ARTICLEdoi:10.1016/j.ymthe.2005.03.024

Gene-Induced Chondrogenesis of PrimaryMesenchymal Stem Cells in Vitro

Glyn D. Palmer,1 Andre Steinert,1 Arnulf Pascher,1 Elvire Gouze,1 Jean-Noel Gouze,1

Oliver Betz,1 Brian Johnstone,2 Christopher H. Evans,1 and Steven C. Ghivizzani1,*

1Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA 02115, USA2Department of Orthopaedics, Case Western Reserve University, Cleveland, OH 44106, USA

*To whom correspondence and reprint requests should be addressed at current address: Department of Orthopaedics and Rehabilitation, University of Florida,

P.O. Box 100137, JHMHC, 1600 SW Archer Road, Gainesville, FL 32610–0137, USA. Fax: +1 352 273 7247. E-mail: ghivisc@otho.ufl.edu.

Available online 17 May 2005

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Adult mesenchymal stem cells (MSCs) have the capacity to differentiate into various connectivetissues such as cartilage and bone following stimulation with certain growth factors. However, less isknown about the capacity of these cells to undergo chondrogenesis when these proteins aredelivered via gene transfer. In this study, we investigated chondrogenesis of primary, bone marrow-derived MSCs in aggregate cultures following genetic modification with adenoviral vectorsencoding chondrogenic growth factors. We found that adenoviral-mediated expression of TGF-B1and BMP-2, but not IGF-1, induced chondrogenesis of MSCs as evidenced by toluidine bluemetachromasia and immunohistochemical detection of type II collagen. Chondrogenesis correlatedwith the level and duration of expressed protein and was strongest in aggregates expressing 10–100ng/ml transgene product. Transgene expression in all aggregates was highly transient, showing amarked decrease after 7 days. Chondrogenesis was inhibited in aggregates modified to express N100ng/ml TGF-B1 or BMP-2; however, this was found to be partly due to the inhibitory effect ofexposure to high adenoviral loads. Our findings indicate that parameters such as these areimportant functional considerations for adapting gene transfer technologies to induce chondro-genesis of MSCs.

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Key Words: gene therapy, mesenchymal stem cell, chondrogenesis, adenovirus,growth factor, bone marrow

INTRODUCTION

Articular cartilage is a highly specialized tissue thatenables almost frictionless motion between the articu-lating surfaces of diarthrodial joints. Although remark-ably durable, it is vulnerable to injury and has alimited capacity for self-repair. Experimental ap-proaches toward treatment of damaged articular carti-lage have increasingly focused on cell-based therapies[1]. In this regard, adult mesenchymal stem cells(MSCs) provide an attractive alternative to maturechondrocytes that must be isolated from a very limitedsupply of healthy articular cartilage. MSCs can beobtained relatively easily from bone marrow and othertissue sources and have the capacity for differentiationinto the cell types characteristic of various mesenchy-mal tissues, including cartilage and bone [2–4]. Undercertain culture conditions, MSCs will maintain theirmultilineage potential with passage, making themamenable to ex vivo applications [5,6].

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Delivery of MSCs to cartilaginous lesions has notyielded satisfactory regeneration of articular cartilage.One possible problem is that there is insufficient localstimulation of the implanted cells by the protein factorsnecessary to drive differentiation in vivo [7]. Gene transfermight be adapted as a means to provide sustainedsynthesis of bioactive transgene products within cartila-ginous lesions; the delivery of the appropriate stimula-tory factor(s) in this manner may enable synthesis of animproved cartilaginous repair tissue [8].

The development of in vitro systems of chondrogenesishas been important to the identification of proteinfactors that can promote chondrocyte differentiation ofadult MSCs and improved cartilage repair in vivo [9–11].Johnstone et al. demonstrated that chondrogenesis isinduced in MSCs when cultured as aggregates in adefined medium containing dexamethasone and trans-forming growth factor-h1 (TGF-h1) [9]. In this system, theaggregates synthesize an extracellular matrix character-

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FIG. 1. Adenoviral-mediated transgene expression in primary rabbit MSCs and

chondrocytes. (A) Monolayer cultures of MSCs or chondrocytes were infected

with increasing doses of Ad.GFP, indicated as viral particles (vp) per cell. At 72

h, GFP+ cells were counted in three fields under light and fluorescence

microscopy. Results are presented as the mean percentage of fluorescent cell

per field at each viral dose. (B) Representative fluorescence of first-passage

MSCs infected with 1000 and 10,000 vp/cell as indicated. (C) TGFh-1 levels in

media conditioned by MSCs or chondrocytes infected with increasing

amounts of Ad.TGFh-1. At 48 h postinfection the media were changed

Twenty-four hours later the media were collected and TGF-h1 production wa

assayed by ELISA. The data are represented as the means of triplicate

experiments. Error bars represent FSD.

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istic of cartilage, containing proteoglycan and type IIcollagen. Related studies have been useful to the eluci-dation of the chondrogenic potential of other growthfactors, including TGF-h2, TGF-h3, bone morphogeneticprotein-2 (BMP-2), BMP-6, and insulin-like growth factor-1 (IGF-1) [10–14]. For example, administration ofrecombinant IGF-1 has been found to induce chondro-genesis of limb bud mesenchymal cells [15] as well asperiosteal mesenchymal cells from rabbits [16].

Toward the development of gene-based methods foreffective delivery of stimulatory proteins, such as thoseabove, several studies have shown that viral vectors,including lentivirus [17,18], retrovirus [19–21], andadenovirus [22–24], and to a lesser degree nonviral DNAformulations [25] can be used to modify MSCs geneticallyto express various transgene products. Moreover, inanimal models, delivery of autologous mesenchymalprogenitor cells genetically modified to secrete certainbiological factors has been reported to stimulate theformation of bone [26,27] and cartilage [28,29]. Whilethese studies indicate that gene transfer can be used todeliver proteins to MSCs and influence their biology, thefunctional parameters of gene-induced mesenchymalchondrogenesis have not been reported and remainpoorly understood.

Using cDNAs encoding TGF-h1, BMP-2, and IGF-1, wehave begun to delineate the functional considerationsimportant for the use of gene transfer as a proteindelivery system for induction of chondrogenesis inprimary MSCs. Here, we report that adenoviral-mediateddelivery of certain growth factors can induce chondro-genesis of MSCs in aggregate culture; however, the levelof transgene expression, its duration, and the viral loadinfluence chondroinduction directly.

RESULTS

Transduction Efficiency of Primary BoneMarrow-Derived MSCs with Adenoviral VectorsFor our experiments we used adherent cells cultured fromiliac crest bone marrow aspirates of New Zealand Whiterabbits as a source of MSCs. To determine if these cellscan be efficiently modified to express exogenous trans-genes, we infected first-passage monolayer cultures withincreasing amounts of adenovirus encoding green fluo-rescent protein, Ad.GFP. We also infected first-passagemonolayer chondrocytes in parallel to provide a relativecomparison for transduction efficiency. As shown inFig. 1A, after 72 h a slightly greater percentage ofchondrocytes appeared to be transduced by the adenovi-rus at each dose; however, this difference was notstatistically significant. Consistent with the heterogene-ous composition of the MSC cultures, green fluorescentprotein (GFP)-positive cells appeared with two distinctmorphologies, small, fibroblast-like and larger, polygonalcells. At the lower viral doses, GFP+ cells appeared

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predominantly of the polygonal type, but as the doseincreased, a greater proportion of GFP+ cells appearedfibroblastic (Fig. 1B).

To determine the relative level of synthesis of asecreted transgene product, we infected parallel culturesof chondrocytes and MSCs with increasing amounts ofadenovirus containing the cDNA for TGF-h1. At 72 hpostinfection, we analyzed the conditioned media forTGF-h1 content. As shown in Fig. 1C, consistent with theresults for GFP, TGF-h1 transgene expression increased

y

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with viral dose similarly for both cell types. At the lowerviral doses, however, expression from the chondrocytecultures was somewhat higher, ranging between 1.4- and3-fold greater than for the MSCs. However, at themaximum dose of virus tested, 10,000 viral particles (vp)per cell, expression from the MSCs was slightly greater.

These results indicated that MSCs were amenable togenetic modification via recombinant adenovirus andshowed a modestly reduced capacity of transgenicexpression relative to chondrocytes. Based on theseresults, we used adenoviral doses ranging from 100 to10,000 vp per cell for subsequent experiments. Thepredicted levels of secreted transgene product at thesedoses, between ~1 and 100 ng/ml per 24 h, encompassthe range of recombinant growth factor concentrationstypically used to supplement MSC cultures in models ofin vitro chondrogenesis.

Chondrogenic Differentiation of MSCs FollowingAdenoviral-Mediated Gene Transfer of TGF-B1We performed experiments using MSCs in an aggregatesystem to determine if gene transfer could be used as aneffective means of protein delivery with which to inducechondrogenesis. In this system, the activity of TGF-h1 hasbeen well characterized [9]; therefore we compared itsability to induce chondrogenesis when delivered as arecombinant (r) protein or when supplied as the productof a transgene. For the latter, we infected first- or second-passage monolayer cultures of MSCs with doses ofAd.TGF-h1 that would be predicted to generate low(b10 ng/ml), medium (10–100 ng/ml), and high (N100ng/ml) levels of secreted TGF-h1 transgene productfollowing aggregate formation. At 24 h postinfection,we seeded monolayer cells into aggregates and main-tained them for 21 days in a defined serum-free medium.Control groups consisted of naive and Ad.GFP-infectedaggregates maintained in the absence or presence ofrTGF-h1 protein at a concentration of 10 ng/ml.

Histologic examination indicated distinct evidence oftransgene-induced chondrogenesis of the MSCs, theextent of which correlated with the levels of expressedTGF-h1 protein (Fig. 2). The genetically modified MSCsthat expressed TGF-h1 in the medium range at 3 dayspost-aggregate formation, as well as the unmodifiedaggregates cultured in 10 ng/ml rTGF-h1, were highlycellular and showed extensive metachromatic stainingwith toluidine blue and corresponding positive immu-nostaining for type II collagen, characteristic of cartilagematrix. Typical of that shown in Fig. 2, we found the levelof chondrogenesis to be reproducibly greater and moreconsistent when TGF-h1 was supplied as a transgeneproduct than as a recombinant protein. For example,quantitative estimates indicated a 75 and 110% increasein relative toluidine blue and type II collagen staining,respectively, in the midlevel-expression (10–100 ng/ml)pellets relative to the recombinant protein.

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In contrast, at day 3 MSCs expressing TGF-h1 at lessthan 10 ng/ml or greater than 100 ng/ml were typicallymuch less chondrogenic and showed only limited, some-what focal matrix production as evidenced by staining forproteoglycans and collagen type II (Fig. 2). Often, observ-able chondrogenesis was confined toward the periphery ofthe aggregates, while the central regions resembledundifferentiated control aggregates. We observed positiveimmunostaining for collagen type I in all groups and ittypically occurred in the outermost regions. Interestingly,aggregates of cells preinfected with Ad.GFP showed noevidence of chondrogenesis either in the presence or inthe absence of rTGF-h1 (data not shown).

Analyses of media conditioned by genetically modi-fied aggregates revealed a marked decrease in TGF-h1transgene expression in all groups over the course of theexperiment (Fig. 2D). For cultures that expressed mediumand high levels of TGF-h1, the decrease was mostprominent, approximately 80%, between day 3 and day7 (Fig. 2D). Conversely, the low-level TGF-h1 groupsynthesized approximately 5 ng/ml of growth factor upto the first week of culture, but this was not sufficient toinduce extensive chondrogenesis within the aggregates.

Chondrogenic Differentiation of Primary MSCsFollowing Adenoviral-Mediated Gene Transferof IGF-1 and BMP-2We used the aggregate culture system further to evaluatethe chondroinductive activity of two additional trans-genes, human BMP-2 and IGF-1. Again, we modifiedaggregates to express low (b10 ng/ml), medium (10–100ng/ml), or high (N100 ng/ml) levels of secreted BMP-2 orIGF-1 and cultured them in defined media for 3 weeks.For comparative controls, we cultured similar unmodifiedaggregates of MSCs in the presence of recombinant BMP-2 or IGF-1 at doses of 25, 50, or 100 ng/ml.

As shown in Fig. 3D, the profiles of transgeneexpression in Ad.BMP-2-modified aggregates were sim-ilar to those of Ad.TGF-h1, with a prominent loss ofexpression at days 3 and 7. As with TGF-h1, adenoviral-mediated expression of BMP-2 induced chondrogenesisin a dose dependent manner (Figs. 3A–3C). Aggregatesexpressing low levels (1–10 ng/ml) of BMP-2 werehighly cellular and showed considerable staining forproteoglycan and type II collagen despite a decrease inBMP-2 expression to b1 ng/ml after only 1 week. Inaggregates expressing 10–100 ng/ml BMP-2, weobserved more abundant staining (greater than twofold)for proteoglycan and type II collagen compared to boththe low BMP-2 group and a control Ad.TGF-h1 groupexpressing 10–100 ng/ml TGF-h1 (similar to that shownin Fig. 2). These aggregates were also highly cellular,and the majority of cells had the appearance of hyper-trophic chondrocytes. Similar to that seen with Ad.TGF-h1, the high-expressing group (N100 ng/ml BMP-2)showed little evidence of cartilaginous matrix (Fig. 3),

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FIG. 2. Chondrogenesis of MSCs following adenoviral-mediated gene transfer of TGF-h1. Monolayer cultures of MSCs were infected with doses of Ad.TGF-h1 to

generate 1–10, 10–100, and N100 ng/ml of transgene product at day 3 following aggregate formation. At 24 h after infection, MSCs were seeded into aggregates

and cultured for 21 days. Nontransduced control groups were cultured in parallel in the absence (0 ng/ml) or presence (10 ng/ml) of recombinant TGF-h1. (A)

Toluidine blue staining of representative aggregate sections for detection of matrix proteoglycan. (B) Immunostaining for the presence of type II collagen and (C)

type I collagen. Regions of positive staining show green fluorescence. (D) Corresponding transgene expression profile for each group for the 21-day culture period.

Values represent TGF-h1 levels in the conditioned media over a 24-h period at days 3, 7, 14, and 21. Data are shown as the means + SD of triplicate measurements

with n = 3 pellets per group per replicate.

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suggesting inhibition of chondrogenesis, perhapsthrough overproduction of growth factor or excessiveadenoviral infection. Aggregates cultured in the pres-ence of rBMP-2 similarly showed a dose-dependenteffect, and chondrogenesis increased with dose ofprotein through the 100 ng/ml level. As seen in Fig.2, chondroinduction at this dose was similar to thatobserved with the adenovirus expressing 1–10 ng ofBMP-2. Digital analysis of staining confirmed approx-imately similar levels of toluidine blue and collagentype II reactivity between these two groups.

Somewhat surprisingly, neither the cultures geneticallymodified to express IGF-1 nor those incubated in thepresence of rIGF-1 showed phenotypic evidence of chon-drogenesis in sections stained for type II collagen orproteoglycan (Fig. 3). Sections also showed little stainingfor collagen type I. Despite the absence of chondrogenesisat the phenotypic level, the profile of expression of the

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IGF-1 transgene from the modified cultures was similar tothat observed with Ad.BMP-2 and Ad.TGF-h1 (Fig. 3D).

We determined the expression of cartilage-specificmarker genes for the genetically modified aggregatesusing RT-PCR (Fig. 4). After 21 days, we observedexpression of mRNAs for aggrecan and type II collagenin cells modified with Ad.TGF-h1, Ad.BMP-2, and Ad.IGF-1, but not preaggregate monolayer MSCs. In Ad.TGF-h1-and Ad.IGF-1-modified aggregates RT-PCR products ofboth splice variants of type II collagen, IIA (432 bp) andIIB (225 bp), were present in approximately equalamounts. However, in Ad.BMP-2-modified aggregates,similar to that seen in articular chondrocytes (lane C),type IIB was the predominant form. Comparison ofreaction products between the Ad.TGF-h1 and theAd.BMP-2 aggregates showed approximately equal levelsof aggrecan and collagen type IIB transcripts, but Ad.TGF-h1-infected aggregates showed approximately 4.7- and

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FIG. 3. Chondrogenesis of MSCs following adenoviral-mediated gene transfer of BMP-2 and IGF-1. Similar to those described for Fig. 2, monolayer cultures of

MSCs were infected with doses of Ad.BMP-2 or Ad.IGF-1 to generate 1–10, 10–100, and N100 ng/ml levels of transgene product at day 3 following aggregate

formation as indicated. The infected MSCs were seeded into aggregates and cultured for 21 days. Parallel unmodified aggregate cultures were incubated in the

presence of 25, 50, or 100 ng/ml rBMP-2 or rIGF-1. The aggregates were then fixed, sectioned, and stained. For rBMP2, the maximum level of chondrogenesis

was observed in the 100 ng/ml group. Since there was no difference in the appearance of sections from any of the rIGF-1 or Ad.IGF-1-infected groups, only

sections from the 100 and 10–100 ng/ml groups, respectively, are shown. (A) Toluidine blue staining (B) Immunostaining for type II collagen and (C) type I

collagen. (D) Corresponding recombinant protein levels or transgene expression profiles for each group for the 21-day culture period are shown as indicated.

Values represent levels of BMP-2 or IGF-1 (shown in yellow on the right) in the conditioned media over a 24-h period at days 3, 7, 14, and 21. Sections shown are

representative of a series of three experimental replicates with n = 3 pellets per group per replicate.

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2.1-fold greater levels of collagen type IIA and collagentype I mRNAs, respectively. Despite the apparent expres-sion of mRNAs for aggrecan and type II collagen in theAd.IGF-1 group at levels nearly equivalent to those of theAd.TGF-h1 aggregates, these cultures showed no histo-logic evidence of chondrogenesis. This suggests that therabbit MSCs are capable of recognizing and responding tothe IGF-1 protein in a manner consistent with chondro-cytic differentiation but may be limited by posttranscrip-tional mechanisms in this culture system.

Effect of Culture Conditions on Transgene Expressionof MSCsDespite high transduction efficiencies, results from ourexperiments suggest that transgene expression is rapidlylost from MSCs genetically modified with adenoviral

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vectors in aggregate culture. However, these results weredetermined by measuring the level of transgene productsreleased into the conditioned media and do not representthose within the cell or bound to extracellular matrixcomponents. To generate a more accurate profile oftransgene expression by the cells, we transduced MSCsto express a nonchondrogenic gene, luciferase (Ad.Luc),and cultured them either as aggregates or as monolayersin the presence of rTGF-h1 at 10 ng/ml.

Luciferase transgene expression in both aggregate andmonolayer cultures was characterized by a markeddecrease by day 7 and followed a pattern similar to thatof growth factor expression in the earlier experiments(Fig. 5). Despite the presence of rTGF-h1, there was novisible evidence of chondrogenesis in Ad.Luc-infectedcultures (not shown). The similar expression profiles in

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FIG. 4. Expression of cartilage-specific genes in genetically modified aggre

gates. After 21 days, total RNA was extracted from aggregates (6 per group

and expression of cartilage marker genes was determined using RT-PCR. The

reaction products were resolved on 1.5% agarose gels and visualized by

staining with ethidium bromide. RT-PCR of RNA isolated from rabbit articula

cartilage was used as a comparative control (lane C). Reaction products o

RNA from preaggregate MSCs are shown in lane 1. RNA from Ad.TGF-h1-

Ad.IGF-1-, or Ad.BMP-2-modified aggregates was used in lanes 2–4

respectively. RT-PCR product sizes were as follows: aggrecan, 313 bp; type

II collagen, 432 (IIA splice variant) and 225 bp (IIB splice variant); type

collagen, 702 bp; GAPDH, 293 bp. Differences in staining intensities between

lanes were normalized using the GAPDH reaction products.

FIG. 5. Luciferase transgene expression in monolayer and aggregate MSC

cultures. Monolayer MSCs were infected with 500 vp/cell Ad.Luc and afte

24 h were cultured as aggregates or reseeded into monolayers. Aggregate

and monolayers were cultured with or without rTGF-h1 10 ng/ml as indicated

Aggregates were harvested and analyzed for luciferase activity at days 3, 7, 14

and 21. Values shown are mean levels of luciferase activity for triplicate

samples in relative light units (RLU) + SD.

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monolayer and aggregate culture indicate that loss oftransgene expression in MSCs is not related to eventsassociated with chondrocytic differentiation, but morelikely occurs through the loss of adenoviral genomes tocell division or cell death.

Effect of Adenoviral Load on Chondrogenesis of MSCsNoting a reduction in chondrogenesis in pellets trans-duced to express high levels of TGF-h1 or BMP-2, wewanted to determine whether high levels of adenoviralinfection could inhibit differentiation of MSCs. For these

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experiments, we used the parent adenoviral vector, C5,which does not contain a transgene. Previously, viraldoses of 100, 500, and 5000 vp/cell were used to establish1–10, 10–100, and N100 ng/ml expressed protein product,respectively. Since optimal chondrogenesis was observedin the 10–100 ng/ml range of expressed TGF-h1 or BMP-2following infection with up to 500 particles/cell, thedifferentiation potential of primary MSCs is not compro-mised at this level of virus. Based on this, we first infectedprimary MSCs with 500 particles/cell Ad.TGF-h1 and after24 h infected them with increasing amounts of C5 toyield cumulative viral doses of 1000, 5000, and 10,000vp/cell. After a further 24 h we seeded the cells intoaggregates and cultured them as before.

Histologic examination of the aggregates after 21 daysrevealed that C5 overinfection decreased chondrogenesisin a dose-dependent manner (Fig. 6A). Control aggregatesmodified with 500 vp/cell Ad.TGF-h1, or 500 vp/cell C5cultured in the presence of rTGF-h1, were similarly

FIG. 6. Chondrogenesis of MSCs following exposure to increasing adenoviral

loads and paracrine delivery of a transgenic growth factor. (A) MSCs were

infected with 500 vp/cell Ad.TGF-h1 as indicated, followed 24 h later by

increasing concentrations (0, 500, 4500, and 9500 vp/cell) of the C5

adenoviral vector to yield cumulative viral doses of 500, 1000, 5000, and

10,000 vp/cell. (Aggregates of MSCs infected with a total of 10,000 vp/cell

disintegrated during culture and are not shown.) A control group (rTGF-h1 +

C5) was transduced with C5 only (500 vp/cell) and cultured in the presence

of 10 ng/ml rTGF-h1. Images show toluidine blue staining of representative

aggregate sections after 21 days. Transgene expression (tx exp) levels at day 3

(mean F SD for triplicate samples) for each group are shown below the

images. (B) Donor MSC aggregates were genetically modified with Ad.BMP-2

to express either 10–100 or N100 ng/ml transgene product. Every 24 h, the

media conditioned by these aggregates were removed and fed to Recipient,

untransduced aggregates. Control, untransduced aggregates were cultured in

the presence of rBMP-2 at a final concentration of 25 ng/ml (10–100 lane) or

100 ng/ml (N100 lane).

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chondrogenic as evidenced by metachromatic stainingwith toluidine blue. In aggregates infected with cumu-lative viral doses of 1000 and 5000 vp/cell there was aprogressive decrease in matrix staining with toluidineblue and aggregate size. Type II collagen immunostainingcorrelated with toluidine blue in all aggregates (notshown). After initially forming aggregates, MSCs infectedwith 10,000 vp/cell disintegrated during the 3-weekculture period and thus are not shown. Analysis of mediaconditioned by the aggregates revealed similar levels ofexpressed TGF-h1 at 3 days after aggregate formation andthroughout the culture period, in aggregates either withor without C5 infection. This indicated that the inhib-itory effect of C5 was not due to altered expression of theTGF-h1 transgene, but rather to excessive adenoviralinfection.

Effect of Paracrine Delivery of Transgenic BMP-2 onChondrogenesis of MSCsAlthough we had evidence that high-level infection ofMSCs was inhibitory to chondrogenesis, we performedadditional experiments to determine if excessivegrowth factor production may also contribute. For thisstudy we transduced bdonorQ MSC cultures with dosesof Ad.BMP-2 that would generate medium and highlevels of transgene product following aggregate forma-tion. At 24 h after initiation of aggregate culture, weremoved the conditioned media from both groups andadded them to untransduced, brecipientQ aggregatescultured in parallel. Donor cultures were refed withchondrogenic media and the process was repeatedevery 24 h for 21 days. An additional group ofaggregates cultured in the presence of rBMP-2 at dosesranging from 25 to 100 ng/ml were also generated forcomparison.

ELISA of the conditioned media from donor culturesafter 3 days confirmed that aggregates modified toexpress medium- and high-dose BMP-2 were withinthe predicted range (23 and 152 ng/ml at day 3,respectively). Consistent with Fig. 3, genetically modi-fied donor aggregates expressing medium-dose BMP-2showed extensive chondrogenesis following toluidineblue staining (Fig. 6B). Similarly, chondrogenesis wasinhibited in the high-dose BMP-2-modified donorgroup. Somewhat surprisingly, secreted, transgenicBMP-2 from medium-dose cultures failed to stimulatechondrogenesis in recipient aggregates (Fig. 6B). How-ever, we observed partial chondrogenesis in recipientaggregates receiving transgenic BMP-2 from the high-dose donor cultures. The chondrogenic response ofaggregates treated with rBMP-2 followed a similar trend,with the extent of chondrogenesis increasing with BMP-2 protein concentration.

These results suggested that BMP-2 provided to cellsin aggregate is less potent as a soluble protein than asa gene product. They also indicated that levels of BMP-

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2 in culture medium of N100 ng/ml are not necessarilyinhibitory to chondrogenic differentiation in thissystem.

DISCUSSION

In the present study, we demonstrate that adenoviral-mediated expression of certain chondrogenic growthfactors can serve as an effective means of protein deliveryto induce chondrogenesis of rabbit, primary, bonemarrow-derived MSCs in aggregate culture. Indeed, forTGF-h1 and BMP-2 this method was typically superior todelivery of recombinant protein. Important differences,however, were noted between the two transgenes withregard to the histologic appearance of the pellets andtransgene expression. Within the range of 1–100 ng/mlexpressed transgene product, Ad.BMP-2-infected aggre-gates were larger, had increased cellularity, and showedmore intense staining for proteoglycan and collagen typeII than Ad.TGF-h1 aggregates. This pattern was consistentover three experiments, each with preparations of MSCsfrom different rabbits. In addition, RT-PCR revealeddifferent mRNA splice variants of type II collagen fromeach growth factor, with Ad.BMP-2 aggregates expressinga greater percentage of the type IIB form typicallyassociated with maturing chondrocytes [14,30].

The failure of IGF-1 to stimulate differentiation ofrabbit MSCs effectively in aggregate culture is largelyinconsistent with the literature. For example, Oh andChun report the ability of recombinant IGF-1 to drivechondrogenesis of chicken limb bud mesenchymal cellsin micromass culture [15]; Fukumoto et al. describe IGF-1-mediated mesenchymal chondrogenesis in periostealexplants from rabbits [16] and Gelse et al. the use of ratMSCs genetically modified to overexpress IGF-1 in acartilage repair model in vivo [29]. It could be argued thatadenoviral infection may interfere with the activity ofthis particular protein, but we were unable to observechondrogenesis with the recombinant protein even athigh doses (z100 ng/ml). Ad.IGF-1 was found to stim-ulate transcription of genes associated with chondro-genesis in the aggregate system, indicating that rabbitcells are capable of recognizing and responding to theIGF-1 transgene product. These results suggest that IGF-1is capable of initiating certain biological pathwaysassociated with chondrocytic differentiation, but as achondroinductive agent it likely relies on the presence ofother factors not present in the context of the aggregateculture system.

Typical of those observed with nonintegrative vectors[31], transgene expression levels in the adenovirallytransduced MSC cultures was transient and showed amarked decrease after 7 days. This decline was observedwith both growth factor and marker transgenes andoccurred independent of chondrogenesis or aggregateculture. This transient profile may limit the types of

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genes/factors that can be effectively delivered to MSCs viaan adenovirus. Alternatively, given that many growthfactors are pleiotropic, a vector that provides transienttransgene expression may naturally limit overproductionand the detrimental side effects that can occur whendelivering these agents to articular tissues [29,32,33].

We found that overinfection of MSCs with adenoviruswas inhibitory to chondrogenic differentiation. Alongthese lines, we also observed an absence of chondro-genesis in MSCs modified with adenovirus to expressmarker genes, GFP and luciferase, despite prolongedculture in media containing rTGF-h1. This type ofresponse has not been reported previously and is incon-sistent with the observations of others. Mosca et al.reported that human MSCs retrovirally transduced toexpress GFP were fully capable of chondrogenic differ-entiation in pellet culture [34]. Similarly, transgenic micethat express GFP are viable and develop mesenchymaltissues normally [35,36]. Thus, it is possible that the lackof differentiation we observed is not a direct consequenceof marker gene expression per se, but more due to theroute of gene delivery via recombinant adenovirus.

An interesting observation from these studies is theincreased potency of certain growth factors when sup-plied as transgene products rather than as recombinantprotein. This may reflect differences in the presentationof the protein in the microenvironment of the aggregate.It is possible that within the aggregate the functionalconcentration of the transgene product is manifoldhigher than is reflected by ELISA measurements of theconditioned media. In the experiments described here, asingle aggregate of 2 � 105 cells represents approximately1/1000 of the volume of the culture fluid, yet is capable ofraising the concentration of the total fluid volume togreater than 100 ng/ml transgene product. Therefore,within the immediate microenvironment of the aggre-gate, the concentration of growth factor at certain timesmust be far greater than that of the medium. Further, thealmost direct presentation of synthesized growth factorto immediately neighboring cells may enhance itsstimulatory capacity. An additional consideration is thatthe transgene product is being released from numerouscells within the aggregate, while the recombinant proteinmust diffuse throughout the matrix of the aggregate tostimulate the cells in the interior. Thus, although there isclear evidence that high viral loads will inhibit chondro-genic differentiation, because of the functional differ-ences in the presentation of growth factors whenprovided as recombinant protein supplement to theculture medium or as a transgene product within theaggregate cultures, we were unable to resolve completelythe possibility that excess growth factor may inhibitchondrogenesis.

In conclusion, our experiments provide a furtherdemonstration of the capacity of gene transfer as adelivery system for bioactive proteins and, within certain

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parameters, the ability to use it to guide the chondro-genic differentiation of multipotent cells. Indeed thesystem described here should provide a useful tool toexamine the chondrogenic potential of other candidatetransgenes in vitro. Thus far, our work has entailed theexamination of gene products on an individual basis.However, to fulfill the potential of gene therapy for stem-cell-based cartilage repair, it is possible that far moresophisticated strategies will be required to reproducefaithfully the complex molecular events of chondrocytedifferentiation and then long-term maintenance of thearticular cartilage phenotype. These may require thecoordinate expression of multiple genes using complexregulatory systems. How gene-induced chondrogenesismay be translated into therapeutic applications has yet tobe determined. There are several experimental approachescurrently under investigation that may prove useful, suchas the local implantation into cartilage defects of MSCsgenetically modified ex vivo [28,29] or, as we have recentlydescribed, the delivery of genetically modified bonemarrow coagulates to cartilaginous lesions [37]. Regardlessof the method, a critical first step in development is thecharacterization of candidate gene products in vitro toenable selection of viable reagents to take forward intoanimal studies.

MATERIALS AND METHODS

Generation of vectors. First-generation, E1, E3-deleted, serotype 5

adenoviral vectors carrying the cDNAs for firefly luciferase, GFP, human

TGF-h1, human IGF-1, and human BMP-2 were constructed using the

method of Hardy et al. [38]. The resulting vectors were designated

Ad.Luc, Ad.GFP, Ad.TGF-h1, Ad.IGF-1, and Ad.BMP-2, respectively. To

generate high-titer preparations, the recombinant vectors were amplified

in 293CRE8 cells and purified over three successive CsCl gradients.

Following dialysis the preparations were aliquoted and stored at �808C.

Viral titers were estimated by optical density and standard plaque assay.

Using these methods preparations of 1012–1013 particles/ml were

obtained.

Cell harvest. Rabbit bone marrow was harvested from the iliac crests of

young adult New Zealand White rabbits and plated in monolayer culture

at 2 � 107 cells/75-cm2 flask in DMEM supplemented with 10% FBS and

1% mesenchymal stem cell stimulatory supplements (Stem Cell Tech-

nologies). After 2–3 weeks, adherent colonies of cells were trypsinized and

replated in 25-cm2 tissue culture flasks or 24-well plates depending on the

procedure.

Adenoviral transduction of MSCs in monolayer. Confluent monolayer

cultures of MSCs in 24-well plates were incubated with various doses of

recombinant adenovirus as indicated in the text and figure legends, in

100 Al of serum-free DMEM for 2 h in a tissue culture incubator.

Following infection, the culture fluids were aspirated and replaced with 1

ml DMEM with 10% FBS. After 24 h, the media were replaced and were

collected following an additional further 24 h incubation. Harvested

media were stored at �208C until analysis by specific ELISA. Ad.GFP-

transduced cultures were viewed for fluorescence at 48 h following

infection.

Aggregate culture of MSCs. Following the initial plating, the adherent

cultures of MSCs were seeded into 25-cm2 flasks and grown to confluence,

generating approximately 6 � 105 cells per flask. Individual flasks of cells

were infected with low, medium, or high doses of Ad.TGF-h1, Ad.IGF-1, or

Ad.BMP-2. Afterward, the supernatant was aspirated and replaced with 5

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Copyright C The American Society of Gene Therapy

ARTICLEdoi:10.1016/j.ymthe.2005.03.024

ml DMEM containing stem cell supplements. After 24 h, the cells were

trypsinized and seeded at 2 � 105 cells/ml in 15-ml polypropylene tubes.

The cells were centrifuged for 5 min at 500g to form a pellet. The pelleted

cells were maintained in 0.5 ml of chondrogenic medium consisting of

serum-free DMEM containing pyruvate (1 mM), 1% ITS (Sigma) ascorbate

2-phosphate (37.5 Ag/ml), and dexamethasone (10�7 M) [9]. In parallel

nontransduced cultures, the medium was supplemented with or without

recombinant human TGF-h1 (R&D Systems) at 10 ng/ml. The pelleted

cells formed free-floating aggregates within the first 24 h of culture. The

media were changed every 2–3 days.

RNA isolation and RT-PCR. RT-PCR was used to evaluate qualitatively

transcription of cartilage-specific genes following infection of MSCs with

medium doses of Ad.TGF-h1, Ad.BMP-2, or Ad.IGF-1. Total RNA was

isolated from two 25-cm2 flasks of MSCs grown in monolayer or from six

MSC aggregates cultured for 21 days. For cDNA synthesis, 1 Ag of total

RNA from each group was reverse transcribed using random hexamer

primers and M-MLV reverse transcriptase (Invitrogen). Two microliters of

reaction product containing approximately 80 ng of cDNA was used as a

template for PCR amplification. The primer sets used have been

previously described: human type II collagen a1(II) chain, human type I

collagen a1(I) [9] rabbit aggrecan, and rabbit glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) [39]. PCR products were visualized on 1%

agarose gels containing 0.1 mg/ml ethidium bromide. The relative

intensity of the individual RT-PCR products within the gels was

determined from digital images using the Scion Image program, version

1.63. Reaction products of GAPDH were used to normalize the intensities

between lanes.

Quantitation of transgene expression. Conditioned media were collected

at various time points and assayed for concentrations of the respective

growth factors using the appropriate Quantikine Immunoassay kits (R&D

Systems) for human TGF-h1, IGF-1, and BMP-2.

To measure luciferase activity, monolayer MSC cultures were infected

with 1.5 � 108 particles of Ad.Luc. At 24 h postinfection, the cells were

trypsinized and reseeded at 4 � 105 cells/ml in either aggregate or

monolayer cultures as described. For aggregate cultures, samples were

incubated with 3 mg/ml collagenase and 0.1% trypsin in serum-free

DMEM for 2 h at 378C. The digests were then transferred to Eppendorf

tubes and mixed with an equal volume of 2� reporter lysis buffer

(Promega) and homogenized using a motorized pestle homogenizer.

Homogenates were centrifuged briefly to remove cellular debris, and

aliquots of the supernatant were assayed for luciferase activity by mixing

with an equal volume of Bright-Glo luciferase assay buffer and measuring

light emitted with a Autolumat Plus luminometer. In monolayer cultures,

cells were lysed by incubation with reporter lysis buffer for 15 min at

room temperature and mixed with an equal volume of Bright-Glo

luciferase assay buffer prior to measurement.

Histology and immunohistochemistry. Aggregates were embedded in

0.8% agarose for ease of handling and then fixed in 10% neutral-buffered

formalin for 1 h at room temperature. After dehydration in graded

alcohols, the aggregates were paraffin embedded and sectioned to 5 Am.

Representative sections were stained using toluidine blue (Sigma) for the

detection of matrix proteoglycan, and alternate sections were used for

immunohistochemistry.

For immunohistochemistry, sections were deparaffinized and treated

with 0.1 U/ml chondroitinase ABC in PBS with 1% BSA at room

temperature for 30 min. Sections were then blocked with 5% BSA in

PBS for 30 min. Afterward, the sections were incubated for 1 h at room

temperature with rabbit polyclonal anti-collagen type I and II primary

antibodies (Rockland, Inc., Gilbertsville, PA, USA) diluted in 1% BSA in

PBS. After three PBS washes to remove unbound primary antibody,

sections were incubated with a fluorescein-conjugated anti-rabbit IgG

secondary antibody for 45 min at room temperature. The slides were

washed again and mounted in Fluoromount G, coverslipped, and

analyzed using fluorescence microscopy.

For each experiment described, three replicates were performed, with

n = 3 pellets for each group and replicate. From each pellet three sections

taken at positions throughout were stained and analyzed. To quantify

MOLECULAR THERAPY Vol. 12, No. 2, August 2005

Copyright C The American Society of Gene Therapy

relative toluidine blue or immunofluorescent staining, digital images of

individual sections were taken, and the mean density of staining across

the entire section was determined using the Scion Image program version

1.63. For the respective methods, sections of aggregates cultured in the

absence of specific growth factor stimulation served as negative controls

and were used to establish baseline levels of staining against which the

sections from the experimental groups were evaluated.

ACKNOWLEDGMENTS

This work was supported by Grants AR48566 and AR50249 from the National

Institute of Arthritis and Musculoskeletal and Skin Diseases.

RECEIVED FOR PUBLICATION JULY 23, 2004; ACCEPTED MARCH 11, 2005.

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