Experimental Cell Research 267, 267–274 (2001)doi:10.1006/excr.2001.5257, available online at http://www.idealibrary.com on

Absence of MyoD Increases Donor Myoblast Migrationinto Host Muscle

Gayle M. Smythe and Miranda D. Grounds1

Department of Anatomy and Human Biology, The University of Western Australia, Crawley, Perth, Western Australia, 6009

Donor myoblast migration is a major limiting factorin the success of myoblast transfer therapy, a poten-tial treatment for Duchenne muscular dystrophy. Apossible strategy to promote the migration of donormyoblasts into host muscle is to enhance their prolif-eration and delay their fusion, two properties that aremajor characteristics of myoblasts in regeneratingskeletal muscle in MyoD null (2/2) mice. Here we in-vestigate whether the migration of MyoD (2/2) donormyoblasts into host muscle is enhanced in vivo. Slicedmuscle grafts from male MyoD (2/2) or normal control(Balb/c) mice were transplanted into the muscles offemale normal (Balb/c) host mice. Muscles were sam-pled at 1, 3, and 12 weeks after grafting, and the fate ofmale donor myoblasts within female host muscles de-termined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (2/2) donormyoblasts migrated into host muscle continuouslyover 1, 3, and 12 weeks after grafting, in contrast withBalb/c donor myoblasts, whose overall numbers andmigratory distances did not increase significantly af-ter 1 week. These results strongly support a role forelevated donor myoblast proliferation and/or their de-layed fusion in enhancing migration into host musclein vivo, and endorse the use of either genetically engi-neered donor myoblasts, or the administration of ex-ogenous myoblast mitogens to improve donor myo-blast migration in myoblast transfer therapy. © 2001

Academic Press

Key Words: myoblast transfer therapy; Duchennemuscular dystrophy; skeletal muscle; migration;MyoD; proliferation.

INTRODUCTION

The transplantation of cultured muscle precursorcells (myoblasts) into host skeletal muscle is referred toas myoblast transfer therapy (MTT), and has potentialfor the treatment of skeletal myopathies [1–6]. How-ever, there are several major barriers to successful

1 To whom correspondence and reprint requests should be addressed.

Fax: 161 8 9380 1051. E-mail: mgrounds@anhb.uwa.edu.au.

267

clinical MTT including the limited survival, migration,and fusion of injected cultured donor myoblasts withinhost muscle (reviewed in [2, 7, 8]). The survival ofinjected donor myoblasts can be improved with hostimmunosuppression (reviewed in [7]). We have re-cently demonstrated that certain immunosuppressantscan also enhance the migration of donor myoblasts intohost muscle [9], and the distance moved is greater indystrophic compared with nondystrophic control hostmuscles.

Several studies have investigated various methods ofenhancing donor myoblast dispersal within host mus-cle after injection. Donor myoblast migration throughthe interstitial connective tissue can be facilitated byinducing the expression of enzymes that degrade theextracellular matrix [10–13], or host muscle regenera-tion [14], and by altering the angle or method of injec-tion [14, 15]. However, the damage caused to hostmuscle using the first two of these methods may limittheir clinical significance. The administration of exog-enous growth factors that enhance donor myoblast mi-gration and/or proliferation may be an attractive andclinically viable alternative. This is supported by evi-dence that basic fibroblast growth factor (bFGF) in-creases the number of injected cultured donor myo-blasts that survive in vivo [16, 17], although whetherbFGF acts as a survival, mitogenic, or chemotacticfactor in this case is unclear. Several other growthfactors that enhance myoblast chemotaxis and/or pro-liferation in vitro, including the insulin-like growthfactors, platelet-derived growth factor, and leukemiainhibitory factor (reviewed in [18]), are candidates forpromoting the success of MTT. Alternatively, variousfactors can be administered by genetic modification ofcultured donor myoblasts, since myoblasts are veryeasily and efficiently transformed in vitro to expressnew or altered genes that subsequently influence theirbehavior [19–23].

As a prelude to such potential genetic engineering ofdonor myoblasts, in the present study we investigatethe hypothesis that enhanced myoblast proliferationand delayed fusion, posttransplantation will result in a

larger number of donor myoblasts dispersing within

0014-4827/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

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268 SMYTHE AND GROUNDS

host muscle tissue. It was recently demonstrated invivo that myoblast proliferation is sustained and fusiondelayed by 2–3 days during skeletal muscle regenera-tion in mice with a null (2/2) mutation in the myo-enic regulatory gene MyoD [24]. This confirms in vitrotudies with MyoD (2/2) myoblasts [25, 26]. To deter-ine if elevated endogenous levels of myoblast prolif-

ration and a delayed fusion phase promote donor myo-last migration in MTT, we examined the migration ofyoD (2/2) donor myoblasts within histocompatible

ormal (Balb/c) host muscle tissue. Since the majorityf injected cultured donor myoblasts die within hostuscle in the absence of host immunosuppression

[27]; reviewed in [7]), whereas long-term donor myo-last survival is seen with whole and sliced musclerafts [9, 28, 29], we used the sliced muscle graft modelf MTT to examine donor myoblast migration and fu-ion. Sliced muscle grafts from male MyoD (2/2) andormal control (Balb/c) donor mice were implanted inhe tibialis anterior muscles of female normal (Balb/c)ost mice. Grafted muscles were sampled at 1, 3, and2 weeks after grafting and the number and location ofale donor myoblasts within female host muscle sec-

ions analyzed by in situ hybridization with the Y-hromosome (male)-specific Y-1 probe [9, 28–30]. At 3nd 12 weeks after grafting there were significantlyore male MyoD (2/2) than Balb/c donor myoblastsithin female host muscle, and at 12 weeks MyoD

2/2) donor myoblasts were located much further fromhe graft compared to the Balb/c donors. These resultsemonstrate that MyoD (2/2) myoblasts have areater propensity to migrate out from sliced musclerafts in vivo, and strongly support the proposal thatnhanced proliferation and/or delayed fusion of donoryoblasts can enhance MTT.

MATERIALS AND METHODS

Animals. A total of 6 male MyoD null (2/2) (ages 6–8 weeks)and 6 male Balb/c (ages 6–8 weeks) donor mice and 12 female Balb/c(ages 6–8 weeks) host mice were used. All animals were obtainedfrom specific pathogen-free breeding stock at the Animal ResourceCentre (Murdoch University, Western Australia) and were housed inthe preclinical facility in the Department of Anatomy and HumanBiology at The University of Western Australia. The MyoD (2/2)

ice were originally a generous gift from Dr. Michael RudnickiMcMaster University, Hamilton, Canada). Animals were housed inlean cages with food and water freely available. All animal proce-ures were carried out in accordance with the guidelines of theational Health and Medical Research Council of Australia, andith the prior approval of the Animal Ethics and Experimentationommittee at The University of Western Australia.Sliced muscle grafting. Sliced muscle grafts were performed fromale MyoD (2/2) and Balb/c donor mice into normal (Balb/c) femaleost mice. MyoD (2/2) and Balb/c mice are fully histocompatible.ale donor mice were sacrificed and both extensor digitorum longus

EDL) muscles removed. Donor EDL muscles were sliced longitudi-ally into three segments, and the midsegment was used for the

raft to ensure that the connective tissue barrier on both sides of the

raft had been removed. Female host mice were anesthetized (1.5%alothane delivered in oxygen) and a longitudinal incision in the skinas made from the knee to the ankle on the anterior surface of theindlimb to expose the tibialis anterior (TA) muscle. A longitudinal

ncision was made in the TA muscle to a depth approximately mid-ay through the muscle, and the donor EDL segment embeddedithin the host TA muscle. The incision was sutured closed with a

ingle suture to keep the graft in place, and the skin was suturedlosed. All female host mice received a male donor EDL sliced muscleraft to both TA muscles.A total of 12 sliced muscle grafts were performed from male MyoD

2/2) mice into 6 female Balb/c mice, and 12 control male Balb/crafts were performed into the remaining 6 female Balb/c mice. Twoost mice from each group (i.e., 4 grafts) were sampled at 1, 3, and 12eeks after grafting, and all grafted muscles were immerse-fixed in:1 methanol:acetic acid for 15 min, and then stored in 70% ethanolntil processing through paraffin wax. Paraffin-embedded samplesere cut, sections mounted on silanated slides, and the nuclei ofale donor cells (originating in the grafts) determined by in situ

hybridization with the digoxigenin (DIG)-labeled mouse Y-chromo-some-specific Y-1 probe.

In situ hybridization. Tissue sections were dewaxed and rehy-drated, fixed in fresh 4% paraformaldehyde, and then pretreatedwith Triton X-100 (Sigma; 0.5% in PBS), 0.2 N HCl, and proteinaseK (Roche Biochemicals). Sections were postfixed in 4% paraformal-dehyde and then prehybridized in hybridization solution (deionizedformamide, 20X SSC, dextran sulfate) at 37°C. The DIG-labeled Y-1probe was diluted in hybridization solution and applied to the tissuesections, and the sections were coverslipped and temporarily sealedwith rubber cement. Sections were denatured on a hot plate at 100°Cand then snap-cooled on ice before hybridizing overnight at 42°C.The following day the coverslips were removed, and the sectionsstringently washed in 2X, 1X, and 0.1X SSC. Detection of the DIG-labeled Y-1 probe hybridized to the Y-chromosome of male donornuclei was carried out by antibody reaction (alkaline phosphatase-conjugated anti-DIG, Roche Biochemicals) and color precipitation ofalkaline phosphatase with nitroblue tetrazolium/5-bromo-4-chloro-3-inodyl-phosphate (NBT/BCIP, Roche Biochemicals).

Analysis. The location of Y-1-labeled male donor nuclei withinfemale host muscle was determined on digitized images using MD-Plot software (Microsoft). The total number and location (i.e., dis-tance from the graft) of male donor myoblasts within female hostmuscle were compared for MyoD (2/2) and control Balb/c donors, todetermine if the absence of MyoD affected the migration of donormyoblasts from sliced muscle grafts within normal host muscle.Distances were determined as the shortest straight-line distancethat male donor nuclei were located from the nearest graft border.Two nonserial sections from each graft were analyzed using thismethod, and the data for all individual donor nuclei pooled for eachgroup at each time point. Differences between the two treatmentgroups and between time points were tested for statistical signifi-cance using Kruskall–Wallis one-way analysis of variance, and weredeemed significant at the 95% confidence level. Statistical analyseswere conducted using Minitab 14.0 software (Microsoft).

RESULTS

Graft Morphology

In general, all sliced muscle grafts (from MyoD (2/2)and Balb/c donors) were almost fully regenerated by 1week after grafting, with occasional inflammatory cellsremaining within some grafts, interspersed among

newly formed myotubes. Occasional myofibers sur-

269MIGRATION OF MyoD NULL MYOBLASTS IN VIVO

vived the grafting procedure (not shown), but the ma-jority of donor myofibers underwent necrosis and re-generation. Since muscle graft regeneration in mice isusually complete by 1 week and this was the earliesttime point examined in this study, myotubes had al-ready been formed and there was no evidence of sig-nificant necrosis (or graft rejection) in any of the 24grafts in the present study. At 3 weeks after grafting,no inflammatory cells remained and all grafts werecomposed of many densely packed, newly formed (cen-trally nucleated) myofibers, which contained predomi-nantly male donor (i.e., Y-1-positive) nuclei (Figs. 1aand 1b). Graft morphology did not change significantlybetween 3 and 12 weeks after grafting. There were noobvious differences in the morphology of host muscles

FIG. 1. In situ hybridization analysis using the Y-1 probe for mathe Y-1 probe are clearly shown within the graft (G) and within hdifference in the number of male donor nuclei (arrows) from (a) MyoDmuscle (H). In contrast, at 12 weeks after grafting there were (c) mmuscle some distance from the graft compared with (d) only occasiofrom the graft in female Balb/c host mice. Bars, 50 mm.

either between the groups or over time.

Numbers of MyoD (2/2) and Balb/c Donor Nucleiwithin Host Muscle

In situ hybridization with the Y-1 probe demon-strated male donor nuclei within female host muscle inall samples. At 1 and 3 weeks after grafting there wereonly occasional male donor nuclei located within thefemale host muscle and there were no obvious qualita-tive differences in the presence of male donor myo-blasts within female host muscle between MyoD (2/2)(Fig. 1a) and Balb/c (Fig. 1b) donor sliced muscle grafts.This was confirmed by quantitative analysis, whichshowed that at 1 and 3 weeks after grafting there wereno significant differences in the mean total number(i.e., fused and interstitial) of MyoD (2/2) and Balb/c

onor nuclei within female host muscle. Male nuclei hybridized withmuscle (H) in (a) and (b). At 3 weeks after grafting there was no/2) and (b) Balb/c sliced muscle grafts (G) within female Balb/c hostmale MyoD (2/2) donor nuclei (arrows) within female Balb/c hostmale Balb/c donor nuclei (arrows) that were located some distance

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270 SMYTHE AND GROUNDS

(Fig. 2). At 12 weeks after grafting, the digitized im-ages of the grafts obtained using the MDPlot programalso demonstrated that more MyoD (2/2) (Fig. 3a)han Balb/c (Fig. 3b) male donor nuclei were presentithin female host muscle, and more MyoD (2/2) (Fig.c) male donor nuclei were present within female hostuscle tissue compared to the earlier time points (Fig.

a). This was in striking contrast to the sliced musclerafts from male Balb/c donor mice at 12 weeks (Fig.d), where the number of male donor nuclei withinemale host was similar to that at earlier times (Fig.b). Quantitative analysis confirmed that at 12 weekshere were significantly more MyoD (2/2) donor nucleiithin host muscle compared with Balb/c donor myo-lasts (P 5 0.016; Fig. 2). There was a significantncrease in the mean number of MyoD (2/2) donoruclei within female host muscle between 3 and 12eeks (P 5 0.034), indicating either that donor myo-last migration out from MyoD (2/2) sliced musclerafts continued through these long-term time pointsr that donor myoblasts underwent proliferationithin host muscle.

istance of Migration of MyoD (2/2) and Balb/cDonor Myoblasts

The distance that MyoD (2/2) and Balb/c male donoruclei were located from the nearest graft border wasalculated from digitized images taken from tissue sec-ions. At 1 week after grafting there was no significantifference between MyoD (2/2) and Balb/c donors inhe mean distance that donor nuclei were located fromhe graft (Fig. 4). There was also a similar pattern ofistribution of MyoD (2/2) and Balb/c donor nuclei athis time, as shown by the data for the distance trav-led by individual nuclei in Fig. 4. In contrast, at both

FIG. 2. Total number of MyoD (2/2) (solid bars) and controlBalb/c (open bars) male donor nuclei present within female Balb/chost muscle at 1, 3, and 12 weeks after sliced muscle grafting. Valuesshown are mean total number 6 standard deviation. Differencesbetween the two treatment groups (MyoD (2/2) versus Balb/c do-nors) at each time point were determined by one-way ANOVA (*P ,0.05).

and 12 weeks after grafting, MyoD (2/2) donor nu-

lei had migrated significantly further out from theraft and into host muscle compared with the Balb/conors (Fig. 4). The mean distance for MyoD (2/2)onor nuclei at 3 weeks was elevated by a single donorell that was located approximately 2.7 mm from theraft (Fig. 4), and at 12 weeks by at least eight donoruclei that were located further than 2 mm away fromhe graft (Fig. 4).

MyoD (2/2) donor nuclei continued to migratehrough host muscle over time as indicated by signifi-ant increases in their mean distance from the graftetween 1 and 3 weeks (P 5 0.025), and between 3nd 12 weeks (P 5 0.002), after grafting. Balb/c donoruclei were located significantly closer to the graft at 3eeks compared with the distance at 1 and 12 weeks

FIG. 3. Digitized images of tissue sections showing male donorsliced muscle grafts (small enclosed areas) lying within female hostmuscle. Markers (* and x) indicate where male donor nuclei weredetected within female host muscle. Male donor nuclei within thesliced donor muscle grafts were numerous and not plotted. (a) MyoD(2/2) sliced muscle graft at 12 weeks after grafting showing manymale donor nuclei (*) within female host muscle compared with (b)Balb/c sliced muscle graft at 12 weeks where there were compara-

tively few donor nuclei (x) within host muscle.

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271MIGRATION OF MyoD NULL MYOBLASTS IN VIVO

(P , 0.005), although the mean distance was verysimilar at 1 and 12 weeks, suggesting that overallthere was very little change in Balb/c donor myoblastmigration over time.

DISCUSSION

In this study we have demonstrated unequivocallythat the absence of the myogenic regulatory geneMyoD in donor myoblasts significantly enhances theirmigration into host muscle from sliced muscle grafts.The overall number of MyoD (2/2) donor nuclei withinhost muscle at each sampling time and their meandistance from the graft increased between 3 and 12weeks. This indicates that MyoD (2/2) sliced musclerafts provide a supply of donor myoblasts available toigrate into host muscle for a relatively long period of

ime (i.e., 12 weeks) or, alternatively, that once MyoD2/2) donor myoblasts entered the host muscle theynderwent extensive proliferation.Although this study has clearly shown that myo-

lasts transplanted from MyoD (2/2) donor muscleave a greater capacity for dispersal within host mus-le in vivo, the mechanisms by which this occurs areot clear. Skeletal muscle regeneration in MyoD (2/2)ice in vivo is transiently delayed, owing to a pro-

onged phase of myoblast proliferation and a delay inhe onset of myoblast fusion [24]. Since the prolongedhase of MyoD (2/2) myoblast proliferation occurs

during the regeneration phase within the first weekafter muscle grafting [24], an early (i.e., 1 week) effecton myoblast migration was predicted. However, whilemale MyoD (2/2) donor myoblasts showed a greater

ropensity for migration into female host muscle com-

FIG. 4. Distance of individual male MyoD (2/2) and Balb/c do-nor nuclei (}) from the nearest sliced muscle graft border in femaleBalb/c host muscle. The mean (small bars) 6 standard deviation(large bars) for each group are indicated. Differences between thetwo treatment groups at each time point were tested for statisticalsignificance by Kruskal–Wallis one-way analysis of variance (*P ,0.05; **P , 0.005).

ared to normal (Balb/c) donor myoblasts, this effect t

was seen only at 3 and 12 weeks after grafting. Thissuggests that the majority of both MyoD (2/2) andcontrol Balb/c donor myoblast migration into host mus-cle occurred within the first week after grafting, andthat the MyoD (2/2) donor myoblasts subsequentlyunderwent further proliferation to result in largernumbers of donor myoblasts, and a greater mean mi-gratory distance. While the sustained proliferationmay simply reflect a disturbance of myoblast cell cycleregulation [24], it has also been proposed that theMyoD (2/2) myoblasts may have similarities to stemcells [31, 32], which are characterized by an extendedcapacity for proliferation. This issue remains to beresolved. The results for the Balb/c donor grafts are inaccordance with our previous studies that showed nosignificant changes over time (up to 12 weeks) in thetotal number and the mean migratory distance of do-nor muscle nuclei out from normal sliced muscle grafts[9, 29].

MyoD (2/2) donor myoblasts were located up to 3mm from the graft, in contrast to the controls and ourprevious studies [33], where normal donor myoblastswere observed no further than 2 mm from the graft.Therefore, we have demonstrated that MyoD (2/2)donor myoblasts have the potential to migrate up to 1mm further into host muscle compared with normalcontrol donor myoblasts (over 3 weeks). These dis-tances, while highly significant in the mouse, are lessstriking in terms of the size of human muscles, and thisemphasizes the importance of parallel studies in largeranimal models. It is not clear whether the enhancedmigration of MyoD (2/2) donor cells observed in thepresent study mainly reflects greater overall numbersof cells, or whether it truly reflects enhanced migrationof individual myoblasts. It should be noted that al-though we have not specifically distinguished betweenmyoblasts and other male donor cell types (e.g., fibro-blasts), the specificity of the MyoD gene for myogeniccells indicates that the influence of the absence of thisgene is reflected only in behavioral changes by myo-blasts. It should be noted that the in situ hybridizationtechnique with the Y-1 probe underestimates the totalmale donor nuclei present, as the probe binds to DNAin only a portion of the nucleus in the 5-mm-thickection [34]. In this study we did not examine whetherale donor cells migrated into other host muscles ad-

acent to the TA muscle. Such migration was not an-icipated, since previous studies have demonstratedhat donor myoblast migration between donor and hostuscles is limited unless the donor and host muscle

pimysia are disrupted [28, 35, 36] or host muscleegeneration is induced, which results in the release ofyoblast chemoattractants [37–39]. However, several

f these studies used immortalized C2C12 myoblastsather than normal donor myoblasts in primary cul-

ures [37, 38], and the superior migration of C2C12

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272 SMYTHE AND GROUNDS

myoblasts was recently attributed to high levels ofexpression of matrix metalloproteinases comparedwith normal transplanted donor myoblasts [11].

While we have not established exactly how the ab-sence of MyoD promotes donor myoblast migration invivo, the results of this study strongly support theproposal that enhancing donor myoblast proliferationincreases myoblast dispersal throughout host muscle.This approach may be applied clinically, using the “exivo” approach, whereby cultured donor cells are ge-etically engineered to alter or induce the expression ofertain genes prior to transplantation in vivo [19–23].astry and colleagues [19] demonstrated that transfec-ion of cultured myoblasts with antisense vectors tontegrin a6 resulted in enhanced myoblast prolifera-

tion and decreased differentiation. This ex vivo use ofntisense therapy could similarly be applied to blockther molecules whose absence has been shown to cor-elate with enhanced myoblast proliferation such asyoD (as shown here), desmin [39b], and transforming

rowth factor-b (TGF-b) [40, 41]. A modification of thisapproach is to induce the overexpression of autocrine/paracrine myoblast mitogens, such as hepatocytegrowth factor [42, 43], insulin-like growth factor-1 [21,44–46], fibroblast growth factors [47, 48], or leukemiainhibitory factor [49, 50].

The most widely used technique to modify such geneexpression in myoblasts is to apply viral vectors suchas retroviruses [51, 52] and adenoviruses [23, 53–55].Another way to deliver small peptide and nucleotidesequences into myoblasts is using “trojan” peptidessuch as penetratin and TAT, which can readily entercells and transiently transfect target cells with “cargo”proteins (reviewed in [56]).

An alternative approach is to administer exogenousgrowth factors that are known myoblast mitogensand/or chemoattractants. Culturing donor myoblastsin the presence of bFGF has been reported to increasemyoblast dispersal in vivo; however, this may be aresult of enhanced survival or proliferation [16, 17].The enhanced myoblast proliferation resulting fromneutralizing antibodies to TGF-b during skeletal mus-cle regeneration in vivo [40] suggests another methodby which the growth factor environment in host musclecan be manipulated to promote the proliferation ofinjected cultured donor myoblasts in MTT.

In summary, this study has demonstrated that theabsence of the myogenic regulatory factor MyoD indonor sliced muscle grafts results in enhanced migra-tion of donor myoblasts into host muscle. This is mostlikely due to sustained endogenous proliferation of thetransplanted donor myoblasts so that more donor myo-blasts are available to emigrate into the host muscleprior to fusion. These results strongly support the no-tion that increased donor myoblast proliferation re-

sults in enhanced migration into host muscle, and en-

dorses the use of techniques to improve donor myoblastproliferation in conventional MTT.

This work was generously supported by a fellowship (G.M.S.) andresearch grant (M.D.G.) from the Association Francaise contre lesMyopathies. The authors acknowledge the assistance and provisionof digitizing equipment by A/Prof Alan Harvey (Department of Anat-omy and Human Biology, The University of Western Australia).

Note added in proof. Another factor possibly contributing to theenhanced migration of MyoD null myoblasts might be reduced at-tachment of these myoblasts to the ECM due to altered expression ofcell adhesion molecules on myoblasts lacking MyoD. (Julis Huijbre-gets, Jason D. White, and Miranda D. Grounds, 2001) The absence ofMyoD in regenerating skeletal muscle affects the expression patternof basement membrane, interstitial matrix and integrin molecules,consistent with delayed myotube formation. Acta Histochem., inpress.

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eceived December 18, 2000evised version received April 18, 2001