regeneration of adult newt skeletal muscle tissu in …mincing destroys the muscle fibres and...

J. Embryol. exp. Morph. 77, 255-271 (1983) 2 5 5Printed in Great Britain © The Company of Biologists Limited 1983

Regeneration of adult newt skeletal muscle tissue in

vitro

By JOAN A. SCHRAG1 AND JO ANN CAMERON1

From the Department of Anatomical Sciences and University of Illinois Collegeof Medicine at Urbana-Champaign, University of Illinois, Urbana

SUMMARY

Explants and cells of forelimb muscle from adult Notophthalmus viridescens were culturedfor periods up to 160 days in MEM-based medium supplemented with serum, hormones, andantibiotics. Explants which were not minced prior to culture contained muscle fibres withhealthy myonuclei and no evidence of dedifferentiation after four weeks. Explants which wereminced prior to culture contained degenerated muscle fibres after 1 day and no evidence ofdedifferentiation after four weeks. Mononucleated cells from both minced and non-mincedexplants proliferated. Cell proliferation and myotube formation was greater in the mincedmuscle cultures. Proliferation and fusion of myoblasts and subsequent formation of myofibrilswere observed on the plate in primary cultures. Secondarily transferred cells proliferated andfused into myotubes. Although adult newt muscle does not contain satellite cells, myogenesisin this amphibian followed the same course as all other vertebrate skeletal muscle: prolifera-tion of mononucleated myogenic cells, fusion of the myoblasts to form syncytia, and eventualaccumulation of myofibrils. The ultimate source of the myogenic cells was not identified;however, the absence of dedifferentiation of the mature fibres and the occurrence ofmyogenesis in cultures of minced muscle explants demonstrated that the regenerated muscledeveloped from a population of mononucleated cells whose origin did not depend upondedifferentiation of intact fibres.

INTRODUCTION

Regeneration of skeletal muscle is accomplished through a process which veryclosely resembles embryonic myogenesis. Mononucleated myoblast cellsproliferate beneath the remaining external laminae of the injured fibres, initiatefusion and form syncytia which differentiate into new skeletal muscle fibres(Carlson, 1973; Konigsberg, Lipton & Konigsberg, 1975). It is extremely dif-ficult to trace the origin of potentially myogenic cells in vivo. Since they are firstdiscernible under the external laminae, it has been suggested that these cells mayhave been members of a minor population of cells in skeletal muscle tissue, thesatellite cells (Mauro, 1961).

Satellite cells are mononucleated cells which lie under the external lamina butoutside the sarcolemma of the skeletal muscle fibre (Mauro, 1961). In the rat

1 Authors' address: 190 Medical Sciences Bldg, 506 S. Mathews, University of Illinois,Urbana, IL 61801, U.S.A.

256 J. A. SCHRAG AND J. A. CAMERON

these cells have been shown to synthesize DNA, divide and fuse with adjacentmuscle fibres in growing animals (Moss & Leblond, 1970). These cells arepresent in skeletal muscle of vertebrates and could serve as a source of myoblastswhich give rise to the regenerated muscle fibre following injury. Recently,several investigators have provided evidence to support the hypothesis thatsatellite cells have myogenic capabilities during muscle regeneration (Snow,1977a,b, 1978; Hsu, Trupin & Roisen, 1979). The most convincing evidence hasbeen obtained from observations of a single regeneration unit, an individualmuscle fibre and its associated satellite cells, in tissue culture (Konigsberg et al.1975; Bischoff, 1979, 1980).

A second hypothesis of the origin of myogenic cells, muscle fibre dedif-ferentiation, has been proposed as a result of studies on regenerating amphibianlimbs (Thornton, 1938, 1968; Hay, 1959; and Lentz, 1969). After the wound-healing phase dedifferentiation of the stump tissues liberates cells whichproliferate and form the regeneration blastema at the distal tip of the limb.During dedifferentiation the cells of differentiated tissues become free of theirintercellular associations, lose their differentiative cell products and displayembryonic characteristics such as an increased nucleocytoplasmic ratio,prominent nucleoli, and increased amounts of free ribosomes. Although dedif-ferentiation of limb muscle fibres has been described in detail (Thornton, 1938;Hay, 1959; Lentz, 1969), the evidence for myogenic capabilities of the resultingnucleated cytoplasmic compartments is circumstantial. In addition, severalspecies of amphibians that have the ability to regenerate their limbs have beenshown to have a population of satellite cells (Popiela, 1976; Carlson & Rogers,1976; and Flood, 1971). Therefore, in amphibian skeletal muscle regenerationthere are three possible sources of myogenic cells: the satellite cells, the myo-nuclei of the muscle fibres which have not been directly injured by the traumathat stimulated the regenerative response and metaplasia of other cell types.

Since adult newt muscle does not have satellite cells (Popiela, 1976), one mightpredict that minced-muscle regeneration would be reduced or absent becausemincing destroys the muscle fibres and effectively prevents dedifferentiation.The process of muscle fibre dedifferentiation is not prerequisite for muscleregeneration in the adult newt. Following mincing of a single muscle in the newt,that muscle regenerates from mononucleated cells which lie outside the externallamina of the muscle fibre (Cameron, submitted). The mononucleated cellspresent in the adult muscle are fibroblasts, Schwann cells, endothelial cells andperivascular cells which lie outside the external laminae of the muscle fibres andare surrounded by their own external laminae (Hay & Doyle, 1973).

In order to observe the process of newt muscle regeneration more closely wedeveloped a tissue culture system which promotes myogenesis within a timecomparable to the in vivo regeneration period. We substantiated in vitro ourobservation that muscle fibre dedifferentiation in adult newts is not aprerequisite for muscle regeneration. In our system myogenesis occurred within

Regeneration of newt skeletal muscle tissue in vitro 257

unminced muscle explant cultures while the old myonuclei and fibres remainedhealthy appearing. In addition newt minced-muscle regeneration in vitrofollowed the same cellular events observed in other vertebrates thus far:myonuclei of the fibres degenerated and cells which originated outside the mus-cle fibres fused to form myotubes. Observations of cultured mononucleated cellsderived from explants revealed that these cells closely resemble blastema cellsfrom adult newt (Jabaily, Blue & Singer, 1982). There were three cell mor-phologies present but the myogenic cells were not morphologically distinguish-able in aggregations of fusing cells.

MATERIALS AND METHODS

Animals

Adult newts, Notophthalmus viridescens, were obtained from Lee's NewtFarm (Oak Ridge, Tennessee) and housed in aquaria containing 1 % Holtfreter'ssolution. They were kept at 23 °C and fed grated liver twice each week.

Explants

Newts were anaesthetized in 0-075% chloretone (Kodak). Their forelimbswere removed at the distal humerus and sterilized by soaking for 90 s in 1 %sodium hypochlorite solution followed by several rinses in sterile Earle's balan-ced salt solution (EBSS, Gibco), pH7-4 (Vethamany-Globus & Liversage,1973). Muscle dissections were done in sterile EBSS. The skin was peeled backand stripped from the forearm. The forearm muscles were cut at their origin andinsertion, carefully removed from the bones and cut into 3 mm3 pieces withiridectomy scissors. In some experiments the pieces were minced with iridectomyscissors into 1 mm3 pieces. The muscle pieces were placed in a sterile solution of0-05 % crystalline trypsin (1:250, Gibco) in Puck's calcium- and magnesium-freesalt solution at pH7-6 (Konigsberg et al. 1975). The solution of trypsin andmuscle tissue was kept 12-24 h at 8 °C in order to allow penetration of the trypsininto the muscle tissue. After the incubation at 8 °C the muscle pieces were stirredin the same solution at room temperature for 30min, treated with 0-03 % soy-bean trypsin inhibitor (Sigma) and transferred to collagen-coated 35 mm Petridishes (Michalopoulis & Pitot, 1975).

Tissue culture media

The composition of the basic medium and additives was based on the mediadescribed by Konigsberg (1971) for chick muscle, Pollack & Koves (1976) forfrog muscle, and Freed & Mezger-Freed (1970) for amphibian cells. The mediumwas tailored to the special needs of amphibian cells which include low osmolar-ity, low pCO2, and pH7-2-7-4 (Balls, Brown & Fleming, 1976). The osmolarityof amphibian cells (260 mOsmol) is approximately 65 % of mammalian cells


(Freed & Mezger-Freed, 1970; Heilbrun, 1943). Minimal Essential Medium withEarle's salts (MEM, Gibco) was used at 89 % to meet this requirement. Variousconcentrations of the base medium plus additives were tested with a vaporpressure osmometer (Wescor) to determine this percentage. The pCO2 of am-phibian blood is 1 % compared to 5 % for mammals (Balls et al. 1976; Prosser,1973). Using the Henderson-Hasselbach equation, it was determined that thecorrect amount of sodium bicarbonate needed to maintain pH7-4 in mediumbuffered with 1 % CO2 was 0-32mg/ml. The cultures were kept in an incubatorgassed with a 1 % CO2-99 % air mixture and kept at 20-23 °C (Freed & Mezger-Freed, 1970).

The MEM was supplemented with 5 % foetal bovine serum (Flow); 100 i.u./ml penicillin; 50/ig/ml streptomycin; 1-25/ig/ml gentamycin sulphate solution(Sigma); 1/ig/ml thymidine (Sigma); 2-5/ig/ml Fungizone (Gibco); and292 jUg/ml L-glutamine (Sigma). Aqueous solutions of four hormones were alsoadded to each 100 ml of medium at the concentrations based on the work ofVethamany-Globus & Liversage (1973): 28i.u. bovine insulin (Sigma), 20/igsomatotropin (ICN), 20 /ig hydrocortisone (ICN), and 1 ng L-thyroxine (Sigma).Dibutyryl cyclic AMP (Sigma) was added at a concentration of 500 jug/ml (Car-lone & Foret, 1979). The calcium ion concentration was adjusted to 1-8HIM topromote fusion of the myoblasts (Cox & Gunter, 1973). The complete mediumwas filtered through a 0-45 ^m Millipore filter, stored at 8 °C, and used within oneweek. Medium was replaced and cultures examined every three days for periodsup to 160 days.

Secondary cell transfer

Primary cultures which showed dense cellular outgrowth without fusion bythree to four weeks were used for secondary cultures. The explants were not usedfor secondary cell transfers. The primary cultures were rinsed two times withEBSS then treated with 0-05 % trypsin made in Puck's calcium- and magnesium-free salt solution, pH7-6. After 1-5 min the cells on the plate rounded up anddetached. The cell suspension were transferred to a 0-03 % solution of soybeantrypsin inhibitor (Sigma) made in culture medium. The cells were pelleted bycentrifugation, resuspended in medium and seeded on multi-well plates of clonaldensity, 200 cells per 16 mm well. The cells were not cultured beyond the firstpassage.

Cytology

Explants and proliferating cells were fixed on the plate with 2-5 % glutaral-dehyde in 0-lM-phosphate buffer at pH7-4. The explants were removed andembedded in glycol methacrylate (Polysciences). Methacrylate blocks were sec-tioned at 1 fim and the sections were stained with a triple stain (Bennet, Wyrick,Lee & McNeil, 1976). New cell growth on the plate was stained withhaematoxylin and eosin, and mounted in 5% polyvinyl alcohol (Sigma). For

Regeneration of newt skeletal muscle tissue in vitro 259electron microscopy, explants and cells on the plates were also fixed as describedabove, postfixed in 2 % osmium tetroxide in 0-1 M-phosphate buffer at pH7-2,dehydrated and embedded in Epon. Thin sections were examined with a SiemensElmiskop 102.

RESULTS

Histological observations of the unminced explants

Since few cells migrated onto the plates in unminced explant cultures, 15explants were sectioned to determine the condition of the cells. After 1 to 2weeks in culture one notable result was the observation of many healthy-appearing intact muscle fibres within the explants which had not been mincedprior to culture (Figs 1, 2 and 4). Some fibres contained pyknotic myonuclei,degenerating mitochondria and indistinct myofibrils, resembling fibres from ex-plants which had been minced. However, many myonuclei appeared healthy andthere was no evidence of dedifferentiation of the muscle fibres within the ex-plants during culture times of up to 4 weeks. The fibres were surrounded by intactexternal laminae and contained euchromatic nuclei with nucleoli, myofibrils andhealthy-appearing mitochondria. The size of the fibres and the peripheral loca-tion of myonuclei revealed that these fibres survived explantation and were notnewly regenerated. New fibres were smaller in diameter and the nuclei werecentrally located (compare Figs 4 and 7). Mononucleated cells with a highnucleocytoplasmic ratio and rough endoplasmic reticulum were often presentamong the fibres (Fig. 2). Few mitotic figures were observed in the explants.Mononucleated cells migrated onto the plate but did not proliferate efficientlyenough to form myotubes.

Histological observations of the minced explants

Explants that had been minced prior to culture exhibited signs of degenerationalmost immediately. The time course for fibre degeneration was based on ob-servation of 25 explants. After 24 h the fibres were swollen and myonuclei werepyknotic (Fig. 3). After 72 h cytoplasm was without organized myofibrils andthere were many nuclear ghosts in place of myonuclei. The minced fibres werenot cleared away by macrophages as occurs in vivo. This preservation of theinjured fibres allowed us to infer the non-myonuclear origin of the mono-nucleated cells which migrated onto the plate (Fig. 5). Mitotic figures werepresent in less than 1 % of the mononucleated cells within the explant. Theminced explants gave greater amounts of cellular outgrowth than did unmincedexplants and many cells fused to form myotubes on the plate by 4 weeks.

Mononucleated cells on the plates in primary culture

The following results were observed in each of 50 plates of minced explant


wgWvfe.' .." -v »;•'' *-**• «£^"

Fig. 1. A myonucleus from a 9-day muscle explant not minced prior to culture. Notethe prominent nucleolus and euchromatin. The cytoplasm of this cell has healthy-appearing mitochondria, glycogen, intact myofibrils, and external lamina. Scalebar =


sv*****^

Fig. 2. Mononucleated cell (arrow) with high nucleocytoplasmic ratio and roughendoplasmic reticulum from a 9-day muscle explant not minced prior to culture.Notice adjacent intact muscle fibres. Scale bar =


• t .

-* cA

f'' ^ '•*>

.**».Fig. 3. Fibre from 4-day muscle explant minced prior to culture. The nucleus ispyknotic, mitochondria are degenerating, no glycogen is present, and the myofibrilsare indistinct. Scale bar = 0-5 /im.


Fig. 4. Intact mature muscle fibres cultured for 5 days. The presence of myonuclei(arrows) and myofibrils demonstrates that the muscle fibres have not degenerated ordedifferentiated. Scale bar = 20 jum.Fig. 5. Muscle explant minced prior to culture for 4 weeks. The injured fibres areintact and nuclear ghosts have replaced the myonuclei (arrows). Mononucleatedcells lie outside the fibres (m). Scale bar = 20jum.


0 % ^^^r

Fig. 6. Large prefusion aggregation of mononucleated cells adjacent to a mincedexplant after 56 days in minced explant culture. Three configurations ofmononucleated cells were present: epithelial (e), stellate (s), and bipolar (b). Scalebar =Fig. 7. Myotubes (arrows) within an aggregation of mononucleated cells after 56days in minced muscle explant culture. Scale bar = 30/im.


. ,)U »-„ . •> . * »

Jk 9 . ••;. *\ «•• : '• L4- • • .• i / fa.

Fig. 8. A myotube formed in an explant culture after 57 days. The alignment ofmyofibrils (arrows) is evidence of differentiation which was not visible at the lightmicroscopic level. Scale bar =


Figs 9-10


cultures. Cellular outgrowth from the minced explants began on days 5 throughto 10. Outgrowth continued and the cells on the plate were studied from day 5to day 160. Three configurations of cells were seen in the cultures: flat cells,stellate cells and elongated bipolar cells. The number of stellate cells was greaterthan either of the other types. After one to two weeks in culture, the cells on theplate began to increase in number and many mitotic figures were seen on theplate. After 3 weeks the cells formed aggregations (Fig. 6). Many cells in theaggregations lined up and after 4 weeks in culture began to fuse into multi-nucleated tubes located away from the explant (Fig. 7). Thin sections throughthese myotubes revealed alignment of myofilaments (Fig. 8). Proliferation ofcells and their fusion into myotubes continued for as long as 160 days. Nodegeneration of the myotubes was observed once they had formed.

Secondary cell transfers

Secondary cell transfers were made from seven primary cultures. Proliferatingmononucleated cells that had migrated onto the plate were seeded into secon-dary cultures at 200 cells per plate. The plating efficiency averaged 70 %. Thecells attached to the plate randomly but migrated to form aggregations beforethey began to proliferate. Thus they did not proliferate as distinct clones. Thesecondary cultures were maintained up to 6 weeks during which time they con-tinued to proliferate and doubled on the average every 6 days. The three cellconfigurations which were seen in the primary cell cultures were present in thesecondary cultures and here too the stellate configuration was predominant. Thesecondary cells formed prefusion aggregations as described for the primary cul-tures and fusion was observed in the secondary cultures by 3 to 4 weeks (Figs 9,10). It was impossible to determine if a particular cell configuration was morefrequently associated with fusion, since the prefusion aggregates contained cellsof more than one shape and not all of the cells fused to form myotubes.

DISCUSSION

We refined a tissue culture medium which, for the first time, permitted an invitro analysis of amphibian myogenesis from primary and secondary cultures ofadult newt forelimb muscle. Our culture conditions enhanced survival of unin-jured explanted muscle fibres. Despite the absence of muscle satellite cells innewts, muscle fibre dedifferentiation was found not to be a prerequisite formyogenesis in culture. Proliferation and differentiation of myogenic cellsoccurred in both primary and secondary cultures. Differentiation of myotubes

Fig. 9. A myotube formed in secondary culture after 39 days. Scale bar = 20jum.Fig. 10. A myotube formed in secondary culture after 39 days. Note the lack oforganized myofilaments. Some thin filaments are present in the cytoplasm (arrows).Scale bar = 0-5jum.


was observed within a time which corresponds to the in vivo regeneration ofminced-muscle fragments in Ambystoma mexicanum (Carlson, 1970) and Noto-phthalmus viridescens (Cameron, submitted). The sequence of cellular eventsduring skeletal muscle regeneration in adult newt was the same as that of othervertebrate muscle which has been studied in vitro.

Jabailey et al. (1982) have recently described a culture system for dissociatednewt blastema cells using supplemented Leibovitz L-15 medium. Theirdissociated blastema cells show similar morphologies to those of the cellsdescribed here. The blastema cells showed a period of proliferation at 2 weeks,aggregations at 3 weeks, and formation of multinucleated tubes at 8 weeks whichdid not show striations even after 16 weeks. Our own attempts at culturing adultnewt muscle in supplemented Leibovitz L-15 yielded cultures which proliferatedand differentiated more slowly than cultures in identically supplemented MEM(Schrag, unpublished results).

It is important to keep in mind the possibility that muscle fibre dedifferentia-tion may give rise to myogenic cells by budding into nucleated cytoplasmicfragments. Although it has not been proven, it has been postulated that musclefibre dedifferentiation occurs in stump fibres which have not been directly in-jured following limb amputation in newts (Hay, 1959; andLentz, 1969). If it doesoccur in vivo the factors which promote it are unknown. Konigsberg etal. (1975)suggest that rapid (8-24 h) myonuclear degeneration observed in vitro aftermechanical isolation of single quail muscle fibres may occur more slowly in vivofollowing injury, thus allowing cytoplasmic budding to occur. Survival of musclefibres in vitro has been observed previously. Bischoff (1980) has reported thatgentle collagenase digestion of adult rat muscle yields single fibres that surviveup to three weeks in culture, and myogenesis occurs from satellite cells. NeitherBischoff nor we have observed cytoplasmic budding in culture, and in bothsystems myoblasts originate from a population of mononucleated cells within themuscle tissue. Perhaps newt muscle would not be expected to dedifferentiate inculture. Recent experiments in our laboratory strongly suggest that muscle fibrededifferentiation does not occur in newts following limb amputation (Cameron,in preparation), or muscle mincing (Cameron, submitted).

The possibility of metaplasia in cultures containing several cell types must alsobe addressed. Carlson (1972) has shown that intact stump muscle is not requiredfor normal morphogenesis of muscle during limb regeneration in Ambystomamexicanum. After removing 99 % of the stump muscle, normal limb regenerateswith a full complement of muscles are obtained. The source of myogenic cells hasnot been identified. Schwann cells also may be capable of forming a regeneratedlimb which contains normal muscles (Maden, 1977). Namenwirth (1974) testedthe developmental potential of blastema cells derived from whole muscle.Triploid marked cells could be traced to regenerated muscle, connective tissue,and cartilage. It is not known which of the cell types found in whole muscleeventually became muscle cells in the regenerate. The extent of metaplasia


during normal regeneration is not known. Although bipolar cells have beenconsidered myogenic in vitro, Holtzer et al. (1980) have shown that stellate cellscan participate in myogenesis. Jabaily et al. (1982) report that individual newtblastema cells can assume the three configurations which we observed in ourcultures. Our observation that stellate cells form aggregations within whichsyncytia are found suggests that stellate cells in our system may also be myogenic.Presumably these aggregations are not clones since the majority of the cellswithin each aggregation did not fuse into myotubes. A direct confirmation ofthis awaits the capability of cloning the cell types found in amphibian muscle.

One problem which has affected previous attempts to culture proliferatingamphibian cells is the long cell-cycle time. Wallace & Maden (1976) haveestimated the cell-cycle time for limb blastema cells in Ambystoma mexicanumto be about 53 h. It is clear that in vitro studies of regenerating tissues must bemaintained for long periods to allow appreciable growth and cellular interactionsto take place along a time course similar to that occurring in vivo. Severalinvestigators have cultured intact limb blastemas. Studies dealing with thegrowth and differentiation patterns in amphibian limb blastemas have employedculture times from 12 h to 25 days (Vethamany-Globus & Liversage, 1973; Conn,Dearlove & Dresden, 1979; and Carlone & Foret, 1979; Bromley & Angus,1971; and Stocum, 1968). Jabaily et al. (1982) have cultured dissociated newtblastemal cells for 4 months, although they did not report differentiation ofmyotubes with striations. The culture conditions described here permit growthand differentiation of amphibian skeletal myoblast cells for at least 160 days.Additional experiments in our laboratory demonstrate that modifications of thismedium can be used to study differentiation of fibroblasts and muscle in Xenopuslaevis, and cartilage and muscle in Ambystoma mexicanum limb blastema ex-plants and cells (Hinterberger & Cameron, 1983).

This work was supported by NSF Grant PCM 79-19338 and Biomedical Research GrantNIHRR7030 awarded to the School of Life Sciences, University of Illinois. We thank MsRebecca Snyders and Mr Allen Hilgers for expert technical assistance. Special thanks are dueto Drs Allen W. Clark and John F. Fallon for helpful discussions during the preparation of thismanuscript.

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(Accepted 16 May 1983)

regeneration of adult newt skeletal muscle tissu in …mincing destroys the muscle fibres and...

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