A human induced pluripotent stem cell line (TRNDi007-B) from an infantile onset Pompe patient carrying p.R854X mutation in the GAA gene

Pompe disease is an autosomal inherent genetic disease caused by mutations in the GAA gene that encodes acid alpha-glucosidase. The disease affects patients in heart, skeletal muscles, liver, and central nervous system. A human induced pluripotent stem cell (iPSC) line was generated from the skin dermal fibroblasts of a Pompe patient with homozygosity for a C.2560C > T (p.R854X) mutation in exon 18 of the GAA gene. This human iPSC line provides a useful resource for disease modeling and drug discovery.

of life including muscle weakness, hypotonia, respiratory insufficiency and hypertrophic cardiomyopathy (Kishnani et al., 2006). On the other hand, late-onset Pompe disease may present at any age with slowly progressive proximal myopathy.
In this study, we generated a human iPSC line (TRNDi007-B) from patient skin fibroblasts (GM00248) which was isolated from a five-month-old male patient carrying a homozygous mutation (c.2560C > T, p.R854X) in exon 18 of the GAA gene. R854X mutation is a well-documented African-American mutation and the homozygous mutation is responsible for severe infantile-onset Pompe disease (Becker et al., 1998). Patient fibroblasts were reprogramed into iPSC by transduction a set of non-integrating Sendai virus vectors encoding OCT3/4, KLF4, S0X2 and C-MYC genes (Beers et al., 2015). A single iPSC colony, termed TRNDi007-B, was isolated and characterized for future applications. Fig. 1A show that the iPSC line presents a standard iPSC morphology and expressed major pluripotent protein markers including SOX2, NANOG, OCT4, and SSEA-4 in the immunocytochemistry assay. The pluripotency markers including TRA-1-60, SSEA-4, and NANOG were also detected and quantified by the flow cytometry analysis (Fig. 1B). The mutation (c.2560C > T) in the GAA gene was confirmed by targeted Sanger sequencing where the PCR product harbors a single nucleotide variation (SNV) in both alleles ( Fig. 1D). At passage 7, cells showed a normal human karyotype (46, XY) (Fig. 1C). The clearance of Sendai virus vectors and the exogenous reprogramming factor genes were verified by reverse transcription polymerase chain reaction (RT-PCR) at passage 17 (Fig. 1E). The iPSCs were free of mycoplasma ( Supplementary Fig. S1) and the cell identity was proved by the short tandem repeat analysis (information available with the authors). Furthermore, the three-germ-layer differentiation capacity was demonstrated by the teratoma formation assay (Ectoderm, pigment epithelium; Mesoderm, cartilage; Endoderm, gut-like endoderm) in vivo ( Fig. 1F).

Cell culture
Human skin fibroblasts (GM00248) were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for medical research and maintained in DMEM containing 10% fetal bovine serum (HyClone), 100 units/mL penicillin, and 100 units/mL streptomycin. Human iPSCs were cultured in StemFlex medium (Thermo Fisher Scientific) on Geltrex-coated plates at 37 °C in humidified air with 5% CO 2 and 5% O 2 . Dulbecco's Phosphate Buffered Saline (DPBS) containing 0.5mM ethylenediaminetetraacetic acid (EDTA) was employed during passaging ( Table 1).

Reprogramming of human skin fibroblasts
Patient fibroblasts were reprogrammed into iPSCs using a Sendai virus based kit (A16517, Thermo Fisher Scientific) as described previously (Beers et al., 2015).

Mutation analysis
The genomic mutation analysis was carried out by Applied StemCell (Milpitas, CA). Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen).

Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 20 min, rinsed with DPBS, and permeabilized with 0.5% Triton X-100/DPBS for 10 min at room temperature in a 96-well format. After blocked in cell staining buffer (BioLegend, CA) for 1 h at room temperature, cells were stained with primary antibodies/staining buffer (Table 2) for overnight at 4 °C. Cells were washed three times with DPBS and incubated with matched secondary antibodies (Table 2) for 1 h at room temperature. Cells were washed with DPBS again and stained with Hoechst 33342 for 20 min. After another wash, cells were imaged by an INCell Analyzer 2200 imaging system (GE Healthcare).

Flow cytometry analysis
The iPSCs were dissociated using TrypLE Express enzyme (ThermoFisher Scientific), fixed with 4% paraformaldehyde for 10 mins at room temperature and then rinsed with DPBS. Before analysis, cells were permeabilized with 0.2% Tween-20 in DPBS for 10 min at room temperature and stained with fluorophore conjugated antibodies for 1 h at 4 °C on a shaker. Cells were then analyzed on a BD Accuri C6 Flow Cytometry system (BD Biosciences). Antibodies used are listed in Table 2.

G-banded karyotyping
iPSCs were sent to WiCell Research Institute (Madison, WI) for G-banded karyotyping analysis. Experiments followed the standard cytogenetic protocol; results were concluded from 20 metaphase cells.

Mycoplasma test
Mycoplasma detection was performed using the MycoAlert mycoplasma detection kit (Lonza). Ratio B/A > 1.2 indicates mycoplasma positive. Ratio between 0.9-1.2 indicates ambiguous results. Ratio B/A < 0.9 indicates mycoplasma negative.

Testing for Sendai reprogramming vector clearance
Total RNA was isolated by RNeasy Plus Mini Kit (Qiagen) and cDNA was synthesized from 1 μg of total RNA and random hexamers using Superscript™ III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). PCRs were performed with Platinum II Hot-Start PCR Master Mix (Thermo Fisher Scientific) on Mastercycler pro S (Eppendorf) following a universal amplification setting: 94 °C for 2 min; 30 cycles of (94 °C for 15 s, 60 °C for 15 s) and 68 °C for 15 s. The primers are listed in Table 2. The positive control was derived from human fibroblasts (GM05659, Coriell Institute) infected with Sendai virus for four days.

Teratoma formation assay
Teratoma was generated by a subcutaneous injection procedure. Approximately 1 × 10 7 hiPSCs were constituted in 400 μL of 25mM HEPES (pH 7.4) solution. Prior to injection, cells were mixed with 200 μL of cold Matrigel (Corning, 354277). The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at two sites (150 μL per injection site). After 6-8 weeks, visible tumors were removed and fixed in 10% Buffer Balanced Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  Characterization and validation.