br Results and Discussion br Experimental

Results and Discussion
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant genetic disorder in which acute inflammation may trigger the formation of a second skeleton of heterotopic bone. Classic FOP is caused by gain-of-function mutation (617G > A; R206H) in the activin receptor-like kinase 2 (ACVR1/ALK2) gene, encoding the bone morphogenetic protein (BMP) type I receptor (Shore et al., 2006). Enhanced BMP signaling in patients with the ALK2 R206H mutation has been attributed to loss of inhibitory activity of the ALK2-interacting protein FK506-binding protein-12 (FKBP12) (Chaikuad et al., 2012; van Dinther et al., 2010). Previous publications indicated that Tie2+ endothelial EPZ-6438 (ECs) and mesenchymal cells (MCs) contributed as progenitor cells to the episodic heterotopic ossification (HO) in FOP (Medici et al., 2010; Wosczyna et al., 2012). Other cells like circulating osteogenic precursors, skeletal myoblasts, and vascular smooth muscle cells also were found in FOP lesions and may contribute to HO in FOP (Hegyi et al., 2003; Lounev et al., 2009; Suda et al., 2009). Despite recent advances in understanding of the disease (Hatsell et al., 2015), to date there is no cure or even treatment for HO in FOP. A comprehensive understanding of the molecular mechanisms underlying abnormal behavior of bone-forming progenitor cells in FOP could be one approach toward effective treatment for HO in FOP, and to other more prevalent situations with HO that, for example, may occur after traumatic accidents or deep tissue burns. The traditional way of obtaining human biopsy tissues from FOP patients is limited as physical and surgical injury can induce HO. New protocols to produce well-characterized FOP bone-forming progenitor cells for research and therapeutic drug screening are needed. The ability to generate human induced pluripotent stem cells (hiPSCs) (Takahashi et al., 2007) from adult tissues provides new opportunities for research on FOP. If derived from patients with genetic disease, hiPSCs allow production of large numbers of diseased target cells for basic research and drug development since they are immortal and pluripotent (Sterneckert et al., 2014).
Results
Discussion We have shown that FOP hiPSCs could be established from cells in urine using non-integrating episomal vectors. BMP signaling contributes to the initial stage of iPSC reprogramming in mouse (Samavarchi-Tehrani et al., 2010), but induces differentiation of hESCs (Xu et al., 2002). Consistent with prior research (Matsumoto et al., 2013), our FOP hiPSCs were generated without the addition of exogenous BMP inhibitors during the reprogramming process. We did not observe activated SMAD1/5 signaling in FOP hiPSCs maintained in hESC medium mTeSR1 unless these cells were placed in differentiation culture conditions. This may explain why FOP hiPSCs could be maintained and passaged in defined medium. Besides, we observed heterogeneous BMP/SMAD signaling and mineralization in FOP hiPSCs and derivative cells, indicating that intrinsic genetic and/or epigenetic features of donor cells may influence properties of hiPSCs and the progeny. To eliminate the heterogeneity, the isogenic correction FOP hiPSCs can help to facilitate more stringent screening for effects arising from clonal variations in hiPSCs (Matsumoto et al., 2015). We found that the generation and maintenance of ECs from FOP hiPSCs were impaired. One explanation is the elevated BMP signaling in FOP hiPSC-ECs, which resulted in downregulation of VEGFR2 expression that mediated VEGF-induced proliferation and survival of ECs; this may have caused the decrease in EC viability. Inhibition of BMP receptor kinase activity by LDN-212854 during the vascular specification stage (from day 3 of hiPSC differentiation) did not rescue the impaired FOP hiPSC-EC phenotypes (unpublished data). BMPs are indispensable for the formation of mesoderm where ECs originate, but they may function as a context-dependent regulator in vascular morphogenesis (Kim et al., 2014). In addition, a recent publication indicated activin A signals through the mutant ALK2 R206H to stimulate HO in FOP conditional-on knockin mice (Hatsell et al., 2015). Of note, in our EC differentiation protocol, we used activin A and BMP4, both of which were shown to induce SMAD1/5 signaling through mutant ALK2 R206H (Hatsell et al., 2015). These ligands may thus combine with the SMAD1/5 signal to contribute to the FOP hiPSC-EC phenotypes that we observed. The other explanation of low EC yields is the increased EndoMT in ALK2-mutated ECs. Consistent with a previous publication (Medici et al., 2010), FOP EC-MCs showed higher expression of EndMT markers (N-cadherin and TWIST), which might be due to the interaction of mutant ALK2 R206H and VEGF signaling in these cells. Further investigation of the crosstalk between BMP signaling and VEGF signaling might contribute to the better understanding of the EndMT mechanism in FOP lesions and also help in identifying new drug-treatable targets to prevent HO in FOP patients.