Generation of an induced pluripotent stem cell line (TRNDi012-B) from Fibrodysplasia Ossificans Progressiva (FOP) patient carrying a heterozygous mutation c. 617G > A in the ACVR1 gene

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of progressive ossification of skeletal muscle, fascia, tendons, and ligaments. Most FOP cases are caused by a heterozygous c. 617G > A mutation in the ACVR1 gene which encodes a gain-of-function of bone morphogenetic protein type I receptor. A human induced pluripotent stem cell (iPSC) line was generated from the dermal skin fibroblasts of a FOP patient who carries the c. 617G > A mutation in the ACVR1 gene. This iPSC line provides an attractive resource for FOP disease modeling.


Resource utility
This human induced pluripotent stem cell line is a useful tool for establishing in vitro disease model and for applications in drug development to treat FOP.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Resource details
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by congenital malfunctions of great toes and progressive heterotopic ossification of soft tissues, that results in severe restriction of joint function and premature mortality (Kaplan et al., 2009). The majority of FOP patients carry a heterozygous missense mutation (c.617G > A; p.R206H) in the ACVR1 gene, which encodes activin A type I receptor /activin receptor-like kinase 2 (ACVR1/ALK2), a morphogenetic protein (BMP) type I receptor (Shore et al., 2006). The mutation causes a gain-of-function of activation of ACVR1, leading to the transformation of connective tissue and muscle tissue into a secondary skeleton (Hatsell et al., 2015). FOP has a prevalence of approximately 1 in 2 million people worldwide (Baujat et al., 2017). Currently, no approved treatment exists for FOP patients.
In this study, we generated a human iPSC line (TRNDi012-B, also named as NCATS-CL7989) from patient skin fibroblast (GM00513) which was isolated from a 16-yearold female FOP patient carrying a heterozygous mutation (c.617G > A; p.R206H) in ACVR1 gene. (see Table 1). A non-integrating CytoTune-Sendai viral vector kit (A16517, ThemoFisher Scientific) containing OCT3/4, KLF4, SOX2 and C-MYC pluripotency transcription factors was employed to transduce the patient cells using a previously described method (Beers et al., 2015). A single iPSC colony, termed NCATS-CL7989, was isolated and characterized. Fig. 1A show that the iPS cells displayed a standard pluripotent stem cell morphology under phase contrast microscopy and expressed pluripotent markers SOX2, OCT4, NANOG and SSEA4 in the immunocytochemistry assay. In addition, flow cytometric analysis showed that the expression levels of NANOG, SSEA4 and cell surface marker TRA-1-60 were over 98% (Fig. 1B). The mutation (c.617G > A) of the ACVR1 gene in the NCATS-CL7989 iPSC line was confirmed by Sanger sequencing the PCR product harboring the single nucleotide variation (SNV) (Fig. 1C). At passage 7, cells showed a normal karyotype (46, XX), as confirmed by the G-banded karyotyping (Fig. 1D). The Sendai virus vector (Sev) clearance was verified with reverse transcription polymerase chain reaction (RT-PCR) using Sev-specific primers. The Sev vector disappeared at passage 19 (Fig. 1E). The iPSCs was not contaminated with mycoplasma ( Supplementary Fig. S1), and 16 loci from NCATS-CL7989 iPS cells and GM00513 FOP fibroblasts determined by the short tandem repeat (STR) DNA profile were identical (information available from the authors). Furthermore, the pluripotency of this iPSC line was confirmed by a teratoma formation experiment that exhibited its ability to differentiate into all three germ layers (Ectoderm, Mesoderm and Endoderm) in vivo (Fig. 1F).

Mutation analysis
Genomic mutation analysis was contracted to Codex BioSolutions (Gaithersburg, MD, USA). Briefly, genomic DNA was extracted from the cell pellet of FOP iPSCs with a PureLink™ Genomic DNA Mini kit (K182002, ThermoFisher Scientific). The mutation in the ACVR1 gene was analyzed by a DNA sequencing of PCR amplified product. With the specific primers listed in Table 2 and JumpStart Taq ReadyMix (P2893, Sigma-Aldrich), 40 cycles of PCR were performed with annealing temperature at 56 °C and extension time of 70 s. Sanger sequencing analysis was then employed for genotyping the heterozygous mutation, resulting in confirmation of the mutation of c. 617G > A in the ACVR1 gene.

Immunocytochemistry staining
The iPSCs in a 96-well plate were fixed in 4% paraformaldehyde for 15 min followed by a plate wash with Dulbecco's phosphate-buffered saline (DPBS) and cell permeabilization with 0.1% Triton X-100 in DPBS for 15 min. The Image-iT™ FX Signal Enhancer (I36933, ThermoFisher Scientific) was added to the fixed cells and incubated for 40 min at room temperature. The primary antibodies against SOX2, OCT4, NANOG and SSEA4 diluted in the Image-iT™ FX Signal Enhancer were then added and incubated at 4 °C overnight. After washing with DPBS, a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 as these listed in Table 2 was added to the cells for 1 h incubation at room temperature. After the cells were washed and stained with Hoechst 33342 for 15 min, an INCell Analyzer 2200 imaging system (GE Healthcare) was used imaging analysis with 20X objective lens and Texas Red, FITC and DAPI filter sets.

Flow cytometry analysis
The iPSCs were harvested using TrypLE Express enzyme (12605010, ThermoFisher Scientific) and fixed with 4% paraformaldehyde for 10 min at room temperature. After the cells were washed with DPBS and permeabilized with 0.2% Tween-20 in DPBS for 10 min at room temperature, fluorophore conjugated antibodies (Table 2) were incubated with the cells for 1 h at 4 °C on a shaker. A BD AccuriC6 Flow Cytometer (BD Biosciences) was used for the flow cytometry analysis.

G-banded karyotyping
The G-banding karyotype analysis of the iPS cells at the passage-7 was performed at the WiCell Research Institute (Madison, WI) using a standard cytogenetic protocol. Cells on a slide were incubated with ethidium bromide and colcemid, followed by a fixation in hypotonic solution. Leishman's stain was then used to stain the metaphase cells. A total of 20 randomly selected metaphases were analyzed.

Short tandem repeat (STR) DNA profile analysis
The Short Tandem Repeat (STR) analysis of original patient fibroblasts and derived iPSCs was carried out by the Translational Research Initiatives in Pathology (TRIP) Laboratory at University of Wisconsin-Madison. A PowerPlex® 16 HS System (DC2101, Promega) was employed in multiplex PCR to amplify fifteen STR loci (D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, D18S51, D21S11, D3S1358, D8S1179, FGA, Penta D, Penta E) and Amelogenin (AMEL, a gender determining marker). Capillary electrophoresis of the PCR products was carried out on an ABI 3500xL Genetic Analyzer (Applied Biosystems) using the Internal Lane Standard 600 (ILS 600) (Promega). A GeneMapper® v 4.1 software (Applied Biosystems) was used for data analysis.

Mycoplasma detection
Using a MycoAlert ™ Mycoplasma Detection Kit (LT07-218, Lonza), mycoplasma detection was performed following the manufacturer's instruction. Ratio B/A < 0.9 indicates mycoplasma negative, Ratio between 0.9 and 1.2 indicates mycoplasma ambiguous results, and Ratio B/A > 1.2 indicates mycoplasma positive.

Testing for Sendai reprogramming vector clearance
Total RNA was isolated from the iPSCs (NCATS-CL7989) at passage 19 using a RNeasy Plus Mini Kit (74034, Qiagen). The total RNA from human fibroblasts (Coriell Institute, GM05659) transduced with Sendai virus for 4 days was used as a positive control. A SuperScript™ III First-Strand Synthesis SuperMix (18080400, ThermoFisher Scientific) was used for the cDNA synthesis from 1 μg RNA and random hexamers, followed by a PCR (primers listed in Table 2) performed using a Platinum II Hot-Start PCR Master Mix (14000012, ThermoFisher Scientific) with the following program: 94 °C, 2 mins; 30 cycles of (94 °C, 15 s, 60 °C, 15 s and 68 °C, 15 s). The PCR products were loaded to an E-Gel® 1.2% with SYBR Safe™ gel, ran at 120 V electric field, and imaged by G: Box Chemi-XX6 gel doc system (Syngene, Frederick, MD).

Teratoma formation assay
After the patient iPSCs (NCATS-CL7989) were dissociated with DPBS containing 0.5 mM EDTA, the cells (approximately 1 × 10 7 ) were resuspended in 400 μl culture medium supplied with 25 mM HEPES pH 7.4. The cells, stored on ice prior to injection, were mixed with 200 μl of cold Matrigel (354277, Corning) and injected subcutaneously into NSG mice (JAX No. 005557) at 150 μl per injection site. Visible tumors were removed after 6-8 weeks that were fixed in 10% Neutral Buffered Formalin, and then embedded in paraffin for staining with hematoxylin and eosin.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Advancing Translational Sciences for mycoplasma testing service. This work was supported by the Intramural Research Program at the National Center for Advancing Translational Sciences, National Institutes of Health, USA.