Lastly we demonstrated that the mutant

Lastly, we demonstrated that the mutant ALK2 R206H contributed to the increased mineralization of FOP hiPSC-pericytes and, as such, is a useful human in vitro disease model for identifying and evaluating the bioactivity of ALK2 inhibitors. More evidence indicates that MCs are the major contributors of HO (Wosczyna et al., 2012), while ECs indirectly contribute to osteogenic differentiation by acting in a paracrine manner via crosstalk between ECs and MCs (Bidarra et al., 2011; Lin et al., 2014). Even though Matsumoto et al. partly exhibited FOP phenotypes by directly differentiating hiPSCs into osteoblast third (Matsumoto et al., 2013), they provided little evidence of how the specific cell types contributed to the increased mineralization. To further clarify the mineralization capacity of FOP hiPSCs in vitro, we differentiated hiPSCs into pericytes. ALP activity of FOP hiPSC-pericytes can be inhibited by pretreating with BMP inhibitor LDN-212854. As the ALP assay has been used as a high-throughput screening (HTS) readout for screening regulators in osteogenic differentiation (Alves et al., 2011), our platform could be used for drug screening and further verifying the bioactivity of ALK2 inhibitors in the future.
Experimental Procedures Primary human cells were obtained with informed consent. Experiments involving human subjects were approved by Institutional Review Board (IRB) GIBH-IRB02-2009002 at Guangzhou Institutes of Biomedicine and Health (GIBH) and 12/467 (2013 January) at VU University Medical Center. The animal research was approved by the IRB at GIBH (2010012). The generation and differentiation of hiPSCs were described previously (Orlova et al., 2014; Xue et al., 2013). Differences between the control group and the FOP group were evaluated by t test or one-way ANOVA with Tukey’s multiple comparison tests (ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). For further information, see the Supplemental Experimental Procedures.
Author Contributions
Acknowledgments
Introduction Mouse embryonic stem cells (ESCs) that lack microRNAs (miRNAs) due to Dicer1 or Dgcr8 deficiency do not proliferate well and display severe differentiation defects (Kanellopoulou et al., 2005; Murchison et al., 2005; Wang et al., 2008). The most highly expressed miRNAs in mouse ESCs belong to the miR-290 family, a cluster of nine miRNAs (also referred to as miR-290∼295), six of which share the same “seed” sequence (Houbaviy et al., 2003). The orthologous human families are miR-302 and miR-371 (Suh et al., 2004). In mice, the miR-290 cluster is transcribed from a single locus on chromosome 7 by the core ESC transcriptional network (Marson et al., 2008) and can rescue defective proliferation in ESCs that lack miRNAs (Wang et al., 2008). While the importance of the miR-290 family is clear, how it contributes to the gene expression program in ESCs is not fully known. The Hox family of transcription factors governs the anterior to posterior axial body plan of vertebrates (Pearson et al., 2005). In mouse and human, the Hox genes are found in four chromosomal clusters (A, B, C, and D). Hox genes are transcriptionally inactive in ESCs due to the action of Polycomb repressive complexes (PRC) (Bracken et al., 2006; Lee et al., 2006), but the role, if any, of miRNAs in this process has not been established. Polycomb group (PcG) proteins are transcriptional repressors that regulate embryonic development and function in ESC pluripotency and induced pluripotent stem cell (iPSC) generation (Bernstein et al., 2006; Boyer et al., 2006; Onder et al., 2012). There are two Polycomb complexes, PRC1 and PRC2, that differ biochemically (Di Croce and Helin, 2013). PRC2 catalyzes the trimethylation of histone H3K27 (H3K27me3), which is recognized by PRC1, although PRC1 can be recruited to chromatin independently of PRC2 and H3K27me3 (Schwartz and Pirrotta, 2014). Overall, despite extensive study, it remains unclear how Polycomb repressive chromatin domains are established in ESCs and reversed during development to allow the expression of differentiation genes.