Generation of an induced pluripotent stem cell line (TRNDi005-A) from a Mucopolysaccharidosis Type IVA (MPS IVA) patient carrying compound heterozygous p.R61W and p.WT405del mutations in the GALNS gene

Mucopolysaccharidosis type IVA (MPS IVA) is a rare genetic disease caused by mutations in the GALNS gene and is inherited in an autosomal recessive manner. GALNS encodes N-acetylgalactosamine-6-sulfatase that breaks down certain complex carbohydrates known as glycosaminoglycans (GAGs). Deficiency in this enzyme causes accumulation of GAGs in lysosomes of body tissues. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a MPS IVA patient that has compound heterozygous mutations (p.R61W and p.WT405del) in the GALNS gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.


Resource details
Mucopolysaccharidosis type IVA (MPS IVA, also called Morquio syndrome type A) is a rare autosomal recessive disorder caused by mutations in the GALNS gene, which encodes Nacetylgalactosamine-6-sulfatase (EC 3.1.6.4). It breaks down certain complex carbohydrates known as glycosaminoglycans (GAGs). Deficiency in N-acetylgalactosamine-6-sulfatase results in accumulations of GAGs in the lysosomes in body tissues and causes clinical symptoms including heart disease, skeletal abnormalities, vision and hearing loss, difficulty breathing, and early death (Khan et al., 2017;Peracha et al., 2018).
In this study, a human iPSC line was established from dermal fibroblasts of a 43-year-old male patient (GM01361, Coriell Institute) carrying compound heterozygous mutations of a p. R61W variant (c.181G > A) in exon 2 and a p.WT405del variant (c. 1213_1218del) in exon 11 of the GALNS gene (Table 1, Fig. 1A). A non-integrating CytoTune-Sendai viral vector kit (A16517, Thermo Fisher Scientific) containing OCT3/4, KLF4, SOX2 and C-MYC pluripotency transcription factors was employed to transduce the fibroblasts using the method described previously (Beers et al., 2012;Beers et al., 2015). The resulting iPSC line was named TRNDi005-A that exhibited a classical embryonic stem cell morphology (Fig.  1C), normal karyotype (46, XY), as confirmed by the G-banding karyotype at passage 11 ( Fig. 1B), and expressed the major pluripotent protein markers of NANOG, SOX2, OCT4, SSEA4 and TRA-1-60 ( Fig. 1C, D) evidenced by both immunofluorescence staining and flow cytometry analysis. Sendai virus vector (SeV) clearance was detected with reverse transcription polymerase chain reaction (RT-PCR) using SeV-specific primers that confirmed the vector elimination in the iPSCs by passage 15 (Fig. 1E). This iPSC line was not contaminated with mycoplasma ( Supplementary Fig. S1) and were authenticated using a short tandem repeat (STR) DNA analysis, which demonstrated matching genotypes at all 16 loci examined (information available with the authors). Furthermore, the pluripotency of this iPS cell line was confirmed by a teratoma formation experiment that exhibited its ability to differentiate into cells of all three germ layers (Ectoderm, neural tube and pigmented epithelium; Mesoderm, cartilage; Endoderm, gut) in vivo (Fig. 1F).

Cell culture
Patient skin fibroblasts were obtained from Coriell Cell Repositories (GM01361) and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified incubator with 5% CO 2 at 37 °C. The iPS cells were cultured in StemFlex medium (Thermo Fisher Scientific) on Matrigel (Corning, 354,277)coated plates at 37 °C in humidified air with 5% CO 2 and 5% O 2 . The cells were dissociated with 0.5 mM Ethylenediaminetetraacetic acid (EDTA) and passaged when they reached 80% confluency.

Reprogramming of human skin fibroblasts
Patient fibroblasts were reprogrammed into iPS cells using the non-integrating Sendai virus technology following the method described previously (Beers et al., 2012;Beers et al., 2015).

Genome analysis
The genome analysis of variants in GALNS was conducted through ACGT, Inc. (Wheeling, IL, USA). Briefly, genomic DNA was extracted from MPS IVA patient fibroblasts using MasterPure Complete DNA and RNA Purification kit (Epicentre, Madison, WI, USA) as per manufacture instruction, followed by PCR amplification of all the exons using PrimeSTAR GXL DNA Polymerase (Takara, Mountain View, CA, USA). Equal quantities of each PCR amplicon were polled and fragmented to an average of size of 400 bp for library preparation. Sequencing of human GALNS exons by Next-Generation Sequencing (NGS) and bioinformatics data analysis was performed to identity the variants in the GALNS gene of MPS IVA patient sample. Gene variants were analyzed by using the Variant Annotation Integrator (VAI) tool from the University of California Santa Cruz Genome Browser (Casper et al., 2018). The specific primers for amplification of GALNS exons are listed in Table 2.

Immunocytochemistry
For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 mins, rinsed with Dulbecco's Phosphate Buffered Saline (DPBS), and permeabilized with 0.3% Triton X-100 in DPBS for 15 mins. The cells were then incubated with the Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 40 mins at room temperature in a humidified environment and then followed by incubation individually with primary antibodies including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. After washing with DPBS, a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 was added to the cells and incubated for 1 h at room temperature (Antibodies used are listed in Table 2). Cells were then washed and stained with Hoechst 33342 nucleic acid stain for 15 mins and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20× objective lens and Texas Red, FITC and DAPI filter sets.

Flow cytometry analysis
The iPSCs were harvested using TrypLE Express enzyme (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde for 10 mins at room temperature and then washed with DPBS. Before fluorescence-activated cell sorting analysis, cells were permeabilized with 0.2% Tween-20 in DPBS for 10 mins at room temperature and stained with fluorophore-conjugated antibodies for 1 h at 4 °C on a shaker. Relative fluorophoreconjugated animal nonimmune Immunoglobulin were used as the negative control (Antibodies and nonimmune immunoglobulin used are listed in Table 2). Cells were then analyzed on a BD Accuri™ C6 Flow Cytometry system (BD Biosciences).

G-banding karyotype
The G-banding karyotype analysis was conducted at WiCell Research Institute (Madison, WI, USA). A total of 20 randomly selected metaphases were analyzed by G-banding for each cell line.

Mycoplasma detection
Mycoplasma testing was performed and analyzed using the Lonza MycoAlert kit following the instructions from the company. Ratio B/A > 1.2 indicates mycoplasma positive. Ratio B/A 0.9-1.2 indicates ambiguous results and Ratio B/A < 0.9 indicates mycoplasma negative.

Testing for Sendai reprogramming vector clearance
Total RNA was extracted from TRNDi005-A iPSCs at passage 15 using RNeasy Plus Mini Kit (Qiagen). Human fibroblasts (GM05659, Coriell Institute) after transduction with Sendai virus for 4 days was used as the positive control. A total of 1 μg RNA/reaction was reverse transcribed with SuperScript™ III First-Strand Synthesis SuperMix kit and PCR was performed using Platinum II Hot-Start PCR Master Mix (Thermo Fischer Scientific). The amplifications were carried out using the following program: 94 °C, 2 mins; 30 cycles of [94 °C, 15 s, 60 °C, 15 s and 68 °C, 15 s] on Mastercycler pro S (Eppendorf) with the primers listed in Table 2. The products were then loaded to the E-Gel® 1.2% with SYBR Safe™ gel, run at 120 V electric field, and then imaged by G: Box Chemi-XX6 gel doc system (Syngene, Frederick, MD).

Teratoma formation assay
Patient iPSCs cultured in 6-well plates were dissociated with DPBS containing 0.5 mM EDTA and approximately 1 × 10 7 dissociated cells were resuspended in 400 μl culture medium supplied with 25 mM HEPES (pH 7.4) and stored on ice. Then, 50% volume (200 μl) of cold Matrigel (Corning, 354277) was added and mixed with the cells. The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at 150 μl per injection site. Visible tumors were removed 6-8 weeks post injection, and were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin.