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Generation of two heterozygous MYBPC3 mutation-carrying human iPSC lines, SCVIi001-A and SCVIi002-A, for modeling hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an inherited heart disease that can cause sudden cardiac death and heart failure. HCM often arises from mutations in sarcomeric genes, among which the MYBPC3 is the most frequently mutated. Here we generated two human induced pluripotent stem cell (iPSC) lines from a HCM patient who has a familial history of HCM and his daughter who carries the pathogenic non-coding mutation. All lines show the typical morphology of pluripotent cells, a high expression of pluripotency markers, normal karyotype, and in vitro capacity to differentiate into all three germ layers. These lines provide a valuable resource for studying the molecular basis of HCM and drug screening for HCM.

due to vast differences in variant-specific HCM mechanisms (Mosqueira et al., 2019;Wu et al., 2019). MYBPC3 is one of the most common mutated genes found in HCM patients (Seeger et al., 2019). MYBPC3 encodes the myosin binding protein C (MyBP-C), a sarcomeric protein that regulates cardiac contractility by modulating myofilament sliding velocity (Seeger et al., 2019). The non-coding variant (c.3330 + 2 T > G) in MYBPC3 is known to be both pathogenic and prevalent (Morita et al., 2008). Peripheral blood mononuclear cells (PBMCs) were isolated from a 60-year-old male HCM patient with this pathogenic variant in one allele and his 24-year-old daughter who was genotype-positive but phenotype-negative at that time (Table 1). The PBMCs were reprogrammed using the Sendai virus. The iPSC clones displayed typical morphology and normal karyotype (passages 12 and 13, respectively) as assessed by the KaryoStat assay. Immunostaining showed a high expression of pluripotency markers OCT3/4, NANOG, and SOX2. Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) confirmed the comparable expression of OCT3/4 (also named as POU5F1) in these two iPSC lines with that of the widely used iPSC line SCVI273, which is 100-fold higher than that in differentiated cardiomyocytes. Additionally, the iPSCs were able to differentiate into derivatives of all three germ layers. The heterozygous c.3330 + 2 T > G mutation was confirmed by Sanger sequencing ( Fig. 1 and Table 2). All lines were mycoplasma-negative. The origin of these lines was confirmed by short tandem repeat (STR) analysis showing that the profile of the lines fully matched that of the donors' somatic cells.

Reprogramming
Peripheral blood mononuclear cells (PBMCs) were isolated from blood by Percoll separation (GE Healthcare), purified by multiple rounds of washing using DPBS buffer (Thermo Fisher), and plated in a 24-well plate. Then, 1-2 × 10 6 PBMCs were cultured in 1 mL of complete PBMC medium made up of complete StemPro-34 medium (Thermo Fisher) combined with supplements including 100 ng/mL SCF (Peprotech), 100 ng/mL FLT3 (Thermo Fisher), 20 ng/mL IL-3 (Peprotech), 20 ng/mL IL-6 (Thermo Fisher), and 20 ng/mL EPO (Thermo Fisher). The medium was replaced every other day until the cell number could remain stable for few days (PBMCs include a heterogeneous population of cells, the medium is targeting a small population). About 2 × 10 5 PBMCs resuspended in 300 μl of complete PBMC medium were infected with a Sendai virus reprogramming cocktail based on the instructions of the CytoTune®-iPSC Sendai Reprogramming Kit (Thermo Fisher). The following day, the cells were washed, resuspended in 1 mL of complete PBMC medium, and plated in one well of a Matrigel-coated 12-well plate. On day 3, the cells were resuspended in 1 mL of StemPro-34 medium (Thermo Fisher) consisting of the StemPro ™ -34 SFM basal medium supplied with the StemPro ™ -34 SFM supplement and plated in one well of a Matrigel-coated 12-well plate. The StemPro ™ -34 medium was replaced every two days. On Day 7, 1 mL of StemMACS medium (Miltenyi Biotec) was added on top of the StemPro ™ -34 medium and the medium was replaced with fresh StemMACS on Day 8. The medium was replaced every other day with 1 mL of fresh StemMACS until Day 10-15 after Sendai virus infection when colonies appeared and were ready to be picked up. The picked colonies were expanded when they grew out and were frozen.

Cell culture
The iPSCs were cultured in StemMACS medium (Miltenyi Biotec), and they were passaged in StemMACS medium combined with 10 μM Y27632 (Selleck Chemicals). Subsequently, the medium was changed every other day with StemMACS medium. All cells were cultured at 37 °C, 5% CO 2 , and 85% relative humidity in an incubator.

Trilineage differentiation
The ability of iPSCs to differentiate into the three germ layers (ectoderm, mesoderm, and endoderm) was assessed using the STEMdiff ™ Trillineage Differentiation Kit (STEMCELL Technologies), following the manufacturer's instructions.

Immunofluorescent staining
Cells were fixed in 4% PFA (EMD Millipore) for 15 min at room temperature, permeabilized with 50 μg/mL digitonin for 10 min, and blocked for 30 min at room temperature with 1% of BSA (Sigma-Aldrich) plus 5% of serum (Thermo Fisher) from host species raised for secondary antibodies. Primary antibodies were incubated with 1% of BSA overnight at 4 °C followed by an incubation with secondary antibodies in 1% of BSA solution for 30 min at room temperature. Nuclei were counterstained with DAPI (Vector Laboratories).

RT-qPCR
Total RNA from iPSCs and differentiated iPSC-CMs was extracted using the Direct-zol RNA Miniprep Kit (ZYMO RESEARCH). Reverse transcription of RNA was performed using iScript ™ cDNA Synthesis Kit (Bio-Rad). cDNA production was amplified on CFX96 Real-Time PCR Detection System using Applied Biosystems TaqMan Fast Advanced Master Mix (Thermo Fisher). Pou5f1 was amplified using POU5F1 (OCT3/4) TaqMan ™ Gene Expression Assay (Thermo Fisher, Hs00999634). Other primers are shown in Table 3.

Mycoplasma detection
Mycoplasma was assessed using the MycoAlert Detection Kit (Lonza) following the manufacturer's instructions.

Short tandem repeat analysis
Genomic DNA was isolated from iPSCs and PBMCs using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. STR-analyses were performed by Stanford PAN Facility, using CLA Identifiler ™ Plus and Identifiler ™ Direct PCR Amplification Kits (Thermo Fisher).

Sanger sequencing
Genomic DNA was isolated from iPSCs using DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions. MYBPC3 mutation analysis was performed on a purified PCR product obtained from genomic DNA amplification using Phusion High-Fidelity PCR Kit (Thermo Fisher). The PCR product was purified using QIAquick PCR Purification Kit (Qiagen) and sent for Sanger sequencing. Primers are shown in Table 3.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  Table 2 Characterization and validation.

Classification Test Result Data
Morphology Photography Visual record of the line: normal Fig. 1