Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo

Abstract

Osteocytes are the most abundant cells in bone yet are the most challenging to study as they are embedded in a mineralized matrix. We generated a clonal cell line called IDG-SW3 (for Immortomouse/Dmp1-GFP-SW3) from long bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T-antigen regulated by IFN-γ. Cells from these mice can be expanded at 33°C in the presence of IFN-γ and then allowed to resume their original phenotype at 37°C in the absence of IFN-γ. IDG-SW3 cells are Dmp1-GFP-negative and T-antigen-positive under immortalizing conditions but Dmp1-GFP-positive and T-antigen-negative under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type I collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and FGF23, regulated by PTH and 1,25-dihydroxyvitamin-D3. When cultured on 3D matrices, they express Dmp1-GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast-to-osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF23.

Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

“…There are many signalling molecules secreted by osteocytes in response to mechanical stimulation, including NO, ATP, PGE2, SOST, DKK1, IGF-1, SDF-1, and FGF23, all of which have been linked to bone anabolism [11]. Interestingly, two key osteocyte secreted factors (sclerostin and FGF23) are not produced by MLO-Y4s, indicating they these are not contributing to our findings [31]. Osteoblasts produce a wealth of soluble factors that play diverse roles in bone growth and repair [32,33].…”

Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice

“…The mouse osteoblastic-late-osteocytic cell line IDG-SW3 was kindly provided by Prof Linda Bonewald and cultured as previously described (49) in MEM containing L-glutamine supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. 50 U/ml of recombinant Mouse Interferon-gamma (INF-γ) (Invitrogen) was added to induce expression of the SV40 large tumor antigen and maintain proliferation of this cell line.…”

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

“…However, investigation of osteocyte biology has been suffering from the limited availability of in vitro models to such an extent that osteoblasts have been used as surrogate cells over a long period before the osteocyte-like cell line MLO-Y4 was made available. More recently, a conditionally immortalized cell line was introduced by the same group 16 , which can be differentiated to ramified late osteocytes expressing sclerostin and FGF23. Though the extreme value of these cell lines is not under question, it is very well known that primary cells resemble more the in vivo situation and offer the possibility to obtain primary osteocytes from transgenic animals to perform functional studies on specific molecular pathways.…”