Chapter 2 Cellular Aspects of Muscle Differentiation in Vitro

CHAPTER 2

CELLULAR ASPECTS OF MUSCLE DIFFERENTIATION in Vitro David Yufe DEPARTMENT OF CELL BIOLOGY, THE WEIZMANN INSTITUTE OF SCIENCE, REHOVOT, ISRAEL

I. Introduction . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 11. Differentiation of Primary Cultures . . . , . . . . . . . . . . . . A. Formation of Multinucleated Muscle Fibers . . . . . . B. Fusion of Myoblasts of Different Species Origin .. . C. I n Vioo “Hybridization” of Muscle Cells . . . . . . . . 111. Myogenic Cell Lines . . . , . .. . . . . . . . . , . . . . . . . . . . . . . A. Effect of Continuous Cell Multiplication on the Retention of Differentiation Potentialities . . . . . . . . B. Cessation of DNA Synthesis at Fusion . . . . . . . . . . . C. Fusion Specificities . .. .. .. .. .. .. .. .. .. .. . . . . . . D. The Inheritance by Single Cells of the Capacity to Differentiate ................................ E. The Effect of Collagen . . . . . . . . . . . . . . . . . . . . . . . . F. Growth Characteristics and Ploidy of Myogenic Cell Lines ...................................... G . Enzymic Manifextations of Differentiation iri Vitro IV. Induction of DNA Synthesis and Mitosis in Nuclei within Muscle Fibers . .. .. .. .. .. .. .. .. . . .. .. . .. .. . V. Comments ...................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

37 39 39 41 44 45 45 47 48 49 52 54

57 60

70 75

1. Introduction

The complex interrelationships between cells in the multicellular organism seriously limit the types of questions about differentiation phenomena that can be analyzed in the intact organism. The hetero37

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geneity of cell types and the existence of homeostatic regulation mechanisms complicate the interpretation of many observations. Working with simple unicellular organisms as model systems proved to be useful in studying some aspects of cellular differentiation. However, the applicability of such systems is also limited, since they lack one of the most fundamental features of differentiation, i.e., the stability of the differentiated state, manifested in the apparently hereditary perpetuation of differencesbetween cell lines within the same organism. Therefore, the study of some of the main aspects of differentiation necessitates the development of experimental systems in which the inherent properties of isolated and defined cell types can be investigated under controlled conditions. As a result of intensive work on the requirements for the cultivation of somatic cells in uitro many obstacles to the growth of isolated cells have been overcome. While the methods thus developed have contributed to our knowledge of the metabolism of animal and plant cells, considerably less successful have been attempts to elucidate questions related to the nature of differentiation processes. The phenomenon of dedifferentiation, i.e., loss of tissue-specific characteristics after a short period of growth in tissue culture, was so universal as to be almost accepted as a characteristic of cells multiplying in uitro, thus raising doubts about the concept of the stability of differentiation as an inherent property of differentiated cells. However, experiments in recent years have shown that this need not be the case. Retention of differentiated traits in uitro has been achieved in the cultivation of several kinds of cells (Cahn and Cahn, 1966; Coon, 1966; Sato and Yasumura, 1966). Although thesc systems arc still few, they show that in spite of the high interdependency of cells in uiuo, isolated cells can manifest a surprisingly high degree of autonomy in their capacity to differentiate. These observations open thc way to more detailed studies on retention of the capacity for differentiation and its expression at the cellular level. Muscle tissue culture had been included in the repertoire of tissue culturists since the first days of this art (Lake, 1915; Lewis, 1915). Some of the unique properties of this system make it especially attractive for studying various aspects of cell biology; differentiation of muscle tissue is associated with very distinct steps which can be readily analyzed, such as formation of multinucleated fibers, cessation of DNA synthesis, crossstriation, and contractility. While early studies were made on explants of small fragments of muscle tissue, the introduction of techniques for the dissociation of tissue into single cell suspensions by enzymic treatment

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39

and for the cultivation of cells in monolayers in partially defined media facilitated direct microscopic observations of single living cells, and permitted a more detailed analytical approach ( Moscona, 1952; Konigsberg et al., 1960; Konigsberg, 1961). This article reviews a series of experiments which utilize muscle cell cultures as a model for studying some of the inherent properties of differentiating cells. The following account is intended to familiarize the reader with the experimental system, describe our approach and results, and point out some of the possibilities which this cell system offers for future research. II. Differentiation of Primary Cultures

A. FORMATION OF MULTINUCLEATED MUSCLEFIBERS Monolayer cultures of muscle cells are prepared from embryonic or newborn skeletal muscle. At these stages of development, the tissue consists of mononucleated muscle cell precursors and partially differentiated multinucleated cells; the tissue is dissociated into a suspension of single cells by treatment with trypsin. Most of the fibers are destroyed or lost in this process. After removal of the trypsin, followed by decantation or filtration of the suspension to remove the large tissue debris, the suspension is seeded into tissue culture containers (usually plastic petri dishes), The cells settle down and form a monolayer which consists of a heterogeneous population of mononucleated cells; the majority of these are spindleshaped myoblasts, the mononucleated precursor cells of muscle fibers. The culture also contains fibroblasts and, possibly, other cells from the original muscle. The cells multiply and on day 3-4 of culture, when a confluent cell layer has been formed, the myoblasts begin to aggregate nnd differentiate into thick multinucleated muscle fibers containing hundreds of nuclei. One or two days later, these fibers begin to contract spontaneously and typical cross-striation can be observed (Figs. 1and 2 ) . The mode of formation of these fibers has been a matter of controversy for many years. Most of the earlier workers attributed the multinucleated state to multiplication of nuclei within the growing fibers. However, inorc recent observations, mostly in tissue culture systems, have supported the notion that the multinucleated fibers were formed by fusion of mononucleated myoblasts into ribbonlike syncitia (for reviews, see Betz et al., 1966; Altschul, 1962; Murray, 1960). Even simple observations on the kinetics of fiber formation in uitro made the earlier proposed mechanisms very unlikely; under proper culture conditions one can often

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DAVID YAFFE

see within a matter of 6 to 12 hours the conversion of a monolayer of mononucleated cells into a network of multinucleated fibers containing hundreds of nuclei. Assuming formation of the fibers by nuclear replication would imply a generation time too short to be plausible. In spite of

FIG. 1. Seventy-hour-old primary rat muscle cell culture fixed at the onset of formation of multinucleated cells. Note aggregation of spindle-shaped myoblasts (arrows) which can be distinguished from the fibroblastic cells.

the rapid formation of the fiber and its increasing content of nuclei, no mitotic figures have been observed within multinucleated cells. Furthermore, the formation of multinucleated fibers could be shown to take place even in the presence of inhibitors of DNA synthesis such as nitrogen mustard and myleran (Bassleer et al., 1963; Konigsberg et al., 1960). More direct evidence that mononucleated cells fuse into multinucleated fibers has been obtained by cinematography (Capers, 1960) and autoradiography ( Stockdale and Holtzer, 1961; Yaffe and Feldman, 1965). Autoradiography of cultures exposed to the tritium-labeled DNA precursor revealed the following features: 1. When myoblasts obtained from rat thigh muscle were exposed to th~rnidine-~H during the first 48 hours of cultivation (before the fonnation of multinucleated fibers), the muscle fibers which subsequently

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formed contained 90-100% thymidine-3H-labeled nuclei. However, when such labeled myoblasts were mixed with unlabeled myoblasts, the resulting fibers contained both labeled and unlabeled nuclei in a random arrangement.

FIG.2. Twelve-day-old rat muscle cell culture consisting of differentiated multinucleated fibers with fibroblastic mononucleated cells in the background.

2. Supply of the labeled nucleotide to cultures during the process of fiber formation, or later on, resulted in the labeling of nuclei within the mononucleated cells but not in the multinucleated fibers. The presence of both labeled and unlabeled nuclei within the same fibers (in the first experiment) indicated that the in oitro formation of multinucleated muscle fibers takes place by fusion of mononucleated cells and not by nuclear replication within the fiber. The lack of incorporation during and after cell fusion (in the second experiment) showed that the nuclei within the fibers are postmitotic and do not synthesize DNA.

B. FUSION OF MYOBLASTS OF DIFFERENT SPECIES ORIGIN The formation of multinucleated fibers via the fusion of mononucleated cells raises questions concerning cell type specificity in the process of

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DAVID YAFFE

fusion. Experiments performed several years ago ( Holtfreter, 1947; Moscona, 1956) have shown that when cells obtained from different tissues are “co-mingled,” the cells usually sort out and aggregate according to their tissue of origin. Even when cells of different species are mixed together, sorting out into aggregates usually takes place according

FIG.3. Hybrid fibers formed in mixed culture of rat and calf thigh muscle cells. Labeled nuclei are of rat origin. (From Yaffe and Feldman, 1965.)

to tissue type rather than species identity ( Moscona, 1957). These experiments were interpreted as indicating that similarities in tissue-specific cell surface properties can extend across wide taxonomic differences. The fusion property of muscle cells offered another approach for testing the functional similarity of homologous specialized cells. Can inyoblasts from different species fuse with each other? Will fibers having both a cytoplasmic and a nuclear contribution from different species be able to differentiate and function? Will myoblasts fuse with cells from other tissues? To answer these questions, primary cultures of thigh muscle cells were prepared from calf fetuscs, newborn rabbits, and rats. Thc rat cells werc

2.

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MUSCLE DIFFERENTIATION IN VITRO

labeled by growing them in the presence of th~midine-~H. After 3 days secondary cultures were prepared in which labeled rat cells were mixed with nonlabeled rabbit or calf cells. In both systems differentiation of multinucleated contracting muscle fibers took place. Autoradiographs of cultures of muscle cells produced in cultures of the mixed myoblasts revealed that in the two combinations tested, the muscle fibers contained both labeled and unlabeled nuclei in a pattern indicating that myoblasts of different species origin fused to form hybrid muscle fibers (Fig. 3, Table I). TABLE I FORMATION O F HYBRID MUSCLE FIBERS IN CULTURES OF THIGH MUSCLECELU OF DIFFERENTGENETICORIGIN^ Sources of cells

Thymidine-3Htreated cultures Rat Rat Rat Rat Rat Rat

Untreated cultures

-

Ralhi t

-

Calf

-

Chick

Labeled nuclei in fibers of secondary cultures ( % ) go+ 2b 55 12b 99f 2 37* 9b 9 3 & 6b 51 + 26b

*

From Yaffe and Feldman (1965). The standard deviation is calculated from weighted values according to the number of nuclei in each fiber. a b

In order to test whether thigh myoblasts of mammalian and avian origin will also fuse to form hybrid fibers, rat and chicken cells were mixed under conditions similar to those described above for the ratcalf mixtures. Hybrid cells formed containing nuclei of the two different genetic origins. However, a certain proportion of fibers contained either labeled or unlabeled nuclei, i.e., nuclei either of rat or of chicken origin. This seems to indicate that although rat myoblasts can fuse with chicken myoblasts to form multinucleated fibers, in this situation there is also a tendency toward fusion according to the genetic origin of the cells. No incorporation of labeled nuclei into fibers was observed when myoblasts were mixed with labeled cells of kidney or heart origin, indicating nonparticipation of the latter in muscle fiber formation.*

* Preliminary experiments have suggested the possibility that, in fact, cardiac myoblasts may sometimes fuse into muscle fiber of skeletal origin, but this is restricted to very specific physiological conditions.

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DAVID YAFFE

These experiments demonstrated that myoblasts derived from as genetically remote organisms as rat, calf, and chicken possess enough similarity in the finer details of differentiation to enable them to fuse and form hybrid cells capable of following an apparently normal course of differentiation within a common cytoplasm. Differentiation of the fiber, following fusion, is associated with various metabolic changes resulting in the formation of the contractile mechanism and appearance of enzymes involved in energy supply for the energetics and biochemistry of contraction (see Section II1,G). Thus, the question arises whether the nuclei of both origins within the hybrid fiber contribute to the differentiation of the cells. If so, how does this affect the synthesis and organization of specialized proteins? Do subunits of structural proteins formed under the direction of the different genomes segregate or form mixed structural elements? C. I n Viuo “HYBRIDIZATION” OF MUSCLECELLS Conclusive evidence that muscle formation in viuo also takes place by cell fusion has recently been produced by Mintz and Baker (1967). Artificial chimeric mice (“allophens”) were formed by aggregating blastomeres dissociated from cleavage stage embryos of different genotypes and reimplanting these combined aggregates into appropriate female hosts for further development. By this procedure these authors obtained adult mice which consisted of a mixture of cells of two genotypes. These allophenic mice were composed of cells originating from two strains of mice, each homozygous for a different allele for an enzyme (isocitric dehydrogenase) . The isozymes could be differentiated by their electrophoretic mobility. Extracts of tissues of genetic F1 hybrids of the parental strains produced on electrophoresis three bands of isozymes: two of the parental type and an intermediate one, the result of the combination of subunits of the two parental isozymes. However, extracts of tissues of the allophenic mice produced only the two parental bands, save for extracts of skeletal muscle tissue which also produced the intermediate band-identical to the pattern produced by the Fl hybrids. Thus, the appearance of a hybrid type of enzyme only in muscle extracts indicates that, unlike in other tissues, in the muscle active nuclei of both genotypes reside within the same muscle cell cytoplasm. Therefore, subunits of the electrophoretically different alleles combine randomly, at status nascendi, to form a hybrid type of isozyme molecules.

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111. Myogenic Cell lines

CELLMULTIPLICATION ON THE RETENTION OF A. EFFECTOF CONTINUOUS DIFFERENTIATION POTENTIALITIES Champy, in a series of articles, has maintained that most of the cells in the body dedifferentiate in tissue cultures. They return, he claims, to a completely indifferent type of cell that no longer shows the imprint of its origin. In explants from late fetal stages he finds that cells of the kidney tubules, of the thyroid, of the parotid and of the submaxillary glands, of the smooth muscle, of the mesenchyme, etc. dedifferentiate into an indifferent embryonic type indistinguishable from each other. This dedifferentiation, he claims, is associated with the phenomena of cell division. The rapidity of dedifferentiation is a function of the rapidity of the celldivision. Furthermore, according to Champy, all cells differentiated for a special function lose or tend to lose during mitosis, their characteristic function. In the animal organism they recover immediately after the telophase, since they are subject to the same functional excitation as before division (Lewis and Lewis, 1917, p. 189).

This phenomenon of dedifferentiation has since been repeatedly observed by many investigators who attempted to grow differentiating cells in culture (Davidson, 1964; Eagle, 1965). Muscle cells in tissue culture manifest a higher capacity to differentiate than other cell types. However, they undergo only a limited number of cell divisions and then fuse into postmitotic multinucleated fibers. Further differentiation of these cells thus takes place in the absence of cell division. Konigsberg has shown that muscle cells can be cloned in tissue culture and still retain the capacity to differentiate ( Konigsberg, 1961). Under these conditions myoblasts multiply and form colonies, each originating from a single cell. However, after several cell divisions these cells also fuse into multinucleated fibers. The capacity of muscle cells to differentiate in vitro may therefore be attributable to their restricted number of cell divisions. Thus, differentiation of these cells may take place on the basis of the utilization of a preexisting stock of informational molecules which were produced in uiuo. Experiments were designed to determine if the fusion and differentiation of myoblasts into postmitotic multinucleated fibers was a rigidly programmed phenomenon which must occur within a definite number of cell divisions. What will be the result of growing the myoblasts under conditions of prolonged multiplication in vitro? Can the capacity to differentiate be retained over extended periods of multiplication? Will the cells continue to grow in the undifferentiated form or will they degenerate? Attempts were made to establish culture conditions which would

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DAVID YAFFE

promote multiplication of myoblasts and prevent cell fusion. An essential requirement was to start out with cultures which would contain predominantly myoblasts. It was noted that after dispersing primary muscle cell cultures with trypsin, on replating the cells in the standard medium (Medium 199 supplemented with 2% chick embryo extract; 0.5% bovine serum albumin, and 10% horse or fetal calf serum), the myoblasts attached to the plate much more slowly than other cell types present in the suspension. Accordingly young cultures, before the onset of cell fusion, were dispersed with trypsin; after removal of the enzyme, the cells were incubated in growth medium for 30 minutes at which time most of the nonmyoblastic cells had begun to attach to the bottom of the plate. The medium containing the floating cells was collected and placed in dishes which were first coated with a collagen film and seeded with a small number of lethally irradiated cells ( 1 X lo6 irradiated cells per a 60 mm diameter plate). The irradiated cells, which are unable to multiply, served as a feeder layer and thus enabled very dilute populations of myoblasts to survive and continue to multiply. After 3-4 days the cultures consisted of a network of myoblasts with very few nonniyogenic cells. The cultures were repassaged in the above manner at the onset of fusion. In some experiments a carcinogenic agent, methylcholanthrene, was introduced into the culture medium during the first two cell passages. This was done to test whether neoplastic transformation of the cells would facilitate their serial propagation in tissue culture (Earle and Nettleship, 1943; Benvald and Sachs, 1985) and prevent degeneration of the cells ( a phenomenon commonly observed in experiments to maintain cells of primary cultures by serial passaging in uitro (Hayflick, 1965) ) . Several independent experiments for the serial passaging of myoblasts were initiated; most of them resulted in loss of the cells after 4-6 passages by gradual cessation of multiplication. However, two cell lines originating from cultures exposed to methylcholanthrene during the first two passages could be maintained by serial passage without loss of their capacity to replicate and differentiate. One of these lines (designated Lo) has now been maintained in uitro for more than 2 years and the other ( Mdl) for 11 months. The differentiation properties of the two lines are virtually indistinguishable. In the course of the first few passages the cells were cultured on a collagen film and feeder layer for good growth and differentiation; however, after several serial passages they grew readily and differentiated into a very dense network of contracting fibers in the absence of collagen and feeder layer (Figs. 4-6) (Yaffe, 1968). It is not clear whether the exposure of the cells during the first two

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47

passages to methylcholanthrene had any effect on them. However, in further attempts to establish myogenic cell lines, four lines were finally established, three of which were obtained from cultures not treated with carcinogens (Table I1 ) , It can therefore be stated that whatever the effect of the carcinogen on these cultures, it clearly does not play an essential role in the establishment of myogenic cell lines.

FIG.4. Muscle fiber formation by cells of the line L, fixed on day 8. X 9. (From Yaffe, 1968.)

B. CESSATION OF DNA SYNTHESIS AT FUSION Experiments were performed to test whether the prolonged multiplication of myogenic cells in vitro affects the cessation of DNA synthesis which normally takes place at fusion. Cultures of the line L, were allowed to differentiate. At different stages after the onset of fusion, the cells were then fixed and processed for exposed to a 5-hour pulse of th~midine-~H, autoradiography. In all preparations, th~rnidine-~H was incorporated only into the nuclei of mononucleated cells-in no case were nuclei within multinucleated fibers labeled. Thus, cells after prolonged main-

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tenance in a state of continuous multiplication retain their capacity to stop DNA synthesis at fusion.

C. FUSION SPECIFICITIES Another parameter which was considered in relation to possible changes in these cells was fusion specificity. It was shown that fusion of myoblasts

FIG. 5. As Fig. 4 but X 130. Note homogeneity of inononucleated cell type and the paucity of fibroblasts (compare to Figs. 1 arid 2 ) .

in primary cultures is tissue-specific and takes place only between myogenic cells (see Section I1,B). In order to see whether myoblasts of primary cultures will fuse with cells of the established lines, undifferentiated 2-day-old cultures of the line Lo were exposed to a 48-hour pulse of thymidine-8H. The cultures were then trypsinized and the cells were added ( 2 X lo6 cells per plate) to 48-hour primary rat thigh muscle cultures ( 4 X lo6 cells per plate). Two days later, i.e., after formation of inultinucleated fibers, the cultures were fixed and processed for autoradiography and the number of labeled nuclei within the fibers was counted. It was found that whereas in cultures prepared from the

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49

th~midine-~H-treated Lo cells alone, 98% of the nuclei within the fibers were labeled, the fibers in the mixed cultures contained on the average only 14 2 6% of labeled nuclei. These results indicate that the labeled myoblasts of the La line formed multinucleated fibers with the primary myoblasts.

FIG.6. Cross-striated muscle fibers of line L, fixed on day 12. X 325. (From Yaffe, 1968.)

D. THE INHERITANCE BY SINGLE CELLSOF

THE

CAPACITY TO DIFFER-

ENTIATE

The inheritance of the capacity to differentiate within the population of the myogenic cell line was investigated. Do all cells carry this capacity? Do single cells transmit it to all their progeny? Since the differentiation of these cells is manifested by fusion into multinucleated fibers, differentiation capacity cannot be studied on the single cell level. Therefore, isolated cells were grown under cloning conditions and their potentialities for differentiation were assayed by determining the muscle-forming capacity of colonies derived from single cells. Cultures of the myogenic cell lines Le and Lpl were dispersed by

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trypsinization and the cell suspensions seeded at cell densities of 25 to 150 single cells per plate on a feeder layer contained in 60 mm collagencoated dishes. Under these conditions 2040% of the cells multiplied and formed small colonies. It was found that in most experiments all the colonies formed muscle fibers ( Figs. 7-9). Cell suspensions prepared

FIG.7. Muscle-forming colonies derived from single cells of line L,. All colonies contain muscle fibers. X 1.7.

from single clones isolated from such cultures were plated again under cloning conditions. This serial reclonization was repeated several times. In most experiments 100% of the clones produced muscle fibers; only occasionally was a clone obtained which did not form muscle fibers. Some of the few clones which did not form muscle fibers were isolated and the cells were again plated under cloning conditions. One hundred percent of muscle-formipg colonies were obtained in all cases, indicating that even cells of the few clones which for some reason did not form muscle

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FIG.8. ( a ) As Fig. 7 but X 6. Note high homogeneity of colony structure. Compare with colonies formed by cells of another myogenic line, M,, ( b ) .

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fibers in these experiments possessed the potentiality to differentiate, as did cells of muscle-forming colonies. Thus, myogenic cells maintained in exponential growth in tissue culture for several months pass on the capacity to differentiate to virtually all their progeny. These experiments

FIG. 9. Colony from Fig. 7 at higher magnification. Note the organization and spatial interrelationships between myoblasts and multinucleated fiber. X 35.

do not exclude the possibility that loss of the capacity to differentiate may be linked with loss of the ability of the cell to multiply and form a colony. However, even if this is so, the high plating efficiency obtained in most experiments demonstrates that the capacity to differentiate is inherited by the mu/ority of progeny. E. THE EFFECTOF COLLAGEN Experiments on cloning chick muscle cells have shown (Konigsberg, 1963) that the culture medium collected from crowded cultures (con-

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53

ditioned medium) in the stationary phase is necessary for the development and differentiation of muscle-forming colonies. It was later found that coating the bottom of the culture plate with a collagen film obviates the need for the conditioned medium. The possibility that collagen serves as an inducer for muscle differentiation was therefore raised (Konigsberg and Hauschka, 1965; Hauschka and Konigsberg, 1966). Experiments with rat muscle cultures in our laboratory also indicated a considerable improvement in growth and differentiation (amount of fibers formed) when cultures were grown on collagen-coated plates. This procedure was therefore adopted and all our myogenic lines were isolated and grown in collagen-coated dishes during their first passages. When the role of collagen was later investigated, it was found that all lines could also be grown in uncoated dishes, although this resulted in a decreased degree of differentiation which varied considerably among the different lines. When cells of lines Ls and M,, were plated for cloning in plates not coated with collagen, 100% of the clones were found to produce muscle fibers (Richler and Yaffe, to be published). Since these plates did contain a small number of feeder layer cells (initially 1 X lo6 cells per plate) which could produce some collagen, an attempt was made to clone cells in petri dishes containing neither collagen nor feeder layer cells. Under these conditions the cells grew poorly but eventually formed colonies. These colonies, after several serial passages, were assayed for muscle forming cells under proper cloning conditions. One hundred percent of the clones produced muscle fibers. Although we do not yet know the effect of very prolonged cultivation of the myogenic cells in the absence of collagen, the experiments thus far indicate that coating the dishes with collagen is not necessary for the maintenance of differentiation capacity by the muscle precursor cells. Thus, although collagen undoubtedly has a very distinct effect on the promotion of both growth and differentiation of cells of myogenic lines, it does not seem to act in this system as a specific inducer for muscle differentiation. It appears that collagen enhances in a non-specific way growth and expression of differentiation potentialities of predetermined myoblasts. Promoting effects of collagen were also observed in other differentiating cell systems such as liver (Ehrman and Gey, 1956), pigment, and cartilage cells (Yaffe, unpublished). In cloning experiments (including those with collagen-treated plates) the source of serum was also observed to have an important effect on growth and differentiation of the clones. Sera obtained from different horses vaned considerably in their effect on both plating efficiency and differentiation. There was no obvious relationship between these two

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DAVID YAFFE

properties; some sera supported growth and differentiation equally well, whereas others lent better support to one or the other. Coon and Cahn (1966) recently reported the fractionation of embryo extract into fractions which differed qualitatively in their effect on the promotion of growth and differentiation in clones of pigment- and cartilage-forming cells. It is also of obvious interest to try to isolate factors responsible for the effect of serum on muscle-forming cells. One might still contend that factors which induce the differentiation of muscle cells are supplied to the cells by one of the medium components. However, the main point to be emphasized is that under the standard conditions which are widely used at present to grow and clone many cell types, only myogenic cells retain the capacity to manifest the specific synthetic pathways of muscle differentiation. Why is it that descendants of muscle cells which have been maintained in culture for more than 600 cell generations continue to form contracting muscle fibers and do not synthesize, say, melanin or hemoglobin?

F. GROWTH CHARACXFZUSTICSAND PLOIDY OF MYOCENICCELL LINES The fact that cell lines in culture retain high differentiation potentialities does not necessarily imply that these cells resemble myoblasts of primary cultures with respect to other parameters, especially as regards their capability to survive culture conditions. Serial passages and serial clonizations were carried out routinely on all the myogenic lines established and maintained in this laboratory. However, repeated attempts to obtain myogenic cell clones from single cells isolated from primary cultures under identical conditions have so far invariably failed due to cessation of multiplication and degeneration of the cells after very few serial passages. All the lines which were successfully established origa s cultures and not inated from serial passages initiated with primary m from single cells. Also, these lines usually went through a period during which generation time increased and pronounced degenerative phenomena occurred. This generally took place between the fourth and eleventh serial passages. This suggests that the evolution of the line may involve some kind of selection and that starting with a large cell population facilitates this process. Cell lines capable of surviving in vitro for a prolonged time have very often been found to undergo changes in chromosome number (Hsu, 1961). Therefore, the chromosomal constitution of the myogenic lines was investigated (Richler and Yaffe, to be published). Results of such ex riments are summarized in Table 11. In all lines the diploid cells are p"

2.

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MUSCLE DIFFERENTIATION IN VITRO

predominant, and in three cell lines the chromosome number does not vary significantly from that of the primary cultures. This includes the oldest line, L6, maintained in continuous growth in culture for almost 2 years. The lines MS8 and L29 (both of which manifest high differentiation capacity) were found to have a high proportion of polyploid and heteroploid cells. Mb8 had 30% polyploid cells. After repeated cloning TABLE I1 PLOIDY OF MYOCENICCELLLINES

Cell line

MCa

Time cells niaintained in vitro

Diploid cellsb

3 days 18 months 9 months 8 months 10 months 8 months 7 months

96 92 98 70 81 85 91

Methylcholanthrene treatment during the first two cell passages. Number of cells containing 42 chromosomes out of 100 cells counted in metaphase. c First passage of a diploid clone isolated from MS8. d A subline of L, obtained by isolating a polyploid clone. Chromosome numbers range between 60 and 84. a b

this was reduced to 15% (Table 11) which is still significantly higher than the primary cultures. It would seem that there is some kind of inherent chromosomal instability in this line which results in the continuous production of polyploid cells. La, is a subline of L6 obtained by isolation of a polyploid clone. This line is tetraploid and heteroploid with no diploid cells (60-84 chromosomes). In spite of this cultures of this line differentiate into a network of contracting fibers. Thus, the drastic change which occurred in the number of chromosomes of the cells did not interfere with the retention and manifestation of differentiation potentialities by these cells. It is puzzling why the lines are not entirely converted into polyploid cell populations, especially in the case of MS8which has been shown to produce continuously high proportions of new polyploid cells. This suggests that the polyploid cells are also continuously eliminated from the

56

70 DAVlD YAFFE

.c c 0

primary Cultures

2000 2

a2 600

-a

.

0

5001

\

1500 N h

c

W

I000; .

500

z

I00

0

0

5-1

0 2

4 Days o f t e r ploting

n

b M,,

F

-2-

4

25

5

41

50

Day8 a f t e r plating

FIG. 10 ( a ) Enzymic activity in differentiating priniary rat thigh muscle cell culture. Cultures, prepared as usual, were collected at different stages of differentiation. Extracts were assayed for enzymic activity. Each value represents three assays. Blocks

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population. The possibility that the diploid cells have a selective advantage over polyploids should be considered.

G. ENZYMIC MANIFESTATIONS OF DIFFERENTIATION in Vitro The structural proteins which constitute the contracting apparatus are the major and most specific product of the developing muscle. The most direct approach to the study of the process of differentiation on the level of protein synthesis is to follow the kinetics of formation of these proteins. However, the quantitative manipulation of these proteins on a microscale is very difficult compared with the relative ease of quantitative assay of many enzyme proteins. Experiments were therefore carried out to study the correlation between morphological differentiation and the synthesis of specific enzymes in primary thigh muscle cultures as compared to that in myogenic cell lines, attempting to utilize these enzyme activities as biochemical parameters for the study of the synthetic processes which occur during the differentiation of myogenic cell lines. Three enzymes were explored in this study: ( 1 ) creatine phosphokinase (CPK, EC 2.7.3.2); ( 2 ) myokinase (EC 2.7.4.3;adenylate kinase); and ( 3 ) glycogen phosphorylase ( E C 2.4.1.1). These enzymes are involved in the provision of the energy required for the contraction of muscle fibers, and they appear in relatively large amounts during muscle differentiation in vivo (Cosmos, 1966; Kendrick-Jones and Perry, 1967; Reporter et al., 1963). Creatine phosphokinase has also been found to appear in chick muscle cultures ( Reporter et al., 1963). The results of experiments to assay enzyme activity during the differentiation of rat thigh muscle primary cultures are summarized in Fig. 10a (Shainberg et al., to be published). It can be seen that very low levels of all three enzymes are found during the first 2 days of culture, i.e., before fusion starts. However, at onset of fusion, the activity of the enzymes begins to rise and continues to do so at a more or less constant rate. Between days 3 and 7 CPK increases in specific activity more than tenfold. The activity per plate rises up to day 9 reaching a plateau when are m-units activity per mg protein and curves are m-units activity per plate. ( M K ) myokinase; (CPK) creatine phosphokinase; (Ph) glycogen phosphorylase; ( F ) onset of fusion. The values of phosphorylase activity have been multiplied by 20 for convenience of presentation. ( b and c ) Enzymic activity in differentiating cultures of the myogenic cell lines M,, and L,, respectively. "indicates no measurable amount of phosphorylase.

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fusion ceases. Very similar behavior is observed with phosphorylase and myokinase. The rise in activity is linked with the process of fusion. When fusion is experimentally delayed, for example, by seeding the cells at low density or by resuspending the culture before fusion and replating, a parallel delay in the onset of elevated enzyme activity is observed. This conclusion is also supported by cytochemical examination. Cultures stained for phosphorylase activity showed staining of multinucleated fibers but not of mononucleated cells. TABLE I11 OF PROTEIN SYNTHESIS ON ENZYMEACTWITYO EFFECTOF INHIBITION

Time (hours)b

CPK Untreated

CH

Phosphorylase Untreated CH

Myokinase Untreated CH

0 4400 27 250 10 490 450 32 300 250 360 250 37 24 440 22 800 400 45 24 430 260 33 680 0 2.8 pg/ml cycloheximide ( C H ) applied to 4-day-old differentiating primary rat thigh muscle cells in culture. Incorporation of leucine-3H into protein was inhibited by 95%. b After addition of the inhibitor. 0 Expressed as m-units activity per plate (one unit of activity represents the amount of enzyme which causes the formation of 1 pmole NADPH per minute at 30°C). Each value represents an average of three assays. Application of inhibitors of protein synthesis like puromycin or cycloheximide at any stage of development stops further elevation in enzyme activity (Table 111);this suggests that the elevation of activity during differentiation represents de nouo synthesis of enzyme and not just activation of preexisting molecules. The experiment with cycloheximide showed also that all three enzymes are relatively stable. Decrease in enzyme activity is not apparent until degeneration of the cycloheximide-treated cells starts. Essentially similar results were obtained when cultures of the myogenic cell lines were examined (Fig. lob, c). Very low levels of activity of CPK and undetectable activity of the phosphorylase were found in young cultures consisting only of mononucleated myoblasts. However, a constantly increasing rise in activity of all three enzymes took place as soon as fusion started. The ratio between phosphorylase and CPK in these cultures remained constant through most of this process

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and resembled that found in primary cultures. The activity of myokinase in these cultures exceeded that of CPK. Also, relatively higher levels of myokinase were found in cultures before fusion. Cytochemical examination of cultures of the La line stained for phosphorylase at various stages of differentiation showed that only multinucleated fibers stained for enzyme activity (Fig. 11).

FIG.11. Cytochemical demonstration of phosphorylase activity in differentiated culture of the line La. Note localization of staining in mukinucleated fibers.

These experiments, although preliminary in nature, indicate the following conclusions: (1) Quantitative increase in activity of at least two out of the three enzymes starts at fusion and takes place in the multinucleated cells. ( 2 ) A similar pattern of enzymic differentiation takes place during the differentiation of cultures of myogenic cell lines after prolonged cultivation in uitro. These results encourage the application of this methodological approach to the study of the regulation of macromolecular synthesis and turnover during differentiation.

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IV. Induction of DNA Synthesis and Mitosis in Nuclei within Muscle Fibers

Cessation of DNA synthesis during differentiation is common to many differentiating cell types (e.g., epidermis, erythropoiesis, neural cells). In some systems, this phenomenon is obviously associated with degenerative changes in the nucleus ( epidermis and mammalian red blood cells). It has been pointed out that the formation of multinucleated muscle fibers is associated with a distinct change in the metabolism of DNA. While mononucleated myoblasts synthesize DNA and divide, incorporation of DNA precursors or mitotic figures have not been observed within multinucleated cells, even during the earliest stages of fusion of myoblasts. In fact, it has been shown that myoblasts stop DNA synthesis at least 6-8 hours preceding fusion (Okazaki and- Holtzer, 1966). Autoradiographic experiments have shown also that the formation of multinucleated cells is associated with a pronounced decrease in thc incorporation of labeled RNA precursors (Yaffe and Fuchs, 1867). Experiments were performed to test whether the cessation of DNA synthesis at fusion is irreversible and associated with a change in the structural integrity of the genetic material (Yaffe and Gershon, 1967a,b). Can nuclei within muscle fibers resume DNA synthesis and undergo mitosis? Since oncogenic viruses have been shown to stimulate DNA synthesis in a variety of experimental systems the effect of the oncogenic virus polyonia (PV) on the differentiation of rat muscle cells was studied. Cells of this species are particularly suitable for such a study since rat cells have been shown to undergo malignant transformation by PV without the synthesis of infectious virus particles. It was therefore assumed that rat muscle cells would not sipport PV multiplication and thus facilitate observation of host DNA synthesis, avoiding the complication of cell lysis. Differentiating primary rat thigh muscle cultures were infected with PV at the onset of fusion. In order to measure DNA synthesis in differentiated fibers, duplicate samples of infected and noninfected culture: were exposed to thyn~idine-~H for 6 hours at various times after the introduction of the virus. The cultures were then processed for autoradiography and stained for detailed cytological examination. DNA synthesis was not observed in multinucleated fibers exposed to virus for less than 30 hours, as judged by lack of incorporation of the tritium-labeled thymidine into their nuclei. However, after 30 hours following infection an increasing proportion of the fibers was found to

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synthesize DNA. After 40 hours as many as 15% of the fibers had labeled nuclei. Usually when DNA synthesis was observed in a fiber, all of the nuclei were found to have incorporated the labeled precursor; however, some fibers were found in which only a fraction of the nuclei was labeled. In the latter case the labeled nuclei were clustered in one section of the fiber.

FIG. 12. Early morphological changes caused by PV infection. Cultures in the process of fusion were infected with PV and fixed 50 hours later. Affected fibers are characterized by the presence of swollen, lightly stained nuclei.

Mitotic figures began to appear in multinucleated fibers about 50 hours after infection. After 70 hours about 3% of the fibers contained nuclei in various stages of mitosis. In many such fibers advanced metaphases could be observed at one end of a group of dividing nuclei; distal to these nuclei progressively earlier stages of mitosis were observed which terminated with early prophases and resting nuclei. In other fibers all the nuclei undergoing mitosis were found to be in metaphase (Figs. 12 and 13). In most cases it was difficult to ascertain whether the chromosomes originated from one nucleus or from several nuclei. However, when this was possible the mitotic figures were found to be diploid.

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Division of nuclei within the fibers did not proceed beyond late metaphase. Instead, the fusion of several metaphase figures into large clumps of chromosomes was often observed (Fig. 14). This phenomenon was frequently associated with rounding of the fibers and loss of their

FIG. 14. Late metaphase in a multinucleated fiber. Note the absence of mitotic spindles and clumping of chromosomes from many nuclei; fixed at 72 hours after PV infection. (From Yaffe and Cershon, 1967b.)

elongated form. In cultures fixed 90 hours after infection or later, fibers and rounded cells containing huge abnormal nuclei were frequently found. Judging from the sequence of events these were most probably FIG. 13. Mitosis in a multinucleated muscle fiber. All nuclei are in prometaphase;

86 hours after PV infection. (From Yaffe and Gershon, 1967a.)

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the result of the clumping of the dividing nuclei observed in earlier stages. Infectivity tests and methylated albumin kieselguhr (MAK) column chromatography of DNA extracted from infected cultures did not reveal any detectable production of infective particles or synthesis of polyoma DNA in the cultures. The results thus suggest that the fusion of myoblasts is not necessarily associated with an irreversible block of DNA synthesis, and that nuclei within the fibers retain their structural integrity and can be induced to synthesize DNA and undergo mitosis. The fusion of dividing nuclei into abnormal clumps of chromosomes may not be due to aberrations in the chromosomes but rather to the unusual situation of the close proximity of many mitotic apparatuses within the same cytoplasm. Fusion of mitotic figures to form giant nuclei was observed in other cell systems where several nuclei were dividing in a common cytoplasm (Harris et al., 1966). There is also a possibility of interference of the contractile proteins of the specialized muscle cell with the formation of mitotic spindles. Often, chromosomes were found to be arranged in long parallel rows similar to the myofibril arrangement. We have seen that there was no incorporation of th~midine-~H into muscle fibers when exposure to the label was made during the first 30 hours postinfection, while exposure of the cultures at later stages did result in the incorporation of the label into nuclei within fibers. This indicates that the introduction of the virus had induced resting nuclei to resume DNA synthesis. It therefore appears that the effect of PV infection is an induction of DNA synthesis in cells already in a postmitotic state rather than the delay or prevention of the establishment of the mitotic block. However, a conclusion that multinucleated muscle cells are sensitive to direct infection by polyoma might be misleading. At the time of infection the cultures were in the stage of cell fusion, i.e., myoblasts were still joining the multinucleated fibers. It was found that the number of fibers induced by PV to synthesize DNA dropped considerably if infection was delayed by a few days, and almost no response could be found if infection was delayed until fusion ceased. This suggests that the cells susceptible to PV infection are mononucleated myoblasts and that infection of the multinucleated fibers takes place by the subsequent fusion of infected myoblasts into the growing multinucleated fiber. Since mononucleated myoblasts can multiply this assumption is in accordance with the supposition that transformation of cells by oncogenic viruses requires cell division (Todaro and Green, 1966).

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In most of the fibers which were induced to synthesize DNA all the nuclei were synthesizing DNA or undergoing mitosis. The probability that all the myoblasts which participated in the formation of these fibers were infected by PV is very low. This phenomenon leads one, therefore, to assume that the incorporation of a few PV-infected myoblasts into a growing fiber induces DNA synthesis and mitosis in other nuclei present

FIG. 15. Autoradiographic demonstration of incorporation of thymidine-3H into multinucleated fibers formed in cultures prepared from a 1:1 mixture of PV-infected rat myoblasts and untreated bovine embryo myoblasts. All nuclei are labeled.

in the same fiber. This conclusion is strongly supported by the following experiment carried out recently (Yaffe and Gershon, unpublished). One-day-old rat myoblast cultures were infected with PV before the onset of fusion. Twelve hours later the cells were suspended, washed several times, and mixed with myoblasts obtained from bovine embryo thigh muscle. The mixed population was plated in the usual manner and 48-72 hours later, when hybrid fibers had been formed, the cultures were exposed to a 6-hour pulse of th~midine-~H and fixed for autoradiography. It was found that although muscle fibers in cultures of bovine muscle cells alone were not affected by PV infection, in the cultures wntaining both types of myoblasts huge hybrid fibers containing hun-

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dreds of labeled nuclei were found (Fig. 15). One day later inany mitotic figures could be observed within such fibers. Unfortunately the large number and great proximity of the mitotic figures within the fibers did not permit conclusive karyotypic examination (Figs. 16 and 17). However, in the autoradiographs many labeled nuclei containing several

FIG. 16. Culture mnditions identical to Fig. 15. Note gradient of progressive stages of mitosis.

nucleoli-very typical of bovine nuclei-could be observed ( Fig. 18). These experiments suggest that the presence of PV-affected rat nuclei within the hybrid fibers resulted in the induction of DNA synthesis and mitosis in the nuclei of bovine origin. Recently, Lee et al. (1988) reported that DNA synthesis in chick muscle fibers could be induced by Row sarcoma virus (RSV) which is an RNA-containing virus. These authors showed that when muscleforming colonies containing myoblasts and differentiating fibers were infected with RSV, fibers incorporating th~midine-~H could be observed 20 hours later. Similar to our observations with PV in many affected fibers all the nuclei were synthesizing DNA. No mitotic figures within

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the fibers were found in these experiments. Unlike the experiments with PV-infected rat muscle cells, fluorescent antibody techniques showed the production of RSV coat antigen in the chick multinucleated muscle cells. Since the effects of RSV on the chick muscle fibers were observed after 20 hours or longer postinfection, the question of whether the RSV af-

FIG.17. As Fig. 16. Hypotonic treatment ruptured the fibers but chromosomal details are accentuated. Most nuclei are in metaphase or late prophase.

fected muscle fiber directly or via the fusion of mononucleated myoblasts remains open (Ebert and Kaighn, 1966). Fogel and Defendi (1967) applied fluorescent antibody techniques to study the appearance of T antigen in PV-infected hamster and rat thigh muscle cultures. This antigen accompanies transformation of cells by PV (but is not related to the production of virus particles ) . Their results are very reminiscent of our results in three main points: ( 1 ) infection of young differentiating cultures resulted in the appearance of T antigen within multinucleated cells in which either large groups of adjacent nuclei or aZZ the nuclei contained T antigen; ( 2 ) the production of viral antigen (which is an indication of virus production) was very rarely

FIG. 18. As in Fig. 15. The light labeling in this branch of the fiber allows the identification of labeled nuclei containing many nucleoli which are assumed to be of calf origin ( arrow )

.

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found in cultures of these species; (3) when cultures of fully formed myotubes were infected by PV no effect could be observed on the fibers whatsoever. Similar results were obtained with SV40-infected human muscle cells. The results thus show that the muscle fiber reacts to oncogenic virus infection as one functional unit. Any explanation of the mechanism by which several PV-infected myoblasts induce the resumption of DNA synthesis and mitosis in other nuclei would at the moment be highly speculative. In experiments on artificial cell fusion with the aid of killed Sendai virus (Harris et al., 1966) cells which synthesize DNA or RNA were hybridized with differentiated cells which usually do not synthesize DNA or RNA. It was found that in all cases the capacity to synthesize the macromolecules was dominant and induced synthesis in the resting nuclei. However, this does not show unequivocally that synthesis of nucleic acid is under positive genetic control. In these experiments the fused cells did not multiply and therefore the nuclei of these cells resided in the original cytoplasm of the donor cells. Under these circumstances one cannot exclude the possibility that genetic control of the synthesis of nucleic acids was negative (i.e., when the regulating genes which control the activity of the structural genes for nucleic acid synthesis are active, the latter are repressed); but in this specific situation the parent cells which synthesized nucleic acids before fusion introduced into the common cytoplasm the enzymes necessary to initiate nucleic acid synthesis ( or residual mRNA which coded for these enzymes). Similarly, when myoblasts affected by PV fuse into a multinucleated muscle fiber DNA synthesis may be "imposed" on the other nuclei in the fiber by factors brought in as a result of fusion with affected myoblasts or by the continued production of such factors (i.e., mRNA for the involved proteins) by the PV-transformed nuclei which were no longer responsive to the mechanism that inhibits DNA synthesis at fusion. It should be mentioned that in both the PV and the RSV systems it was observed that, for the most part, the extent of labeling of individual nuclei within the myotubes was lower than that of nuclei in mononucleated cells. This may imply that DNA synthesis proceeds more slowly in the former cells because of a lower level of DNA synthesizing enzymes. This would be expected if the information for these enzymes is produced only by a small number of nuclei derived from PV-infected myoblasts. Synthesis of viral DNA was not detected in the PV-infected cultures; however, the production of minute amounts of viral DNA which are transmitted intracellularly from PV-infected to uninfected nuclei would not be detectable with the methods employed. Therefore, such an ex-

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planation for the almost all-or-none response of muscle fiber, although unlikely, cannot be excluded. Experiments with a different cell system may have some relevance to this question. When SV4O-transformed cells containing T antigen were fused artificially with nontransformed ones, the T antigen appeared also in the nuclei originating from the nontransformed donors. Steplewski et al. (1W) have recently shown that the appearance of T antigen in these nuclei, originating from the nontransformed donor, takes place in the presence of 5-fluorodeoxyuridine, which suppresses DNA synthesis. However, it is dependent on new RNA and protein synthesis. Without the biological function of the T antigen being known, this experiment shows that the synthesis or acquisition by noninfected nuclei of a new protein is induced by the presence of a transformed nucleus, and that this may take place without the production of viral DNA. The resumption of DNA synthesis and mitosis of nuclei within multinucleated fibers has been induced by an oncogenic virus. However, since it has been demonstrated that this activity is not irreversibly blocked in these cells, the possibility that nuclei within muscle fibers can, under nonmalignant but specific physiological conditions, give rise to dividing cells should be considered. The repeated claims that during muscle regeneration in duo mononucleated cells are formed by fragmentation of injured multinucleated muscle fibers should be mentioned here (for reviews see Field, 1961; Betz et al., 1966). The property of myoblasts to fuse into multinucleated cells and the characteristic cessation of DNA synthesis at fusion may offer new experimental approaches to further exploration of the interrelations between malignant and normal cells and throw light on some open questions in carcinogenesis. V. Comments

A main outcome of the experiments reviewed here is the establishment of myogenic cell lines which are able to multiply for very extended periods (apparently indefinitely) in culture and retain their capacity to differentiate. It is presently not clear why some of the attempts to establish myogenic lines resulted in the loss of the cells by degeneration and cessation of multiplication whereas others were successful. This experience is common for many kinds of mammalian or avian cells serially passaged in vitro (Hayflick, 1965; Kuroki et al., 1967; Lithner and Ponten, 1966; Sanford, 1965). In such experiments the cells were either

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lost altogether or a line was established by the appearance of a new type of cell population which differed in its growth characteristics from the original one. The establishment of myogenic cell lines is of special interest with relevance to this phenomenon since it shows that the changes which take place in the growth characteristics during establishment of cell lines may be distinct from the differentiation characteristics of these lines. Although these lines differ in their capacity for growth from populations of freshly isolated myoblasts they nevertheless preserve very similar differentiation properties. Since we do not know the exact nature of the differences between the cell lines and the primary cultures the possibility exists that some of the characteristics that have been studied in the cell lines reflect qualitative or quantitative properties specific to these established lines rather than to normal differentiation; however, the availability of many parameters unique to the differentiation of muscle cells minimizes the likelihood of this happening. The possibility of cloning and thus performing analyses and experiments on homogeneous populations of one kind of cell makes this system valuable and versatile for studying many aspects of cell differentiation. Differentiated primary cultures always contain, in addition to the network of contracting fibers, a population of mononucleated cells which do not participate in fusion. This was attributed to the presence of nonmyogenic cells in the initial cell population derived from the embryonic muscle tissue. However, even in pure populations of cloned muscle cells in which all the cells were supposed to be myoblasts, almost never did all the mononucleated cells become incorporated into fibers. This is clearly seen in the myogenic cell lines; differentiated cultures maintained for weeks always contained mononucleated cells. When these cells were trypsinized and seeded under cloning conditions all the clones formed muscle fibers and no difference could be observed between clones obtained from these residual mononucleated cells and cells obtained from young cultures trypsinized before fiber formation. Even repeated cloning of mononucleated cells from fully differentiated clones did not result in any selection of cells of lower capacity for differentiation. These experiments suggest that an equilibrium exists in these cultures between mononucleated myoblasts and the differentiated fibers. The relative proportion between the amount of mononucleated and multinucleated cells is variable and is influenced by culture conditions. Also, under identical culture conditions, clones produced by different cell lines differ considerably in their mononucleated cell content. These observations may be relevant to a mechanism which regulates

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cell populations in viuo; in most tissues there exists throughout adult life a dynamic equilibrium between proliferation of precursor cells and differentiation. This is most clearly exemplified in erythropoietic and epidermal tissue in which the differentiated cells are dead ends which do not divide and proceed into progressive stages of degeneration. However, the tissue is maintained by a continuous regulated multiplication of a stock of precursor cells (stem cells) and production of new differentiating cells. Adult muscle tissue consists predominantly of highly differentiated multinucleated fibers with very few mononucleated cells of unidentified nature. No quantitative replacement of muscle fiber takes place; however, damaged muscle can repair by regeneration and production of new muscle tissue. The cellular origin of this regenerated tissuc is not yet clearly understood. However, most investigations support the notion that mononucleated cells which accompany muscle fibers do multiply and fuse into new fibers (Betz et al., 1966). The observation that mononucleated myoblasts and differentiated fibers are always present together in cultures of myogenic cells suggests that fundamentally a similar situation exists in viuo-some of the mononucleated cells which accompany muscle fiber are indeed muscle precursor cells which participate in replacement and regeneration of muscle tissue. Thus, muscle may not differ qualitatively from tissues which are continuously renewed, such as skin and erythropoietic tissue, but does differ quantitatively in the lifespan of the differentiated cells and in the rate of their replacement. How do precursor cells which multiply continuously retain their capacity to differentiate? The experimental evidence emphasizes the stability of the retention of this capacity. Myogenic cells have been maintained as pure populations in culture for more than 2 years without losing their capacity for differentiation which clearly demonstrates that this capacity is an inherent property, reproduced continuously within these cells and independent of interaction with other cell types, There is no antagonism between cell replication in culture and retention of differentiation potentialities. The mechanism by which the capacity for differentiation is reproduced must be very stable; this is indicated by the fact that regardless of a variety of culture conditions and continuous multiplication, 100% of clones formed by plating single cells differentiated into muscle-forming colonies. In essence, similar results were obtained in experiments with cartilage cells and retina pigment cells (Cahn and Cahn, 1966; Coon, 1966). Experiments on the retention of differentiation capacity in uitro in these two types of cells were made during

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the first passages in tissue culture, i.e., on cells which presumably have not changed or undergone selection in uitro. This shows that retention of differentiation potentialities in vitro in lines of myoblasts undergoing continuous multiplication is not a property which has been fixed during the establishment of such lines but rather a phenomenon of general significance. Since the differentiated multinucleated muscle cells did not divide, they were continuously selected out and the line was maintained only by the precursor cells which did not differentiate beyond the myoblastic level. Thus, the ability to transmit differentiation potentialities to progeny cells is altogether independent of the expression of the traits. The property which is preserved during multiplication of the determined cells is not the differentiated trait itself but the capacity to express this pattern under the right conditions. The cells differ from other cells in their pattern of response to the environment and to signals from other cells. The actual expression of differentiation is flexible and adaptive. Most reported cases of “dedifferentiation” refer, in fact, to the disappearance of manifestations of the differentiation traits (“modulation” according to P. Weiss) but not to a change in the intrinsic capacity of the cells. When cartilage or retina pigment cells, for example, are grown in culture they soon lose their tissue-specific characteristics and acquire a fibroblastic morphology. However, even after many cell divisions these cells still retain a capacity to resume their morphological and biochemical differentiation. As soon as the proper growth conditions are supplied, under identical conditions, fibroblast cells of cartilage origin will form cartilage, and cells of retina pigment origin will become epithelial and pigmented (Cahn and Cahn, 1966; Coon, 1966). Therefore, what condition is perpetuated in precursor cells which preserves their capacity to differentiate? What is the state of activity of the genes which code for muscle-specific proteins? Are they repressed in the same way as other genes which do not participate in differentiation or are they subject to another type of control, tuned for the specific signals which appear at fusion? Can it be that the muscle-specifk genes are active in these cells and produce their mRNA, but the timing of the synthesis of muscle-specific proteins is regulated at the translational level? In many differentiated cell types proteins have been shown to be produced on stable mRNA (De Bellis et al., 1964; Reeder and Bell, 1965; Wilt, 1965; Humphreys et al., 1964; Kafatos and Reich, 1968). The low incorporation of RNA precursors into multinucleated muscle cells and the

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relative resistance of muscle cells to the toxic effect of actinomycin D suggests also the existence of stable mRNA in these cells (Yaffe and Feldman, 1964; Yaffe and Fuchs, 1967). At first sight it is tempting to attribute stability of differentiation to the presence in the cell of stablc macromolecules which were formed at earlier stages. However, this cannot apply to the retention of differentiation capacity in continuously multiplying cells, since preformed macromolecules would be diluted out during replication. Furthermore, in most systems, it has been found that stable mRNA appears only in the late stages of differentiation. The existence of stable templates at these stages has an obvious economic advantage for cells which continuously produce large amounts of few proteins. It seems, therefore, more likely that the stable forms of mRNA described in these experiments play a role in the regulation and stabilization of the expression of differentiation traits rather than in the retention of the capacity to differentiate. However, it is also easy to visualize that stable informational macromolecules may participate as components of a complex, self-perpetuating, dynamic system and play a role in stabilizing it during cell division, temporal inhibition of macromolecule synthesis, etc. Jacob and Monod (1963) suggested a model of stable circuits which can maintain specific genes in a continuous state of activity or repression as a modification of the model generally accepted for the regulation of synthesis of adaptive enzymes in bacteria. Such models are appealing for their simplicity and analogy to well-studied regulation mechanisms in microorganisms. However, the greater complexity of phenomena and structures in cells of multicellular organisms as compared to the bacteria suggests also the possibility of new regulation mechanisms which may replace or amplify the regulatory systems suggested by Jacob and Monod. At present too little is known of the molecular events to enable the construction of a model based on experimental data which would explain the stability of cell differentiation. A number of observations may be mentioned here to illustrate the existence of a large variety of mechanisms which may control specific changes in the informational content of a cell: ( a ) the ability of specific genes to replicate and form extra copies. This has been demonstrated to occur in the genes which code for ribosomalRNA in amphibians (Brown and Dawid, 1968); ( b ) the establishment during embryogenesis of replicatioiial or transcriptional differences between chromosomes. A regular loss or inactivation of specific chromosomes during the embryogenesis of some invertebrates was observed at the morphological level many years ago. The phenomenon of inherited

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differences in expression of genetic activity between the two X chromosomes within clones of mammalian somatic cells (De Mars, 1967) has already been pointed out as a possible model for studies of cellular differentiation ( Lederberg, 1966); ( c ) the demonstrated ability of RNA molecules to control their own replication. Thus, replication of RNA viruses is accomplished by the synthesis of a specific replicase. This replicase is coded by the viral RNA and replicates specifically only the viral RNA (Spiegelman and Haruna, 1966). It is not meant to suggest that these phenomena as such play a general role in differentiation. However, their existence even in special situations should be taken into account since an understanding of their underlying mechanisms may give some insight into processes which take place regularly in differentiation. The capability of isolated cells to retain and manifest in uitro their capacity to differentiate, and the possibility to analyze the inheritance of these properties by single cells, make the possible relevance of these phenomena to the mechanism of differentiation more amenable to experimental analysis. The stable retention of the capacity to differentiate demonstrated by cells maintained in uitro in a state of continuous replication and the clear distinction in several experimental systems between the retention of this capacity and its actual expression indicate some of the requirements for a working hypothesis. ACKNOWLEDGMENTS Th'anks are due to Professor M. Feldrnan for his interest and helpful discussions. The participation of Dr. D. Gershon, Dr. G. Yagil, MI. A. Shainberg, and Miss C. Richler in the experiments is gratefully acknowledged, as is the excellent technical assistance of Mrs. S. Neuman, Miss M. Debby, Mr. E. Mor, and Mr. M. Gabbai, and the photographic assistance of Mr. E. Thum. The studies reported in this review were supported in part by the DBlhgation GnCrale B la Recherche Scientifique et Technique, France, and by Grant DRG 1007 from the Damon Runyon Memorial Fund for Cancer Research. REFERENCES Altschul, R. (1962). 2.Zellforsch. Mikroskop. A w t . 56, 425. Bassleer, R., Colignon, P., and Matague-Dhossche, F. (1963). Arch. Biol. (Liege) 74, 79. Benvald, Y., and Sachs, L. (1965). 1. Natl. Cancer Inst. 35, 641. Betz, F. H., Firket, H., and Reznik, M. (1966). Intern. Reu. Cytol. 19, 203. Brown, D. D., and Dawid, I. B. (1968). Science 160, 272. Cahn, R. D., and Cahn, M. B. (1966). Proc. Natl. Acad. Sci. US. 55, 106 Capers, C. R. (1960). I. Biophys. Biochem. Cytol. 7 , 559. Coon, H. G. ( 1966). Proc. Natl. Acad. Sci. U S . 55, 66. Coon, H. G., and Cahn, R. D. (1966). Science 153, 1116.

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