An induced pluripotent stem cell line (TRNDi009-C) from a Niemann-Pick disease type A patient carrying a heterozygous p.L302P (c.905 T > C) mutation in the SMPD1 gene

Niemann-Pick disease type A (NPA) is a rare autosomal recessive lysosomal storage disease caused by mutations in the SMPD1 gene, which encodes for the protein acid sphingomyelinase. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a 21-fetal-week-old female patient with NPA that has a heterozygous mutation of a p.L302P variant (c.905 T > C) 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 NPA.


Resource utility
This human induced pluripotent stem cell (iPSC) line is a useful tool for studies of disease phenotype and pathophysiology, and use as a cell-based disease model for drug development to treat patients with NPA.

Resource details
Niemann-Pick disease type A (NPA) is a rare autosomal recessive, neuropathic lysosomal storage disease that is caused by mutations in the SMPD1 gene, leading to a deficiency in acid sphingomyelinase (< 10% enzyme activity) in the cells (Schuchman and Wasserstein, 2015). This deficiency causes an accumulation of sphingomyelin in the lysosome, resulting in hepatosplenomegaly, developmental delay, interstitial lung disease, and cherry-red maculae (Schuchman and Wasserstein, 2015;Vanier, 2013). What differentiates NPA from Niemann-Pick disease type B (NPB) is the decrease in neurological function, such as the loss of acquired motor skills and a decrease in interest in their surrounding. These neurological symptoms are not commonly seen in NPB, which makes NPA the more severe form of the disease (Vanier, 2013). So far, there are no effective treatments or cures for NPA.
In this study, a human iPSC line was established from skin dermal fibroblasts of a 21-fetal week-old female patient (GM03252, Coriell Institute) (Table 1). To generate the iPSC line, the integration-free CytoTune-Sendai viral vector kit containing OCT ¾, 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). The iPSC line named TRNDi009-C was generated and the mutation of p.L302P (c.905 T > C) in exon 2 described by the Coriell Cell Repositories was confirmed in the TRNDi009-C iPSC line by Sanger sequencing of the PCR products harboring the single nucleotide variation (SNV) (Fig. 1C). The patient iPSCs exhibit a classical embryonic stem cell morphology ( Fig.  1A) and carry normal karyotype (46, XX), as confirmed by G-banding karyotype analysis (Fig. 1B). Immunohistochemistry staining and flow cytometry analysis demonstrated high expression level of major pluripotency protein markers of NANOG, SOX2, OCT4, SSEA4, and TRA-1-60 ( Fig. 1A and D). The Sendai virus (SeV) clearance was determined with reverse transcription polymerase chain reaction (RT-PCR) using SeV-specific primers and the vectors disappeared by passage 15 (Fig. 1E). Mycoplasma status was confirmed to be negative ( Supplementary Fig. S1) and the cell line was authenticated using STR DNA profiling analysis, which demonstrated matching genotype at all 16 loci examined (information available with the authors). Furthermore, the pluripotency of this iPSC line was confirmed by teratoma formation experiment, which exhibited the ability to differentiate into cells/tissues of all three germ layers (ectoderm, neural epithelium; mesoderm, cartilage; endoderm, gut tissue) 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 method previously stated (Chen et al., 2011;Beers et al., 2015).

Immunocytochemistry staining
For immunofluorescence staining, iPSCs cultured in 96-well plate were fixed with 4% paraformaldehyde for 30 mins at RT, permeabilized with 0.3% Triton X-100 in Dulbecco's phosphate-buffered saline (DPBS) for 15 mins, and washed with DPBS. Cells were blocked using Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 30 mins at room temperature 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 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 fluorophore-conjugated animal nonimmune Immunoglobulin was used as the negative control. Antibodies and nonimmune immunoglobulins 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 performed at WiCell Research Institute (Madison, WI, USA). Cell harvest, slide preparation, and G-banding karyotype were performed using standard cytogenetic protocols. Cells were incubated with ethidium bromide and colcemid and placed in a hypotonic solution, followed by fixation. Metaphase cell preparations were stained with Leishman's stain. A total of 20 randomly selected metaphases were analyzed by G-banding.

Gene analysis of SMPD1
The gene analysis of SMPD1 was conducted through Applied StemCell (Milpitas, CA, USA  Table 2.

Testing for Sendai reprogramming vector clearance
Total RNA was isolated from iPSCs TRNDi009-C of passage 15 using RNeasy Plus Mini Kit (Qiagen). Human fibroblasts (Coriell Institute, GM05659) after transduction with Sendai virus for 4 days were used as 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 Fisher 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).

Mycoplasma detection
Mycoplasma testing was performed and analyzed using the Lonza MycoAlert kit, 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
Human 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 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 (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, and were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin. Images were collected using the NanoZoomer Digital Pathology software (Hamamatsu).

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