Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

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.     

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. 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.     

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.     

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.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

CHANGES IN THE CAPACITY FOR CLONAL GROWTH AND DIFFERENTIATION IN VITRO OF THE VERTEBRAL CARTILAGE CELLS WITH EMBRYONIC DEVELOPMENT II. VITALIZING EFFECT OF CONDITIONED MEDIUM ON THE CELLS OF YOUNGER EMBRYOS

Most of the younger cartilage cells taken from chick embryos at the stage 35 were known to regress after the initial cell multiplication in in vitro clonal culture. A partial supplementation of conditioned medium (CM) to the standard medium was effective to permit colony formation from many of these younger cells. Most colonies thus derived from the younger cells by the aid of CM expressed differentiative traits as cartilage, as the cells from older embryos did. CM exerted multiple effects on clonal development of the younger cells. It vitalized the cells destined to regress, to promote cell multiplication and to flatten the shape of the colony. The experimental results suggested that these effects were associated with an independent factor each.     

A clonal approach to the problem of neural crest determination.

A fundamental question regarding neural crest development is the possible pluripotential nature of this embryonic tissue. As a first step in examining this problem, clonal techniques are used to produce homogeneous populations of crest cells. Primary cultures of these cells are obtained by explanting neural tubes from Japanese quail in vitro and allowing crest cells to migrate away. The explant is removed, the outgrowth is isolated, dissociated with trypsin, and the cells plated at clonal density. Colonies derived in this manner fall into the following categories: all cells of the colony pigmented; none of the cells pigmented; and some of the cells pigmented, the remainder unpigmented. Pigmented colonies generally arise from small, round cells whereas the non-pigmented colonies usually originate from large, flattened polymorphous cells. Differentiation of melanocytes does not preclude their continued proliferation. The pigment phenotype, in addition, is stable through at least 25 generations. That the mixed colonies, in fact, are clonally derived is shown by physically isolating single cells. The identity of the non-pigment cells was not established in the present work. A possible neural fate is suggested, however, since nerve-like cells develop after the petri plates become overgrown. Neural clones did not form even though nerve growth factor activity is present as a normal constituent of the culture medium and was added as a supplement in some instances. These techniques permit the preparation of large, homogeneous populations of neural crest cells and afford an opportunity to examine aspects of crest determination heretofore impossible to study.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Differences in the Expression and Distribution of Flotillin-2 in Chick,Mice and Human Muscle Cells

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.     

The ultrastructural differentiation of the clonal myogenic cell line L6 in normal and high K+ medium

Cells of the clonal myogenic line L6 were examined electron microscopically at successive stages of growth. L6 cells are developmentally similar to those of chick and rat primary skeletal muscle cultures and skeletal muscle in vivo, with respect to myofibrillogenesis and sarcomere organization. However, the sarcoplasmic reticulum and T systems of L6 myotubes are not as well differentiated as those of primary muscle cultures and adult skeletal muscle. Finally, L6 myotubes show precocious sarcomere differentiation when cultured in medium containing 25 mM potassium.     

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo.

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived.Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [³H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.     

The in vitro cell fusion of embryonic chick muscle without DNA synthesis

A system has been developed for the in vitro development of chick skeletal muscle monolayers, in which a burst of synchronous fusion occurs, such that some 40% of the spindle-shaped cells fuse in a 10-hr period. Cells inhibited from synthesizing DNA by ara-C do fuse, but at a later time than the normal burst. If ara-C is added to cultures 6 hr or more before the normal fusion time, fusion is delayed, but no delay results when the drug is added after this time. A medium change will delay the fusion if done 4 hr or more before fusion, but gives no delay if done later. Cells grown in conditioned medium fuse some 10 hr earlier than controls, even in the presence of ara-C, as do cultures prepared at higher than normal cell densities. The data suggest that muscle cell fusion is independent of DNA synthesis in vitro, but depends upon a modification of the culture medium to a sufficient degree required for initiating the synthetic program for fusion.     

Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.     

Electrophysiologic and morphologic properties of neurons in dissociated chick spinal cord cell cultures

Neurons dissociated from embryonic chick spinal cords mature in relatively sparse cell culture and survive in vitro for several weeks. They generate action potentials and form both excitatory and inhibitory chemical synapses with one another. By electrophysiologic and morphologic criteria, it appears that the neuronal population (after 2–3 weeks) is made up of a variety of different cell types; few, if any, are motoneurons. Neuron cell bodies are not covered by glia or satellite cells and nerve processes are not myelinated. Thus, the cultures should permit more direct microelectrode and pharmacologic analysis of differentiation of cell specific properties and of synapse formation than is possible in the intact central nervous system.     

Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.     

Microencapsulated human bone marrow cultures: a potential culture system for the clonal outgrowth of hematopoietic progenitor cells

Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate-poly-L-lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24-fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst-forming units erythroid (BFU-E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.     

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.