Mark Nameroff

Isolation and clonal analysis of satellite cells from chicken pectoralis muscle

Satellite cells, liberated from the breast muscle of young adult chickens by sequential treatment with collagenase and trypsin, were fractionated by Percoll density centrifugation to remove myofibril fragments and cell debris which otherwise heavily contaminate the preparation. This procedure allowed direct measurements of cell yields (1.5-4 X 10(5) cells/g tissue), plating efficiencies (27-40%) and identification of single cells in culture. In mass cultures, satellite cells gave rise to myotubes on the fifth day, and the progeny of these cells were sequentially passaged several times without losing myogenic traits. In clonal studies, over 90% of the satellite cells gave rise to large clones of which more than 99% were myogenic as demonstrated by the appearance of myotubes. The results obtained with satellite cells differ from observations made using embryonic muscle cell preparation from chicks. In the embryonic system massive formation of myotubes was observed following the third day of culture; sequential subculturing led to overgrowth of fibroblast-like cells following the first passage; and cells gave rise to both small myogenic clones (up to 16 terminally differentiated cells per clone) and non-myogenic clones in addition to large myogenic clones. We conclude that the isolated satellite cells represent a homogeneous cell population and reside in a stem cell compartment.

Inhibition of cellular differentiation by phospholipase C: II. Separation of fusion and recognition among myogenic cells☆

In cell culture, a partially purified commercial preparation of phospholipase C (PLC) from Clostridium welchii inhibited fusion of myoblasts at concentrations of 12–50 µg per ml. At lower concentrations, PLC-treated cultures were indistinguishable from controls, and at concentrations above 100 µg per ml, PLC-treated cells detached from their substrates. The effect was reversible and fusion resumed approximately one cell cycle time after removal of the enzyme. Neither the percent of cells in the mitotic cycle nor the duration of the different phases of the cycle were altered by PLC at concentrations which inhibited fusion. Cell motility was not reduced by the enzyme. Unfused, PLC-treated myoblasts were virtually indistinguishable in ultrastructure from untreated cells just before fusion. In the presence of PLC, mononucleated myogenic cells did not synthesize thick (150 Å) filaments. Treatment of culture medium with insolubilized commercial PLC did not abolish the capacity of the medium to support myogenesis. Chondrocytes treated with PLC divided repeatedly but failed to synthesize metachromatic matrix and failed to incorporate labeled sulfate into chondroitin sulfate. PLC was further purified by chromatography on Sephadex G-100. The resulting preparation was free of detectable protease, yielded one band on SDS-acrylamide gel electrophoresis, and displayed all of the biological activities of the less pure material.

Myoblast differentiation in vitro: Morphological differentiation of mononucleated myoblasts

Abstract Phospholipase C from Clostridium perfringens has been shown previously to inhibit the fusion of cultured chick myoblasts without affecting recognition or cell cycle parameters. In this paper we report that the mononucleated myoblasts, in phospholipase C, synthesize thick and thin filaments and organize them into myofibrils, and that T-tubules and sarcoplasmic reticulum differentiate and join in morphologically typical junctions. The structurally differentiated myoblasts can then fuse with one another to form myotubes. We conclude that cell fusion is not necessary for muscle differentiation.

Age-dependent changes in myogenic precursor cell compartment sizes. Evidence for the existence of a stem cell.

Individual myogenic cells were isolated from the pectoralis muscles of chick embryos from days 8-14 of embryogenesis. When separately cloned, these cells produced three types of colonies in culture: (1) Positive: all cells in the clone were terminally differentiated muscle cells; (2) negative: no cells in the clone were terminally differentiated muscle; (3) mixed: some cells in the clone were terminally differentiated muscle. Positive clones from all ages tended to contain 2n cells (n = 0, 1, 2, 3, 4). Negative clones were found in all sizes and did not cluster around powers of 2 in cell number. Mixed clones were, by far, the most common type among those clones larger than 24 in cell number. Estimates of cell numbers in embryonic muscle tissue revealed that, while the numbers of cells in all myogenic compartments increased steadily with embryonic age, the number and percentage of precursor cells that produced large mixed clones increased dramatically. Subclones, prepared from populations of cells equivalent to large mixed clones, yielded both small positive and large mixed colonies. This indicated that the precursors to the large mixed clones were also precursors to the smaller positive clones. These observations suggest a model for the myogenic lineage in which there exists a stem cell that can generate, by a series of asymmetric divisions, cohorts of terminally differentiated muscle cells. The model can explain the asynchrony of production of terminally differentiated muscle cells both in vitro and in vivo.

Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle

SummaryNon-myogenic cells were isolated from the breast muscle of 10-day-old chicken embryos employing Percoll density centrifugation. In culture, these cells exhibited the spread out, stellate morphology of fibroblast-like cells. They also exhibited receptor-mediated binding of plateletderived growth factor (PDGF). Such binding was not detected in cultures of predominantly myogenic cells isolated by the Percoll density centrifugation from the same muscle. Percoll-isolated myogenic and fibrogenic cell populations were also analyzed by two-dimensional polyacrylamide gel electrophoresis immediately after removal from the muscle. This analysis revealed at least six polypeptides specific to the fibroblasts but not detected in the myogenic cell population. In addition, at least eight polypeptides found in the myogenic population were barely detectable, or lacking altogether from the fibroblast-like cells. Ultrastructural analysis of the freshly isolated cells demonstrated that the fibroblasts were larger than the myoblasts and that their cytoplasm contained many vesicles. We conclude that the fibrogenic and myogenic cells isolated by Percoll from embryonic muscle express cell type-specific characteristics. Moreover, based on the PDGF binding studies, the fibrogenic cells can be categorized as “true” fibroblasts.

Analysis of the myogenic lineage in chick embryos: II. Evidence for a deterministic lineage in the final stages☆

Abstract Myogenic cells from the breast muscles of 11-day chick embryos were cultured in clones for intervals up to 96 h. Individual cells in these clones were assayed for terminal differentiation by staining with antiserum to muscle-type creatine phosphokinase. Terminal differentiation was expressed in these clones in characteristic patterns which are indicative of a deterministic lineage during the last 4 or 5 cell division. This lineage is composed of discrete compartments culminating in the post-mitotic myoblast. The pattern of transition through these compartments is not altered by growth in media of various nutritional composition, indicating that cells must undergo lineage-dependent changes before the terminal phenotype can be expressed. Thus, myogenic precursors are not a homogeneous population. Stem cells (self-renewing cells) may occur before these last few divisions.

Analysis of the myogenic lineage in chick embryos: I. Studies on the terminal cell division

Abstract An antibody prepared against the MM isozyme of creatine phosphokinase (M-CK) stained multinucleated myotubes and post-mitotic mononucleated myoblasts in mass cultures of myogenic cells taken from the breast muscles of 11-day chick embryos. No cycling cells bound the antibody. Single cells isolated either directly from the embryo or from mass cultures were seeded at clonal density and allowed to undergo one division. The resulting pairs of cells were stained with the antibody and were scored as ( a ) both members of the pair M-CK + ; ( b ) both M-CK − ; or ( c ) mixed (one M-CK + and one M-CK − ). No mixed pairs were observed. Conditioned medium did not induce all myogenic pairs to differentiate and growth medium did not keep myogenic pairs in the cell cycle. About 10% of clonal pairs established from 10 h cultures were M-CK + , while about 27% of pairs established from 30 h mass cultures were M-CK + . These results indicate that (1) the myogenic lineage ends in a symmetrical division whose products are two post-mitotic M-CK + cells; (2) the expression of the muscle phenotype is not determined exclusively by the environment; (3) the terminal cells are the product of an intrinsic program or cell lineage in which only the last cells can synthesize muscle-specific proteins.

Characterization of a Marker for Tracheal Basal Cells

An IgM monoclonal antibody (1D9/B3) is characterized, which specifically recognizes basal cells of the upper airway epithelium. Although morphological features have been used to follow cell lineage and differentiation, an objective assessment of differentiation can be enhanced by characterizing the expression of specific antigens that form the phenotypic profile of specialized cells. Mice were immunized with rabbit tracheal basal cells that had been obtained by pronase digestion and purified into a subpopulation of basal cells by flow cytometry. Six immunization experiments produced five hybridomas specific to epithelial cells. A hybridoma whose supernatant immunocytochemically stained the basal cell subpopulation of rabbit tracheal cells was selected. The antibody reacted with tracheal basal cells in rabbit, rat, sheep, pig, and human tracheal sections, and in cultured monolayers of tracheal epithelial cells of the same species. The antibody did not react with the basal cells of other rabbit tissue, including the skin, or other rabbit epithelia. Confocal microscopy and exposure of tracheal epithelial cells to fluorescent-tagged monoclonal antibody 1D9/B3 prior to loading on to flow cytometry showed that the basal cell antibody recognized an intracellular epitope. The epitope for the 1D9/B3 antibody was characterized by Western blotting. The 1D9/B3 antibody appears to be a distinct and specific marker to the airway epithelial basal cell and will be useful in studies of airway epithelial differentiation, injury, and regeneration.

The Arithmetic of Skeletal Myogenesis in the Chick Embryo

Attention is often focused on the molecular mechanisms of cell differentiation before an adequate understanding of the biology of the cells has been attained. Cells that are precursors to any particular end-stage, differentiated cell type, for example, are sometimes considered to he a population of identical cells, all of which express the same genes. Although this is a convenient assumption and may be true for certain cell lines, it is demonstrably not true for cells in the embryo. The point is that the biology of the precursor cells is what must eventually be explained in molecular terms. Without the biology, proposed molecular mechanisms may be misleading, and important events in the differentiation process may be missed entirely. This point is especially important in view of the different mechanisms used by various organisms to generate the same type of differentiated cells. With this issue in mind, my colleagues and I have undertaken to determine whether all skeletal muscle precursor cells in the chick embryo are equivalent. We approached this question in the simplest possible way; that is, by counting the total number of cells and terminally differentiated muscle cells produced by individual myogenic precursors in clonal cultures under various experimental conditions. The markers we used for terminal differentiation were cytoplasmic staining with antibodies against skeletal muscle myosin heavy chain (MHC) and/or muscle type creatine phosphokinase (MCK). This simple protocol was used on myogenic cells from the pectoral muscles of embryos that were 10 to 18 days old and from 4to 6-week-old posthatch juveniles. In our standard culture conditions (200-500 cells in 60 mm dishes with 3 mL of medium (85% MEM. 10% horse serum, 5% embryo extract)), we found two general classes of myogenic precursor cells.’ The first class consisted of cells that produced only terminally differentiated muscle cells. In clones derived from these cells, the number of nuclei was nearly always a power of 2 ( i . e . , 1, 2, 4, 8, or 16). We called these “small” clones. The second class consisted of cells that produced hundreds of descendants, only some of which were terminally differentiated cells at the times we stained the cultures. I n clones derived from these cells, the number of nuclei in the terminally differentiated cells was nearly always an integer multiple of 16.2 These were “large” myogenic clones. In 10-day embryos, cells that produced small myogenic clones represented approximately 90% of the myogenic cells; the remaining 10% produced large myogenic clones. By contrast, in IS-day embryos and in posthatch juveniles,) 4-10% of precursor cells produced small myogenic clones: 90-96%’ produced large myogenic clones. There was a gradual and roughly linear shift in the relative percentages of small and large clone precursors between 10 and 18 days of in~ubat ion .~ Culturing the cells in conditioned medium did not affect either the types of clone classes observed or the final percentages of the small and

Fusion, Phospholipase C, and Myogenesis

Our experiments with phospholipase C (PLC) have focused on several aspects of terminal skeletal muscle differentiation: how does fusion occur and how is fusion related to other events of terminal differentiation. Elsewhere (Leung et al., 1973; Leung et al., 1975) we have reported that PLC releases a number of proteins from the surfaces of myogenic cells and we have suggested that at least one of these proteins is involved in the fusion process. Since the theme of this meeting is the relationship of cell division to cell differentiation, I shall briefly describe several experiments which suggest that (1) mononucleated myogenic cells withdraw from the cell cycle prior to fusing and (2) there exists, prior to and separable from the fusion event, a recognition step in which mononucleated cells line-up and remain in close association with one another.