J. Cell Sci. 38, 201-210 (1979) 201Printed in Great Britain © Company of Biologists Limited 1979

AN ABNORMALITY OF CELL BEHAVIOUR IN

HUMAN DYSTROPHIC MUSCLE CULTURES:

A TIME-LAPSE STUDY

ROSE YASIN," G. VAN BEERS,* P. N. RIDDLE.f D. BROWN.fG. WIDDOWSON.f AND E. J. THOMPSON**Muscular Dystrophy Research Laboratories, The National Hospital, Queen Square,London WCiN 3BG, and^Imperial Cancer Research Fund, Lincoln's Inn Fields,London WCzA 3PX, England

SUMMARY

The initiation, of monolayer mass cell cultures from adult human biopsies has revealed astriking abnormality in the growth and development pattern of muscle cultures from Duchenne-type dystrophy. This abnormality in cell behaviour was seen as early as 4 days in culture, wellbefore myotube formation or confluence, and consisted of areas where cells clustered togetherin a multilayered mass rather than showing the typical monolayer distribution normally ob-served.

To gain some insight into the mechanism of cell cluster development, we have examined sucha culture by time-lapse cinematography and also the cell behaviour of other control cultures.The results of this study show that the clusters enlarged primarily by cell division and, to a lesserextent, by the acquisition of neighbouring cells. Furthermore, none of the single cells surround-ing the clusters exhibited contact inhibition of movement. This behaviour was not observed inthe other cultures examined. These findings could be indicative of an abnormality in the cellsurface or cell-locomotory machinery of dystrophic cells.

INTRODUCTION

Muscular dystrophies are genetic diseases in which symptoms predominantly affectskeletal muscle. There is some evidence, however, that other organs may be involved(Jerusalem, 1976). Although a number of biochemical, morphological and physiologi-cal abnormalities have been described (Jerusalem, 1976), a basic understanding of theprimary lesion involved is still lacking. Currently, there are 4 major hypotheses on theaetiology, with most of the research centring on the Duchenne-type dystrophy. Theseare: (1) a defect in the cell membranes (Kunze, Reichman, Leuschner & Eckhardt,1973; Mokri & Engel, 1975; Bosmann et al. 1976; Mawatari, Miranda & Rowland,1976); (2) a primary lesion of the motor neurons (McComas, Sica & Currie, 1970);(3) a lesion in the muscle microcirculation (Demos, Place & Chereau, 1970; Mendellet al. 1972); and (4) an anomaly of the connective tissue (Bourne & Golarz, 1963;Lahoda, 1975). Most of the evidence at present favours the first possibility.

Studies on diseased muscle in culture have been carried out to try to elucidate thespecific defects involved, but attempts to show differences in morphology and growthpatterns in vitro from explanted biopsy material, in the presence or absence of nerve,

202 R. Yasin and others

have so far yielded equivocal results (Morgan & Cohen, 1974; Witkowski & Dubowitz,1975; Askanas & Engel, 1977).

Recently, Thompson et al. (1977) have described a highly distinctive behaviouralpattern in populations of cells obtained from patients with Duchenne dystrophy andalso, to a lesser extent, from Becker dystrophy. These cultures were initiated frommononucleated cells released enzymically from muscle biopsies (Yasin et al. 1977).This abnormality in cell behaviour was observed as early as 4 days in culture, beforeconfluence or myotube formation, and consisted of areas where cells clustered togetherin a multilayered mass rather than showing the typical monolayer distribution normallyobserved.

In order to gain some insight into the mechanism of cluster development, we haveanalysed a similar culture by time-lapse cinematography before the appearance ofmyotubes. This investigation describes the temporal mode of formation of the clustersin Duchenne dystrophy and also the cell behaviour of other control cultures for com-parison. The results of this study show that clusters enlarge primarily by cell divisionand, to a much lesser extent, by the incorporation of neighbouring cells. This be-haviour was not observed in the other cultures examined.

MATERIALS AND METHODSMuscle biopsies were obtained from patients being examined at the Hospital for Sick Child-

ren at Great Ormond Street, London, and The National Hospital, Queen Square, London.Mononucleated cells were released from freshly excised human muscle biopsies and cultured aspreviously described by Yasin et al. (1977), except for a minor modification. The muscle wasdissociated with an enzyme mixture consisting of o-i % (w/v) collagenase (Sigma Type II),O-IS % (w/v) trypsin (Difco 11250) and 01 % (w/v) bovine serum albumin (twice crystallized,from BDH) in phosphate-buffered saline, pH 7-1, at 37 °C (Yasin et al. 1977). This alterationwas found necessary for the release of viable cells with a new batch of trypsin. The weight ofmuscle dissociated and cell yields obtained are listed in Table 1. 50-70 x 103 cells were seeded in35-mm Lux plastic tissue-culture dishes (Lux Scientific Corp., Los Angeles, CA., U.S.A.)coated with rat tail collagen. Rat tail tendon collagen was prepared aseptically according to theprocedure described by Wood & Keech (i960) for calf skin collagen. First 0-5 ml of collagensolution (containing 50 /*g/ml protein, as measured by the procedure of Lowry, Rosebrough,Farr & Randall, 1951) was added to one side of a 35-mm Lux dish. Then an equal volume ofbuffer (40 mM NaH2PC>4 and 200 mM NaCl, pH 7-2) was added to the other side of the dish andthe mixture was spread uniformly over the surface with a glass rod. After allowing the collagento precipitate for at least 2 h at room temperature, the buffer was removed by washing the plates3 times with 2 ml of sterile glass-distilled water. The plates were dried in a laminar flow cabinetand stored for a maximum of 2 weeks at 37 °C in a humidified incubator.

After 20 h in vitro the cells were washed and fed fresh medium 3 times weekly for the remain-der of the growth period, and incubated at 37 °C in a humidified atmosphere containing 8 %(v/v) CO2 in air. The medium was similar to that used previously (Thompson et al. 1977), andconsisted of 100 parts Dulbecco's Modified Eagle's Medium (Gibco-Biocult Ltd. 10 timesconcentrate, without pyruvate) supplemented with glutamine, NaHCO3 and antibiotics; 10parts horse serum (Flow) pretested for seeding efficiency and differentiation using adult hamsterskeletal muscle as the test system; and 2 parts chicken embryo extract (Flow) after detoxification(Skrbic et al. 1975). With the exception of case 4, at least 2 plates were generally prepared fromeach biopsy. The cells were allowed to settle for at least 3 days before analysis, and one of theculture dishes was transferred to a plastic chamber with an open base and a clean window, andplaced in an inverted microscope. 10 % (v/v) humidified CO2 in air was supplied to the chamberand the whole microscope was encased in a Perspex box kept at 37 °C (+ o-i °C). Cinefilms

Abnormal muscle cells 203

were prepared with a 2-min interval between exposures and the microscope light was shutteredbetween these exposures (Riddle, 1977). Processed films (usually as a direct negative) wereanalysed using an LW projector, with the following criteria taken as indicators of cell behaviour:(1) intermitotic interval - the length of time between mitosis of parent and daughter cells; (2) cellnuclear crossovers - the time for the nucleus of a cell within the culture to cross over (or to becrossed) by any visible projection of another cell; and (3) formation and growth of a cell cluster.

Creatine phosphokinase specific activity (CPK) and proteins were measured as describedpreviously (Yasin et al. 1977).

Table 1. Comparative data on growth and differentiation ofcultures analysed by time-lapse cinematography

Caseno.

1

2

3

4

5

6

Clinicaldiagnosis

Laminectomy,normal muscleMusculardystrophy

Musculardystrophy

Duchennecarrier

Duchennecarrier

Mini co remyopathy

Wet wtof biopsy

dissociated,mg

315

J53

1 2 1

1 1 2

43°

254

Cell-yield

X IO~°

0-85

3-11

1-17

o-49

0 2 6

456

Seeding6

efficiency,%

28-5 (3)

393 (6)

353 (3)

4-0 (1)

17-5 (2)

29-5 (16)

Cells/plate

X IO~3

DIVi

1 8 2

25-5

1 8 8

2 1

lO'I

1 9 3

Appear-ance of

first myo-tubes DIV

9

9

8

1 2

9

1 0

Specificactivity

0 ofCPK"

o-s at DIV15026 at DIV14Notdetermined6

Notdetermined/

09 at DIV141-3 at DIV18

" Cell yield: no. of cells released per g muscle wet wt.6 Seeding efficiency: % of viable cells remaining from the original inoculum after 20 h in

culture. Results are represented as mean values except for case 4.0 DIV: days in vitro.a CPK: /tmol creatine formed per min per mg protein at 30 °C." Myotubes were short, undifferentiated and generally sparse./ Morphology was similar to previous cultures which yielded CPK specific activity values of

o-8-io at peak differentiation.Nos. of plates established from each biopsy are given in parentheses.

RESULTS

Fig. 1 illustrates the development of a cell cluster in a muscle culture from Duch-enne dystrophy. Several groups, consisting of 3-4 cells, were first observed in allplates at day 3 in culture. Filming of one of these groups was commenced on day 4 andcontinued for 70 h (Case 2, Table 2). Initially, a group of 9 cells (Fig. 1 A)-a putativecluster, as judged by previous experience of their formation (Thompson et al. 1977)-was surrounded by 21 scattered single cells. The cluster increased in area and depthduring the next 28 h (Fig. ic) but no accurate assessment of movement or multi-plication of parent cells or progeny was possible within the cluster because of themultilayered cells in the image. However, the cluster moved as a single mass and

R. Yasin and others

' D •

Fig. i . Sequence showing the growth of a cell cluster in a culture from muscular dys-trophy (Case 2, Table i) starting from 4-5 days in culture, times in. h: A, a-6; B, 6 5 ;C, 27-2; D, 50-5. Scale bar represents 100 /tm.

Abnormal muscle cells 205

migrated from the centre region to the edge of the field within the first 30 h of filming(Fig. 1 A-C). The surrounding cells also increased in number, exhibiting a mean inter-mitotic interval of 23-1 ± 7-8 h (Case 2-Table 2).

Table 2. Quantitative analysis of the initial time-lapse sequence ofmuscle cultures

Caseno.

1

2

3

4

S

6

Clinicaldiagnosis

Laminectomy,normal muscle

Musculardystrophy

Musculardystrophy

Maternalcarrier

Maternalcarrier

Minicoremyopathy

Ageand sex

16 yearsMale

4 yearsMale

3 yearsMale

32 yearsFemale

35 yearsFemale

29 yearsFemale

h undertime-lapse

72-2

from DIV°

3'570

from DIV4'5

91-6from DIV

3'573-3

from DIVf.D

6 6 2from DIV

545-6

from DIV

Initialcell no.

37

3°9 in cluster

21 single cells

34

15

IS

17

Intermitotictime, h

22-3 ±s-o6

(27)°

23-1 ±7-8(")'

21-7 + 4-1(21)

20-9 + 4-6(5)

n.d/

16-6 + 2-9*(18)

Nuclearcrosses, h

2-7±i-81>

(i7)d

Cluster -continuous

single cells -none

1-17 + 0-89*(4i)

2-4±o-6(3)

56 ±7-2"(8)

2-5 ±i-4(S)

0 DIV: days in vitro.6 ± standard deviation.0 No. of cells.d No. of nuclear crosses.0 Cells outside the cluster.f Not determined - many cells left the field after initial mitosis." See Results for explanation of this high standard deviation.* Significant difference compared to Case 1, as determined by the Student t-test, P<o-ooi.

While the qualitative difference in movement between the cluster and surroundingsingle cells was most pronounced, no numerical assessment of nuclear crossoverscould be made within the cluster. Once the cluster had become multilayered, allprogeny were overlapped nearly from the outset (Fig. 1 B). Only one daughter cell wasseen to escape from the mass after the parent cell divided, at the edge of the cluster. Asmaller group of cells adjacent to the cluster (Fig. 1 A, arrow at bottom left) was incor-porated after 6 h (Fig. 1 B) and could not be distinguished subsequently. In contrast,the surrounding single cells showed frequent contact with one another but crossing ofthe cell nucleus was not observed during this time-lapse sequence. However, thesecontacts were strikingly different from those observed in the other cultures in thatnone of these cells exhibited the contact inhibition of movement described by Aber-crombie & Ambrose (1958). Instead they remained in continuous contact, formingbridges in the open spaces or sliding alongside each other. This latter behaviour was

14 CEL38

206 R. Yasin and others

Fig. 2. Sequence showing growth pattern of a culture from muscular dystrophy (Case3, Table i) starting from 3-5 days in culture, times in h: A, O; B, 6'6; C, 60; D, 63-9.Scale bar represents 100 fim.

Abnormal muscle cells 207

normally observed only in densely populated cultures and/or prior to fusion of myo-blasts.

Table 2 summarizes the results for intermitotic interval and crossing times in the 6cultures analysed. The intermitotic times were similar for all the cells examined,except in the minicore myopathy (Case 6, Table 2) which exhibited the significantlower mean time of 16-6 h as compared with the normal culture (P< o-ooi). This dif-ference may have resulted from the presence of different proportions of cell types(such as fibroblasts, myoblasts at various stages of differentiation). In comparison withthe first case of dystrophy, nuclear crossing times were short and frequently transient in

Fig. 3. Sequence showing the growth pattern of a culture from normal muscle (Case1, Table 1) starting from 3-5 days in culture, times in h: A, O; B, 12; C, 72. Scale barrepresents 100 /tm

208 R. Yasin and others

nature in the other cultures examined. It may, however, be important that longerperiods were observed for one of the cells from a carrier (Table 2, Case 5), thuscausing a standard deviation which was higher than the mean value. While 6 cross-overs were of a similar transient nature to the crosses in the other cultures (except forthe Duchenne dystrophy), one lasted for 14 h and another 16 h. Further analysis ofcells from maternal carriers is needed to confirm the significance of this observation.

To illustrate and compare the cell behaviour in some of the other cultures listed inTable 1, frames of time-lapse sequences of cells from the second muscular dystrophy(Case 3) and normal muscle (laminectomy) are shown in Figs. 2 and 3 respectively.These cultures did not display cluster formation at any time during the full growthperiod (2 weeks). Although frequent nuclear crossings were observed in Case 3, thesewere of a significantly shorter duration than those observed for the other cultures(P<o-ooi) shown in Table 2. The reason for this behaviour is at present unknown.This dystrophy has been diagnosed primarily on the basis of muscle histology andclinical symptoms as possibly of a Duchenne type, but it is recognized that clinicaldifferentiation is difficult where there is no family history, or other factors are notfound. An alternative diagnosis of autosomal recessive dystrophy has been suggested asa possibility (J. Wilson, personal communication).

Figs. 1-3 also demonstrate the varying cell morphologies present in these cultures.In the normal muscle culture (Fig. 3) many of the cells exhibited the typical bipolarspindle morphology described for myoblasts (Morris & Cole, 1972) while in thedystrophies (Figs. 1, 2) as well as in other diseases (not shown) large, flat, less-refractilecells with numerous cytoplasmic granules were frequently present. The time ofappearance of first myotubes, however, was similar in all these cases (Table 1) and alsoto that previously reported (Yasin et al. 1977; Thompson et al. 1977). A large variationwas observed in the degree of differentiation of these cultures, as measured by theactivity of the muscle enzyme, creatine phosphokinase with a lower value beingobtained for Duchenne dystrophy as compared to the other cultures described here(Table 1) and also to the values previously reported for normal muscle (Yasin et al.1977; Thompson et al. 1977).

DISCUSSION

The formation of multilayered cell clusters in muscle cultures from Duchennedystrophy and also, to a lesser extent, in Becker dystrophy, were first described byThompson et al. (1977), but the origin and significance of the cells within the clustersremained obscure. They were never seen in a series of control cultures from variousneuromuscular disorders. We have now prepared cultures from 29 Duchenne dys-trophies and 23 of these have exhibited cluster formation. One of the cultures whichdid not exhibit this abnormality is shown in Fig. 2. The differentiation of Duchennefrom other progressive muscular dystrophies by histological techniques is difficultsince most of the dystrophies have a similar appearance. Also, muscle sections maydisplay marked dystrophic changes adjacent to other areas which could be interpretedas having signs of denervation (B. Lake, personal communication). In view of these

Abnormal muscle cells 209

difficulties in classification, other forms of dystrophy such as the congenital or auto-somal recessive types might be considered. The present investigation has shown thatclusters enlarged primarily by cell division, but also, to a limited extent, by incorporat-ing adjacent cells. Only one cell - the product of a division at the edge of the mass -escaped. Such behaviour suggests that these cells are abnormally 'sticky' to oneanother, and could be indicative of an abnormality in the cell membrane or cell loco-motory machinery. The failure of other studies to observe this abnormality may havebeen due to the tissue-culture techniques employed. These were based upon explantsof small muscle pieces which require at least 1 week for the outgrowth of single cellsfrom the original tissue pieces and several additional weeks before myotube formationis seen (Askanas & Engel, 1975; Witkowski, Durbidge & Dubowitz, 1976). Thepresent observation suggests that only the free 'non-sticky' cells would be able tomigrate out of the explant, while the clustering cells would remain inside the tissueexplant. The sticky behaviour found in dystrophic cells might result from an increasein synthesis and secretion of some protein, e.g. collagen. This possibility is not in-consistent with the increased proliferation of connective tissue observed in vitro withdystrophic muscle. Furthermore Lipton (1977 a, b) has demonstrated a phenotypicalteration of myoblasts under certain in vitro conditions, which results in a reversiblealteration of cells into collagen-producing and/or fat cells.

An important question relates to the origin of the cells within the clusters. In anattempt to answer this, we are now carrying out an electron-microscopic investigationof the cells within the clusters, and preliminary results show that these clusters con-tain fibroblasts, myoblasts and myotubes. In addition, some of these cells displayabnormalities in ultrastructure which are consistent with an increase in synthesis ofsubstances for both the cell membranes and for export and may be indicative of anabnormal form of fibroblast and/or aberrant myoblasts.

We thank Drs J. Wilson and E. Brett of the Hospital for Sick Children, Great Ormond Street,London, and Dr J. A. Morgan-Hughes of The National Hospital for allowing us to study theirpatients.

We also thank Dr B. D. Lake for his helpful discussions.This work was supported by the Muscular Dystrophy Group of Great Britain.

REFERENCES

ABERCROMBIE, M. & AMBROSE, E. J. (1958). Interference microscopy studies of cell contacts in.tissue culture. Expl Cell Res. 15, 332-345.

ASKANAS, V. & ENGEL, W. K. (1975)- A new program for investigating adult human skeletalmuscle grown aneurally in tissue culture. Neurology, Mineap. 25, 58-67.

ASKANAS, V. & ENGEL, W. K. (1977). Diseased human muscle in tissue culture. A new approachto the pathogenesis of human neuromuscular disorders. In Pathogenesis of Human MuscularDystrophies (ed. L. P. Rowland), pp. 856-871. Amsterdam: Excerpta Medica.

BOSMANN, H. B., GERSTEN, D. M., GRIGGS, R. C , HOWLAND, J. L., HUDECKI, M. S.,KATYARE, S. & MCLAUGHLIN, J. (1976). Erythrocyte surface membrane alteration. Findingsin human and animal muscular dystrophies. Archs Neurol. 33, 135-138.

BOURNE, G. H. & GOLARZ, M. N. (1963). Histochemical evidence for a possible primary bio-chemical lesion in muscular dystrophy. J. Histochem. 11, 286-288.

DEMOS, J., PLACE, T. & CHEREAU, H. (1970). Myopathy-a disorder of the microcirculation?Excerpta Med. Int. Congr. Ser. 199, 408-411.

210 R. Yasin and others

JERUSALEM, E. (1976). Hypothesis and recent findings concerning aetiology and pathogenesis ofthe muscular dystrophies. .7. Neurol. 213, 155-162.

KUNZE, D., REICHMAN, G., LEUSCHNER, E. & ECKHARDT, H. (1973). Erythrozytenlipide beiprogressvvier Muskeldystrophie. Clin. chim. Acta 43, 333-341.

LAHODA, F. (1975). Zum Stoffwechse bei der Dystrophia musculorum progressiva ERB.Deutscher neurologenkongress, Hamburg (Referat).

LIPTON, B. H. (1977a). A fine-structural analysis of normal and modulated cells in myogeniccultures. Devi Biol. 60, 26-47.

LIPTON, B. H. (19776). Collagen synthesis by normal and bromodeoxyuridine-modulated cellsin myogenic cultures. Devi Biol. 61, 153-165.

LOWRY, O. H., ROSEBROUGH, W. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measure-ments with the folin phenol reagent. J. biol. Chem. 193, 265-275.

MAWATARI, S., MIRANDA, A. & ROWLAND, L. P. (1976). Adenyl cyclase abnormality in Duchennemuscular dystrophy: muscle cells in culture. Neurology, Minneap. 26, 1021-1026.

MCCOMAS, A. J., SICA, R. I. & CURRIE, S. J. (1970). Muscular dystrophy: evidence for a neuralfactor. Nature, Lond. 226, 1263-1264.

MENDELL, J. R., MURPHY, D. L., ENGEL, W. K., CHASE, TH. N. & GORDON, E. (1972). Cate-cholamine and indoleamine in patients with Duchenne muscular dystrophy. Arch. Neurol.27. 518-S20.

MOKRI, B. & ENGEL, A. G. (1975). Duchenne dystrophy: electron microscopic findings point-ing to a basic or early abnormality in the plasma membrane of the muscle fiber. Neurology,Minneap. 25, 1111-1120.

MORGAN, J. & COHEN, L. (1974). Crystalline perinuclear inclusions in myoblasts from humandystrophic muscle. New Engl. J. Med. 290, 863.

MORRIS, G. E. & COLE, R. J. (1972). Cell fusion and differentiation in cultured chick musclecells. Expl Cell Res. 75, 191-199.

RIDDLE, P. N. (1977). Beam deflection as an alternative to light shuttering in cine-microscopy.Lab. Pract. 11, 866.

SKRBIC, T. R., YASIN, R., VAN BEERS, G., BULIEN, D. & THOMPSON, E. J. (1975). Optimizationof tissue culture conditions for differentiation of muscle from dissociated single cells. Biochem.Soc. Trans. 3, 496-498.

THOMPSON, E. J., YASIN, R., VAN BEERS, G., NURSE, K. & AL-ANI, S. (1977). Myogenic defectin human muscular dystrophy. Nature, Lond. 268, 241-243.

WITKOSWKI, J. A. & DUBOVVITZ, V. (1975). Growth of diseased human muscle in combinedcultures with normal mouse embryonic spinal cord. J. neurol. Sci. 26, 203-220.

WITKOWSKI, J. A., DURBIDGE, M. & DUBOWITZ, V. (1976). Growth of human muscle in tissueculture. In Vitro 12, 98-106.

WOOD, G. C. & KEECH, M. K. (i960). The formation of fibrils from collagen solutions. 1, Theeffect of experimental conditions: kinetic and electron-microscope studies. Biochem. J. 75,588-598.

YASIN, R., VAN BEERS, G., NURSE, K., AL-ANI, S., LANDON, D. N. & THOMPSON, E. J. (1977).A quantitative technique for growing human adult skeletal muscle in culture starting frommononucleated cells. J. neurol. Sci. 32, 347-360.

{Received 11 December 1978)