Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation mineralization potential

A series of subclonal cell lines with high or low differentiation/mineraliz ation potential after growth in the presence of ascorbic acid (AA) were der ived from murine MC3T3-E1 cells. Subclones were characterized in terms of t heir ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes, When compared with nonmi neralizing cells, mineralizing subclones selectively expressed mRNAs for th e osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the p arathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) recept or, In contrast, alkaline phosphatase mRNA was present in certain nonminera lizing as well as mineralizing subclones, suggesting that its expression ma y be subject to different controls from other osteoblast markers, Only high ly differentiating subclones exhibited strong AA-dependent induction of a t ransiently transfected OCN promoter-luciferase reporter gene, indicating th at there was a good correlation between mRNA levels and transcriptional act ivity. Consistent with its postulated role in biomineralization, BSP as mea sured by Western blotting was only present in mineralizing subclones, After implantation into immunodeficient mice, highly differentiating subclones f ormed bone-like ossicles resembling woven bone, while poorly differentiatin g cells only produced fibrous tissue. Interestingly, subclones with both hi gh and low differentiation potential produced similar amounts of collagen i n culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1 , an osteoblast-related transcription factor. Although some strongly differ entiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and i nduction of mRNAs for other differentiation markers. Thus, the mere presenc e of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentia tion. These subclones will be very useful for studying critical events in o steoblast differentiation and mineralization.