New Protocol to Optimize iPS Cells for Genome Analysis of Fibrodysplasia Ossificans Progressiva

Abstract

Successful in vitro disease-recapitulation using patient-specific induced pluripotent stem cells (iPSCs) requires two fundamental technical issues: appropriate control cells and robust differentiation protocols. To investigate fibrodysplasia ossificans progressiva (FOP), a rare genetic disease leading to extraskeletal bone formation through endochondral ossification, gene-corrected (rescued) iPSC clones (resFOP-iPSC) were generated from patient-derived iPSC (FOP-iPSC) as genetically matched controls, and the stepwise induction method of mesenchymal stromal cells (iMSCs) through neural crest cell (NCC) lineage was used to recapitulate the disease phenotype. FOP-iMSCs possessing enhanced chondrogenic ability were transcriptionally distinguishable from resFOPiMSCs and activated the SMAD1/5/8 and SMAD2/3 pathways at steady state. Using this method, we identified MMP1 and PAI1 as genes responsible for accelerating the chondrogenesis of FOPiMSCs. These data indicate that iMSCs through NCC lineage are useful for investigating the molecular mechanism of FOP and corresponding drug discovery.

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

“…The induction and maintenance of induced neural crest cells (iNCCs) and iMSCs derived from iPSC were previously described (43). FOP-iPSCs used in this study [FOP-iPSCs from patient 1 and 2, previously described as vFOP4-1 and vFOP5-22 (25), respectively] harbor the R206H heterozygous mutation in ACVR1, and gene-corrected resFOP-iPSCs were generated by BAC-based homologous recombination (26). All experiments shown in Figs.…”

“…All experiments shown in Figs. 1-5 were performed using FOP-iPSCs from patient 1 and resFOP-iPSCs (cl1) (26). and treated with TGF-β superfamily ligands.…”

“…To screen noncanonical BMP ligands that activate BMP signaling through FOP-ACVR1 but not through WT-ACVR1, we focused our attention on FOP-iMSCs from FOP patient-derived iPSCs as test cells and mutation-rescued FOP-iMSCs (resFOP-iMSCs) as genetically matched control cells (26). A BMP-specific luciferase reporter construct (BRELuc) was transfected into both FOP-iMSCs and resFOP-iMSCs, and detection of luminescence was made 16 h after ligand stimulation (Fig.…”

“…To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12)(13)(14)(15)(16)(17)(18)(19)(20), mouse embryonic fibroblasts derived from Alk2 R206H/+ mice (21,22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24,25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2 R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter.…”

“…Among these cells, Alk2 R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22,26). In addition, despite the essential role of BMP signaling in development (27)(28)(29)(30)(31), the pre-and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1)(2)(3)(4)(5)(6).…”