Background Mesenchymal stem cell derived from bone marrow have already been used for bone regeneration. A number of studies have confirmed that MSCs cultured in scaffolds can induce osteogenesis in vivo and improve healing of critical-size defects.
Objectives In the present study, we generated mesodermal cell lineages from hiPSCs following treatment of embryoid bodies (EBs) with retinoic acid. Subsequently, we induced osteogenic differentiation from EBs using osteogenic medium and also test the in vivo repair of osseous defect using these osteo-induced cells. To avoid the potential safety issues associated with using viruses, we used the hiPSC line by direct delivery of four proteins fused to a cell penetrating peptide in present study.
Methods Cell culture and differentiation of EB – We used the hiPSC line (SBI, cat# SC802A-1) generated by direct delivery of four proteins fused to a cell penetrating peptide.
Osteogenic differentiation was induced by trypsinizing EB to a single cell suspension, diluting cells to a final concentration of 5x105 cells/ml and being cultured on gelatin-coated plates for 7 and 14 days.
Analysis for osteogenic differentiation – After 7 and 14 days of in vitro culture, osteogenic differentiation was demonstated by Alizarin red S staining, alkaline phosphatase (ALP) staining, and the expression of osteogenic markers.
Transplantation – The fibrin-hiPSCs and HA+β-TCP-hiPSCs constructs were implanted in the calvarial defect (f 4mm) in immunosuppressed rat. Also, these hiPSCs in fibrin were implanted in the segmental long bone defects created in the ulna of immunosuppressed of rat.
Results After 7 and 14 days of osteogenic culture, alizarin red S staining and ALP staining identified calcium in these cells and osteogenic differentiation. The osteogenic marker gene and protein expression including COL1A1, Runx2, and bone sialoprotein (BSP) increased after 7 days and expression level of gene and protein after 14 days was higher than 7 days.
When hiPS cells in fibrin and HA+β-TCP were implanted in calvarial defects created in the parietal bone of immunosuppressed rats, the defects implanted with osteo-induced hiPS cells showed a significantly better quality of bone repair than the control defects. Goldner's trichrome staining confirmed that bone regeneration in those treated with hiPSCs-fibrin and HA+β-TCP. Immunofluorescent imaging showed that human nucleus antigens were observed in the cytoplasm of implanted hiPSC-Osteoblasts.
In addition, hiPSC-Osteoblasts or hBMMSC-Osteoblasts in fibrin were implanted in the 4mm segmental defects created in the ulna of immunosuppressed rats. The defects with osteo-induced hiPS cell implantation achieved rapid bone healing compared with the control defects.
Conclusions The cell culture protocol presented herein affords high yield of pure populations of hiPSC-EBs with high osteogenic capability. The osteoblasts derived from these hiPSC-EBs were able to form new mineralized and vascularized bone matrix in vivo. Once the functionality of these derived cells will be thoroughly examined in a bone repair model, the potential of the described method as a source of osteoblasts for bone tissue regenerating can be established.
Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005 May;36(5):758-69.
Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012M3A9B4028566 & 2013R1A1A2062961).
Disclosure of Interest None declared