Finally we cultured hiPSCs according to the present strategy

Finally, we cultured hiPSCs according to the present strategy with some modifications (Figure 4A; Supplemental Experimental Procedures). hiPSCs formed colonies on day 0. In the mesoderm induction phase, these colonies were disrupted, but the neurokinin receptor antagonist generated some clusters upon CHIR and Cyc treatment. The remaining cells proliferated and formed colonies by days 19 and 23 of the osteoblast induction and maturation phases (Figure 4A). Gene expression patterns in the cultured hiPSCs are shown in Figure 4B. NANOG was downregulated during induction. T and MIXL1 were transiently upregulated on day 5. RUNX2 and SP7 were also upregulated on day 5; their expression levels were maintained and statistically significant on day 23 compared with day 0. IBSP and COL1A1 were significantly upregulated on days 19 and 23, respectively. The contribution of Hh signaling to osteoblast-related gene expression might cause differences in the gene expression pattern between human and mouse cells, as previously reported by Plaisant et al. (2009). On day 23, RUNX2 protein was evenly expressed in cell clusters, and SP7 was expressed in the periphery, where RUNX2 and SP7 were colocalized (Figure 4C). Calcification on days 19 and 23 was significantly higher than that on day 5 (Figures 4D and 4E). These results suggest that the present strategy is capable of differentiating hiPSCs into osteoblasts. This study demonstrates the robust generation of osteoblasts from pluripotent stem cells using four small molecules under serum-free and feeder-free conditions. This strategy requires no or very little use of confounding factors derived from serum and feeder cells. Such a simple, small molecule-based system offers significant benefits for skeletal research and regenerative medicine by minimizing costs and maintaining the stability of the inducers. However, this study has two major limitations. First, the target molecules of TH are unclear; we are currently screening for these targets using a proteomic approach with a modified TH carrying a moiety that binds to magnetic beads via an amide bond. Second, we used Matrigel, which was not a fully defined reagent (Hughes et al., 2010), for hiPSCs to maintain cell viability. Because we observed a substantial level of hiPSC death on the Matrigel-coated plates during the initial 5-day induction, this strategy might be deleterious for the survival and differentiation of hiPSCs. In future studies, there will be a need for defined reagents suppressing the cell death of hiPSCs as well as reagents that will eliminate residual pluripotent cells (Ben-David and Benvenisty, 2011; Tang et al., 2011); the 2.3 kb Col1a1-GFP may be used to sort out osteoblasts. In conclusion, the present stepwise differentiation strategy offers a tool for in vitro mechanistic studies of osteoblast development and stem cell-based therapies for massive bone defects, although the strategy will need to be further optimized to enable broader application.
Experimental Procedures
Acknowledgments We thank Dr. M.J. Owen for his kind gift of the Runx2 knockout ESCs and Dr. Andrew P. McMahon for his helpful input. This work was supported by Grants-in-Aid for Scientific Research (#23689079), the Center for Medical System Innovation, the Graduate Program for Leaders in Life Innovation, Core-to-Core Program A (Advanced Research Networks), the Funding Program for World-Leading Innovative R&D on Science and Technology, the Center for NanoBio Integration, the S-innovation program, Nakayama Foundation for Human Science and the Nakatomi Foundation Research Grant.
Introduction Stem cell niches are specialized local microenvironments that are able to house and maintain stem cells (Fuchs et al., 2004; Morrison and Spradling, 2008). Previous studies have shown that stem cell niches are composed of supporting cells and their associated extracellular matrix (ECM) (Chen et al., 2013; Jones and Wagers, 2008; Lander et al., 2012). Supporting cells can regulate stem cells by secreting diffusible factors or through adheren junctions (Chen et al., 2013; Jones and Wagers, 2008; Xie and Spradling, 2000). However, roles of ECM in niches are less understood.