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Generation of an induced pluripotent stem cell line (TRNDi004-I) from a Niemann-Pick disease type B patient carrying a heterozygous mutation of p.L43_A44delLA in the SMPD1 gene

Niemann-Pick disease type B (NPB) is a rare autosomal recessive lysosomal storage disease caused by mutations in the SMPD1 gene, which encodes for acid sphingomyelinase. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a 1-year old male patient with NPB that has a heterozygous mutation of a p.L43_A44delLA of SMPD1 using non-integrating Sendai virus technique. This iPSC line offers a useful resource to study the disease pathophysiology and as a cell-based model for drug development to treat NPB.

In this study, a human induced pluripotent stem cell (iPSC) line TRNDi004-I was established from dermal fibroblasts of a 1-year-old male patient (Table 1). The integration free CytoTune-Sendai viral vector kit (A16517, Thermo Fisher Scientific) containing OCT 3/4, KLF4, SOX2, and c-MYC pluripotency transcription factors was used to transduce the patient fibroblasts using methods previously stated (Chen et al., 2011;Beers et al., 2015). Genetic analysis shows that this iPSC line carries a heterozygous gene mutation of p.L43_A44delLA in exon 1 of SMPD1 (Fig. 1B). The cells exhibit a classical embryonic stem cell morphology (Fig. 1A) and carry a normal karyotype (46, XY), as confirmed by G banding karyotype analysis (Fig. 1D). Immunohistochemistry staining and flow cytometry analysis demonstrated high expression levels of major pluripotency protein markers of NANOG, SOX2, OCT4, SSEA4, and TRA-1-60 on those cells ( Fig. 1A and C). Sendai virus (SeV) clearance was verified with reverse transcription polymerase chain reaction (RT PCR) using SeV-specific primers with no virus present by passage 15 (Fig. 1E). The iPSC line was not contaminated with mycoplasma ( Supplementary Fig. S1) and was authenticated using STR DNA analysis, which demonstrated matching genotype at all 18 loci examined (information available with the authors). Furthermore, the pluripotency of this iPSC line was confirmed by the teratoma formation experiment that exhibited its ability to differentiate into cells/tissues of all three germ layers (ectoderm, mesoderm and endoderm) in vivo (Fig. 1F).

Reprogramming of human skin fibroblasts
Fibroblast cells were reprogrammed into iPSCs using the integration-free CytoTune Sendai viral vector kit (A16517, Thermo Fisher Scientific) following the methods previously stated (Chen et al., 2011;Beers et al., 2015).

Immunocytochemistry staining
For immunofluorescence staining, patient iPSCs were fixed with 4% paraformaldehyde for 30 min at RT, permeabilized with 0.3% Triton X-100 in Dulbecco's phosphate-buffered saline (DBPS) for 15 min, and washed with DPBS. Cells were blocked using Image iT™ FX signal enhancer (Thermo Fisher Scientific) for 1 h and incubated with primary antibodies, including SOX2, OCT4, NANOG and SSEA4, overnight at 4 °C. Cells were then washed and incubated with corresponding secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 for 1 h at room temperature (antibodies used are listed in Table 2). Cells were washed and stained with Hoechst 33342 for 15 min and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20× objective lens and Texas Red, FITC and DAPI filter sets.

Genome analysis of variant in SMPD1 gene
The genome analysis of variants in the SMPD1 gene was conducted through Applied StemCell (Milpitas, CA, USA). Genomic DNA was extracted from iPSC line TRNDi004 I followed by PCR amplification using MyTaq™ Red Mix (Bioline, Taunton, MA). Amplifications were carried out using the following program: 95 °C, 2 min; 35 cycles of [95 °C, 15 s; 60 °C, 15 s; 72 °C, elongation duration varies by amplificon size], 72 °C, 5 min; 4 °C, indefinite. PCR products were subsequently sequenced by using Sanger sequencing analysis to identify potential mutations. The specific primers for gene amplification and sequencing are listed in Table 2. The heterozygous gene mutation of p.L43_A44delLA in exon 1 of SMPD1 was further confirmed by Codex BioSolutions, Inc. (Gaithersburg, MD).

Flow cytometry analysis
The iPSCs were harvested using TrypLE Express enzyme (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde for 10 min 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 min at room temperature and stained with fluorophore conjugated antibodies for 1 h at 4 °C on a shaker. Relative fluorophore-conjugated animal nonimmune immunoglobulin was used as the negative control (antibodies and nonimmune immunoglobulin used are listed in Table 2). Cells were then analyzed on a BD AccuriC6 Flow Cytometry system (BD Biosciences).

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

Testing for Sendai reprogramming vector clearance
Total RNA was isolated from iPSCs TRNDi004-I of passage 15 using RNeasy Plus Mini Kit (Qiagen). Human fibroblasts (Coriell Institute, GM05659), after infection with Sendai virus for 4 days, were used as a 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) with the primers listed in Table 2. The products were then loaded into an E-Gel® 1.2% with SYBR Safe™ gel and run at 120 V electric field. Finally, the image was collected using G: Box Chemi-XX6 gel doc system from Syngene (Frederick, MD).

Short tandem repeat (STR) DNA profile analysis
NPB patient fibroblasts and iPSCs were sent to the John Hopkins University Genetic Resources Core Facility for STR DNA profile analysis using a Promega PowerPlex 18D Kit. The PCR product was electrophoresed on an ABI Prism® 3730xl Genetic Analyzer and data were analyzed using GeneMapper® v 4.0 software (Applied Biosystems).

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

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 collected and re-suspended 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 (354277, Corning) 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 that were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin. Images were collected and analyzed using the NanoZoomer Digital Pathology software (Hamamatsu).

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