Abstract
Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.
Highlights
As it is the case of other primary cells, primary osteoblasts undergo a finite number of cell divisions in culture before they enter a state, known as replicative senescence [1], where they can no longer divide
After isolation and expansion of the primary cells, they were transduced with four different lentiviral vectors encoding for (i) SV40 large T antigen (TAg), (ii) cmyc, (iii) E7 and (iv) inhibitor of DNA binding 2 (Id2), a dominant negative helixloop-helix protein
Osteoporosis is a disorder characterized by a low bone mass
Summary
As it is the case of other primary cells, primary osteoblasts undergo a finite number of cell divisions in culture before they enter a state, known as replicative senescence [1], where they can no longer divide. Primary human bone cells often show alterations of their phenotypic characteristics with increasing passage number Other model systems, such as osteoblastic cell lines of rodent origin [3,4] or human osteosarcoma cell lines [5], have been used to try to avoid this problem. Regarding the osteoblastic lines of rodent origin, their limitations come from the existence of species-specific characteristics To overcome these problems, researches have developed human primary osteoblasic cell lines with extended replicative capacity [6,7]. In order to become useful in experimental models, theses cells should be able to increase the number of cell divisions that can undergo in culture, but they require the genotypic and phenotypic characteristics of the cell line to be preserved throughout the expansion process
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