Enhanced Osteogenesis of iPS Cells by Id2 Gene Knockout

Induced pluripotent stem (iPS) cells are expected to be useful for alveolar bone augmentation/regeneration in dental implant and prosthodontic treatments; however, the mechanisms of iPS cell osteogenesis remain unclear.  Id2 is an inhibitor of DNA binding/differentiation factor that regulates the expression of several tissue-specific genes.  We hypothesized that iPS cells generated from Id2-knockout mice could be a powerful tool to investigate novel functions of Id2 in iPS cell osteogenesis.  Objective: To investigate the role of Id2 in iPS cell osteogenesis by establishing Id2-deficient model iPS cells.  Methods: The induction of pluripotent stem cells from gingival fibroblasts of Id2-knockout (Id2^-/--iPS cells) or wild-type mice (WT-iPS cells) was performed by retroviral transduction of the Yamanaka Factors.  The pluripotency of the generated cell clones was analyzed by alkaline phosphatase (ALP) staining, RT-PCR analysis for embryonic stem (ES) cell markers and the teratoma assay.  Embryoid bodies were generated from Id2^-/-- and WT-iPS cells and maintained for 28 days in osteogenic induction medium.  The expression of osteogenic marker genes and the mineralized nodule formation of the iPS cells were evaluated by real-time RT-PCR analysis and von Kossa staining, respectively.  Results: The Id2^-/--iPS cells expressed ES cell marker genes (Nanog, Oct3/4, ERas) and ALP activity at levels comparable to those in WT-iPS cells.  Id2^-/--iPS cells exhibited a significantly higher generation rate than WT-iPS cells (Student’s t-test; P<0.01).  Id2^-/--iPS cells produced teratomas containing a various tissues from all three germ layers.  Osteogenically induced Id2^-/--iPS cells formed robust mineralized nodules and showed significant up-regulation of collagen1a2, osterix, bone sialoprotein and osteocalcin genes compared with WT-iPS cells (ANOVA; P<0.01).  Conclusion: We successfully established an osteogenesis model with Id2-deficient iPS cells, which demonstrated enhanced osteogenic differentiation and represent an important step toward the further analysis of novel mechanisms underlying iPS cell osteogenesis.