Phenotypic differences in subclones and long‐term cultures of clonally derived rat bone cell lines

Abstract

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.

In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo‐like expression

“…We hypothesised that any differences in the nature of differentiation produced by various osteogenic media would be reflected in the gene expression profiles of the cells. We confirmed the differentiation of cells grown in each osteogenic medium by staining for the formation of a collagenous ECM and mineralised bone nodules, generally used as phenotypic markers of mature osteoblasts (Ecarot‐Charrier et al, 1983; Bellows et al, 1986; Owen et al, 1990). While both osteogenic media stimulated the formation of a mineralised ECM, in general, the gene expression profiles induced by the two media were different and varied depending on the stage of morphogenesis of the tissue from which the cells originated (Table 2).…”

β1A Integrin Is a Master Regulator of Invadosome Organization and Function

“…A population of primary mouse osteoblast-enriched cells was isolated from newborn mouse calvaria by using a mixture of 0.3 mg/ml collagenase type I (Sigma-Aldrich) and 0.25% trypsin (Invitrogen), as described previously (Bellows et al , 1986; Bouvard et al , 2007). Cells were grown in α-minimal essential medium containing 10% fetal calf serum (FCS).…”

Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro

“…To this end, this study sought to modulate Mustn1 expression in the pre-chondrogenic RCJ cell line. The RCJ line represents a heterogeneous cell population capable of differentiation from proliferating chondrocytes to ultimately terminally differentiated collagen X producing hypertrophic chondrocytes [5]. Also, since Mustn1 is highly expressed in the early stages of fracture repair and this process is known to recapitulate embryonic development [2,6,7,8], we decided to further investigate Mustn1 expression during embryogenesis.…”