BONE TISSUE SUBSTITUTES ENGINEERED FROM HUMAN PLURIPOTENT STEM CELLS

Summary Statement A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo . Methods hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global gene expression profiles were evaluated prior and after bioreactor culture. Bone development was investigated using biochemical and histological methods, and by micro-computed tomography (μCT) imaging over the duration of bioreactor culture and after 12-week subcutaneous implantation in immunodeficient mice. Results Progenitors with high proliferation potential, expressing typical mesenchymal surface antigens were successfully derived from three hiPSC lines. Differences in mesenchymal surface antigens expression and global gene expression profiles of progenitors from different lines corresponded to their differentiation abilities toward the osteogenic, chondrogenic and adipogenic lineages. Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into the culture medium as compared to static culture. Dense bone matrix formation was evidenced by the positive staining of collagen, osteopontin, bone sialoprotein and osteocalcin. In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce. μCT revealed a significant increase in bone structural parameters, evidencing mineralization of the deposited bone tissue during the 5-week culture in bioreactors. Osteogenesis and bone tissue formation were comparable between hESCs, hiPSCs and hMSCs (3). Bioreactor cultivation resulted in repression of genes involved in proliferation and tumorigenesis, and upregulation of genes associated with osteogenesis and bone development. Engineered bone tissue displayed stable phenotype after 12-week implantation in vivo, with cells of human origin, ingrowing vasculature and osteoclasts, suggesting an initiation of tissue remodeling. Discussion/Conclusion Our biomimetic strategy opens the possibility to construct an unlimited quantity of patient-specific bone grafts for personalised applications, and to generate qualified experimental models to study bone biology under normal and pathological conditions, as well as test new drugs using selected pools of hiPSC lines.