The Phenotypic Complexity of Myogenic Clones (Leg Muscle/Cell Culture/Fibroblasts) J

Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1506-1510, April 1974

The Phenotypic Complexity of Myogenic Clones (leg muscle/cell culture/fibroblasts) J. ABBOTT, J. SCHILTZ, S. DIENSTMAN, AND H. HOLTZER Sloan Kettering Institute for Cancer Research, New York, N.Y.; and the Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia, Pa. Communicated by Hewson Swift, December 26, 1973

ABSTRACT A single cell isolated from cultured 8-day (6). Cells (105) were plated in 35-mm collagen-coated Falcon leg muscle may, when subcultured, yield a myogenic dishes; and 4 days later the medium (7) was removed and clone. A myogenic clone consists of myotubes and mononucleated cells. When such a myogenic clone is sub. used as "conditioned medium" for the clones to be described. cultured, large numbers of mononucleated cells are re- medium covered. These mononucleated cells histologically and Primary Clones. A trypsin-collagenase digestion biochemically are indistinguishable from authentic (15) was added to the above primary cultures and the cells fibroblasts cultured under the same conditions: they syn- were suspended in F-10. A single cell was removed under a thesize (al)2a2 chains of collagen, large amounts of hyal- microscope with an oral pipette and placed into a collagen- uronic acid, and modest amounts of chondroitin sulfate. coated well of a Microtest plate (Falcon), containing 0.5 ml of These mononucleated cells, however, will not chondrify when grown under culture conditions known to permit conditioned medium. These clones were not fed for 8 days, presumptive chondroblasts to differentiate terminally. and thereafter fed 1-2 drops of conditioned medium every 4 These findings demonstrate that there is a population of days. Only clones consisting of many multinucleated myo- single cells in 8-day muscle that is neither a myoblast tubes and mononucleated cells were used to set up secondary nor a fibroblast, but is the common progenitor for cells in yield of positive "muscle clones" the myogenic and fibrogenic lineages: this progenitor, monolayer cultures. The however, is beyond the point of readily yielding chon- prepared from primary mass cultures was considerably greater drogenic cells. These findings are discussed in terms of than when prepared from cells directly from the embryo (e.g., the limited number of phenotypic options open to dif- ref. 9). ferentiating cells in each of the successive compartments of their respective lineages. Secondary Monolayer Cultures. Large numbers of cells derived from one cell were obtained by suspending a "muscle Only a modest number of cells from stage 17-18 chick somites clone," and introducing all the cells from that clone into one or limb buds have the option in vitro to differentiate into collagen-coated 60-mm Falcon dish. At all times the progeny myoblasts, chondroblasts, and fibroblasts (1-3). The bulk of of one cell were separated fronfthe progeny of other single early somite and limb-bud cells operationally can only be "muscle clones." By subculturing secondary monolayer cul- defined as precursor cells. Some of these precursor cells belong tures, many millions of cells, all derived from a single "myo- to early compartments of their respective lineages, whereas genic clone," were readily obtained. others may be still more primitive and belong to the compartment prior to the diversification into committed myogenic, Collagen and Glycosaminoglycan Analysis. The values or chondrogenic, or fibrogenic cells. These observations have [(hydroxyproline)/(hydroxyproline + proline)] X 100 were led to the following proposals: (1) Committed cells in the early obtained by treatment of confluent secondary monolayer compartments of their definitive lineages do not have the cultures with ['Hlproline (New England Nuclear Corp., 1.25 option to transform directly into terminal myoblasts, chondro- /Ci/ml). The collagen subunits were analyzed as described in blasts, or fibroblasts, though their progeny do (4, 5); and (2) Schiltz et al. (10). The kinds of glycosaminoglyeans syn- populations of one or more types of precursor cells should be thesized in secondary monolayer cultures was followed with present in older limbs to serve as progenitors for cells that will [3S sulfate (Amersham Searle, carrier-free sodium salt) or enter the ultimate compartments of their respective lineages ['H ]glucosamine (New England Nuclear Corp., 3.6 Ci/mmol). at rates required for limb morphogenesis (3). The labeled glycosaminoglycans were separated by high-volt- To learn about committed precursor cells, and about the age electrophoresis as described in Mayne et al. (11). Hex- number of antecedent compartments within the myogenic osamine ratios of the peaks were determined by the method of lineage, we cloned single cells from muscle cultures. These Gardell (12), and analysis with testicular hyaluronidase single cells yield a heterogeneous progeny that includes, at a (HSEP, Worthington Biochemicals, 100 ug/ml) was performed minimum, presumptive myoblasts, myoblasts, and fibro- as in Abbott et al. (13). Degradation of ["Slsulfate-labeled blasts, but not chondroblasts. These findings emphasize the materials by chondroitinase ABC and AC (Miles Labs, phenotypic complexity of "myogenic clones," and suggest the Elkart, Ind.) was by the method of Saito et al. (14). existence of a compartment of precursor cells, which contains cells ancestral to myogenic and fibrogenic cells, but which Nomenclature. A "myogenic clone" was a population de- does not contain cells ancestral to chondrogenic cells. rived from a single cell containing at least two well-defined myotubes. "Chondrogenic clone" was as described in Chacko MATERIALS AND METHODS et al. (15). Cells in the ultimate compartment of the fibro- Primary Mfass Cultures. Thigh muscles of 8-day chick genic lineage were mononucleated and synthesized (al),a2 embryos were prepared as described in Bischoff and Holtzer collagen subunits (16-18), hyaluronic acid, and various 1506 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Phenotypic Complexity of Myogenic Clones 1507

amounts of chondroitin sulftate (19, 20). There is no un- ambiguous method of distinguishing, in mixed populations, among precursor myogenic cells, precursor chondrogenic

cells, and fibrogenic cells. The term "primitive mesenchyme" E

rise ,cm cell is reserved for the putative progenitor that will give 0.-: to cells of the early compartments of the myogenic, chondro- 0-) genic, and fibrogenic lineages. RESULTS Primary Clones. Approximately 50% of the single cells from primary mass cultures failed to survive, or gave rise to clones FRACTION NUMBER of less than six cells (Table 1). Such clones could not be scored FIG. 1. Subunit structure of newly synthesized collagen from as either myogenic or fibrogenic. Roughly another 15% of the a confluent secondary monolayer culture. The culture was incu- clones consisted of no more than a dozen cells. In many of bated for 24 hr with ['H]proline together with ascorbic acid (50 these virtually all cells had fused, forming clones of two to four ,ug/ml) and ,-aminoproprionitrile (125 ,ug/ml). After extraction, thin myotubes with only one or two mononucleated cells; it the culture was mixed with lathyritic chick-skin collagen and these mononucleated cells cannot be analyzed by chromatography on a carboxymethylcellulose is to be stressed that column by the procedures described in Schiltz et al. (10). reliably identified as either myogenic or fibrogenic. These (0-0-) A230nm of carrier lathyritic collagen; (0 O) cpm small clones generally degenerated by the second week. The of ['H]proline incorporation. Two peaks of radioactivity can be remaining 35% of the clones displayed a progeny of 50 to over observed corresponding to the al and a2 peaks of the carrier a thousand cells. The large clones displayed a ratio of myo- lathyritic collagen. In this experiment the ratio of radioactivity tubes to mononucleated cells indistinguishable from that ob- in the al/a2 peaks was 2.1. served in mass primary cultures (21, 22). Occasionally, clones with myotubes during the second week were lacking in to dishes with hundreds. In all these secondary cultures there myotubes during the third week; either the myotubes degen- were large numbers of mononucleated cells that histologi- erated or they were obscured by overgrowth of replicating cally were indistinguishable from authentic fibroblasts (10, mononucleated cells. 23). To determine whether these cells had the biochemical It is likely that the number of single cells that can give rise properties of fibroblasts, the cultures were exposed to to sizable myogenic clones diminishes as the embryo ages. In a [3H]proline and analyzed for collagen as shown in Table 2. typical experiment the frequency of sizable 3-week myogenic Clearly some cells synthesize collagen. As shown in Fig. 1, the clones recovered from single cells from mass cultures of 8-day, quantity and types of collagen subunits were indistinguish- 14-day and 18-day muscles was 54%, 12%, and 0%, respec- able from those synthesized by authentic fibroblasts cultured tively. under the same conditions (10). The mononucleated cells not only synthesize the common collagen chains but they Secondary Monolayer Cultures Derived from "M31yogenic are assembled into typically striated collagen fibrils as ob- Clones." Only myogenic clones were used as a source of cells served by electron microscopy. for the secondary monolayer cultures. The number and size of Fig. 2 shows the profile of radioactivity of the glycosamino- myotubes in secondary cultures varied from dishes with few, glycans from secondary monolayer cultures after labeling with that chondroitinase AC alone TABLE 1. Primary clones from mass cultures of 8-day muscle [H3]glucosamine. The fact cleaved the second peak suggests that no chondroitin sulfate B 50 or more cells was present. In experiments with [S35]sulfate (not shown) only the second peak was present. The ratios of hyaluronic acid to Total no. of 2 or more after 3 weeks chondroitin sulfate varied from the approximately 1 shown in single cells cells after With No Fig. 2 to 0.20. Authentic fibroblasts grown for different periods Exp no. cloned 1 week myotubes myotubes and at different densities display different ratios of hyaluronic M6 96 50 17 3 acid to chondroitin sulfate (10, 11, 19, 20, 23). Myotubes were .M7 91 55 10 8 rarely present in the second subculture of the monolayers and MI8 96 51 29 5 AI111 88 38 13 12 TABLE 2. Synthesis of collagen by cells in secondary monolayer cultures All clones were initiated by introduction of a single cell into the well of a Microtest plate. The variability in numbers of clones Exp. no. Cell layer Medium scored as not having myotubes is due to: (1) myotubes often degenerated leaving only mononucleated cells; (2) often the A7 2.21 13.36 small myotubes were obscured by overgrowth of mononucleated C6 1.51 10.47 cells; (3) different batches of medium promoted fusion to dif- G1 2.75 11.92 ferent degrees; and (4) mechanical damage associated with G9 2.81 9.25 trypsinization affects the viability of the cloned cells. These unknowns render a more rigorous quantitation of this kind of Confluent, secondary monolayer cultures were exposed to material dubious. Some myogenic clones displayed a few, others ['H]proline for 6 hr. Incorporation into trichloroacetic acid- over 100, multinucleated myotubes, plus goodly numbers of precipitable material was determined for cells and medium after mononucleated cells. Only myogenic clones with sizable numbers hydrolysis and separation on Dowex 50 resin as described in of both mononucleated cells and myotubes were used to initiate Schiltz et al. (10). The figures in this table are in terms of: secondary monolayer cultures. (hydroxyproline)/(hydroxyproline + proline) X 100. Downloaded by guest on September 24, 2021 1508 Cell Biology: Abbott et al. Proc. Nat. Acad. Sci. USA 71 (1974) chondroblasts in all these experiments suggests that in 8-day muscle there are modest numbers of cells that will give rise to myoblasts and fibroblasts, but there are no cells that will give rise to: (1) myoblasts, and fibroblasts, and chondroblasts; (2) E fibroblasts and chondroblasts; (3) myoblasts and chondroblasts; or (4) chondroblasts alone. DISCUSSION A cell from cultures of leg muscles from 8-day embryos may yield a myogenic clone. This clone may yield progeny that are indistinguishable from fibroblasts both cytologically and in terms of the collagens and glycosaminoglyeans they synthesize. However, the cell that replicates and yields a mixed progeny of myogenic and fibrogenic cells will not also yield 2 4 6 8 12

CENTIMETERS chondrogenic cells when grown in media known to permit pre- FIG. 2. Glycosammnoglycan synthesis in a confluent secondary sumptive chondroblasts to move into the terminal compart- monolayer culture. ['H] Glucosamine was added for 24 hr. The ment of the chondrogenic lineage. glycosaminoglycans were isolated and fractionated by high- These findings raise issues regarding (a) the homogeneity of voltage electrophoresis as described in Mayne et at. (11). cells in "pure" muscle clones, and (b) the type of cells responsi- The position of the hyaluronic acid (HA) and chondroitin sul- ble for the propagation of myogenic lines (28-31). Just as it is fate (CS) standards are indicated at the top of the figure. That difficult, if not impossible, to propagate a pure chondrogenic the first peak is hyaluronic acid is demonstrated by: (1) hydrolysis of the peak in 0.05 N HCl and separation of glucosamine clone in which each cell is equally committed to the terminal

from galactosamine showed 92% of the labeled hexosamihe to be chondrogenic phenotype (15, 36), so the myogenic clones glucosamine, and (2) on treatment with testicular hyaluronidase described in this report exhibit phenotypic diversity. Are 81% of the peak was destroyed. That the second peak is chon- myogenic lines propagated by replicating cells in the penulti- droitin sulfate is shown by: (1 ) hydrolysis of the peak showed 94% mate or even antepenultimate (3, 22) compartment of the of the labeled hexosamine to be galactosamine, and (2) the peak myogenic lineage, or by precursors that are antecedent to is completely sensitive to chondroitinase ABC and AC. Similar the commitment to either the myogenic or fibrogenic line- digestion experiments with similar results were performed with ages? The fact that some of these myogenic lines do not con- [35S]sulfate-labeled glycosaminoglycans. Values on the ordinate tinue to yield myoblasts is consistent with the latter possibility. should be multiplied by 103. In experiments reported elsewhere, we have found that a collagen-like, a hyaluronic acid-like, and a chondroitin-

were never present in the third or fourth subculture. The sulfate-like molecule are synthesized in the primary muscle

mononucleated cells in the third and fourth subcultures dis- clones, as well as in cultures of pure myotubes in which cyto- played the same profiles of collagen and glycosaminoglycan sine'arabinoside was used to kill replicating mononucleated

synthesis as described above. After 4-6 weeks of vigorous cells (39). However, it remains to be determined whether or growth these mononucleated descendents of a "myogenic these collagens glycosaminoglyeans are identical, or only or clone" entered a declining phase (24). similar, to those produced by muscle fibroblasts, whether they are of a kind associated with the basement membranes Are There Cells in the "Primitive Mesenchyme" Compartment of mature muscle fibers. The collagen chains and glycosamino- in 8-Day M11uscle? A single presumptive chondroblast from 3- glycans synthesized by myotubes are largely released into day limb buds or 3-day somites, or a single terminally- the medium. differentitaed chondroblast from 10-day embryos may yield a Can myogenic clones emerge without fibrogenic cells? This characteristic chondrogenic clone when grown in appropriate issue cannot be settled by the claim that myogenic cells can be media (3, 15, 25-27). These culture conditions allow detection distinguished from fibrogenic by virtue of the former being of one such committed chondrogenic cell out of many thou- spindle-shaped, for many kinds of fibrogenic cells (11, 32), sands of nonchondrogenic cells by permitting either the pre- and chondrogenic cells, are spindle-shaped when grown on sumptive chondroblast, or the differentiated chondroblast, to collagen. Nevertheless, there is circumstantial evidence that replicate and differentiate terminally (13). To determine cells committed to one particular compartment within the whether among the cells used for these experiments there were myogenic lineage yield only myogenic progeny. In occasional cells that had the option to differentiate into chondroblasts, clones, and most often in small clones, virtually every cell had the following experiments were performed: Suspended cells been incorporated into myotubes (see Fig. 4 in ref. 3). This from 8-day muscle were grown in F-10 plus 10%t calf serum in type of clone may have been initiated by a cell of a compart- densities ranging from 1 cell to 2 X 106 cells per dish under ment subsequent to the bifurcation at which purely myogenic conditions known to promote replication and terminal progeny separate from the fibrogenic progeny. Differentiation chondrogenesis. Similar experiments were performed with 4- based upon this interpretation postulates that the bifurcation day-old muscle cultures. In addition, efforts were made to of purely myogenic and fibrogenic lineages occurs in a com- clone cells from primary "muscle clones" and from secondary partment antecedent to the compartment of presumptive myo- monolayers. In other experiments these cells were grafted to blasts. According to this scheme the myogenic lineage proper, the chorioallantoic membrane. The results of all these experi- at a minimum, must consist of an ultimate compartment, the ments are quickly summarized: chondroblasts were never ob- myoblast, and a penultimate compartment, the presumptive served in any of these cultures or grafts. The failure to observe myoblast. Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Phenotypic Complexity of Myogenic Clones 1509

A provocative finding was that the cell yielding myoblasts Ms and fibroblasts did not yield chondroblasts. Clearly the cells in this compartment are not equivalent to those in the PPMbFb PPCbFb "primitive mesenchyme" compartment which are believed to be present in early somites and limb buds and which yield myoblasts, fibroblasts, and chondroblasts. In experiments with PMb... Fb ... Fb ... PCb orCb ... limb buds and somites in which the lineage of myogenic and chondrogenic cells was analyzed, it was concluded that at a Mb minimum, there from the FIG. 3. A proposed lineage based on binary decisions beginning were three compartments "primi- with cells in the "presumptive mesenchyme" (Ms) compartment tive mesenchyme" cell to the myoblast (2-4, 34). In order that leads to myoblasts (Mb), to fibroblasts (Fb), and to chon- to accommodate these crucial observations into a lineage, a droblasts (Cb). The presumptive myoblast (PMb) compartment scheme at least of the complexity shown in Fig. 3 is re- consists of cells yielding post-mitotic, Mb cells. Ellipses indicate quired. that cells within that compartment are replicating and capable Two obvious variations on the lineage in Fig. 3 are: (1) Cul- of either increasing the population within that compartment or of ture conditions or other experimental factors that block fusion yielding progeny for the next compartment. As some terminally permit presumptive myoblast cells to differentiate into fibro- differentiated Fb cells emerge very early in development (1, 4) genic cells; this would be a form of dedifferentiation (25, 36); and readily replicate, it may be there is no presumptive fibroblat- (2) after a finite number of cell divisions, presumptive myo- compartment. Currently there is no way of identifying presumps blast cells throw off fibrogenic cells with greater frequency tive chondroblasts (PCb). While the small clones described in this paper may have been derived from cells within the PMb than myogenic cells; this would be a form of transdetermina- compartment, the larger clones are presumed to be from the tion (37, 38). Either of these events might also operate in vivo PPMbFb compartment. It is also suggested that the PP\IbFb and be the means of generating normal muscle fibroblasts. compartment is greatly depleted in day-18 muscles, whereas Evidence that our culture conditions need not per se dedif- the Ms compartment must be depleted before day 8. Indeed, ferentiate these myogenic cells derives from experiments from the data on limb buds (3) and on somites (2, 4), there is with BrdU. BrdU-suppressed presumptive myoblasts main- nothing to rule out the possibility that the Ms compartment is tained in vitro over 6 weeks yield terminal myoblasts if depleted even in 3-day embryos, and that by stage 17-18 all allowed to replicate in our normal medium (4, 6). mesenchymal cells have at least entered the PPMbFb or PPCbFb Worth stressing is that the fibroblastic cells from muscle compartments. The arrows and question, marks indicate that the clones have been grown for many generations, but they do not progeny of chondroblasts will form fibroblasts (8, 15), and that the progeny of fibroblasts will form to chondroblasts (10, 23, 25, 3.5); yield myoblasts though grown under conditions known per- fibroblasts, however, do not form myoblasts (22, 34, 44). mit movement from the presumptive myoblast compartment into the myoblast compartment. Furthermore, these cells have of cells depicted in Fig. 3 have the properties of a "multi- not simply lost their capacity to fuse; they do not display the potential" system, any one cell in any one compartment is at capacity to assemble cross-striated myofibrils as normal the most only "bipotential." A given presumptive mesen- mononucleated myoblasts do (34, 39). These findings suggest chyme cell does not have the option itself of directly changing that irrespective of whether the fibroblastic cells arise from the into, or within one generation of giving rise to, myoblasts, presumptive myoblast or the presumptive myoblast-fibroblast or chondroblasts, or fibroblasts. Similarly, the only option, compartment, the genetic shunt to this compartment ijvolves in terms of phenotypic diversification, open to a given pre- a relatively irreversible decision. An intriguing problem posed sumptive mesenchyme cell is to yield presumptive myoblast- by these considerations is the relationship among fibroblasts fibroblast and/or presumptive chondroblast-fibroblast cells. that emerge via the chondrogenic lineage (10, 23), those emerg- Our prediction is that a multipotential cell, i.e. a single cell ing via the myogenic lineage, and those that emerge elsewhere. having many options of itself diversifying into many pheno- The notion that fibroblasts from different progenitors might types, does not exist in most eukaryotes, and that step- have different synthetic programs is not a novel suggestion wise diversification is the sum of many binary decisions or (10, 19, 35). quantal cell cycles (4, 5). The similarities between this scheme If lineages are to be useful, their nodal points must be for generating diversity in mesenchymal tissues and those translated into specific generations of cells and the restricted proposed for hematopoietic cells (40. 41) and for cells medi- synthetic options open to cells in any one compartment ating antigen-antibody reactions (42) require no elaboration explained. For example, where in Fig. 3 is cell division dis- here. pensible, facultative, or obligatory in moving cells from one We are indebted to Dr. R. Mayne for help in the glycosamino- compartment to the next? Presumptive myoblast cells have glycan determinations. Supported by grants from the American been shown not to have the option of assembling cross-striated Cancer Society (VC-45), National Science Foundation (GB- myofibrils although their daughter cells do (33, 34). On the 27933), National Institutes of Health (HD-00030 and HL- other hand, is the presumptive mesenchyme cell obligated to 15835), and Muscular Dystrophy. divide asymmetrically, always yielding one presumptive 1. Matheson, D. & Holtzer, H. (1970) in Chemistry and myoblast-fibroblast and one presumptive chondroblast- Molecular Biology of the Intercellular Matrix, ed. Balazs, E. (Academic Press, New York), Vol. 3, pp. 1753-1764. fibroblast cell, or is the event a probabilistic one, each division 2. Holtzer, H. & Mayne, R. (1973) in Pathobiology of Develop- being symmetrical, but cell divisions, in response to exogenous ment, eds. Perrin, E. & Finegold, M., pp. 52-64. fluctuations, leading to either two presumptive myoblast-fibro- 3. Dienstman, S., Biehl, J., Holtzer, S. & Holtzer, H. (1974), blast or two presumptive chondroblast-fibroblast cells? Develop. Biol., in press (July issue). 4. Holtzer, H., Weintraub, H. & Mayne, R. 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