Kanke, K., Masaki, H., Saito, T., Komiyama, Y., Hojo, H., Nakauchi, H., ... & Ohba, S. (2014). Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions. Stem Cell Reports, 2(6), 751-760.
DOI: 10.1016/j.stemcr.2014.04.016. 

In this study, an effective and mass-producing strategy is reported for the stepwise differentiation of mouse embryonic stem cells and mouse and human induced pluripotent stem cells into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. 

For the differentiation of mESCs into Osteoblasts, different components are supplemented to 2i-medium as specified in the Experimental Procedures. 2i-medium is a proprietary product.