/. Embryol. exp. Morph. Vol. 28, 3, pp. 559-570,1972
Printed in Great Britain
559
Modification of muscle cell phenotype in
monolayer culture by different media
By OSCAR RAMIREZ 1 AND VICTOR ALEMAN 2
SUMMARY
Conditions for monolayer cultures of chick skeletal muscle in a rich medium showed that
non-muscle contaminants were only 20 % of the population. Although the calcium content
of this rich medium was 0-95 mM, multinucleate and long fibres were observed after
8 days in culture.
Either in rich or in restricted media, gelatin organized the pattern of cell and fibre development in the Petri plates. Cells grown outside the gelatin boundaries looked fibroblastshaped and many were vacuolated. Gelatin did not fully prevent the growth of fibroblast-like
population.
Calcium in restricted media appeared to be very important for the acquisition of a definite
elongated shape.
The possibility of the existence of myofibro-myoblasts was supported by the finding of
multinucleated fibroblast-like cells during culture in restricted medium. Epigenetic factors,
such as different media in a given culture or the origin of a serum batch utilized as a component of those media, affected the fate of the cultures and might also explain the myoblastic
variance observed in this and other studies reported.
The capacity of phenotypic expression during the modulation change of muscle cells, in
vitro, depends not only upon their genotypic origin but also on parameters such as the cell
stage during this process, the type of nutrient media used and the interplay of both
parameters; in vivo it depends upon specific cytological interactions.
INTRODUCTION
The retention or change of characteristic properties of cell-multiplying
populations has gained the attention of several groups of investigators during
the past decade. This is a consequence of the development of experimental
techniques that permit the various aspects of this phenomenon to be analysed
at the cellular level (Konigsberg, 1962; Reporter, Konigsberg & Strehler, 1963;
Konigsberg, 1963; Coon, 1966; Cahn & Cahn, 1966; Coleman & Coleman,
1968; Richler & Yaffe, 1970).
Primary skeletal-muscle cultures in monolayer are usually initiated with
suspensions of mononucleated cells which, after adhesive plating, appear to
consist of two main classes - 'spindle-shaped' and 'fibroblast-like' cells.
1
Author's address: Departamento de Bioquimica, Instituto Nacional de Cardiologia,
Mexico 7, D.F. Mexico.
2
Author's address: Departamento de Bioquimica, Centro de Investigation y de Estudios
Avanzados del I.P.N., Mexico 14, D.F. Mexico.
560
O. RAMIREZ AND V. ALEMAN
The photographs appearing in most publications of the muscle field, whether
clonally or mass cultured, usually show more than one cell type at any of the
stages studied (Konigsberg, 1963; Yaffe & Feldman, 1965; Hauschka &
Konigsberg, 1966; De La Haba, Cooper & Elting, 1966; Yaffe & Fuchs, 1967;
Coleman & Coleman, 1968; Reporter, 1969; Cox & Simpson, 1970; Richler &
Yaffe, 1970). By using clonal analysis the last authors have commented upon the
capacity of various muscle cell lines to differentiate, but stressed that there are
differences in the phenotype of the same cell line along passages.
Since the beginning of the clonal analysis (Konigsberg, 1962) it was observed
that there was a low efficiency (10 %) of the colonies growing in a rich medium,
which eventually produced well developed and fully differentiated muscle
clones. From such findings, two important questions arise: firstly, what is the
significance of the percentage of the differentiated clones; and secondly, what are
the origin and stage(s) of the colonies that remain morphologically nondifferentiated ? Thus in muscle tissue there is a claim that, both in vitro and in
vivo, the same stem line of cells give rise to myoblasts and myotubes and on the
other hand some presumptive myoblasts are generated and remain as l satellite
cells' for many years (Holtzer, 1970).
Modification of the muscle cell phenotype by differences in the composition
of the culture media has not been considered extensively, but recently, in relation
to the myotube formation from spindle-shaped cells, attention has been paid to
the role that calcium plays in this phenomenon (Shainberg, Yagil & Yaffe,
1969; Ozawa & Ebert, 1970).
The present work reports, correlates and discusses what is known of the
culture of muscle, either in rich or restricted media. The role of factors involved
in the composition of those media is also analysed in terms of time-specificity
during muscle development. With this type of analysis an explanation for the
coexistence of differentiated and undifferentiated cells within the same culture
could be provided.
MATERIALS AND METHODS
Skeletal-muscle cultures
Rhode Island chick embryos of 11-12 days were employed routinely. Cells
were obtained from the thigh muscle as described elsewhere (Ramirez & Aleman,
1972) and grown 9 days in the following media.
(1) Fresh Medium (FM) (Konigsberg, 1963) containing 0-95 mM-CaCl2.
Horse serum was from Myn Laboratories, Mexico, D.F.; chick embryo extract
was prepared in our laboratory.
(2) Eagle's Minimum Essential Medium for suspension culture supplemented
with non-essential amino acids (Sigma Co.) plus glutamine (MEM) and 10 %
Fetal Calf Serum (FCS) from Difco, containing 0-3 mM-CaCl2 (MEMS).
(3) National Cancer Institute solution (NCI) prepared in this laboratory
contained 1-12 or 2-24mM-CaCl2 as properly indicated. It was supplemented
Modification of muscle cell phenotype
561
with 10%FCS. Unless otherwise indicated, all media contained 100 i.u. of
penicillin and 50 /tg of streptomycin per ml.
Coating of the slides and Petri plates was with gelatin.
Collagen was obtained from adult rat tails (Gallop, 1955). Stock solutions of
collagen were tyndallized and kept frozen at - 2 0 °C; water dilute solutions
were succesively filtered through Selas 01 and 02 candles for sterilization.
Electron micrographs showed characteristic striations in collagen samples, but
a randomly oriented material appeared in gelatin samples. With a 50 fi\ Hamilton
microsyringe, narrow and broad strips of gelatin were gently aligned on slides
washed previously with neutral detergent. On the other hand, after several
assays with different doses, 35 jug of gelatin were routinely applied to 10 cm
diameter Falcon Petri dishes, diluted with 2-5 ml of double distilled water to
completely cover the bottom of the plate. Petri dishes were gently rotated to
homogenize the solution and left overnight at 45 °C to evaporate the liquid.
Embryo extract
Nine-day-old chick embryos were decapitated, completely eviscerated and
homogenized in Saline G solution (Konigsberg, 1963) for 45 s in a Sorvall OmniMixer at 16000 rev/min, dispersed with 10 mg of hyaluronidase (Worthington)
per 100 ml of the suspension for 1 h, centrifuged 20min at 31000g- and the
resultant supernatant spun down for 3 h in a Beckman SW 25-1 (or SW 25-3)
rotor at 64000 g. The supernatant was sterilized by successive filtration through
Selas 01 and 02 and kept frozen until used.
Gelatin stripped slides were seeded with hundreds of cells and covered with
FM; the development of the culture was followed daily by direct observation
under the microscope.
Stain and observation of cultures
The plates were rinsed with Saline G solution and fixed in 4 % formaldehyde.
Aniline blue orange and Delafield haematoxylin were used to stain the monolayers and covered with 5 % polyvinylic alcohol. A Zeiss POL microscope was
utilized for the microphotographs and a Honeywell 80S Repronar for the
panoramic views.
RESULTS
During the early stages of development in FM there was a definite orientation
of bipolar cells and myotubes were formed along their major axis which ran
parallel, mainly at the edges of the strips of gelatin (Fig. 1 A). It is known, and
we have confirmed, that myoblastic embryonic cells will grow in a similar
manner in vivo (10-11 days); these are aligned along the connective tissue fibres
(Fig. 1B). At a later stage, in vitro as well as in vivo, the ends of the cells
approximate to each other and fuse to form myotubes. Remarkably, the strip
guide in both cases is either connective tissue fibres or the gelatin lanes. Cells
36
EMB 28
562
O. RAMIREZ AND V. ALEMAN
2C
Fig. 1. Alignment and muscle fibre organization in gelatin lanes growing in vitro in
FM (A) and connective tissue in vivo (B). x 250.
Fig. 2. (A, B) Initiation of myotube formation from bipolar cells at 24 and 48 h of
culture in FM; x 160 and x 63. (C, D) Monolayer muscle cultures grown in FM
during 8 days; x 1-2 and x 12.
Modification of muscle cell phenotype
563
growing on the slides looked healthy and were bipolar within the boundaries
of the strips, whereas the cells outside these limits were ftbroblast-shaped,
grew very poorly and many of them were vacuolated. It must be noticed that the
alignment of cells in later stages of the culture, formed curls inside the strips of
gelatin and they tended to remain straight along the border.
Another series of experiments in FM were performed in plastic Petri dishes
that were previously covered with gelatin: the fibre pattern developed was a
curled one, whose behaviour resembled that observed in the slides.
Although collagen or gelatin can organize the fibre pattern in vitro, it is known
that during clonal analysis the critical time for cloning muscle cells in the
presence of such protein is a very short one, namely the first 24 h of culture.
However, it is also known that in mass cultures of fibroblasts the period for
active synthesis of collagen for 'conditioned medium' in muscle development is
the stationary phase; and it is the same period when contracting muscle fibres
appear in primary cultures of chick embryo thighs (Konigsberg & Hauschka,
1965).
Because of these facts, it was thought that in a muscle mass culture the
number of myogenic cells would be improved if the gelatin could be maintained
at a fixed concentration in a rapidly dividing population, which had been
started with 106 cells per plate. On the other hand, this precaution would also
'prevent' excessive contamination from other cell types. Thus we scheduled
medium changes which included the addition of 35 /.tg of gelatin per plate every
time the medium was replaced. With cell densities of about 3 x 106 per Petri dish
(10 cm in diameter), it was observed that around 24 h of culture the cells were
bipolar and mononucleated (Fig. 2 A) and at 48 h myotube formation was
clearly observed (Fig. 2B). Only 20 % contaminant by non-characteristic cells
appeared and these were interspersed mostly at the edges of the plate at the end
of one week of the culture (Fig. 2C, D). This type of cell appeared even though
the 'differential adhesion' technique was employed for cultivation of the muscle
cells (Ozawa & Ebert, 1970).
Worthy of mention is the fact that Ca2+ concentration in FM measured by
atomic absorption spectrometry was only 0-91 mM instead of the calculated
1-12 mM. Nevertheless, the average number of nuclei per fibre was higher than
100 (Fig. 3 A, B), although previous reports (Shainberg et ah 1969) have shown
that l-4mM-Ca2+ was the minimum for the onset of fusion and formation of
multinucleated fibres in rat cultures. However, under conditions of low Ca2+
spontaneous contraction of the fibres was rather exiguous.
In some experiments Ca2+ was increased in FM to 2-24 mM. During the first
2 days of culture the growth of the cells was faster than at the lower Ca2+
concentration. Furthermore myotubes were observed before 48 h of culture
when 2-5 x 106 cells were plated by using the 'differential adhesion' technique.
Nevertheless, despite the established routine of medium replacement every
other day, the plates began to show a cloudy aspect after the third day in
36-2
564
O. RAMIREZ AND V. ALEMAN
Fig. 3. A, B. Monolayer muscle cultures grown in FM during 8 days, x 160.
Fig. 4. Dusty aspect of muscle cells grown in FM containing double amount of
CaCl2, after third day. x 63.
Fig. 5. A,B- Muscle cells grown in MEMS during 2 and 3 days; myotube formation
and atypical syncytium. x 160.
Fig. 6. Atypical fibroblast-like cells containing more than one nuclei grown in
MEMS during 4 days, x 250.
Modification of muscle cell phenotype
565
culture. Obviously, these were not optimal conditions for muscle culture,
although fibres were wider and multinuclearity was found along the fibre (Fig. 4).
In order to test the components of FM and its capability to differentiate
muscle cells, an experiment was performed employing FM with 2-24 mM Ca21",
but horse serum was replaced by 10 % FCS. The cells grew and differentiated
poorly, but when medium was replaced by complete MEM containing 10 %
of FCS, the culture improved and myotubes began to appear in the plates (even
though Ca2+ was 0-3 mM). This suggests that NCI in presence of FCS impairs
the phenotypic expression of myotube formation.
The differentiation of embryonic muscle cells was tested in several media and
the schedules used were as follows:
(a) FM was renewed on days 2, 4 and 6.
(b) FM was used on the first day and was replaced by MEMS containing
0-3 mM-Ca2+ on days 2, 4 and 6.
(c) MEMS containing 0-3 mM-Ca2+ was renewed on days 2, 4 and 6.
(d) NCI containing 7 % FCS and 1-2 mM-Ca2+ (NCIS) was renewed on days
2, 4 and 6.
(e) FM was used on the first day and was replaced by NCIS on days 2, 4
and 6.
During the first day of culture the MEMS and NCIS plates contained highly
elongated bipolar cells and the myotube formation was initiated well in advance
of the 50-60 h previously reported for a primary culture of rat skeletal muscle
(Yaffe, 1971).
After the first replacement of media, plates growing in MEMS and those
first grown in FM and replaced by MEMS showed the initiation of myotube
formation with elongated mononucleated cells and eventually both showed a
progressive development (Fig. 5 A). However, more myotubes appeared in the
plates initiated with FM, although the cells were one-fifth shorter than those
grown with MEMS alone. Remarkably, after 78 h in culture the plates containing MEMS showed very wide fibres with an axial ratio of approximately
5:1. It was also observed that their cytoplasm had at least 20 nuclei contained
in three or four rows. On the other hand, the total length of such fibres was
approximately four times that of an elongated myoblast (Fig. 5B). Disorganization within the syncytium was also observed. In comparison with the
FM containing plates the population of MEMS plates was healthier but
seemed exiguous.
Cells grown in MEMS, whether or not initiated in FM, developed into
long-shaped myotubes that looked very healthy. Plates which were started with
FM and subsequently fed with MEMS after the sixth day of culture had
predominantly a well-developed population of fibres.
On the contrary, in FM there were abundant fibres but with very shortlooking myoblasts. After the renewal of the medium the cell pattern followed
prior observations. Cells which were changed to or remained in FM, were more
566
O. RAMIREZ AND V. ALEMAN
abundant and formed as much as 70 % more fibres than cells growing in poorer
media. In FM fibres became very wide.
Cultures grown in NCIS or in FM followed by NCIS were covered by a dusty
medium and only isolated cells and few myotubes were observed.
From the above-mentioned results it seems apparent that a rich medium is
needed for culture initiation in order to preserve the capability of myotubes to
develop into fibres after a week, since the addition of gelatin every time the
medium was changed was not enough to accomplish such effect.
DISCUSSION
In the first experiments evidence was obtained to support the role of gelatin
in organization of the pattern of fibre growth. On the other hand, the replacement of medium containing gelatin scheduled every other day, permitted the
muscle-type population to predominate over the 'fibroblast-like' cells within
the same plate. The 80 % proportion of the muscular population at the end of
one week in culture must be considered in relation to the factors involved in
such a culture. Thus conditioning of the surface of the plates prevents, delays
or diminishes the probability that the non-muscle cell types will divide at the
normal rate. This effect was reinforced in our cultures every time the medium
was changed, since gelatin was added on each occasion in a rapidly growing
population.
It is possible that gelatin in these culture experiments is playing a role in
myogenesis, which is similar to that of collagen in connective tissue; that is,
collagen may be important in the modulation of myogenesis in vivo.
With FM, collagen was utilized for clonal analysis of chick muscle and 43 %
of the colonies obtained were myogenic ones. Nevertheless, the clonal efficiency
was not augmented whether or not more collagen was added to the plates
(Konigsberg & Hauschka, 1965).
In regard to the remaining fibroblast-like population, several points must be
mentioned. Thus it is important to determine if these cells are true fibroblasts
or are atypical myoblastic cells. We have observed trapezoidal cells from a
primary muscle cell culture grown in MEMS contain three or four nuclei in
their cytoplasm (Fig. 6), which supports the idea that cells other than spindle
shaped are capable of forming a syncytium. Ca2+ appears to be very important
for the acquisition of a definite shape of the cells possibly by affecting their
membrane (Ambrose, 1967). A Ca2+ concentration of only 0-9 mM in FM was
enough to sustain fusion and formation of long jnurtinucleated fibres although
contractility was impaired. An increase in Ca2+ may be necessary to develop the
non-typical myogenic cells to a spindle-shaped population. Thus at a Ca2+
content of 2-2 mM almost 100 % of the population at the end of the first day was
fusiform.
In regard to the Ca2+ concentration in a given medium, it should be
Modification of muscle cell phenotype
567
mentioned that NCI as compared with MEM does not permit addition of Ca2+
above 1-3 mM, since precipitation occurred presumably because of the phosphate
salts already present for buffering action.
Recent observations regarding the behaviour of spindle- or fibroblast-shaped
cells in several environmental conditions have led to the hypothesis of myofibro-myoblastic cells (Lappano-Colleta, 1970).
It is possible that some cells of a given inoculum have to pass through
proliferative mitosis before they acquire the capability for the so called 'quantal
mitosis' (Holtzer, 1970). Our findings with cells cultured 24 h in MEMS and
NCIS give support to this hypothesis since myotubes developed before 48 h. In
earlier work which led to the discovery of the conditioned-medium containing
collagen (Konigsberg, 1962), it was observed that with different cell densities of
trypsinized muscle grown within the same medium, myotubes were formed at
the same rate irrespective of the cell confluency shown by the culture. In other
words, a high-density myoblastic population divides less rapidly, then a lower
population can give rise to a permissive 'quantal' myoblastic cell. Similar
findings with rat muscle have been reported recently (Yaffe, 1971). The interpretation was that at the onset of fusion there are myoblasts differing in their
capacity to fuse and this is only dependent on the length of time the cells were
maintained in vitro and was irrespective of their plating density. It is likely that,
in our case, cells grown in MEMS and NCIS for 24 h could already contain
some 'mature' cells to initiate myotube formation. Since the number of
myotubes within a given plate is low at the beginning of the culture, it is likely
that the distribution of such 'permissive' myoblasts is random in the plate, so
the probability of meeting each other is low. However, we cannot exclude the
possibility of local outbursts of myotubes.
The influence of a given serum batch must be pointed out. In our initial
experiments with FM, cells and fibres were perfectly normal and accounted for
the highest proportion of muscle cells type in a Petri plate ever obtained. On the
other hand, a fresh batch of horse serum gave rise to short myoblasts grown
initially or all the time with FM. Remarkably, FM initiated cultures which
were subsequently transferred to MEMS began to change their myoblastic
shape to highly elongated (4-5:1 axis ratio) mononucleated cells, thus favouring
the hypothesis of myofibro-myoblastic cells. However, these cells were not as
elongated as those cultivated in MEMS alone.
In regard to cells cultivated in MEMS there are some points to be discussed.
Firstly, culture in these conditions always gave rise to a very elongated and
bipolar cell population within the first 48 h, in contrast to cells growing in FM
during the same time. Secondly, in MEMS plus gelatin after 78 h the width of
the fibre, as well as the unusual disposition of the nuclei (three or four rows of
nuclei in the same fibre) along its major axis, is a clear example of changes of
phenotypic expression of cells under different environmental conditions.
However, the length of the fibre in comparison with a mononucleated cell does
568
O. RAMIREZ AND V. ALEMAN
not agree with the number and pattern of this nuclear distribution within the
fibre. This finding suggests that there is an alternative explanation to that of the
myoblast-ends fusion. One possibility is the multiplication of nuclei in the fibre
without cytoplasmic participation; however, this possibility seems unlikely in
view of the existing evidence in favour of fusion as the mechanism for syncytium
formation in vitro (Konigsberg, 1963) and in vivo (Mintz & Baker, 1967). One
further alternative is lateral fusion of the highly elongated cells, even though two or
three terminal cell fusions are in accordance with the length of the formed fibre.
In culture, once a strain cell achieves a definite stage it is not irreversibly
obligated to remain in it (Cahn & Cahn, 1966; Richler & Yaffe, 1970). There
is evidence to show the role played by certain epigenetic factors in the regulation
of phenotype expression, such as intra- and inter-cellular metabolite interactions, cell contiguity, cell products of different strain(s), population densities
(Konigsberg, 1963; Grobstein, 1967), inducers or factors for strain recognition
(Tiedemann, 1968; Humphreys, 1963). Since some of these factors can be
changed in cell cultures, this provides a convenient method for the study of the
mechanisms involved during changes in phenotypic expression.
It appears difficult to explain the periodical behaviour of muscle-cell lines
cultivated in clones that failed to form fibres in spite of their proved capacity to
produce them previously. They were also capable of regaining such an ability
after various passages. Such findings were difficult to explain since it was
shown that changes in ploidy do not affect the phenotypic expression of skeletal
muscle cells (Richler & Yaffe, 1970). In the light of our present work we can
speculate that different batches of horse serum could be responsible for such a
myoblastic variance. The retention of the capacity of phenotypic expression of
muscle cells appears to depend not only on their genotypic origin but on some
other parameters: the stage of phenotypic expression during the modulation
process, which includes specific cyto- and tissue-architecture; the type of nutrient
media used, and the time-specificity in the action of a given medium to a given
cell during development.
RESUME
Nous decrivons des conditions pour faire des cultures de muscle squelettique de Poulet
dans monocouches. Dans un milieu enrichie on n'a pas trouve que 20 % de contaminants
par rapport a la population totale. Malgre la faible concentration de calcium dans ce milieu
(0,95 mM), apres huit jours de culture nous avons observe desfibreslongues et multinucleaires.
Aussi bien que dans le milieu enrichie, dans le milieu restreint, l'organisation du
developpement des cellules et des fibres a ete guidee par la gelatine dans le boites de Petri.
Les cellules qui ont pousse hors de la gelatine avaient forme fibroblastique et beaucoup
d'entre elles ont ete vacuolees. La gelatine n'a pas empeche completement la croissance des
cellules fibroblastiques.
Dans le milieu restreint, la calcium semble important pour l'acquisition d'une forme
elongue definitive.
Le fait d'avoir trouve cellules semblables au fibroblastes multinucleaires pendant la culture
dans le milieu restreint, soutient la possible existence de myofibre-myoblastes.
Certains facteurs epigenetiques ont montre l'affectation des cultures. Nous avons observe
Modification of muscle cell phenotype
569
par exemple, q'un milieu different dans une culture donnee ou la variation de la source
d'obtention de la meme espece d'un serum utilise dans dite milieu, ont montre Faffectation
des cultures et ceci peut aussi expliquer la varietee de myoblastes observes au cours de ce
travail et des autres publies.
La capacite d'expresion phenotipique pendant la differentiation du muscle en culture
depend, non seulement de son origine genotipique, mais des parametres tels que: l'etape de
differentiation des cellules, le sort du milieu de culture utilise, ou tous les deux. In vivo cette
capacite depende aussi des interactions entre cellules du meme ou de type differente.
The authors wish to thank Dr C. S. Corker for correcting the manuscript and Dr E. A.
Newsholme for helpful discussions.
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(Manuscript received 25 January 1972, revised 1 June 1972)