Clonal analysis of vertebrate myogenesis. 3. Developmental changes in the muscle-colony-forming cells of the human fetal limb

The cell lineage of developing human limb muscle has been investigated by means of an in vitro clonal assay. Single cells capable of forming differentiated muscle colonies have been detected within the prospective leg muscle region as early as the 36th day of human development (Streeter's Horizon XVI). At this stage clonable myoblasts account for 14% of the total colony-forming cells. The relative proportion of clonable myoblasts increases rapidly during subsequent fetal development and attains a plateau level of approximately 90% by the 100th day of development. The 90% plateau level persists at least until day 172. By correlating the percent muscle colony differentiation with clonal plating efficiency and with the number of single cells derived from the total limb muscle region of fetuses of different ages, an estimate of the actual number of clonable myoblasts within the developing limb musculature is obtained.Sequential changes within the muscle cell lineage have been further dissected by the temporal analysis of age-dependent medium effects on muscle colony differentiation. The analysis indicates that clonable myoblasts derived from early fetuses are sensitive to medium conditions to which older muscle-colony-forming cells are relatively insensitive. In addition, fusing and nonfusing colonies have been classified into recognizable morphological types whose relative proportions are observed to change during limb development. The results are correlated with human limb morphogenesis and skeletal muscle histogenesis and an operational model of muscle cell lineage is proposed.     

Clonal analysis of vertebrate myogenesis. II. Environmental influences upon human muscle differentiation

In vitro procedures for obtaining the differentiation of human fetal muscle colonies were developed, and the sensitivity of clonal differentiation to environmental influences was examined. Human muscle colonies are capable of differentiating in the absence of an exogenous collagen substrate. The dependence of clonal diffeentiation upon the addition of chick embryo extract to the culture medium is determined by the serum type used in the medium and by the substrate upon which the colonies are grown. Clonal differentiation also depends upon conditioning of the medium by the colonies. The rate of medium conditioning is affected by clonal density and initial medium composition. The required medium modification is not species specific since medium conditioned by chick muscle cells also permits the early differentiation of human muscle clones. By manipulating the various environmental parameters described above it has been possible to define a number of in vitro conditions which permit a normal rate of cell proliferation but do not permit cell fusion. Results from these experiments are discussed in terms of their developmental implications.     

The retention of differentiated properties by lens epithelial cells in clonal cell culture

A small number of cells of lens epithelium from newly hatched chickens were cultured at clonal density to investigate the retention of differentiated properties during cellular growth in vitro. Singly plated cells proliferated to produce colonies, at least some of which were considered to be true clones of single cell origin. The differentiation of lens fibers occurring in many colonies was identified through observations by electron microscopy as well as immunofluorescence utilizing specific antiserum against lens fibers. Primary or secondary mass cultures of cells of lens epithelium contained cells which produce differentiated colonies when cultured at clonal density. Colony-producing cells can be differentially dissociated from monolayers by EDTA treatment without using tyrpsin. For successful culture of cells of lens epithelium at clonal density, the use of conditioned medium is necessary.     

Clonal analysis of vertebrate myogenesis. VI. Acetylcholinesterase and acetylcholine receptor in myogenic and nonmyogenic clones from chick embryo leg cells.

Myogenic clones grown in vitro from cells of 4-, 6-, and 12-day chick embryo leg buds demonstrate reproducible stage-specific characteristics of morphology, extent of myotube formation, and culture medium requirements for differentiation, suggesting heterogeneity in the myogenic cell populations of the developing limb. To determine whether there is heterogeneity in the cytodifferentiation of different muscle colony types, clones have been examined for the appearance of two muscle-specific gene products—acetylcholinesterase (AChE) and acetylcholine receptor (AChR). AChE (detected by cytochemical reaction) and AChR (detected by autoradiography of [¹²⁵I]α-bungarotoxin binding) appeared in myotubes of all muscle colony types, and also appeared in about 5% of the mononucleated cells of all muscle colonies; but neither were detectable in cells of nonfused clones (colonies containing no myotubes). The results suggest that all muscle colony-forming cell types have equivalent capacities to elaborate muscle-specific gene products once the process of differentiation is initiated. However, when putative muscle colony-forming cells are grown under certain conditions that do not permit cell fusion (e.g., conditioned medium-requiring clones grown in fresh medium), mononucleated cells do not accumulate AChE or AChR. Conditioned medium-dependent differentiation thus differs from the fusion-specific processes affected by Ca²⁺ deprivation and phospholipase C treatment, since in these cases mononucleated cells exhibit differentiated functions. The apparent cytodifferentiation (without fusion) of some mononucleated cells within muscle colonies in which most mononucleated cells continue to proliferate raises questions concerning the control of myoblast differentiation and its relationship to the cell cycle and to fusion.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.     

Transient Tumor-Fibroblast Interactions Increase Tumor Cell Malignancy by a TGF-β Mediated Mechanism in a Mouse Xenograft Model of Breast Cancer

Carcinoma are complex societies of mutually interacting cells in which there is a progressive failure of normal homeostatic mechanisms, causing the parenchymal component to expand inappropriately and ultimately to disseminate to distant sites. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumor microenvironment and then to normal fibroblasts as it traverses the underlying connective tissue towards the bloodstream. The interaction of tumor cells with stromal fibroblasts influences tumor biology by mechanisms that are not yet fully understood. Here, we report a role for normal stroma fibroblasts in the progression of invasive tumors to metastatic tumors. Using a coculture system of human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts, we found that medium conditioned by cocultures of the two cell types (CoCM) increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. Transient treatment of MCF10CA1a cells with CoCM in vitro accelerated tumor growth at orthotopic sites in vivo, and resulted in an expanded pattern of metastatic engraftment. The effects of CoCM on MCF10CA1a cells were dependent on small amounts of active TGF-β1 secreted by fibroblasts under the influence of the tumor cells, and required intact ALK5-, p38-, and JNK signaling in the tumor cells. In conclusion, these results demonstrate that transient interactions between tumor cells and normal fibroblasts can modify the acellular component of the local microenvironment such that it induces long-lasting increases in tumorigenicity and alters the metastatic pattern of the cancer cells in vivo. TGF-β appears to be a key player in this process, providing further rationale for the development of anti-cancer therapeutics that target the TGF-β pathway.     

The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. II. Cell sensitivity to the lethal effect of the drug as a function of the cell age and of the nature of the culture medium

We have tested the sensitivity of KB cells to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), measured as cell loss within the 20-h period following a 1-h drug treatment, as a function of culture age and of the medium in which treated cells were incubated after elimination of MNNG. We showed that KB cell sensitivity to the lethal effect of the drug decreased with time after seeding when the treated cells were post-incubated in drug-free medium conditioned by untreated cells of the same age as treated ones but not when they were post-incubated in fresh drug-free medium. This difference was due in part to the fact that the conditioned medium had acquired with time a protective activity for treated cells and in part to an increased competence of aging cells to be protected by this medium. By post-incubating treated stationary cells sequentially in both media, we showed that a brief (15 min) post-incubation of the cells in fresh medium was sufficient to trigger cell death even if the cells were afterwards transferred to conditioned medium. In contrast, long post-incubation in fresh medium did not cause cell death if the cells were first post-incubated in conditioned medium for about 3 h. We conclude that: the medium acted on cell sensitivity to the lethal effect of MNNG through its growth regulatory ability; quiescent cells were less sensitive to the drug than growing cells; the sensitive phase of the cell was located before S; cell hypersensitivity might be due to deficient repair of cellular lesions rather than to increased lesion formation.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Clonal analysis of vertebrate myogenesis. I. Early developmental events in the chick limb

Early developmental events occurring in the prospective muscle tissue region of chick embryo leg buds have been subjected to an in vitro clonal analysis. Colony-forming cells are present at stage 20 (72 hr incubation), but none of the colonies exhibit morphological signs of muscle differentiation. After an additional 8 hr of incubation (stage 21), approximately 10% of the colony-forming cells have acquired the capacity to form multinucleated cells in vitro, and the percentage of clonable myoblasts increases to a level of approximately 60% during the next 3 days of incubation. Clonal analysis of myoblast populations within regions of the developing limb have indicated that, between stages 21 and 27, the dorsal and ventral segments of the myogenic region contain appreciably more clonable muscle cells than the anterior and posterior segments. In addition, during stages 21 and 22 there is a 3-fold difference in muscle-colony-forming cells between the proximal and distal halves of the dorsal-ventral segments, as well as between the proximal and distal halves of the anterior-posterior segments. Thus at least two temporal and regional gradients—proximal to distal and medial to lateral—of clonable myoblast content can be delineated within the developing chick limb. In addition to changes in the proportions of muscle-colony-forming cells, the extent of multinuclearity within individual muscle colonies increases with the developmental age of the embryo from which the clonable myoblasts are derived. The progressive changes in the relative proportions of muscle-colony-forming cells and in clonal morphology are discussed in terms of their possible cell lineage implications.     

Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts

The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes. We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome. © 1996 Wiley-Liss, Inc.     

A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts

Stable myoblast cell lines were isolated after a brief exposure of mouse fibroblasts (10T1/2 cells) to 5-azacytidine. We show that transfection of 10T1/2 cells with DNA from these azacytidine-induced myoblasts (or from mouse C2C12 myoblasts) results in myogenic conversion of approximately 1 in 15,000 transfected colonies. In contrast, transfection of 10T1/2 cells with DNA from nonmyogenic cells (parental 10T1/2 cell DNA) does not give rise to myoblast colonies. These results indicate that an azacytidine-induced structural modification (presumably demethylation) in the DNA of a single locus is sufficient to convert 10T1/2 cells into determined myoblasts.     

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Spatial analysis of limb bud myogenesis: A proximodistal gradient of muscle colony-forming cells in chick embryo leg buds

The regional distribution of myogenic cells in developing chick leg buds has been investigated using an in vitro clonal assay. Leg buds were embedded in gelatin and sectioned at intervals of 100–300 μm utilizing a vibratome, and cells dissected from prospective myogenic areas were analyzed for their ability to form colonies containing multinucleated myotubes. The results show that muscle colony-forming (MCF) cells from stage 23 ( to 4-day incubation) are exclusively of the early morphological type, and are found in the proximal two-thirds of the bud. Late-type MCF cells are first obtained from the proximal sections of stage 24–25 (4- to day) buds; in succeeding stages (26–29), late MCF cells supercede the early MCF cell type in the proximal regions, and extend into progressively more distal sections in a graded fashion. Results from sequential sections suggest that early and late MCF cells are located within the same muscle groups. The proportion of late MCF cells continues to increase throughout this period, until by stage 31 (7 days) only the most distal myogenic regions (the toe muscle regions) have an appreciable proportion of early MCF cells. Clonal plating efficiencies increase throughout the period of analysis, and by stage 31 precisely dissected myogenic regions yield plating efficiencies as high as 36% with greater than 95% of these colonies differentiating as muscle.     

Myogenic differentiation in permanent clonal mouse myoblast cell lines: Regulation by macromolecular growth factors in the culture medium

A permanent clonal cell line of mouse myoblasts (MM14) has been used to study the transition from proliferation to terminal differentiation. Results indicate that the transition is strictly dependent on the culture medium environment. Evidence from clonal density cultures suggests that (1) specific macromolecular mitogenic components of the culture medium stimulate mouse myoblast proliferation and prevent differentiation, (2) mouse myoblasts eliminate mitogenic activity from the culture medium before differentiating, and (3) lowered activity of specific mitogens stops mouse myoblast proliferation and triggers the program of terminal differentiation leading to the elaboration of muscle specific gene products and formation of myotubes. Evidence for the regulatory role of specific mitogens is the stimulation of proliferation and delay of differentiation by the addition of nanomolar concentrations of fibroblast growth factor to mitogen-depleted, differentiation-promoting, culture medium, whereas the addition of other purified mitogens has no effect. The results support and extend evidence from other muscle culture systems that stimulation of proliferation delays myoblast differentiation, and they provide an experimental basis for controlling the synchronous differentiation of pure populations of clonally derived mouse myoblasts.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.