Collagen distribution in endochondral cartilage obtained in vitro from dedifferentiated cells.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R Cancedda
Publications and source records attributed to R Cancedda.
Dimethyl sulfoxide interferes with in vitro differentiation of chick embryo endochondral chondrocytes.
Dedifferentiated chondrocytes derived from 6-day-old chick embryo tibiae when transferred on agarose, revert to the chondrocytic phenotype and mature to hypertrophic, type X collagen-producing chondrocytes (Castagnola et al. (1986). J. Cell Biol. 102, 2310-2317). The continuous presence of 180 mM dimethyl sulfoxide (DMSO) during the culture specifically inhibited synthesis of type X collagen and accumulation of its mRNA. The synthesis of the cartilage-specific type II collagen and the level of its mRNA were essentially unchanged in treated and control untreated cells.
Animals↗
Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro.
This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary, types II and IX collagen mRNAs sharply increased within the first week of suspension culture, peaked in the second week, and thereafter began to decrease. This decrease was particularly significant for type IX collagen mRNA. The level of type X collagen mRNA progressively increased during the course of the culture, reached its maximal value after 3-4 wk, and decreased only at a later stage of cell differentiation. As determined by in vitro run-off transcription assays, all these changes in collagen mRNA levels could be attributed to parallel modifications in the relative rate of transcription of the corresponding collagen genes. We suggest that chicken chondrocyte differentiation proceeds through at least two different steps: (a) first, transition from a stage characterized by a high level of type I collagen mRNA to a stage characterized by predominance of types II and IX collagen mRNAs; (b) later, transition to a stage characterized by the highest level of type X collagen mRNA.
Animals↗
Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis.
Human keratinocytes isolated from a skin biopsy and cultured in vitro reconstitute a stratified squamous epithelium suitable for grafting on burned patients. Melanocytes coisolated from the same skin biopsy also proliferate under these culture conditions and maintain differentiated functions (i.e., synthesize melanin granules, regularly intersperse in the basal layer of the cultured epidermis, and transfer melanosomes in the cytoplasm of contiguous keratinocytes) (De Luca, M., A. T. Franzi, F. D'Anna, A. Zicca, E. Albanese, S. Bondanza, and R. Cancedda. 1988. Eur. J. Cell Biol. 46:176-180). Isolated melanocytes in culture grow in the presence of specific growth factors with a mean population doubling time of 4-10 d. In this paper we show that (a) human keratinocytes and oral epithelial cells possess strong and specific melanocyte growth stimulating activity (doubling time, 24 h); (b) melanocyte growth is not autonomous but requires close keratinocyte contact and is regulated to maintain a physiological melanocytes/keratinocytes ratiol and (c) pure skin keratinocytes, but not oral epithelial cells, have all the information required for the proper physiological location and differentiation of melanocytes in the epidermis.
Cell Communication↗
Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes.
When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by immunohistochemistry, we show that in the chick embryo tibia the Ch 21 protein first appears at the boundary of the cone of hypertrophic cartilage and in the newly formed bone between the 6 and 10 d of embryo development and localizes in calcifying hypertrophic cartilage thereafter. The Ch 21 protein synthesized by the cultured chondrocytes is closely related and possibly identical to a 21K transformation-sensitive protein associated to the cell substratum of chick embryo fibroblasts.
Age Factors↗
Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium.
Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.
Cell Differentiation↗
[Separation, by elutriation, of human keratinocytes cultured in vitro].
Explore the source record for details and available documents.
Cell Separation↗
Type X collagen synthesis by cultured chondrocytes derived from the permanent cartilaginous region of chick embryo sternum.
In the developing chick embryo sternum, type X collagen is synthesized by chondrocytes from the cephalic region (presumptive mineralization zone) but not by chondrocytes from the caudal region (permanent cartilaginous zone) (Gibson et al., 1984, J. Cell Biol. 99, 208-216). To distinguish between two possibilities, the presence of a nonpermissive microenvironment in the permanent cartilage or the intrinsic inability of caudal chondrocytes to become hypertrophic, type X-producing cells, we have isolated chondrocytes from the caudal third of stage 44 chick embryo sterna and grown them in suspension on agarose-coated dishes. We have found that in these conditions chondrocytes from the caudal zone differentiate to hypertrophic chondrocytes and synthesize large amount of type X collagen, as revealed by the electrophoretic pattern of labeled proteins made in vitro and by slot blot analysis of mRNAs with specific cDNA probes.
Animals↗
In vitro morphogenesis of chick embryo hypertrophic cartilage.
Dedifferentiated chick embryo chondrocytes (Castagnola, P., G. Moro, F. Descalzi-Cancedda, and R. Cancedda, 1986, J. Cell Biol., 102:2310-2317), when transferred to suspension culture on agarose-coated dishes in the presence of ascorbic acid, aggregate and remain clustered. With time in culture, clusters grow in size and adhere to each other, forming structures that may be several millimeters in dimension. These structures after 7 d of culture have the histologic appearance of mature hypertrophic cartilage partially surrounded by a layer of elongated cells resembling the perichondrium. Cells inside the aggregates have ultrastructural features of stage I (proliferating) or stage II (hypertrophic) chondrocytes depending on their location. Occurrence and distribution of type I, II, and X collagens in the in vitro-formed cartilage at different times of culture, show a temporal and spatial distribution of these antigens reminiscent of the maturation events occurring in the cartilage in vivo. A comparable histologic appearance is shown also by cell aggregates obtained starting with a population of cells derived from a single, cloned, dedifferentiated chondrocyte.
Animals↗
In vitro translation of chicken type X collagen in the presence of pancreas microsomes.
Total RNA from epiphysis of 17-day-old chick embryo tibiae was used to direct protein synthesis in a wheat germ cell free system. The type X collagen chain, identified on the basis of its electrophoretic migration and of peptides obtained by S. aureus V8 protease digestion, was the major translation product. The newly synthesized chain included a signal sequence that was removed when dog pancreas membranes were added at the time of the protein synthesis.
Animals↗
Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes.
In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375-381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the cells attach and dedifferentiate, as revealed by their fibroblastic morphology. Dedifferentiated chondrocytes, after several passages, can still reexpress the differentiated phenotype and continue their development to hypertrophic, type X collagen-synthesizing chondrocytes. Hypertrophic chondrocytes, when plated in anchorage permissive dishes, attach, maintaining the differentiated phenotype, and continue the synthesis of type X collagen.
Animals↗
Purification and characterization of the low-molecular-mass (type X) collagen from chick-embryo tibial cartilage.
Type X collagen, synthesized in large amount by cultured tibial chondrocytes, is deposited in vivo in the epiphyseal cartilages of 17-day-old chick embryo tibiae. Here we report the extraction of this collagen from these cartilages by limited pepsin digestion and its purification to electrophoretic homogeneity by salt precipitation followed by agarose gel filtration. Identity of the collagen purified from cartilage with the type X collagen synthesized by cultured chondrocytes is confirmed by comparison of the amino acid compositions. The high glycosylation extent of type X collagen is reminiscent of the glycosylation extent of pericellular collagens. The possible role of type X collagen is discussed.
Amino Acids↗
Deposition of type X collagen in the cartilage extracellular matrix.
In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.
Animals↗
Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes.
Quail embryo chondrocytes in culture display two morphological phenotypes: polygonal epithelial-like and floating cells. Both cell populations synthesize cartilage extracellular matrix proteins (type II collagen and specific proteoglycans), whereas type X collagen, which appears to be a marker of later stages of chondrocyte differentiation, is expressed only by the epithelial-like cells. Avian myelocytomatosis virus strain MC29 does not induce morphological transformation in quail embryo chondrocytes but stimulates these cells to proliferate with a progressively reduced doubling time. MC29-infected chondrocytes can be established in culture as a continuous cell line, whereas control (uninfected) cultures only survive a few months. Rapidly dividing MC29-infected chondrocytes still express type II collagen and cartilage proteoglycans but do not synthesize type X collagen.
Animals↗
In vitro synthesis of the gene coding for the glycoprotein E1 of Sindbis virus.
Ds cDNA of the 42S virionic RNA of Sindbis Virus has been synthesized and cloned in the plasmid pBR 322. Restriction map analysis and hybridization studies show that two clones cover the 3' end non coding region of the RNA, the whole membrane glycoprotein E1 and a peptide of MW 6,000 daltons. The use of these clones in experiments of gene expression in mammalian cell is discussed.
Base Sequence↗
The low molecular weight collagen synthesized by chick tibial chondrocytes is deposited in the extracellular matrix both in culture and in vivo.
The low mol. wt. collagen (64 K) synthesized by chick embryo chondrocytes in culture is deposited in the extracellular matrix; its deposition is strictly dependent upon a correct hydroxylation. In vivo the 64 K collagen has been isolated from the cartilage of tibiae obtained from 17-day-old chick embryos. The turnover of this collagen in the extracellular matrix is very rapid: within a few hours it is matured into a 30-K fragment released in the medium. Also this maturation is dependent upon a correct hydroxylation of the molecule. The underhydroxylated form, synthesized in the absence of ascorbic acid or in the presence of alpha-alpha' dipyridyl, is not deposited in the extracellular matrix and is directly secreted as 64 K collagen in the culture medium.
2,2'-Dipyridyl↗
Cytoskeleton and adhesion patterns of cultured chick embryo chondrocytes during cell spreading and Rous sarcoma virus transformation.
The cytoskeleton and the adhesion complex of chick embryo chondrocytes maintained in vitro have been studied by fluorescence and interference reflection microscopy during the process of cell spreading. The pattern of actin-containing microfilaments and the distribution of vinculin speckles on adhesion plaques have been found to change as a function of the culture time. Newly plated chondrocytes adhere to the substratum mostly around a peripheral ring-like region and show a complex tridimensional array of microfilaments. When chondrocytes flatten, they develop stress fibres and show a diffuse system of vinculin-containing adhesion plaques scattered over the entire ventral side of the cells. Upon infection with Rous sarcoma virus (RSV) chondrocytes display one or more actin-containing ruffles located on the dorsal side similar to the 'actin flowers' earlier described in other cell types. These structures have been found to accumulate vinculin too. In chondrocytes infected with two td-ts mutants of RSV, 'actin flowers' have been found to persist at the restrictive temperature. At this temperature, however, in the majority of cells, stress fibres and adhesion plaques reappear.
Actinin↗
Location of 64K collagen producer chondrocytes in developing chicken embryo tibiae.
The synthesis of a new low-molecular-weight collagen by cultured chicken embryo chondrocytes has been recently demonstrated (Capasso et al., Exp. Cell Res. 142:197-206, 1982; Gibson et al., J. Cell Biol. 93:767-774, 1982; Schmid and Conrad, J. Biol. Chem. 257:12444-12450, 1982). In this paper we report results on the location of chondrocytes synthesizing this new collagen (64K collagen) in the developing chicken embryo. The 64K collagen is synthesized in very large amounts by cells concentrated at the diaphysis of 9-day-old and at the epiphysis of 17-day-old embryo tibiae. These regions are characterized by a remodeling of the cartilage matrix leading to the replacement of the cartilage with bone tissue; therefore, this collagen appears to be a marker of a specific developmental stage of chondrocytes. The origin of cells competent for the synthesis of the 64K collagen is also discussed.
Animals↗