Deferoxamine Induces Self-Organized Osteo-Chondrogenic Differentiation of Induced Pluripotent Stem Cells

Objectives: Induced pluripotent stem cell (iPSC)-derived bioengineered bone/cartilage complex would have great clinical potential for bone augmentation in dental implant/prosthodontic treatment. Hypoxia plays important roles in osteogenesis and chondrogenesis, and deferoxamine (DFX) treatment mimics hypoxia in cell culture. We hypothesized that DFX would facilitate self-organized differentiation of iPSCs into 3-dimensional bone/cartilage-like cell constructs in vitro, which would be a good graft for bone regeneration. Objectives: To investigate the effects of DFX on self-organized differentiation of iPSCs into bone and cartilage tissues, and to evaluate the bone regeneration of these tissues.
Methods: iPSC constructs were fabricated using a micro-space culture plate (Kuraray, Elplasia) and cultured in osteogenic medium with and without 10 μM DFX for 35 days. Osteogenic and chondrogenic gene and protein expression was evaluated by RT-PCR and immunohistochemistry. Subsequently, the cell constructs were transplanted into critical-size femoral bone defects in cyclosporin-immunosuppressed Sprague-Dawley rats and extracted at 3 weeks for histological and micro CT analyses.
Results: DFX significantly upregulated osteogenic [Osterix, Type I collagen and Bone sialoprotein] and chondrogenic [Sox9, Type II collagen, Type X collagen (Col10a1), Osteopontin (Opn) and Matrix metalloprotease-13 (MMP-13)] genes in iPSC constructs within 14 days (ANOVA; P<0.01). After 35 days of induction, DFX-treated iPSC constructs showed an osteo-chondrogenic phenotype with partial calcification and high Opn, Col10a1 and MMP-13 protein expression. In vivo, cortical bone formation was significantly increased in the DFX-treated group (Mann-Whitney U test; P<0.05), in which significantly more blood vessels and TRAP-positive osteoclasts were observed around the Opn- and MMP-13-positive areas of the transplants (Mann-Whitney U test; P<0.05).
Conclusions: DFX induces self-organized differentiation of iPSCs into osteo-chondrogenic constructs, which facilitates efficient bone regeneration in vivo via accelerated angiogenesis and enhancement of osteoclast function by Opn and MMP-13. These findings represent an important step toward the clinical translation of iPSCs for regenerative prosthodontics.