The Phenotypic Complexity of Myogenic Clones (Leg Muscle/Cell Culture/Fibroblasts) J
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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 mono- nucleated 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 compart- ment 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.