Recapitulation of pro-inflammatory signature of monocytes with ACVR1 mutation using FOP patient-derived iPSCs.

Abstract Background Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive heterotopic ossification (HO) in soft tissues due to a heterozygous mutation of the ACVR1A/ALK2 gene (FOP-ACVR1A), which erroneously transduces the BMP signal by Activin-A. Although inflammation is known to trigger HO in FOP, the role of FOP-ACVR1A on inflammatory cells remains to be elucidated. Results We generated immortalized monocytic cell lines from FOP-iPSCs (FOP-ML) and mutation rescued iPSCs (resFOP-ML). Cell morphology was evaluated during the monocyte induction and after immortalization. Fluorescence-activated cell sorting (FACS) was performed to evaluate the cell surface markers, CD14 and CD16, on MLs. MLs were stimulated by lipopolysaccharide (LPS) or Activin-A and the gene expression was evaluated by quantitative PCR (qPCR) and microarray analysis. Histological analysis was performed for HO tissue obtained from wild type mice and FOP-ACVR1 mice which conditionally express human mutant ACVR1 gene by doxycycline (Dox) administration.Without any stimulation, FOP-ML showed the pro-inflammatory signature of CD16+ monocytes with upregulation of INHBA gene, and treatment of resFOP-ML with Activin-A induced the expression profile mimicking those of FOP-ML at baseline. Treatment of FOP-ML with Activin-A further induced the inflammatory profile with up-regulation of inflammation-associated genes, some, but not all, of which were suppressed by corticosteroid. Experiments using an inhibitor for TGFβ or BMP signal demonstrated that Activin-A-induced genes such as CD16 and CCL7 were regulated by both signals, indicating Activin-A transduced dual signals in FOP-ML. Comparison with resFOP-ML identified several down-regulated genes in FOP-ML including LYVE-1 , which is known to suppress matrix-formation in vivo. Down-regulation of LYVE-1 in HO tissues was confirmed in FOP model mice, verifying the significance of in vitro experiments. Conclusion These results indicate that FOP-ML faithfully recapitulated the phenotype of primary monocytes of FOP and the combination with resFOP-ML is a useful tool to investigate molecular events at the initial inflammation stage of HO in FOP.