Transdifferentiation and regeneration in vitro

Striated muscle tissue and endoderm can be isolated from the anthomedusa Podocoryne carnea. The isolates are uncontaminated by other cell types and can be cultivated in artificial seawater for months without undergoing autonomous regeneration. However, if the endoderm is combined with collagenase-treated striated muscle, a regeneration process is initiated which leads to the formation of the sexual and feeding organ (manubrium) of the medusa. The original endoderm and striated muscle are replaced in the regenerate by at least seven new cell types, including gametes. Labeling experiments with [³H]thymidine and experiments in which mitosis is inhibited in either the striated muscle or the endoderm with mitomycin C demonstrate that the striated muscle is able to transdifferentiate into all the cell types found in the regenerate. With the possible exception of ectodermal smooth muscle this statement is also valid for the endoderm.     

Cell transformation in isolated striated muscle of hydromedusae independent of DNA synthesis

Monotypical isolated and cultivated striated muscle was demonstrated to change (metaplasia) into a cell type possessing flagellae and other characteristics of smooth muscle cells [4]. In situ and within the first 48 h following isolation, no DNA synthesis is observed in striated muscle tissue. However, between the 2nd and 3rd day in culture, approximately one day prior to the formation of flagellae, a dramatic burst of DNA synthesis is observed. In the presence of low concentrations of mitomycin-C which inhibited DNA synthesis, the transformation process from striated to smooth muscle was virtually unaffected. Increasingly higher concentrations of mitomycin-C, which completely inhibited DNA synthesis yet inhibited flagellae formation when administered immediately following isolation, had only a graduated effect on flagellae formation which was inversely proportional to the length of time in culture before treatment, and the concentration employed. The results represent the first clear case of transformation of a differentiated cell type to another distinct type without going through the S phase of the cell cycle.     

Isolated, mononucleated, striated muscle can undergo pluripotent transdifferentiation and form a complex regenerate

Isolated, mononucleated, cross-striated muscle of a medusa can be activated by collagenase treatment to transdifferentiate completely to various new cell types and to regenerate autonomously the sexual (without gametes) and feeding organ of the animal. Under these circumstances all isolated muscle fragments produce smooth muscle cells and a glandular cell type (y-cells). When culture conditions are appropriate, endoderm is also formed, followed by regeneration of a complex organ of seven or eight new non-muscle cell types, including nematocytes, digestive, secretory, gland, interstitial, and presumably nerve cells.     

Striated muscle: influence of an aceullular layer on the maintenance of muscle differentiation in anthomedusa.

Striated muscle of different anthomedusae from the Pacific and the Mediterranian was isolated and cultured in normal sea water for up to 15 weeks. The isolates were able to change their state of differentiation, as indicated by renewed DNA synthesis, changes in cytoplasmic ultrastructure, and formation of flagella. Whereas the time of flagellum formation is variable and species specific, DNA synthesis always starts between the first and the second day after isolation. At this time, the cells synthesizing DNA or undergoing mitosis still contain striated myofibrils or their degraded products. The observed changes in striated muscle isolates are strongly influenced by the presence of absence of acellular mesoglea. Mechanically isolated tissue to which portions of the inner mesoglea adhere preserves the differentiated state, i.e., shows no DNA synthesis and flagellum formation. However, when incubated in collagenase or hyaluronidase (enzymes which destroy the integrity of the inner mesoglea), flagellum formation increases in proportion to the length of incubation. Both enzyme treatments stimulate otherwise quiescent cells to synthesize DNA, but collagenase is more effective in triggering the change in DNA synthesis and the differentiated state than hyaluronidase.     

The transformational potential of striated muscle in hydromedusae.

Isolated striated muscle tissue of the Anthomedusa Podocoryne carnea participates in the regeneration of a functional manubrium (the feeding organ of medusae) when it is combined homoclonally with endodermal cells of the medusa umbrella. The morphogenetic potential of striated muscle cells in this regeneration process was evaluated by combining nuclear labeled striated muscle cells with some unlabeled endoderm cells. Histological and autoradiographical results demonstrate that transformation of striated muscle cells into smooth muscle cells of the ectoderm and also into endoderm cells must have occurred in the regenerate. The potential for cell transformation of isolated striated muscle cells of Podocoryne carnea is discussed and it is postulated that under appropriate conditions all cell types necessary for the regeneration of a manubrium can be formed from striated muscle cells.     

The proportion altering factor (PAF) and the in vitro transdifferentiation of isolated striated muscle of jellyfish into nerve cells

The effect of proportion altering factor (PAF) on the transdifferentiation of isolated striated muscle into RFamide-positive nerve cells was investigated. The factor reduces incorporation of 3H-thymidine into replicating DNA; the effect is concentration-dependent and reversible. Transdifferentiation to nerve cells increases by up to 60% if PAF is applied shortly before or at the time of initiation of DNA synthesis. When treatment was terminated 4 h before the start of S-phase or when PAF was applied at the peak of S-phase no increase in nerve cell formation was observed.     

Species specificity in cell-substrate interactions in medusae

A new system is described for the study of ECM-tissue interactions, using the ECM (called mesogloea) of various cnidarians and isolated striated muscle and endodermal tissue of jellyfish. The mesogloea consists mainly of water and collagen. It is present in all cnidarians and can be isolated without enzyme treatment. It can be used as a substrate to which cells and tissues adhere and on which they spread and migrate. Tissues of striated muscle and endoderm adhere and spread not only on mesogloea from regions they normally cover, but also from other regions of the animal. However, adhesion and spreading are highly species-specific. Species-specific adhesion is found throughout the whole mass of mesogloea even at regions where cells do not occur naturally. The cell adhesion factor can be extracted from the mesogloea so that the mesogloea no longer shows any cell adhesion properties. The extract consists mainly of a cysteine-containing collagen.     

Transdifferentiation from striated muscle of medusae in vitro

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.     

Reversible inactivation of cell-type-specific regulatory and structural genes in migrating isolated striated muscle cells of jellyfish.

We have investigated, by RT-PCR and in situ hybridization, expression of genes encoding regulatory and structural proteins in migrating mononucleated striated muscle cells of the medusa Podocoryne carnea. Expression of the three homeobox genes Otx, Cnox1-Pc, and Cnox3-Pc; a specific splice variant of the myosin heavy chain gene (Myo1); and a tropomyosin (Tpm2) is stable in isolated and cultured striated muscle tissue. When grafted onto cell-free extracellular matrix (ECM), muscle cells of the tissue fragments leave their native ECM and migrate as a coherent tissue onto a host ECM until a stretched cell monolayer is formed. Shortly after the first cells of the grafted isolate have made contact with the host ECM, Otx and Cnox1-Pc expression is completely turned off in all cells of the graft, including those still adhering to their native ECM. Myo1 message disappears with a delay while the expression level of Tpm2 is strongly reduced. However, expression of the homeobox gene Cnox3-Pc, a msh-like gene, and of the ubiquitously expressed elongation factor 1 alpha is not affected by the migration process. All genes are reexpressed after 12-24 h, once migration of the cells has ceased. Our results demonstrate that the first few migrating cells induce a change in gene expression which is rapidly communicated throughout the entire tissue. Furthermore, we showed that commitment of striated muscle cells remains stable despite the transient inactivation of cell-type-specific regulatory and structural genes.     

Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.

Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.     

Integrin and talin in the jellyfish Podocoryne carnea

We have isolated an integrin-beta and -alpha subunit from Podocoryne carnea (Cnidaria, Hydrozoa) and studied their expression in the life-cycle and during cell migration, in vitro transdifferentiation and regeneration. Comparison of the integrin expression pattern with a Podocoryne talin homologue by RT-PCR demonstrates that all three genes are maternal messages and continuously expressed in the life-cycle, in medusa development and in all medusae tissues. In situ hybridisation experiments confirm co-expression of both integrin subunits in the different life-stages. Integrin expression was furthermore studied in isolated striated muscle induced to transdifferentiate to new cell types, or grafted on ECM where the muscle adheres and migrates. Integrin expression was maintained continuously throughout both processes. These results suggest that in Podocoryne carnea processes such as cell migration and differentiation are not controlled by up- or downregulation of alternative integrin subunits, but by a single integrin heterodimer which activates different downstream signalling cascades.     

Cell cycles and in vitro transdifferentiation and regeneration of isolated, striated muscle of jellyfish

Isolated, mononucleated, cross-striated muscle cells of a medusa can transdifferentiate in vitro to various new cell types and even form a complex regenerate. The transdifferentiation events follow a strict pattern. The first new cell type resembles smooth muscle and is formed without a preceding DNA replication. This cell type behaves like a stem cell and by quantal cell cycles produces all other new cell types. Some preparations develop an inner and an outer layer separated by a basal lamella. Formation of these layers does not depend on DNA replication. When layers do not form, each division results in nerve cells and smooth muscle cells. If separation into layers occurs, then a regenerate will be formed, and in the course of only two cell cycles all necessary cell types to form a functional regenerate will differentiate.     

Serotonin: an inducer of collagenase in myometrial smooth muscle cells.

Rat myometrial smooth muscle cells in culture actively produce collagenase in medium containing fetal bovine serum, but not in medium containing newborn bovine serum or containing fetal serum adsorbed with dextran-coated charcoal. A dialyzable molecule has been isolated from fetal bovine serum, which restores the ability of the smooth muscle cells to produce collagenase. The molecule has been purified and identified as serotonin (5-hydroxytryptamine). Cells cultured in medium depleted of serotonin for 3 days fail to produce collagenase, as assessed both enzymatically and immunologically. Addition of serotonin promptly restores the ability of the cells to produce the enzyme. The EC50 for serotonin is approximately 2 microM; maximum stimulation of collagenase production is observed at 5 microM. The response is specific for serotonin: a wide variety of compounds tested, either related to serotonin or of potential reproductive significance, were without effect in the induction of collagenase production by the cells. No changes in DNA content, general protein synthesis, or cellular collagen production were observed as a consequence of serotonin depletion or restoration, suggesting a selective effect of the compound on collagenase production. The effect of serotonin was also selective to myometrial smooth muscle cells; collagenase-producing fibroblasts from skin and cervix displayed no serotonin requirement for enzyme production. Studies using specific agonists or antagonists for a variety of serotonin receptor subtypes suggest that the 5-HT-2 receptor mediates the serotonin induction of collagenase in these cells. Preliminary evidence indicates that cultured human myometrial smooth muscle cells are also dependent upon serotonin for collagenase production. The evidence in this study suggests the possibility that serotonin serves as a signal to begin the massive collagen degradation that occurs in the postpartum uterus.     

Affinity-chromatographic isolation and some properties of troponin C from different muscle types.

1. The formation of a complex between troponin I and troponin C that is stable in 6M-urea and dependent on Ca2+ was demonstrated in extracts of vertebrate striated and smooth muscles. 2. A method using troponin I coupled to Sepharose is described for the rapid isolation of troponin C from striated and smooth muscles of vertebrates. 3. Troponin C of rabbit cardiac muscle differs significantly in amino acid composition from troponin C of skeletal muscle. The primary structures of troponin C of red and white skeletal muscle are very similar. 4. The troponin C-like protein isolated from rabbit uterus muscle has a slightly different amino acid composition, but possess many similar properties to the forms of troponin C isolated from other muscle types. 5. The electrophoretic mobilities of the I-troponin C complexes formed from components isolated from different muscle types are determined by the troponin I component.     

Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos

Specification of germ layers is a crucial event in early embryogenesis. In embryos of the ascidian, Halocynthia roretzi, endoderm cells originate from two distinct lineages in the vegetal hemisphere. Cell dissociation experiments suggest that cell interactions are required for posterior endoderm formation, which has hitherto been thought to be solely regulated by localized egg cytoplasmic factors. Without cell interaction, every descendant of posterior-vegetal blastomeres, including endoderm precursors, assumed muscle fate. Cell interactions are required for suppression of muscle fate and thereby promote endoderm differentiation in the posterior endoderm precursors. The cell interactions take place at the 16- to 32-cell stage. Inhibition of cell signaling by FGF receptor and MEK inhibitor also supported the requirement of cell interactions. Consistently, FGF was a potent signaling molecule, whose signaling is transduced by MEK-MAPK. By contrast, such cell interactions are not required for formation of the anterior endoderm. Our results suggest that another redundant signaling molecule is also involved in the posterior endoderm formation, which is likely to be mediated by BMP. Suppression of the function of macho-1, a muscle determinant in ascidian eggs, by antisense oligonucleotide was enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone

RCJ 3.1, a clonally derived cell population isolated from 21-d fetal rat calvaria, expresses the osteoblast-associated characteristics of polygonal morphology, a cAMP response to parathyroid hormone, synthesis of predominantly type I collagen, and the presence of 1,25-dihydroxyvitamin D3-regulated alkaline phosphatase activity. When cultured in the presence of ascorbic acid, sodium beta-glycerophosphate, and the synthetic glucocorticoid dexamethasone, this clone differentiated in a time-dependent manner into four morphologically distinct phenotypes of known mesenchymal origin. Multinucleated muscle cells were observed as early as 9-10 d in culture, lipid-containing adipocytes formed after 12 d, chondrocyte nodules were observed after 16 d, and mineralized bone nodules formed after 21 d in culture. The differentiated cell types were characterized morphologically, histochemically, and immunohistochemically. The formation of adipocytes and chondrocytes was dependent upon the addition of dexamethasone; the muscle and bone phenotypes were also expressed at low frequency in the absence of dexamethasone. The sex steroid hormones progesterone and 17 beta-estradiol had no effect on differentiation in this system, suggesting that the effects of dexamethasone represent effects specific for glucocorticosteroids. Increasing concentrations of dexamethasone (10(-9)-10(-6) M) increased the numbers of myotubes, adipocytes, and chondrocytes; however, when present continuously for 35 d, the lower concentrations appeared to better maintain the muscle and adipocyte phenotypes. Bone nodules were not quantitated because the frequency of bone nodule formation was too low. Single cells obtained by plating RCJ 3.1 cells at limiting dilutions in the presence of dexamethasone, were shown to give rise to subclones that could differentiate into either single or multiple phenotypes. Thus, the data suggest that this clonal cell line contains subpopulations of mesenchymal progenitor cells which can, under the influence of glucocorticoid hormones, differentiate in vitro into four distinct cell types. It is, therefore, a unique cell line which will be of great use in the study of the regulation of mesenchymal stem cell differentiation.