J. Embryol. exp. Morph. 77, 255-271 (1983)
255
Printed in Great Britain © The Company of Biologists Limited 1983
Regeneration of adult newt skeletal muscle tissue in
vitro
By JOAN A. SCHRAG 1 AND JO ANN CAMERON 1
From the Department of Anatomical Sciences and University of Illinois College
of Medicine at Urbana-Champaign, University of Illinois, Urbana
SUMMARY
Explants and cells of forelimb muscle from adult Notophthalmus viridescens were cultured
for periods up to 160 days in MEM-based medium supplemented with serum, hormones, and
antibiotics. Explants which were not minced prior to culture contained muscle fibres with
healthy myonuclei and no evidence of dedifferentiation after four weeks. Explants which were
minced prior to culture contained degenerated muscle fibres after 1 day and no evidence of
dedifferentiation after four weeks. Mononucleated cells from both minced and non-minced
explants proliferated. Cell proliferation and myotube formation was greater in the minced
muscle cultures. Proliferation and fusion of myoblasts and subsequent formation of myofibrils
were observed on the plate in primary cultures. Secondarily transferred cells proliferated and
fused into myotubes. Although adult newt muscle does not contain satellite cells, myogenesis
in this amphibian followed the same course as all other vertebrate skeletal muscle: proliferation of mononucleated myogenic cells, fusion of the myoblasts to form syncytia, and eventual
accumulation of myofibrils. The ultimate source of the myogenic cells was not identified;
however, the absence of dedifferentiation of the mature fibres and the occurrence of
myogenesis in cultures of minced muscle explants demonstrated that the regenerated muscle
developed from a population of mononucleated cells whose origin did not depend upon
dedifferentiation of intact fibres.
INTRODUCTION
Regeneration of skeletal muscle is accomplished through a process which very
closely resembles embryonic myogenesis. Mononucleated myoblast cells
proliferate beneath the remaining external laminae of the injured fibres, initiate
fusion and form syncytia which differentiate into new skeletal muscle fibres
(Carlson, 1973; Konigsberg, Lipton & Konigsberg, 1975). It is extremely difficult to trace the origin of potentially myogenic cells in vivo. Since they are first
discernible under the external laminae, it has been suggested that these cells may
have been members of a minor population of cells in skeletal muscle tissue, the
satellite cells (Mauro, 1961).
Satellite cells are mononucleated cells which lie under the external lamina but
outside 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 adjacent
muscle fibres in growing animals (Moss & Leblond, 1970). These cells are
present in skeletal muscle of vertebrates and could serve as a source of myoblasts
which give rise to the regenerated muscle fibre following injury. Recently,
several investigators have provided evidence to support the hypothesis that
satellite cells have myogenic capabilities during muscle regeneration (Snow,
1977a,b, 1978; Hsu, Trupin & Roisen, 1979). The most convincing evidence has
been obtained from observations of a single regeneration unit, an individual
muscle 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 dedifferentiation, has been proposed as a result of studies on regenerating amphibian
limbs (Thornton, 1938, 1968; Hay, 1959; and Lentz, 1969). After the woundhealing phase dedifferentiation of the stump tissues liberates cells which
proliferate and form the regeneration blastema at the distal tip of the limb.
During dedifferentiation the cells of differentiated tissues become free of their
intercellular associations, lose their differentiative cell products and display
embryonic characteristics such as an increased nucleocytoplasmic ratio,
prominent nucleoli, and increased amounts of free ribosomes. Although dedifferentiation of limb muscle fibres has been described in detail (Thornton, 1938;
Hay, 1959; Lentz, 1969), the evidence for myogenic capabilities of the resulting
nucleated cytoplasmic compartments is circumstantial. In addition, several
species of amphibians that have the ability to regenerate their limbs have been
shown to have a population of satellite cells (Popiela, 1976; Carlson & Rogers,
1976; and Flood, 1971). Therefore, in amphibian skeletal muscle regeneration
there are three possible sources of myogenic cells: the satellite cells, the myonuclei of the muscle fibres which have not been directly injured by the trauma
that stimulated the regenerative response and metaplasia of other cell types.
Since adult newt muscle does not have satellite cells (Popiela, 1976), one might
predict that minced-muscle regeneration would be reduced or absent because
mincing destroys the muscle fibres and effectively prevents dedifferentiation.
The process of muscle fibre dedifferentiation is not prerequisite for muscle
regeneration in the adult newt. Following mincing of a single muscle in the newt,
that muscle regenerates from mononucleated cells which lie outside the external
lamina of the muscle fibre (Cameron, submitted). The mononucleated cells
present in the adult muscle are fibroblasts, Schwann cells, endothelial cells and
perivascular cells which lie outside the external laminae of the muscle fibres and
are surrounded by their own external laminae (Hay & Doyle, 1973).
In order to observe the process of newt muscle regeneration more closely we
developed a tissue culture system which promotes myogenesis within a time
comparable to the in vivo regeneration period. We substantiated in vitro our
observation that muscle fibre dedifferentiation in adult newts is not a
prerequisite 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 remained
healthy appearing. In addition newt minced-muscle regeneration in vitro
followed the same cellular events observed in other vertebrates thus far:
myonuclei of the fibres degenerated and cells which originated outside the muscle fibres fused to form myotubes. Observations of cultured mononucleated cells
derived from explants revealed that these cells closely resemble blastema cells
from adult newt (Jabaily, Blue & Singer, 1982). There were three cell morphologies present but the myogenic cells were not morphologically distinguishable in aggregations of fusing cells.
MATERIALS AND METHODS
Animals
Adult newts, Notophthalmus viridescens, were obtained from Lee's Newt
Farm (Oak Ridge, Tennessee) and housed in aquaria containing 1 % Holtfreter's
solution. They were kept at 23 °C and fed grated liver twice each week.
Explants
Newts were anaesthetized in 0-075% chloretone (Kodak). Their forelimbs
were 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 balanced salt solution (EBSS, Gibco), pH7-4 (Vethamany-Globus & Liversage,
1973). Muscle dissections were done in sterile EBSS. The skin was peeled back
and stripped from the forearm. The forearm muscles were cut at their origin and
insertion, carefully removed from the bones and cut into 3 mm3 pieces with
iridectomy scissors. In some experiments the pieces were minced with iridectomy
scissors into 1 mm3 pieces. The muscle pieces were placed in a sterile solution of
0-05 % crystalline trypsin (1:250, Gibco) in Puck's calcium- and magnesium-free
salt solution at pH7-6 (Konigsberg et al. 1975). The solution of trypsin and
muscle tissue was kept 12-24 h at 8 °C in order to allow penetration of the trypsin
into the muscle tissue. After the incubation at 8 °C the muscle pieces were stirred
in the same solution at room temperature for 30min, treated with 0-03 % soybean trypsin inhibitor (Sigma) and transferred to collagen-coated 35 mm Petri
dishes (Michalopoulis & Pitot, 1975).
Tissue culture media
The composition of the basic medium and additives was based on the media
described by Konigsberg (1971) for chick muscle, Pollack & Koves (1976) for
frog muscle, and Freed & Mezger-Freed (1970) for amphibian cells. The medium
was tailored to the special needs of amphibian cells which include low osmolarity, low pCO2, and pH7-2-7-4 (Balls, Brown & Fleming, 1976). The osmolarity
of amphibian cells (260 mOsmol) is approximately 65 % of mammalian cells
258
J. A. SCHRAG AND J. A. CAMERON
(Freed & Mezger-Freed, 1970; Heilbrun, 1943). Minimal Essential Medium with
Earle's salts (MEM, Gibco) was used at 89 % to meet this requirement. Various
concentrations of the base medium plus additives were tested with a vapor
pressure osmometer (Wescor) to determine this percentage. The pCO2 of amphibian blood is 1 % compared to 5 % for mammals (Balls et al. 1976; Prosser,
1973). Using the Henderson-Hasselbach equation, it was determined that the
correct amount of sodium bicarbonate needed to maintain pH7-4 in medium
buffered with 1 % CO2 was 0-32mg/ml. The cultures were kept in an incubator
gassed with a 1 % CO 2 -99 % air mixture and kept at 20-23 °C (Freed & MezgerFreed, 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); and
292 jUg/ml L-glutamine (Sigma). Aqueous solutions of four hormones were also
added to each 100 ml of medium at the concentrations based on the work of
Vethamany-Globus & Liversage (1973): 28i.u. bovine insulin (Sigma), 20/ig
somatotropin (ICN), 20 /ig hydrocortisone (ICN), and 1 ng L-thyroxine (Sigma).
Dibutyryl cyclic AMP (Sigma) was added at a concentration of 500 jug/ml (Carlone & Foret, 1979). The calcium ion concentration was adjusted to 1-8HIM to
promote fusion of the myoblasts (Cox & Gunter, 1973). The complete medium
was filtered through a 0-45 ^m Millipore filter, stored at 8 °C, and used within one
week. Medium was replaced and cultures examined every three days for periods
up to 160 days.
Secondary cell transfer
Primary cultures which showed dense cellular outgrowth without fusion by
three to four weeks were used for secondary cultures. The explants were not used
for secondary cell transfers. The primary cultures were rinsed two times with
EBSS then treated with 0-05 % trypsin made in Puck's calcium- and magnesiumfree salt solution, pH7-6. After 1-5 min the cells on the plate rounded up and
detached. The cell suspension were transferred to a 0-03 % solution of soybean
trypsin inhibitor (Sigma) made in culture medium. The cells were pelleted by
centrifugation, resuspended in medium and seeded on multi-well plates of clonal
density, 200 cells per 16 mm well. The cells were not cultured beyond the first
passage.
Cytology
Explants and proliferating cells were fixed on the plate with 2-5 % glutaraldehyde in 0-lM-phosphate buffer at pH7-4. The explants were removed and
embedded in glycol methacrylate (Polysciences). Methacrylate blocks were sectioned 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 with
haematoxylin and eosin, and mounted in 5 % polyvinyl alcohol (Sigma). For
Regeneration of newt skeletal muscle tissue in vitro
259
electron microscopy, explants and cells on the plates were also fixed as described
above, 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 Siemens
Elmiskop 102.
RESULTS
Histological observations of the unminced explants
Since few cells migrated onto the plates in unminced explant cultures, 15
explants were sectioned to determine the condition of the cells. After 1 to 2
weeks in culture one notable result was the observation of many healthyappearing intact muscle fibres within the explants which had not been minced
prior to culture (Figs 1, 2 and 4). Some fibres contained pyknotic myonuclei,
degenerating mitochondria and indistinct myofibrils, resembling fibres from explants which had been minced. However, many myonuclei appeared healthy and
there was no evidence of dedifferentiation of the muscle fibres within the explants during culture times of up to 4 weeks. The fibres were surrounded by intact
external laminae and contained euchromatic nuclei with nucleoli, myofibrils and
healthy-appearing mitochondria. The size of the fibres and the peripheral location of myonuclei revealed that these fibres survived explantation and were not
newly regenerated. New fibres were smaller in diameter and the nuclei were
centrally located (compare Figs 4 and 7). Mononucleated cells with a high
nucleocytoplasmic ratio and rough endoplasmic reticulum were often present
among the fibres (Fig. 2). Few mitotic figures were observed in the explants.
Mononucleated cells migrated onto the plate but did not proliferate efficiently
enough to form myotubes.
Histological observations of the minced explants
Explants that had been minced prior to culture exhibited signs of degeneration
almost immediately. The time course for fibre degeneration was based on observation of 25 explants. After 24 h the fibres were swollen and myonuclei were
pyknotic (Fig. 3). After 72 h cytoplasm was without organized myofibrils and
there were many nuclear ghosts in place of myonuclei. The minced fibres were
not cleared away by macrophages as occurs in vivo. This preservation of the
injured fibres allowed us to infer the non-myonuclear origin of the mononucleated cells which migrated onto the plate (Fig. 5). Mitotic figures were
present in less than 1 % of the mononucleated cells within the explant. The
minced explants gave greater amounts of cellular outgrowth than did unminced
explants 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
260
J. A. SCHRAG AND J. A. CAMERON
wgWvfe.' .." -v »;•'' *-**• «£^"
Fig. 1. A myonucleus from a 9-day muscle explant not minced prior to culture. Note
the prominent nucleolus and euchromatin. The cytoplasm of this cell has healthyappearing mitochondria, glycogen, intact myofibrils, and external lamina. Scale
bar =
Regeneration of newt skeletal muscle tissue in vitro
261
sv*****^
Fig. 2. Mononucleated cell (arrow) with high nucleocytoplasmic ratio and rough
endoplasmic reticulum from a 9-day muscle explant not minced prior to culture.
Notice adjacent intact muscle fibres. Scale bar =
262
J. A. SCHRAG AND J. A. CAMERON
•
t .
-* c
A
''
^ '•*>
f
.**».
Fig. 3. Fibre from 4-day muscle explant minced prior to culture. The nucleus is
pyknotic, mitochondria are degenerating, no glycogen is present, and the myofibrils
are indistinct. Scale bar = 0-5 /im.
Regeneration of newt skeletal muscle tissue in vitro
Fig. 4. Intact mature muscle fibres cultured for 5 days. The presence of myonuclei
(arrows) and myofibrils demonstrates that the musclefibreshave not degenerated or
dedifferentiated. Scale bar = 20 jum.
Fig. 5. Muscle explant minced prior to culture for 4 weeks. The injured fibres are
intact and nuclear ghosts have replaced the myonuclei (arrows). Mononucleated
cells lie outside the fibres (m). Scale bar = 20jum.
263
264
J. A. SCHRAG AND J. A. CAMERON
0 %
^^^r
Fig. 6. Large prefusion aggregation of mononucleated cells adjacent to a minced
explant after 56 days in minced explant culture. Three configurations of
mononucleated cells were present: epithelial (e), stellate (s), and bipolar (b). Scale
bar =
Fig. 7. Myotubes (arrows) within an aggregation of mononucleated cells after 56
days in minced muscle explant culture. Scale bar = 30/im.
Regeneration of newt skeletal muscle tissue in vitro
.
,)U »-„
.
•>
265
.*»
Jk 9 . ••;. *\ «•• : '• L4-
• • .• i /
Fig. 8. A myotube formed in an explant culture after 57 days. The alignment of
myofibrils (arrows) is evidence of differentiation which was not visible at the light
microscopic level. Scale bar =
fa.
266
J. A. SCHRAG AND J. A. CAMERON
Figs 9-10
Regeneration of newt skeletal muscle tissue in vitro
267
cultures. Cellular outgrowth from the minced explants began on days 5 through
to 10. Outgrowth continued and the cells on the plate were studied from day 5
to 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 greater
than either of the other types. After one to two weeks in culture, the cells on the
plate began to increase in number and many mitotic figures were seen on the
plate. After 3 weeks the cells formed aggregations (Fig. 6). Many cells in the
aggregations lined up and after 4 weeks in culture began to fuse into multinucleated tubes located away from the explant (Fig. 7). Thin sections through
these myotubes revealed alignment of myofilaments (Fig. 8). Proliferation of
cells and their fusion into myotubes continued for as long as 160 days. No
degeneration of the myotubes was observed once they had formed.
Secondary cell transfers
Secondary cell transfers were made from seven primary cultures. Proliferating
mononucleated cells that had migrated onto the plate were seeded into secondary cultures at 200 cells per plate. The plating efficiency averaged 70 %. The
cells attached to the plate randomly but migrated to form aggregations before
they began to proliferate. Thus they did not proliferate as distinct clones. The
secondary cultures were maintained up to 6 weeks during which time they continued to proliferate and doubled on the average every 6 days. The three cell
configurations which were seen in the primary cell cultures were present in the
secondary cultures and here too the stellate configuration was predominant. The
secondary cells formed prefusion aggregations as described for the primary cultures 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 more
frequently associated with fusion, since the prefusion aggregates contained cells
of 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 in
vitro analysis of amphibian myogenesis from primary and secondary cultures of
adult newt forelimb muscle. Our culture conditions enhanced survival of uninjured explanted muscle fibres. Despite the absence of muscle satellite cells in
newts, muscle fibre dedifferentiation was found not to be a prerequisite for
myogenesis in culture. Proliferation and differentiation of myogenic cells
occurred 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 of
organized myofilaments. Some thinfilamentsare present in the cytoplasm (arrows).
Scale bar = 0-5jum.
268
J. A. SCHRAG AND J. A. CAMERON
was observed within a time which corresponds to the in vivo regeneration of
minced-muscle fragments in Ambystoma mexicanum (Carlson, 1970) and Notophthalmus viridescens (Cameron, submitted). The sequence of cellular events
during skeletal muscle regeneration in adult newt was the same as that of other
vertebrate muscle which has been studied in vitro.
Jabailey et al. (1982) have recently described a culture system for dissociated
newt blastema cells using supplemented Leibovitz L-15 medium. Their
dissociated blastema cells show similar morphologies to those of the cells
described here. The blastema cells showed a period of proliferation at 2 weeks,
aggregations at 3 weeks, and formation of multinucleated tubes at 8 weeks which
did not show striations even after 16 weeks. Our own attempts at culturing adult
newt muscle in supplemented Leibovitz L-15 yielded cultures which proliferated
and differentiated more slowly than cultures in identically supplemented MEM
(Schrag, unpublished results).
It is important to keep in mind the possibility that muscle fibre dedifferentiation may give rise to myogenic cells by budding into nucleated cytoplasmic
fragments. Although it has not been proven, it has been postulated that muscle
fibre dedifferentiation occurs in stump fibres which have not been directly injured following limb amputation in newts (Hay, 1959; andLentz, 1969). If it does
occur in vivo the factors which promote it are unknown. Konigsberg etal. (1975)
suggest that rapid (8-24 h) myonuclear degeneration observed in vitro after
mechanical isolation of single quail muscle fibres may occur more slowly in vivo
following injury, thus allowing cytoplasmic budding to occur. Survival of muscle
fibres in vitro has been observed previously. Bischoff (1980) has reported that
gentle collagenase digestion of adult rat muscle yields single fibres that survive
up to three weeks in culture, and myogenesis occurs from satellite cells. Neither
Bischoff nor we have observed cytoplasmic budding in culture, and in both
systems myoblasts originate from a population of mononucleated cells within the
muscle tissue. Perhaps newt muscle would not be expected to dedifferentiate in
culture. Recent experiments in our laboratory strongly suggest that muscle fibre
dedifferentiation 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 also
be addressed. Carlson (1972) has shown that intact stump muscle is not required
for normal morphogenesis of muscle during limb regeneration in Ambystoma
mexicanum. After removing 99 % of the stump muscle, normal limb regenerates
with a full complement of muscles are obtained. The source of myogenic cells has
not been identified. Schwann cells also may be capable of forming a regenerated
limb which contains normal muscles (Maden, 1977). Namenwirth (1974) tested
the 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 muscle
eventually became muscle cells in the regenerate. The extent of metaplasia
Regeneration of newt skeletal muscle tissue in vitro
269
during normal regeneration is not known. Although bipolar cells have been
considered myogenic in vitro, Holtzer et al. (1980) have shown that stellate cells
can participate in myogenesis. Jabaily et al. (1982) report that individual newt
blastema cells can assume the three configurations which we observed in our
cultures. Our observation that stellate cells form aggregations within which
syncytia are found suggests that stellate cells in our system may also be myogenic.
Presumably these aggregations are not clones since the majority of the cells
within each aggregation did not fuse into myotubes. A direct confirmation of
this awaits the capability of cloning the cell types found in amphibian muscle.
One problem which has affected previous attempts to culture proliferating
amphibian cells is the long cell-cycle time. Wallace & Maden (1976) have
estimated the cell-cycle time for limb blastema cells in Ambystoma mexicanum
to be about 53 h. It is clear that in vitro studies of regenerating tissues must be
maintained for long periods to allow appreciable growth and cellular interactions
to take place along a time course similar to that occurring in vivo. Several
investigators have cultured intact limb blastemas. Studies dealing with the
growth and differentiation patterns in amphibian limb blastemas have employed
culture 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 newt
blastemal cells for 4 months, although they did not report differentiation of
myotubes with striations. The culture conditions described here permit growth
and differentiation of amphibian skeletal myoblast cells for at least 160 days.
Additional experiments in our laboratory demonstrate that modifications of this
medium can be used to study differentiation of fibroblasts and muscle in Xenopus
laevis, and cartilage and muscle in Ambystoma mexicanum limb blastema explants and cells (Hinterberger & Cameron, 1983).
This work was supported by NSF Grant PCM 79-19338 and Biomedical Research Grant
NIHRR7030 awarded to the School of Life Sciences, University of Illinois. We thank Ms
Rebecca Snyders and Mr Allen Hilgers for expert technical assistance. Special thanks are due
to Drs Allen W. Clark and John F. Fallon for helpful discussions during the preparation of this
manuscript.
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PROSSER,
(Accepted 16 May 1983)