In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

Abstract

Abstract. Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of ,,o13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while ,x,50% of them showed positivity for type II collagen. (6,19,24). Chondrocytes from Hamburger and Hamilton stage 28-30 (17) chick embryo tibiae dedifferentiate when cultured on plastic dishes. These cells, as well as dedifferentiated chondrocytes from another source (4), maintain the dedifferentiated phenotype as long as the conditions for the cell attachment to the plastic are kept. When the same cells are transferred into suspension culture, they resume the chondrocytic phenotype and mature to hypertrophic chondrocytes through a transient stage of cell aggregation (7). This process is accompanied by an increase in the length of the cell cycle and in the number of quiescent and degenerating cells (16), as well as by a modulation in the transcriptional activity of the collagen genes (9). We have recently reported the identification, purification, and characterization of a novel, low molecular weight protein, named Ch21, expressed and secreted by the in vitro differentiating chondrocytes at a late stage of development (11). When dedifferentiated cells are cultured in suspension in the constant presence of ascorbic acid, they organize their own extracellular matrix and develop into a tissue closely resembling calcifying hypertrophic cartilage (28,29).The heterogeneity of the dedifferentiated cell population did not allow us to definitely state whether the cells followed successive stages of differentiation or the culture conditions made a selection of cell subpopulations. To answer this important question we selected clones of dedifferentiated cells to follow the respective behavior of each clone in terms of morphology and collagen phenotype in the different culture conditions. We show that up to 45 % of the cell populations obtained from single cloned dedifferentiated cells are still able to differentiate to hypertrophic chondrocytes and to reconstitute hyp...

Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines

“…Repeated passaging of the cells did not merely attenuate their ability to form cartilage, since the time course of nodule formation was the same as in cultures of freshly thawed cells, and no new nodules formed even when the cultures were maintained for up to 36 days. Indeed, the expression of the cartilage phenotype by chondrogenic cell populations can be modulated either by direct cell-cell interactions or via soluble mediators secreted by different subpopulations [38]. 5 Phase-contrast (a) and fluorescence (b) micrographs of a representative RCJ 3.1C5 subclone, C5.25, cultured for 16 days in the presence of 10 −7 M Dex.…”

Co-expression of collagen types II and X mRNAs in newly formed hypertrophic chondrocytes of the embryonic chick vertebral body demonstrated by double-fluorescence in situ hybridization

“…These transitional cells, or what we call newly formed hypertrophic chondrocytes, seem to regulate switching from collagen type II to type X expression. Double-fluorescence immunostaining of cultured chondrocytes has demonstrated that developing hypertrophic chondrocytes stain for both type II and X collagens (Solursh et al, 1986;Quarto et al, 1990). It is expected, therefore, that these newly formed hypertrophic chondrocytes may be simultaneously expressing both type II and X collagen genes.…”

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

“…Although many types of mineralizing chondrocyte cultures have been described, these cultures use either growth plate chondrocytes, embryonic chondrocytes, or embryonic growth plate chondrocytes and not articular chondrocytes. It doesn't seem appropriate to extrapolate, to calcified cartilage, observations generated from these types of mineralizing cultures as these cultures use ceils that generate cartilage which serves as a template for bone formation (3,4,9,10,16,22,25,32,36), whereas the calcified zone of articular cartilage, in mature individuals after growth has ceased, appears to be hyaline cartilage which has undergone mineralization. Furthermore, during the process of mineralization, in vivo, those cell types synthesize an extracellular matrix that will be permeated by vascular ~To whom correspondence should be addressed at Department of Pathology, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada.…”