Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

临床相关的人类诱导性多能干细胞（hiPSC）衍生物需要有效的方案，才能将hiPSC区分为特定谱系。在这里，我们开发了一种完全定义的无异源策略，可在21天内将hiPSC导向成骨细胞。该策略通过顺序使用小分子诱导剂的组合成功实现了四个独立衍生的hiPSC系的成骨诱导。诱导首先产生中胚层细胞，其随后概括了主要成骨细胞基因和蛋白质的发育表达模式。重要的是，接受此策略的Col2.3-Cherry hiPSC强烈表达了在体内成骨成骨细胞中观察到的樱桃荧光。。此外，该协议与三维（3D）支架相结合适用于无异种3D成骨系统的生成。因此，我们的策略为骨骼生物学研究提供了一个具有明显优势的平台，也将有助于hiPSC在骨骼再生医学中的临床应用。