Efficient derivation of osteoprogenitor cells from induced pluripotent stem cells for bone regeneration

There has been great interest in the use of induced pluripotent stem cells (iPSCs) in bone regenerative strategies. To generate osteoprogenitor cells from iPSCs, the most widely used protocol relies on an intermediate using embryoid body (EB) formation. We hypothesized that an osteoprogenitor cell population could be efficiently generated from iPSCs by employing a "direct-plating method" without the EB formation step. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent homogeneous fibroblast-like cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). Expression levels of Oct-3/4 mRNA were analysed by real-time PCR. DPCs were evaluated for cell-surface protein expression using flow cytometry. After osteogenic induction, osteogenic differentiation ability of DPCs was evaluated. The expression level of Oct-3/4 in DPCs was significantly down-regulated compared to that observed in iPSCs, suggesting that the cells lost pluripotency. Flow cytometry analysis revealed that DPCs exhibited cell-surface antigens similar to those of bone marrow stromal cells. Furthermore, the cells proved to have a high osteogenic differentiation capacity, which was confirmed by the significant increase in alkaline phosphatase activity, the expression levels of osteogenic genes, and calcium mineralization after 14-day osteogenic induction. These findings indicate that our novel direct-plating method provides a clinically applicable, simple, and labour-efficient system for generating large numbers of homogeneous iPSC-derived osteoprogenitor cells for bone regeneration.