellingmanet al., 2011; Pelttari et al., 2006). Comparing embryoniclimb development with in vitro differentiation of humanMSCs might therefore provide insights into how we cangain control over MSC differentiation to produce stableand functional cartilage.

MSCs in vitromust be expanded to obtain enough cells forclinical and research use. During development, FGF ligandsare the main factors capable of promoting proliferation ofmesenchymal cells while maintaining the undifferentiatedstate of the cells. Attempts have already been made to mimicthis function of FGF through supplementation of expansion

media with FGF-2. What is surprising, however, is thatalthough Wnt signalling coordinates with FGF signalling toachieve these effects, there is no information availableregarding the use of Wnt in the expansion phase of humanMSCs and subsequent effects on chondrogenic differentia-tion. The combination of these factors during expansionmay improve outcomes in the chondrogenic differentiationof MSCs and might therefore be an interesting topic ofresearch in the field.

Other attempts to mimic in vivo processes come in theform of TGFb supplementation during chondrogenicinduction. While it is clear that TGFb signalling is neces-sary for induction of chondrogenesis once differentiationhas begun, TGFb signalling can enhance hypertrophy(Hellingman et al., 2011). Recently it was demonstratedthat the expression of FGFRs, during both in vitro differen-tiation of MSCs and in vivo embryonic limb development,occurs in a stage-dependent pattern (Hellingman et al.,2010). Clear expression of FGFR-2 and -3 was observedin cells during the condensation phase; all FGFRs wereexpressed by hypertrophic chondrocytes, but none wereexpressed by mature hyaline chondrocytes. It is possiblethat TGFb ALK receptors also exhibit stage-dependentexpression patterns and this would, in part, provide anexplanation as to why TGFb signalling can have contrastingeffects at different stages in chondrogenesis.

It is important to keep in mind, however, that TGFb is aknown inducer of fibrosis in various tissues, with synovialjoints being no exception (Lorda-Diez et al., 2009).Fibrocartilage is observed in the outer layer of MSC-derived chondrogenic pellets treated with TGFb. In vitroredifferentiated chondrocytes do not form hyaline carti-lage but rather fibrocartilage (Bonaventure et al., 1994;Lefebvre et al., 1990; Mayne et al., 1976). TGFb likely playsa role in this process, and has previously been shown tointerfere with the redifferentiation process of chondrocytes(Narcisi et al., 2012) However, its precise function, andthe function of FGF and Wnt in such a process, remains tobe investigated.

Our focus on the interactions between the FGF, TGFb andWnt signalling pathways in chondrogenesis highlights therole of Twist1 during the in vivo process. To our knowledge,this transcription factor has been the subject of only onein vitro study involving chondrogenic differentiation ofhuman MSCs (Isenmann et al., 2009), but could prove aninteresting molecule for future research for understandingand controlling the immature state of chondrocytes. Further-more, by focusing on the interactions between these threepathways, we are aware that the roles of many otherimportant factors, such as parathyroid hormone-relatedprotein and members of the Hedgehog signalling family,and their interactions with these pathways individually, havebeen overlooked. Such interactions, however, were outsidethe scope of this review.

There is no doubt that FGF, TGFb and Wnt contributepositively to the chondrogenic process both in vivoand in vitro; however, the sometimes contrasting or dualroles of different family members within these pathwaysresults in a crosstalk that is very complex between these

M. A. Cleary et al.

Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

pathways. On top of this, timing appears to be crucial. Thepoint at which MSCs are exposed to these factors duringchondrogenesis can determine how they react. Stages ofchondrogenesis are often discriminated in in vivo work;however, this is not the case for in vitro work. This isdifficult to do, because of the rapid onset of hypertrophyduring in vitro pellet culture work; however, more effortshould be made to discriminate between the moreeasily identified condensation, early chondrogenic and latechondrogenic stages, characterized by N-cadherin, collagenII and collagen X expression, respectively. This is important,as the literature points to the need for a staged-basedapproach to in vitroMSC chondrogenic differentiation, with

the application of more tailored culture regimes in whichcells are perhaps only exposed transiently at different stagesto the optimum factor (Barry et al., 2001; Hellingman et al.,2010, 2011; Pelttari et al., 2006).

Acknowledgements

This study was supported by the Science Foundation Ireland(Grant No. 11/RFP/BMT/3150) and a grant from the Dutchgovernment to the Netherlands Institute for RegenerativeMedicine (NIRM; Grant No. FES0908), and was performedwithin the framework of the Erasmus Postgraduate School ofMolecular Medicine.

References

Adachi N, Sato K, Usas A, et al. 2002;Muscle derived, cell based ex vivo genetherapy for treatment of full thicknessarticular cartilage defects. J Rheumatol29: 1920–1930.

van Amerongen R, Mikels A, Nusse R. 2008;Alternative wnt signaling is initiated bydistinct receptors. Sci Signal 1: re9.

Baksh D, Boland GM, Tuan RS. 2007;Cross-talk between Wnt signaling path-ways in human mesenchymal stem cellsleads to functional antagonism duringosteogenic differentiation. J Cell Biochem101: 1109–1124.

Barry F, Boynton RE, Liu B, et al. 2001;Chondrogenic differentiation of mesen-chymal stem cells from bone marrow:differentiation-dependent gene expres-sion of matrix components. Exp Cell Res268: 189–200.

Benavente CA, Sierralta WD, Conget PA,et al. 2003; Subcellular distribution andmito-genic effect of basic fibroblast growth factorin mesenchymal uncommitted stem cells.Growth Factors 21: 87–94.

ten Berge D, Brugmann SA, Helms JA, et al.2008; Wnt and FGF signals interact to co-ordinate growth with cell fate specificationduring limb development. Development135: 3247–3257.

Bonaventure J, Kadhom N, Cohen-Solal L,et al. 1994; Reexpression of cartilage-specific genes by dedifferentiated humanarticular chondrocytes cultured in algi-nate beads. Exp Cell Res 212: 97–104.

Bosch P, Musgrave DS, Lee JY, et al. 2000;Osteoprogenitor cells within skeletal mus-cle. J Orthop Res 18: 933–944.

Cals FL, Hellingman CA, Koevoet W, et al.2012; Effects of transforming growthfactor-b subtypes on in vitro cartilageproduction and mineralization of humanbone marrow stromal-derived mesenchy-mal stem cells. J Tissue Eng Regen Med6: 68–76.

Cancedda R, Castagnola P, Cancedda FD,et al. 2000; Developmental control ofchondrogenesis and osteogenesis. Int JDev Biol 44: 707–714.

Carrington JL, Fallon JF. 1984; The stages offlank ectoderm capable of responding toridge induction in the chick embryo. JEmbryol Exp Morphol 84: 19–34.

Chase LG, Lakshmipathy U, Solchaga LA,et al. 2010; A novel serum-free mediumfor the expansion of human mesenchymalstem cells. Stem Cell Res Ther 1: 8.

Chen ZF, Behringer RR. 1995; twist isrequired in head mesenchyme for cra-nial neural tube morphogenesis. GenesDev 9: 686–699.

Chimal-Monroy J, Montero JA, Ganan Y,et al. 2002; Comparative analysis of theexpression and regulation of Wnt5a,Fz4, and Frzb1 during digit formationand in micromass cultures. Dev Dyn224: 314–320.

Colvin JS, Feldman B, Nadeau JH, et al.1999; Genomic organization and embry-onic expression of the mouse fibroblastgrowth factor 9 gene. Dev Dyn 216: 72–88.

Colvin JS, White AC, Pratt SJ, et al. 2001a;Lung hypoplasia and neonatal death inFgf9-null mice identify this gene as anessential regulator of lung mesenchyme.Development 128: 2095–2106.

Colvin JS, Green RP, Schmahl J, et al. 2001b;Male-to-female sex reversal in micelacking fibroblast growth factor 9. Cell104: 875–889.

Crossley PH, Minowada G, MacArthur CA,et al. 1996; Roles for FGF8 in the induc-tion, initiation, and maintenance of chicklimb development. Cell 84: 127–136.

Dailey L, Ambrosetti D, Mansukhani A, et al.2005; Mechanisms underlying differentialresponses to FGF signaling. CytokineGrowth Factor Rev 16: 233–247.

Dale TC. 1998; Signal transduction by theWnt family of ligands. Biochem J 329(2):209–223.

Dao DY, Yang X, Flick LM, et al. 2010; Axin2regulates chondrocyte maturation andaxial skeletal development. J Orthop Res28: 89–95.

De Bari C, Dell’Accio F, Luyten FP. 2001a;Human periosteum-derived cells maintainphenotypic stability and chondrogenicpotential throughout expansion regardlessof donor age. Arthritis Rheum 44: 85–95.

De Bari C, Dell’Accio F, Tylzanowski P, et al.2001b; Multipotent mesenchymal stemcells from adult human synovial mem-brane. Arthritis Rheum 44: 1928–1942.

De Boer J, Wang HJ, Van Blitterswijk C. 2004;Effects ofWnt signaling on proliferation and

differentiation of human mesenchymalstem cells. Tissue Eng 10: 393–401.

Delezoide AL, Benoist-Lasselin C, Legeai-Mallet L, et al. 1998; Spatio-temporal ex-pression of FGFR 1, 2 and 3 genes duringhuman embryo-fetal ossification. MechDev 77: 19–30.

Deng C, Wynshaw-Boris A, Zhou F, et al.1996; Fibroblast growth factor receptor 3is a negative regulator of bone growth. Cell84: 911–921.

ten Dijke P, Yamashita H, Ichijo H, et al. 1994;Characterization of type I receptors fortransforming growth factor-b and activin.Science 264: 101–104.

Dominici M, Le Blanc K, Mueller I, et al.2006; Minimal criteria for definingmultipotent mesenchymal stromal cells.The International Society for CellularTherapy position statement. Cytotherapy8: 315–317.

Dong Y, Drissi H, Chen M, et al. 2005; Wnt-mediated regulation of chondrocyte matu-ration: modulation by TGFb. J Cell Biochem95: 1057–1068.

Dong YF, Soung do Y, Schwarz EM, et al.2006; Wnt induction of chondrocyte hy-pertrophy through the Runx2 transcriptionfactor. J Cell Physiol 208: 77–86.

Dong YF, Soung do Y, Chang Y, et al.2007; Transforming growth factor-band Wnt signals regulate chondrocytedifferentiation through Twist1 in astage-specific manner. Mol Endocrinol21: 2805–2820.

Dono R, Zeller R. 1994; Cell-type-specificnuclear translocation of fibroblast growthfactor-2 isoforms during chicken kidneyand limb morphogenesis. Dev Biol 163:316–330.

Dorey K, Amaya E. 2010; FGF signalling:diverse roles during early vertebrate em-bryogenesis. Development 137: 3731–3742.

Enomoto H, Furuichi T, Zanma A, et al.2004; Runx2 deficiency in chondrocytescauses adipogenic changes in vitro. JCell Sci 117: 417–425.

Enomoto-Iwamoto M, Enomoto H, Komori T,et al. 2001; Participation of Cbfa1 inregulation of chondrocyte maturation.Osteoarthr Cartilage 9(suppl A): S76–84.

Farrell E, van der Jagt OP, Koevoet W, et al.2009; Chondrogenic priming of human

FGF, TGFb and Wnt crosstalk: embryonic to in vitro cartilage development from MSCs

Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

bone marrow stromal cells: a better routeto bone repair? Tissue Eng Part C Methods15: 285–295.

Fischer S, Draper BW, Neumann CJ. 2003;The zebrafish fgf24 mutant identifies anadditional level of Fgf signaling involvedin vertebrate forelimb initiation. Develop-ment 130: 3515–3524.

Furuhashi M, Yagi K, Yamamoto H, et al.2001; Axin facilitates Smad3 activation inthe transforming growth factor-b signalingpathway. Mol Cell Biol 21: 5132–5141.

Goldfarb M. 1996; Functions of fibroblastgrowth factors in vertebrate development.Cytokine Growth Factor Rev 7: 311–325.

Goldring MB, Tsuchimochi K, Ijiri K. 2006;The control of chondrogenesis. J CellBiochem 97: 33–44.

Gronthos S, Franklin DM, Leddy HA, et al.2001; Surface protein characterization ofhuman adipose tissue-derived stromalcells. J Cell Physiol 189: 54–63.

Handorf AM, Li WJ. 2011; Fibroblast growthfactor-2 primes human mesenchymal stemcells for enhanced chondrogenesis. PLoSOne 6: e22887.

Hellingman CA, Koevoet W, Kops N, et al.2010; Fibroblast growth factor receptorsin in vitro and in vivo chondrogenesis:relating tissue engineering using adultmesenchymal stem cells to embryonicdevelopment. Tissue Eng Part A16: 545–556.

Hellingman CA, Davidson EN, Koevoet W,et al. 2011; Smad signaling determineschondrogenic differentiation of bone-marrow-derived mesenchymal stem cells:inhibition of Smad1/5/8P prevents termi-nal differentiation and calcification. TissueEng Part A 17: 1157–1167.

Hinoi E, Bialek P, Chen YT, et al. 2006;Runx2 inhibits chondrocyte proliferationand hypertrophy through its expressionin the perichondrium. Genes Dev 20:2937–2942.

Hofmann C, Luo G, Balling R, et al. 1996;Analysis of limb patterning in BMP-7-deficient mice. Dev Genet 19: 43–50.

Huelsken J, Behrens J. 2002; The Wnt signal-ling pathway. J Cell Sci 115: 3977–3978.

Isenmann S, Arthur A, Zanettino AC. et al.2009; TWIST family of basic helix-loop-he-lix transcription factors mediate humanmesenchymal stem cell growth and com-mitment. Stem Cells 27: 2457–2468.

ItoT, Sawada R, Fujiwara Y, et al. 2007; FGF-2suppresses cellular senescence of humanmesenchymal stem cells by downregulationof TGFb2. Biochem Biophys Res Commun359: 108–114.

Itoh N. 2010; Hormone-like (endocrine)Fgfs: their evolutionary history and rolesin development, metabolism, and disease.Cell Tissue Res 342: 1–11.

Iwai T, Murai J, Yoshikawa H, et al. 2008;Smad7 inhibits chondrocyte differentia-tion at multiple steps during endochondralbone formation and downregulates p38MAPK pathways. J Biol Chem 283:27154–27164.

Jho EH, Zhang T, Domon C, et al. 2002; Wnt/b-catenin/Tcf signaling induces the tran-scription of Axin2, a negative regulator ofthe signaling pathway. Mol Cell Biol 22:1172–1183.

Jian H, Shen X, Liu I, et al. 2006; Smad3-dependent nuclear translocation ofb-catenin is required for TGFb1-induced

proliferation of bone marrow-derivedadult human mesenchymal stem cells.Genes Dev 20: 666–674.

Johnstone B, Hering TM, Caplan AI, et al.1998; In vitro chondrogenesis of bonemarrow-derived mesenchymal progenitorcells. Exp Cell Res 238: 265–272.

Kawakami Y, Capdevila J, Buscher D,et al. 2001; WNT signals controlFGF-dependent limb initiation andAER induction in the chick embryo.Cell 104: 891–900.

Kawano Y, Kypta R. 2003; Secreted antago-nists of the Wnt signalling pathway. J CellSci 116: 2627–2634.

Kengaku M, Capdevila J, Rodriguez-EstebanC, et al. 1998; Distinct WNT pathways reg-ulating AER formation and dorsoventralpolarity in the chick limb bud. Science280: 1274–1277.

Kharitonenkov A. 2009; FGFs and metabo-lism. Curr Opin Pharmacol 9: 805–810.

Kronenberg HM. 2003; Developmentalregulation of the growth plate. Nature423: 332–336.

Lefebvre V, Peeters-Joris C, Vaes G. 1990;Production of collagens, collagenase andcollagenase inhibitor during the dediffer-entiation of articular chondrocytes byserial subcultures. Biochim Biophys Acta1051: 266–275.

Leijten JC, van Blitterwijk CA, Karperien M,et al. 2012; GREM1, FRZB and DKK1are key regulators of human articularcartilage homeostasis. Arthritis Rheum64: 3302–3312.

Leung JY, Kolligs FT, Wu R, et al. 2002; Acti-vation of AXIN2 expression by b-catenin-Tcell factor. A feedback repressor pathwayregulating Wnt signaling. J Biol Chem277: 21657–21665.

Li TF, Darowish M, Zuscik MJ, et al. 2006;Smad3-deficient chondrocytes haveenhanced BMP signaling and accelerateddifferentiation. J Bone Miner Res 21: 4–16.

Liu Z, Xu J, Colvin JS, et al. 2002; Coordina-tion of chondrogenesis and osteogenesisby fibroblast growth factor 18. Genes Dev16: 859–869.

Liu W, Rui H, Wang J, et al. 2006; Axin is ascaffold protein in TGFb signaling thatpromotes degradation of Smad7 byArkadia. EMBO J 25: 1646–1658.

Lorda-Diez CI, Montero JA, Martinez-Cue C,et al. 2009; Transforming growth factors-bcoordinate cartilage and tendon differentia-tion in the developing limb mesenchyme.J Biol Chem 284: 29988–29996.

Lorda-Diez CI, Montero JA, Garcia-Porrero JA,et al. 2010; Tgfb2 and 3 are coexpressed withtheir extracellular regulator Ltbp1 in theearly limb bud and modulate mesodermaloutgrowth and BMP signaling in chicken em-bryos. BMC Dev Biol 10: 69.

Martin GR. 1998; The roles of FGFs in theearly development of vertebrate limbs.Genes Dev 12: 1571–1586.

Mayne R, Vail MS, Mayne PM, et al. 1976;Changes in type of collagen synthesizedas clones of chick chondrocytes grow andeventually lose division capacity. Proc NatlAcad Sci USA 73: 1674–1678.

Merino R, Rodriguez-Leon J, Macias D, et al.1999; The BMP antagonist Gremlin regu-lates outgrowth, chondrogenesis andprogrammed cell death in the developinglimb. Development 126: 5515–5522.

Miller LD, Park KS, Guo QM, et al. 2001;Silencing of Wnt signaling and activa-tion of multiple metabolic pathways inresponse to thyroid hormone-stimulatedcell proliferation. Mol Cell Biol 21:6626–6639.

Mohammadi M, Olsen SK, Ibrahimi OA.2005; Structural basis for fibroblastgrowth factor receptor activation. CytokineGrowth Factor Rev 16: 107–137.

Moon RT, Kohn AD, De Ferrari GV, et al.2004; WNT and b-catenin signalling:diseases and therapies. Nat Rev Genet5: 691–701.

Mu Y, Gudey SK, Landstrom M. 2012; Non-Smad signaling pathways. Cell Tissue Res347: 11–20.

Muenke M, Schell U. 1995; Fibroblast-growth-factor receptor mutations inhuman skeletal disorders. Trends Genet11: 308–313.

Nakahara H, Goldberg VM, Caplan AI. 1991;Culture-expanded human periosteal-derived cells exhibit osteochondral poten-tial in vivo. J Orthop Res 9: 465–476.

Narcisi R, Signorile L, Verhaar JA, et al.2012; TGFb inhibition during expansionphase increases the chondrogenicredifferentiation capacity of humanarticular chondrocytes. Osteoarthr Carti-lage 20: 1152–1160.

Naski MC, Ornitz DM. 1998; FGF signal-ing in skeletal development. FrontBiosci 3: d781–794.

Niswander L. 2003; Pattern formation:old models out on a limb. Nat RevGenet 4: 133–143.

Niswander L, Tickle C, Vogel A, et al. 1993;FGF-4 replaces the apical ectodermal ridgeand directs outgrowth and patterning ofthe limb. Cell 75: 579–587.

Noth U, Osyczka AM, Tuli R, et al. 2002;Multilineage mesenchymal differentiationpotential of human trabecular bone-derived cells. J Orthop Res 20: 1060–1069.

Nusse R. 2005; Wnt signaling in disease andin development. Cell Res 15: 28–32.

Oh CD, Chang SH, Yoon YM, et al. 2000;Opposing role of mitogen-activated pro-tein kinase subtypes, erk-1/2 and p38, inthe regulation of chondrogenesis of mes-enchymes. J Biol Chem 275: 5613–5619.

Ohbayashi N, Shibayama M, Kurotaki Y, et al.2002; FGF18 is required for normal cellproliferation and differentiation duringosteogenesis and chondrogenesis. GenesDev 16: 870–879.

Ohuchi H, Yoshioka H, Noji S. 1995; [Induc-tion of an additional limb by FGF and dupli-cation of digits by Sonic hedgehog].Seikagaku 67: 1232–1236.

Ohuchi H, Nakagawa T, Yamamoto A, et al.1997; The mesenchymal factor, FGF10, ini-tiates and maintains the outgrowth of thechick limb bud through interaction withFGF8, an apical ectodermal factor. Develop-ment 124: 2235–2244.

Olwin BB, Arthur K, Hannon K, et al.1994; Role of FGFs in skeletal muscleand limb development. Mol Reprod Dev39: 90–100; discussion, 100–101.

Ornitz DM. 2005; FGF signaling in the devel-oping endochondral skeleton. CytokineGrowth Factor Rev 16: 205–213.

O’Rourke MP, Tam PP. 2002; Twist func-tions in mouse development. Int J DevBiol 46: 401–413.

M. A. Cleary et al.

Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

Orr-Urtreger A, Givol D, Yayon A, et al. 1991;Developmental expression of two murinefibroblast growth factor receptors, flg andbek. Development 113: 1419–1434.

Osyczka AM, Noth U, Danielson KG, et al.2002a; Different osteochondral potentialof clonal cell lines derived from adulthuman trabecular bone. Ann NY Acad Sci961: 73–77.

Osyczka AM, Noth U, O’Connor J, et al.2002b; Multilineage differentiation ofadult human bone marrow progenitorcells transduced with human papillomavirus type 16 E6/E7 genes. Calcif TissueInt 71: 447–458.

Pelttari K, Winter A, Steck E, et al. 2006; Pre-mature induction of hypertrophy duringin vitro chondrogenesis of human mesen-chymal stem cells correlates with calcifica-tion and vascular invasion after ectopictransplantation in SCID mice. ArthritisRheum 54: 3254–3266.

Peters KG, Werner S, Chen G, et al. 1992;Two FGF receptor genes are differen-tially expressed in epithelial and mesen-chymal tissues during limb formationand organogenesis in themouse.Development114: 233–243.

Pittenger MF, Mackay AM, Beck SC, et al.1999; Multilineage potential of adult hu-man mesenchymal stem cells. Science284: 143–147.

Reinhold MI, Naski MC. 2007; Directinteractions of Runx2 and canonicalWnt signaling induce FGF18. J BiolChem 282: 3653–3663.

Sacchetti B, Funari A, Michienzi S, et al. 2007;Self-renewing osteoprogenitors in bonemar-row sinusoids can organize a hematopoieticmicroenvironment. Cell 131: 324–336.

Sahni M, Ambrosetti DC, Mansukhani A,et al. 1999; FGF signaling inhibits chon-drocyte proliferation and regulates bonedevelopment through the STAT-1 pathway.Genes Dev 13: 1361–1366.

Saunders JW Jr. 1948; The proximo-distal se-quence of origin of the parts of the chickwing and the role of the ectoderm. J ExpZool 108: 363–403.

Saunders JW Jr, Reuss C. 1974; Inductiveand axial properties of prospective wing-bud mesoderm in the chick embryo. DevBiol 38: 41–50.

SavageMP,Hart CE, Riley BB, et al.1993;Distri-bution of FGF-2 suggests it has a role in chicklimb bud growth. Dev Dyn 198: 159–170.

Sawada R, Ito T, Tsuchiya T. 2006; Changesin expression of genes related to cell prolif-eration in human mesenchymal stem cellsduring in vitro culture in comparison withcancer cells. J Artif Organs 9: 179–184.

Schlessinger J. 2000; Cell signaling by recep-tor tyrosine kinases. Cell 103: 211–225.

Scotti C, Tonnarelli B, Papadimitropoulos A,et al. 2010; Recapitulation of endochondral

bone formation using human adultmesenchymal stem cells as a paradigmfor developmental engineering. ProcNatl Acad Sci USA 107: 7251–7256.

Semenov MV, Habas R, Macdonald BT, et al.2007; SnapShot: Noncanonical Wnt sig-naling pathways. Cell 131: 1378.

Serra R, Johnson M, Filvaroff EH, et al. 1997;Expression of a truncated, kinase-defectiveTGFb type II receptor in mouse skeletal tis-sue promotes terminal chondrocyte differ-entiation and osteoarthritis. J Cell Biol139: 541–552.

Sethi JK, Vidal-Puig A. 2010; Wnt signallingand the control of cellular metabolism.Biochem J 427: 1–17.

Solchaga LA, Penick K, Porter JD, et al. 2005;FGF-2 enhances themitotic and chondrogenicpotentials of human adult bone marrow-derived mesenchymal stem cells. J CellPhysiol 203: 398–409.

Solchaga LA, Penick K, Goldberg VM,et al. 2010; Fibroblast growth factor-2enhances proliferation and delays lossof chondrogenic potential in humanadult bone-marrow-derived mesenchy-mal stem cells. Tissue Eng Part A 16:1009–1019.

Stanton LA, Underhill TM, Beier F. 2003;MAP kinases in chondrocyte differentia-tion. Dev Biol 263: 165–175.

Stoetzel C, Weber B, Bourgeois P, et al.1995; Dorso-ventral and rostro-caudalsequential expression of M-twist in thepostimplantation murine embryo. MechDev 51: 251–263.

Tavares AT, Izpisuja-Belmonte JC,Rodriguez-Leon J. 2001; Developmentalexpression of chick twist and its regulationduring limb patterning. Int J Dev Biol 45:707–713.

Tickle C, Munsterberg A. 2001; Vertebratelimb development – the early stages inchick and mouse. Curr Opin Genet Dev11: 476–481.

Todt WL, Fallon JF. 1984; Developmentof the apical ectodermal ridge in thechick wing bud. J Embryol Exp Morphol80: 21–41.

Tsutsumi S, Shimazu A, Miyazaki K, et al.2001; Retention of multilineage differ-entiation potential of mesenchymalcells during proliferation in responseto FGF. Biochem Biophys Res Commun288: 413–419.

Tuan RS. 2003; Cellular signaling in develop-mental chondrogenesis: N-cadherin, Wnts,and BMP-2. J Bone Joint Surg Am 85A(suppl 2): 137–141.

Tufan AC, Tuan RS. 2001; Wnt regulation oflimb mesenchymal chondrogenesis is ac-companied by altered N-cadherin-relatedfunctions. FASEB J 15: 1436–1438.

Tufan AC, Daumer KM, DeLise AM, et al.2002; AP-1 transcription factor complex

is a target of signals from both WnT-7aand N-cadherin-dependent cell–cell adhe-sion complex during the regulation of limbmesenchymal chondrogenesis. Exp Cell Res273: 197–203.

Tuli R, Tuli S, Nandi S, et al. 2003;Transforming growth factor-b-mediatedchondrogenesis of human mesenchymalprogenitor cells involves N-cadherinand mitogen-activated protein kinaseand Wnt signaling crosstalk. J Biol Chem278: 41227–41236.

Vajo Z, Francomano CA, Wilkin DJ. 2000;The molecular and genetic basis of fibro-blast growth factor receptor 3 disorders:the achondroplasia family of skeletaldysplasias, Muenke craniosynostosis, andCrouzon syndrome with acanthosisnigricans. Endocr Rev 21: 23–39.

Vandenabeele F, De Bari C, Moreels M, et al.2003; Morphological and immunocyto-chemical characterization of culturedfibroblast-like cells derived from adulthuman synovial membrane. Arch HistolCytol 66: 145–153.

Vogel A, Rodriguez C, Izpisua-Belmonte JC.1996; Involvement of FGF-8 in initiation,outgrowth and patterning of the verte-brate limb. Development 122: 1737–1750.

Wang M, Shen J, Jin H, et al. 2011; Recentprogress in understanding molecularmechanisms of cartilage degenerationduring osteoarthritis. Ann NY Acad Sci1240: 61–69.

Xu X, Weinstein M, Li C, et al. 1998; Fi-broblast growth factor receptor 2(FGFR2)-mediated reciprocal regulationloop between FGF8 and FGF10 is essen-tial for limb induction. Development125: 753–765.

Yang X, Chen L, Xu X, et al. 2001; TGFb/Smad3 signals repress chondrocyte hyper-trophic differentiation and are requiredfor maintaining articular cartilage. J CellBiol 153: 35–46.

Yonei-Tamura S, Endo T, Yajima H, et al.1999; FGF7 and FGF10 directly inducethe apical ectodermal ridge in chickembryos. Dev Biol 211: 133–143.

Zhang YE. 2009; Non-Smad pathways inTGFb signaling. Cell Res 19: 128–139.

Zhang X, Ibrahimi OA, Olsen SK, et al.2006; Receptor specificity of the fibro-blast growth factor family. The completemammalian FGF family. J Biol Chem281: 15694–15700.

Zhou S, Eid K, Glowacki J. 2004; Coopera-tion between TGFb and Wnt pathwaysduring chondrocyte and adipocyte differ-entiation of human marrow stromalcells. J Bone Miner Res 19: 463–470.

Zuk PA, Zhu M, Mizuno H, et al. 2001;Multilineage cells from human adiposetissue: implications for cell-based thera-pies. Tissue Eng 7: 211–228.

FGF, TGFb and Wnt crosstalk: embryonic to in vitro cartilage development from MSCs

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