Segregation of the myogenic cell lineage in mouse muscle development

GAKETH E. JONES1, SUSAN J. MURPHY1'2 and DIANA J. WATT2'*

1 Anatomy and Human Biology Group, Division of Biomedical Sciences, King's College, London WC2R 2LS, UK^Department of Anatomy, Charing Cross & Westminster Medical School, Fulham Palace Road, London W6 8RF, UK

* Author for correspondence

Summary

With increasing interest in the idea of therapeuticimplantation of normal muscle precursor cells intomuscle lacking the protein product of the dystrophingene, it has become important to obtain enrichedpopulations of myogenic cells from biopsied musclesources. Myogenic cells for implantation are highlyfavoured as they are the only cells that will fusereadily with host muscle fibres into which they areimplanted, thus carrying the introduced gene intothe target fibre with the maximum of efficiency.Second, myogenic cells appear less immunogenicthan those of a non-myogenic nature; and third, theuse of mononuclear myogenic cells may permit theintroduction of multiple copies of a deficient geneinto the patient's own cells.

From a mixed population of cells obtained by theenzymic disaggregation of neonatal murine musclewe have selected, utilising a modification of thepanning technique, for a cell population rich inmyogenic cells. Segregation was accomplished using

Mab H28, an antibody to the mouse neuronal celladhesion molecule (N-CAM), derived from mouse/rathybridoma cells. Following incubation with Mab1128, disaggregated muscle was applied to the sur-face of a bacteriological grade dish previously coatedwith anti-rat immunoglobulin. Cells segregated intotwo populations; those bearing N-CAM, and hencelabelled with Mab H28, were adherent to the dish,whereas those not expressing N-CAM remained insuspension.

Use of this technique, which involves minimal cellloss, resulted in the segregation of prefusion myo-genic cells together with fibroblasts in the 'non-adherent' fraction, whereas cells in the adherentfraction consisted of a highly enriched population ofactively dividing myogenic cells.

Key words: myogenesis, cell isolation, myoblast proliferation.

Introduction

The recent discovery (Hoffman et al. 1987) of an abnormal'dystrophin' gene product in the muscle fibres of patientssuffering from the Duchenne (DMD) and Becker (BMD)forms of muscular dystrophy, has increased the possibilityof alleviating these primary myopathic diseases of skeletalmuscle by the introduction of normal genes into thediseased muscle fibre. The use of gene therapy in thecorrection of inherited disease has been contemplatedusing other tissue systems, and work on animal modelshas already shown that incorporation of normal genes into

• bone marrow results in raised levels of enzymes that aredeficient in the normal host (Miller et al. 1984; Dick et al.1985; Williams et al. 1986). More recent work has alsoindicated that genetically modified liver cells survivewhen implanted into the host animal, suggesting thatimplantation of such modified cells could well be used toalter an inherited defect in this system (Demetriou et al.1986; Gupta et al. 1987). Such an approach of introducingnormal genes into abnormal muscle cells is now beingcontemplated (Law et al. 1988; Karpati, 1986; Watt et al.1984; Partridge et al. 1989), for the multinucleate musclefibre lends itself very well to this sort of manipulation.Skeletal muscle fibres both develop and regenerate by thefusion of mononuclear muscle precursor cells to formmultinucleate fibres, in which many hundreds of nuclei

Journal of Cell Science 97, 669-667 (1990)Printed in Great Britain © The Company of Biologiata Limited 1990

each export their gene products into the sarcoplasmicenvironment surrounding such nuclei. Further, it is nowwell established from animal models that donor muscleprecursor cells of one genotype introduced into the musclefibres of a genetically different, but immunologicallytolerant, strain of recipient animal, will fuse withrecipient muscle fibres to form mosaic host/donor fibres(Watt et al. 1982,1984), within which donor gene productsare expressed. More recent work has shown (Partridgeet al. 1989) that a deficiency of the gene product dystro-phin, which is lacking in the skeletal muscle fibres of bothDMD patients and their mouse counterparts - the mdxstrain - is compensated for by the incorporation of normaldonor nuclei within the mouse dystrophic muscle fibres; anincrease of up to 70 % of dystrophin gene product beingfound in such modified mdx muscle fibres.

The therapeutic use of allografts of muscle precursorcells in the human raises several problems, including notonly immune rejection, although this may not prove to besuch an unsurmountable problem in the case of muscle(Karpati, 1989; Watt, 1990), but also the source and natureof the donor cells to be implanted. Ideally, to allowmaximum fusion with existing myopathic host fibres,introduced donor cells should be myogenic in nature. Theobvious source of donor cells for implantation is foetalmuscle, but the limitations in the use of such a tissue are:first, the restricted muscle mass that can be harvested;

659

and second, the presence of large numbers of non-myogenic cells, particularly fibroblasts, within the tissue.Clearly, one approach that would compensate for thesedeficits would be to develop a method that could separatedonor muscle precursor cells from contaminants and thenamplify the muscle precursor cells by in vitro culture toboost the available cells for implantation into myopathichost muscle.

From a mixed population of myogenic and fibroblasticcells obtained by enzymically disaggregating neonatalmouse muscle, we have selected for a myogenic cellpopulation using a modification of the panning technique,first introduced by Wysocki and Sato (1978) and success-fully used to enrich selectively for human epidermal cellsubpopulations by Morhenn et al. (1983) and Linge et al.(1989). In this study selection of myogenic cells fromcultures of mouse muscle has been achieved using theantibody to the neuronal cell adhesion molecule N-CAM.Myogenic cells, unlike fibroblastic cells, express receptorto this antibody on their surfaces (Walsh and Moore, 1986).Using such a method a population of cells enriched for themyogenic component has been isolated and cultured invitro.

Materials and methods

Enzymic disaggregation of neonatal mouse muscleMyogenic and fibroblastic cells to be separated using the panningtechnique were prepared by enzymically disaggregating neonatalskeletal muscle from two strains of normal mice: CBA andC57Bl/lOScSn. Muscle was disaggregated as previously described(Watt etal. 1982), with the following modifications: 0.15 g ofmuscle removed from the limbs and back of a neonatal donor wasfinely cut with curved scissors and divided into five equalportions, each of which was placed in a universal containercontaining 5 ml of enzyme solution (0.5 % Pangestin, 1 % trypsinin Ca2+- and Mg2+-free Hanks' salt solution buffered to pH7.4with Hepes buffer). Each universal container was incubated at37°C in a shaking water bath for 7 min, after which the contentswere aspirated in and out of a Pasteur pipette for a further 7 minin order to disaggregate the muscle tissue. The supernatantcontaining any single cells was then removed and the action of theenzymes stopped by pouring the supernatant into 5 ml of coldinhibition medium consisting of 80 % Medium 199 with Earle'ssalts, Hepes buffer and glutamine, 20% foetal calf serum,SOi.u.ml"1 penicillin, SOmgml"1 streptomycin. A further 5 ml ofenzyme solution was added to any remaining muscle fragments ineach of the universal bottles, and the disaggregation repeated. Allbatches of inhibition medium containing the supernatants fromboth disaggregations were filtered through three layers of 45 fanpore size nylon cloth and the resulting pooled cell suspension wasaspirated in and out of a Pasteur pipette in order to disperse anyremaining clumps of cells. The yield of viable cells, i.e. those thatexcluded Trypan Blue, was estimated by counting in a haemocyt-ometer. The cells were resuspended in growth medium consistingof 88% Medium 199 with Earle's salts, Hepes buffer andglutamine, 10 % foetal calf serum, 2 % chick embryo extractcontaining SSi.u.ml"1 penicillin and SSmgml"1 streptomycin.Cells were plated out at 2 x 106 cells ml"1 into 25 cm2 growth-areaculture flasks coated with 0.01 % gelatin and grown in culture for4 days at 37 °C in 5% CO2.

Pre-separation of myogenic and fibroblastic cells, prior topanningMixed cultures of myogenic and fibroblastic cells obtained by theenzymic disaggregation of neonatal mouse muscle are very rich inthe fibroblastic component of the culture. In some of ourexperiments, in an attempt to reduce the number of fibroblastspresent in the population to be panned, differential adhesion wasused as the first step in the separation of fibroblastic and

myogenic cells. Suspensions of mixed fibroblastic/myogeniccultures were initially plated out onto non-gelatinised tissueculture flasks for 40 min. During this time some of the cellsadhered to the substratum, these cells being largely fibroblastic innature (Yaffe, 1968). The remaining non-adherent cells, contain-ing a higher proportion of myogenic cells than those that initiallyadhered to the substratum, were removed with the medium into asecond tissue culture flask. Cultures in which differentialadhesion had been employed prior to panning were designated'separated' cultures, and those in which no such step was initiallyemployed were designated 'unseparated' cultures.

Segregation of myogenic and fibroblastic populationsusing the panning techniqueThe method used to separate myogenic from fibroblastic cells wasadapted from that used by Morhenn et al. (1983) and Linge et al.(1989) with the following modifications: mixed cultures ofmyogenic and fibroblastic cells prepared from the enzymicdisaggregation of neonatal mouse muscle were grown in culturefor 4 days. In some experiments, cells separated by differentialadhesion methods (see Materials and methods) were used as thecultures to be panned. Cells were trypsinised from the cultureplate using 0.25 % trypsin in Hanks' solution (Flow Laboratories),a cell viability count was performed and cells were spun at 350 #for 10 min at 4°C to yield pellets of 4x 106 cells. Following removalof the supernatant, the cell pellet was agitated and thenincubated on ice for 1 h with 100 /.A of hybridoma supernatantanti-N-CAM, designated H28, from a rat/mouse hybrid hybrid-oma, kindly donated by Professor F. Walsh. Meanwhile, thesurface of a 30 mm diameter bacteriological grade Petri dish(Sterilin Ltd), which does not normally support the adhesion ofcells, was evenly coated with 10/jgml goat anti-rat IgG (SigmaChemical Co. Ltd) in 0.05 M Tris-HCl buffer, pH 9.5. Dishes wereincubated for 40 min at room temperature, washed three times inphosphate-buffered saline (PBS) and twice with 5% fetal calfserum (FCS) in PBS.

After incubation in primary antibody H28, cells were washed,resuspended in 2 ml of 5 % FCS/PBS, carefully layered onto thesurface of the prepared bacteriological grade dishes and incubatedat 4°C for 40 min, gently swirled by hand, and then the incubationwas repeated for a further 40 min. Following incubation, non-adherent cells were removed by very carefully pouring off thesupernatant, the surface of the dish was washed twice with 5 %FCS/PBS, and the two washes were pooled with the collectedsupernatant. These cells, which had not adhered to the surface ofthe IgG-coated bacteriological grade dish, were designated the'non-adherent' fraction. To remove cells that had adhered to thesurface of the dish, a further sample of 5 % FCS/PBS was added tothe Petri dish and triturated with a sterile Pasteur pipette. Thedish was washed twice more with 5 % FCS/PBS and these washeswere pooled with the adherent cells collected by trituration. Thisfraction was therefore designated the 'adherent fraction'. Bothfractions were spun in a centrifuge at 4°C for 10 min at 350 # toobtain cell pellets, which were reauspended in 1 ml of growthmedium consisting of 88 % Medium 199 with Earle's salts, Hepesbuffer and glutamine, 10 % fetal calf serum, 2 % chick embryoextract containing 88 i.u. ml"1 penicillin and 88 mg ml"1 strepto-mycin. Cell viability counts were performed on each fraction priorto plating out the cells on tissue culture dishes.

Control cultures used during panningFour types of controls were routinely used. Control cultures weresubjected to all procedures involved in panning, with theexception that: group 1 controls were not incubated with primaryantibody; group 2 controls were incubated with primary antibody,but no secondary antibody was used to coat the Petri dish duringthe 80-min incubation in the bacteriological grade dish; in group 3controls both primary and secondary antibodies were omitted;group 4 controls were not subjected to panning procedures, initialmixed cultures being trypsised from the growth surface andreplated out onto tissue culture dishes.

Culturing of cells following panningFollowing resuspension of both the non-adherent and adherent

660 G. E. Jones et al.

fractions in lml of growth medium as described above, cellviability counts were performed and the number of cells wasadjusted to 10s cells ml"1 of medium. Cell suspensions were platedout at this cell density into 30 mm tissue culture grade (FalconLtd) Petri dishes that had previously been coated with 0.01%sterile gelatin. Cultures were grown at 37.5°C in 5% CCy Inaddition, 50 (A of each cell fraction was added to each well of amultitest slide (ICN-Flow Laboratories Ltd) to enable laterimmunofluorescent labelling with antibodies to intermediatefilament proteins and to anti-N-CAM antibody H28.

Immunofluorescent characterisation of cellsOwing to the unreliability of morphological criteria in assessingthe myogenic and fibroblastic nature of cells in culture, antibodiesto the intermediate filament proteins vimentin and desmin wereused to characterise the nature of cells present in the resultingtwo fractions following panning of cultures. Subsequent to theseparation of the two cell populations, i.e. adherent and non-adherent, 50 jA of cell suspension from each fraction was pipettedinto each of the 10 cavities present in the multitest slide. Afterallowing attachment and spreading of the cells overnight understandard conditions, each fraction was stained using antibodies(Sigma Chemical Co. Ltd) to the two cytoskeletal proteins, thesecondary antibody in each case in the characterisation of celltype being FITC-conjugated anti-IgG (Sigma Chemical Co. Ltd).The cytoskeletal protein desmin is found predominantly inmuscle cells, but is absent from fibroblasts. Vimentin-containingfilaments are found in cultured fibroblasts, although they are notexclusive to this cell, as they are also found within other celltypes, including myogenic cells (Stewart, 1990).

Assay of fusion indicesAfter 3 days in culture, cells were fixed in methanol, and countsperformed according to the method of Konigsberg (1963) toascertain the number of nuclei present in mononuclear cells andthose present in multinuclear cells, in both the non-adherent andadherent fractions separated during the panning technique.

Counts were performed using a 19 mm square grid graticule, atotal of five fields being counted for each culture. The nuclei wereclassified as belonging to one of three groups: (1) nuclei presentwithin mononuclear cells; the latter that by morphologicalcriteria appeared fibroblastic in nature; (2) nuclei present withinmononuclear cells; the latter that by morphological criteriaappeared myogenic in nature; (3) nuclei present within multinuc-lear myotubes.

Detection of N-CAM post-panningAs previously described (Materials and methods), 50 ^1 samples ofcell suspension from both adherent and non-adherent fractionswere applied to the wells of multitest slides (Flow LaboratoriesLtd) and immunocytochemically stained at both 24 and 72 h post-

panning to detect the presence of N-CAM on the cell surface usingimmunofluorescent staining techniques. Cells were incubated forl h in a 1:100 dilution of anti-N-CAM antibody H28, washed inPBS, incubated for a further hour with 1:40 dilution offluorescein-conjugated goat anti-rat IgG, and post-fixed in 2%par aformaldehyde.

Results

Appearance of cultures prior to panningFig. 1 shows a mixed culture of myogenic and fibroblasticcells 4 days after plating out the cell suspension derivedfrom enzymically disaggregated neonatal CBA strainmuscle that had not been subjected to differentialadhesion prior to employing the panning technique, i.e. an'unseparated' culture. Cells that by morphological criteriaappear as fibroblastic predominate, although many mono-nuclear myogenic cells are also evident. In 'separated'cultures, where the original cell suspension was subjectedto 40 min differential adhesion in an attempt to reduce thenumber of fibroblasts present in one of the fractions to bepanned, two populations of cells were achieved: cells thatadhered rapidly to the substratum during this timepredominantly appeared fibroblastic in character; thisseparated culture was designated 'separated fibroblastic'whereas the fraction derived from cells that had notadhered to the substratum during this initial 40 mincontained many more cells that appeared myogenic bymorphological criteria, i.e. 'separated myogenic' culture.Both unseparated cultures and the two fractions of cellsobtained after 40 min of differential adhesion were used inpanning.

Panning of culturesControl groups. Table 1 indicates the cell counts per-

formed on all cultures both prior to (column 3) and after(columns 4 and 5) panning; column 6 indicates thepercentage total recovery after panning and column 7 thepercentage of recovered cells present in the adherentfraction. In all control groups, the cell pellet to be pannedcontained 4 x 10s cells, except in one case where thenumber of cells was very much less. Recovery of cellsfollowing panning of group 1 controls, where onlysecondary, but no primary, antibody was used to coat thebacteriological grade dish, was low, amounting to only 43and 35 % (Table 1); the percentage of the recovered cells

Fig. 1. Mixed myogenic/fibroblastic culture 4 daysafter enzymic disaggregation of donor muscle, butprior to punning Cells are all mononucleate butexhibit both stellate (arrowed) fibroblastic andbipolar (*) myogenic morphology. Bar, 200 /an.

Segregation of myogenic cells 661

Table 1. Cell viability counts prior to and after panning

Strain

CBACBACBACBACBAC57B1C57B1CBACBACBAC57B1CBA

Initial culture

UnaeparatedUnsepa ratedUnsepa ratedSeparated myogenicSeparated myogenicSeparated myogenicSeparated fibroblasticControl group 1Control group 1Control group 2Control group 3Control group 4

No. cellapanned(xlO8)

3.674.004.001.803.203.003.604.004.002.364004.00

Cell counts

Adherent(xlO6)

0.4502.2251.2O01.3501.2002.2000.8000.3750.262

——_

after panning

Non-adherent(xlO6)

187516001.6000.1601.7600,6505.7001,3501.1261.9403.260

—

% Recoveryof cellsafter

panning

6393688392959743368382_

%ofrecovered

cells presentin adherent

fraction

196044904172222219—

—

The initial type of culture and number of cells to be panned IB indicated in columns 2 and 3, respectively. The number of cells present in eaqhfraction subsequent to panning is indicated in columns 4 and 6; the percentage of cells recovered after panning indicated in column 6; and of thoserecovered, the % present in the adherent fraction, in column 7.

that were isolated in the adherent fraction was 22 and19%, respectively. These controls, however, yielded twofractions of cells, i.e. adherent and non-adherent, presum-ably the secondary antibody coating of the bacteriologicalgrade dish provided a substratum for some cells to adhereto. Initially, at plating out, the numbers of cells in theadherent fraction were less than those in the non-adherent. By 72 h in culture the two fractions wereindistinguishable from each other, consisting of a densemat of fibroblasts overlain with developing myotubes(Fig. 2). The omission of primary antibody, as in group 1controls, thus failed to segregate the two cell populations.In group 2 controls, where the initial number of cells to bepanned was less than in other control groups, cells wereincubated with primary antibody, no secondary antibodybeing used to coat the bacteriological grade dish. In suchcases, cells from the adherent fraction, when viewed usingthe haemocytometer, were so few in number that nosatisfactory cell count could be provided. This also provedto be the case for the adherent fraction of group 3 controls,where neither primary nor secondary antibody was used.The lack of secondary antibody in group 2 and 3 controlsthus resulted in the failure of cells to adhere to the surfaceof the bacteriological grade dish. Group 4 controls, wherecells from the initial mixed culture were trypsinised fromthe growth surface and replated out onto tissue culture

grade dishes, without panning, continued to grow asmixed myogenic/fibroblastic cultures. Cell counts werenot performed on such controls subsequent to trypsinisa-tion.

Experimental groups. In the majority of experimentalcases the number of cells to be panned ranged from 3xlO5

to 4xlOB cells (Table 1, column 3), although in one'separated myogenic' culture the number was very muchlower. In three instances where unseparated cultures werepanned, the recovery of cells after panning ranged from 63to 93% (see Table 1); 19, 60 and 44% of such recoveredcells being isolated in the adherent fraction. Thisvariability showed no definite trend (such as withincreasing experience of operators) and we presume thatthis reflects variable proportions of fibroblastic or myo-genic cells present in initial cultures.

Where initial cultures were of the 'separated myogenic'type, recovery of cells post-panning was high, i.e. 83, 92and 95%, respectively, in the three experiments per-formed. The majority of cells, i.e. 90 and 72 %, subsequentto panning were found in the adherent fraction in two ofthree cultures studied, although this was not the case withthe third, where cell numbers were almost equal for eachof the two panned fractions (41 % for the adherent and 59 %for the non-adherent fraction). In the one culture of'separated fibroblastic' cells used for panning, cell recovery

\ '•

Fig. 2. Adherent fraction obtained post-panning ofgroup 1 control culture, 72 h after seeding out.Cultures are characterised by a dense mat ofmononuclear fibroblasts (*) overlain withmultinucleate myotubes. Bar, 200 ;an.

662 G. E. Jones et al.

Fig. 3. Adherent fraction of cells achieved onpanning using H28 N-CAM antibody as primary,followed by goat anti-rat IgG as secondary antibody.The culture, viewed 72 h after plating out of theadherent fraction, consisted of many small foci ofmononuclear cells, which by morphological criteriaappeared to be myogenic. Bar, 200 ;<m.

post-panning was again high, being 97 %, the majority ofcells (78 %) being found within the non-adherent fraction.Subsequently, routine use of this method in our laboratoryhas demonstrated that some 85% of each cell typesegregates into the appropriate fraction following pan-ning.

The appearance of cultures derived from the adherentpost-panned fraction were similar, regardless of the initialculture panned, i.e. unseparated, separated myogenic, orseparated nbroblastic. At 24 h post-panning such culturescontained many isolated foci of cells of 'rounded' morpho-logical appearance. By 48-72 h in culture the majority ofthese cells had assumed a bipolar shape but remained asmononuclear cells, the cultures being characterised bymany small individual clones of cells assuming suchmorphology (Fig. 3). Very few fibroblastic cells wereobserved between the myogenic foci in such fractions. Incontrast, 24 h after plating out, the non-adherent fractionwas characterised by the presence of a mat of fibroblasticcells interspersed with cells of bipolar shape; the latter, bymorphological criteria, appeared to be myogenic in nature.This was further borne out at a later stage in culture, i.e.72 h, when the non-adherent fraction was found to consist,as at earlier stages in culture, of a mat of fibroblastic cells,but at this stage in culture such cells were overlain

with many multinucleate myotubes (Fig. 4). This is asurprising finding in view of the fact that as such cellswere of myogenic character they would have beenexpected to have bound anti-N-CAM to their surface andthus have segregated with the adherent fraction. Thedifference, however, between the myogenic cells concen-trated in the adherent fraction and those found in the non-adherent fraction at this time (72 h) was the lack of fusion,even at 5 days post-panning, between mononuclear cellsthat by morphological criteria appeared myogenic in theadherent fraction and those found in the non-adherentfraction, which by 3 days post-panning had already fusedto form relatively mature multinucleate myotubes. Mono-nuclear cells isolated in the adherent fraction however dideventually fuse to form multinucleate myotubes (Fig. 5)following 7-9 days of active proliferation in culture,showing that these cells were indeed myogenic.

Immunofluorescent characterisation of cellsOwing to the unreliability of morphological criteria inassessing the myogenic and fibroblastic nature of cells inculture, antibodies to the cytoskeletal proteins vimentinand desmin were used to characterise cell types present inthe two fractions following panning of cultures. Fig. 6shows one focus of cells that segregated in the adherent

Fig. 4. Non-adherent fraction of cells achieved onpanning using H28 N-CAM antibody as primary,followed by goat anti-rat IgG as secondary antibody.In contrast to the adherent fraction, these culturesat 72 h consisted of a mat of fibroblasts overlainwith many multinucleate myotubes. Bar, 200 ;an.

Segregation of myogenic cells 663

Fig. 5. Nine days after panning, cultures derivedfrom the adherent fraction contained multinucleatemyogenic cells formed by the fusion of themononuclear cells characteristic of earlier adherentcultures. Bar, 200 [<m.

fraction and stained with anti-desmin antibody 3 dayspost-panning. The majority of these cells, which morpho-logically were rounded or bipolar in shape, stain positivelyfor anti-desmin, indicating that cells segregating in theadherent fraction are of myogenic nature. In contrast, themajority of cells in the non-adherent fraction, which are ofstellate morphology when viewed using phase-contrast(Fig. 4), failed to show fluorescence when stained withanti-desmin antibody, although multinucleate cells, recog-nised by phase-contrast microscopy as multinucleatemyotubes, were positively stained using anti-desmin(Fig. 7). This result indicated that the mat of cellsunderlying the multinucleate cells in the non-adherentfraction were non-myogenic in nature, bearing out theobservation from morphological examination of these cellsthat they were fibroblasts. Staining with antibody to theintermediate filament protein vimentin resulted in cells ofboth the adherent and the non-adherent fractions stainingpositively, vimentin being a cytoskeletal protein commonto both fibroblasts and myogenic cells (Stewart, 1990).

Percentage of cell types in panned fractionsCounts of nuclei present in mononuclear and multi-nucleate cells in both the non-adherent and adherentfractions post-panning are shown in Table 2A and B.Nuclear counts of the non-adherent fractions achieved onpanning of unseparated cultures (Table 2A) indicatedthat, with time in culture, a larger proportion of nucleiwere counted within cells that morphologically, and byusing antibodies to the intermediate filaments vimentinand desmin, were recognised as fibroblasts. At the earlieststages post-panning, i.e. 24 h, 72 % of nuclei were scored asbeing present within mononuclear myogenic cells,although the number present within such cells declinedwith time post-panning as the number of nuclei presentwithin multinucleate myotubes increased. Nuclear countsfrom such a non-adherent fraction indicated that fibro-blastic cells segregated in this fraction as did cells of amyogenic nature, which by 3 days in culture had fused toform multinucleate myotubes. In marked contrast to thisresult, the majority of nuclei (ranging from 71 to 81%) ofthe adherent fraction were found within mononuclearmyogenic cells. Few nuclei were observed within myo-tubes in this fraction. Similar results (Table 2B) wereachieved using initially separated myogenic and fibroblas-tic cultures for panning, in that the majority of nuclei in

the adherent fractions of both initially separated fibro-blastic and separated myogenic cultures were foundwithin mononuclear myogenic cells. Adherent fractionscontained fewer myonuclei within myotubes comparedwith non-adherent fractions, the former being character-ised by actively proliferating myogenic cells. Non-adher-ent fractions from separated cultures contained a highproportion of fibroblasts and, by 3 days in culture, severalmultinucleate myotubes.

Detection of N-CAM post-panningIn view of the unexpected finding of a percentage ofmyogenic cells in the non-adherent fraction followingpanning of the initial cultures, 50 ^1 samples of cellsuspension from each panned fraction were added to thewells of a multitest slide. At 24 and 72 h in culture, wellswere stained immunocytochemically to probe for thepresence of N-CAM on the cell surface. Twenty-four hoursafter panning, cells in the non-adherent fraction werefound to be stained negatively using H28 antibody (Fig. 8),whereas by 3 days post-panning multinucleate myotubesin such cultures were positively, although weakly, stainedfor this antibody (Fig. 9A), although the underlying mat offibroblasts detected by phase-contrast microscopy(Fig. 9B) failed to elicit staining with H28. No satisfactoryresults were obtained for anti-N-CAM staining of theadherent fraction 24 h after panning, due to a failure of thecells to adhere sufficiently to the glase substratum of themultitest slide during the rigours of repeated washingnecessary to the protocol. After 48 h plating out, however,myogenic cells had adhered sufficiently to allow theimmunostaining to be carried out, and such cells allstained uniformly positively with the anti N-CAMantibody (Fig. 10).

Discussion

Over the past few years there has been an increasinginterest in the possible therapeutic implantation ofnormal muscle precursor cells into myopathic muscle toalleviate or correct the underlying defect. Muscle, beingmultinucleate, will incorporate mononuclear precursorcells during growth (Moss and Leblond, 1971) or regener-ation (Carlson, 1973; Carlson and Faulkner, 1983; Liptonand Schultz, 1979; Partridge etal. 1978; Watt, 1982).

664 G. E. Jones et al.

Fig. 6. Cells segregating in the adherent fraction were immunostained with anti-desmin antibodies 3 days after panning. Cellsthat stained positively for the antibody assumed a rounded or bipolar shape. Staining of such cells is intense even when viewed atthe low magnification indicated. X150.Fig. 7. A 72 h culture derived from the non-adherent fraction and stained with anti-desmin antibody. The majority of cells forminga mat of fibroblastic cells failed to stain positively with this antibody, although cells that were recognised as multinucleatemyotubes did fluoresce. X300.Fig. 8. Non-adherent fraction 24 h post-panning stained immunocytochemically with H28 anti N-CAM antibody in order to probefor the presence of N-CAM on the cell surface. At this stage cells in this fraction stained negatively, x 150.Fig. 9. (A) Non-adherent fraction 72 h post-panning stained immunocytochemically with H28 anti N-CAM antibody in order toprobe for the presence of N-CAM on the cell surface. Only the overlying multinucleate myotubes present within the culture stainpositively for N-CAM; the underlying mat of fibroblasts visible in the phase-contrast field of this culture (Fig. SB) failing to elicitstaining with H28. X300. (B) Phase-contrast field of the same 72 h culture derived from non-adherent fraction in whichmultinucleate myotubes stained positively for N-CAM (A). The culture clearly contains both mononuclear fibroblasts andmultinucleate myotubes. Bar, 100 ̂ m.Fig. 10. A 48 h culture derived from the adherent fraction post-panning and stained immunocytochemically to probe for thepresence of N-CAM on the cell surface. Cells that stain positively at this stage in culture are mononuclear assuming a morphologytypical of myogenic cells. Even at low magnification the staining of such cells is intense. X300.

Table 2. Percentages of nuclei present in mononuclear and multinuclear cells in post-panned fractions of initiallyunseparated cultures

B

Strain

C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C67B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1C57B1

Strain

CBACBACBACBACBACBACBACBACBACBACBACBACBACBACBACBACBACBACBA

Age of culturepoat-panning

(days)

33333333333553333333333

Age of culturepoat-panning

(days)

1122274447422224222

Initial typeof separated

culture

FibroblasticFibroblaflticFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenicMyogenic

Fraction '.nchipvcH

No. of nuclei present in cell categories

on panning Myoblast

Non-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentAdherentAdherentAdherentAdherentAdherent

Fractionachieved

on panning

AdherentAdherentAdherentAdherentAdherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentNon-adherentAdherentAdherentAdherentAdherentAdherentAdherentAdherentAdherentAdherentAdherentNon-adherentNon-adherent

74601030482830

748

182931

95145756060

Fibroblast

No

296

5296

113151666485187

10496821218171615

of nuclei

Myoblast

7697837672325617151816754268487678485588902146

Myotube_8

435153

24

10392612575448-—58

-

% Nucleiin

myoblasts

72721017226075849

1515176

8171777280

. present in cell categories

Fibroblast

9161597

162151213142172123

39735637

12115415

Myotube

66

1614_

1664

314660

1715—---——6157

% Nucleiin

myoblasts

84827377911526

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Owing to the fusion of mononuclear cells to produce themultinucleate fibre, normal genes and their products canbe expressed within deficient fibres by the introduction ofnormal precursor cells. This sort of approach has becomeeven more pertinent with the recent discovery of the genedeficiency in both the Duchenne and Becker forms ofmuscular dystrophy (Hoffman et al. 1987), and the obser-vation in experimental models of expression of the missinggene product following implantation of normal precursorcells into myopathic muscle (Morgan et al. 1988; Partridgeet al. 1989). To increase the efficiency of implantation intomyopathic muscle fibres it is necessary to maximise thenumber of myogenic cells within the population to beimplanted, for only myogenic cells fuse readily with hostmuscle (Partridge et al. 1989; Watt, 1990; Watt et al. 1982).Foetal human muscle seems the likeliest source ofprecursor cells for implantation (Partridge, 1990), yet it isknown that the percentage of myogenic cells within

muscle obtained from human embryological sources can belower than 30% (Blau et al. 1983), with a high proportionof cells within the muscle being fibroblastic. Thus ifmaximum efficiency of fusion of introduced precursor cellswith host fibres is to be achieved, enrichment for myogeniccells from foetal sources would be advisable. The mostsuccessful and recently devised method for separatingcells centres on the use of the Fluorescent Activated CellSorter (FACS), which has already been employed to enrichfor myogenic cells from a mixed cell population obtainedfrom fetal muscle (Webster etal. 1988). Such a methodhowever resulted in the loss of 50 % of myoblasts from amixed cell population where, as already reported, theinitial proportion of myogenic cells can be as low as 30 %(Blau et al. 1983). This obviously presents itself as a majorproblem in myoblast implantation, in terms of obtainingsufficient cells for alleviation of the myopathic condition tohave any therapeutic value.

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The panning method has been successfully used toseparate B and T lymphocytes (Wysocki et al. 1978), anddifferent types of epidermal cells (Morhenn et al. 1983;Linge et al. 1989). In the present paper we report the use ofthe anti N-CAM antibody H28, which binds to murine N-CAM of all isoforms (Moore etal. 1987), to separatemyogenic cells from fibroblasts present in a mixed cellpopulation obtained from the enzymic disaggregation ofneonatal donor mouse muscle. This method resulted in therecovery of high percentages of viable cells, which weredivided into adherent and non-adherent fractions. Adher-ent fractions were characterised by the presence ofmononuclear myogenic cells with few contaminatingfibroblasts. Enrichment for myogenic cells was increasedin adherent fractions when cell suspensions obtained fromthe enzymic disaggregation of neonatal muscle weresubjected to a differential adhesion step prior to initialplating of the crude cell suspensions. An unexpectedobservation however was the presence of myotubes withinpanned non-adherent fractions 3 days post-panning.Logically, we would have expected these to be carried overin the adherent fraction, as it has been suggested (Mooreet al. 1987) that there is a gradual increase in the surfaceexpression of N-CAM during the myoblast to myotubetransition. Such multinucleate myotubes were not ob-served within cultures either prior to, or in either of thefractions achieved 24 h after, panning and hence onlyarose in the non-adherent fractions approximately 72 hpost-panning. In contrast, myogenic cells in adherentfractions continued as proliferating mononuclear myo-genic cells for up to 8 days in culture, after which time cellsbegan to aggregate and fuse to form multinucleatemyotubes, thus confirming the myogenic nature of thesecells. It thus appears that the myogenic cells that areselected for by adhesion to the H28 anti N-CAM antibodyactively proliferate in culture for several days prior totheir fusion, whereas myogenic cells isolated in the non-adherent fraction are further terminally differentiatedtowards myogenesis, for these cells form multinucleatemyotubes within 72 h in culture. The expression of N-CAMon myoblasts has been observed in primary culturesderived from a variety of species (Covault and Sanes, 1986;Grumet etal. 1982) including man (Moore and Walsh,1985), and an increased expression of N-CAM has beenidentified in two mouse cell lines with increasingcommitment to myogenesis (Moore et al. 1987).

Previous studies have indicated a change in N-CAMpolypeptides during myogenesis (Covault etal. 1986;Gennarini et al. 1986) and immunoblot studies of N-CAMduring three stages of myogenesis using murine cultures(Moore etal. 1987) indicated a down regulation of a145xlO3Mr isoform of N-CAM with fusion, the otherisoforms of N-CAM being expressed as fusion of myoblastsensues. It would be tempting to suggest that H28preferentially binds to the 145xlO3Mr isoform. However,it is known that H28 recognises all isoforms equally(Moore etal. 1987). The lack of positivity for anti N-CAMstaining at 24 h, and its presence at 72 h post-panning inthe non-adherent fraction suggests to us some down-regulation of N-CAM expression in these cells at thisparticular time.

The elimination of such prefusion cells from theadherent fraction at panning indicates that the isolation ofa highly enriched and rapidly proliferating myogenicpopulation of cells is a practical proposition. When oneconsiders therapeutic implantation of normal cells intomyopathic host fibres, the highly myogenic nature of the

cells achieved on panning first ensures maximum ef-ficiency of fusion with host fibres, for only myogenic cellsfuse readily with such fibres (Partridge etal. 1989; Wattet al. 1982; Watt, 1990). Second, the proliferative capacityof these panned cells may also be of great benefit in termsof obtaining increased cell numbers for implantation,compared with the limited number of actively prolifer-ating myogenic cells obtained from biopsy material. Workis now in progress to amplify further this highly enrichedpopulation of myogenic cells by the addition of selectedgrowth factors to the cell culture system. Growth factorssuch as basic FGF (DeMario and Strohman, 1988) andIGF-like growth factors (Allen and Boxhorn, 1989) arepotential candidates for study, but others (Leibovitch et al.1989) are also under active investigation in our labora-tories.

This work was supported by the Medical Research Council andthe Muscular Dystrophy Group of Great Britain and NorthernIreland. We thank Professor Frank Walsh for the gift of thehybridoma cells and for his encouragement and support.

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(Received 10 July 1990 - Accepted 12 September 1990)

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