Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying TNNI3 mutations

Hypertrophic cardiomyopathy (HCM) is a common inherited heart disease with a prevalence of about 0.2%. HCM is typically caused by mutations in genes encoding sarcomere or sarcomere-associated proteins. Here, we characterized induced pluripotent stem cell (iPSC) lines generated from the peripheral blood mononuclear cells of three HCM patients each carrying c.433C > T, c.610C > T, or c.235C > T mutation in the TNNI3 gene by non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, and the potential of trilineage differentiation. The cardiomyocytes differentiated from these iPSC lines can serve as useful tools to model HCM in vitro.


Resource utility
Three induced pluripotent stem cell (iPSC) lines were generated from three hypertrophic cardiomyopathy (HCM) patients each carrying different heterozygous mutation in the TNNI3 gene.These fully characterized iPSC lines can be differentiated into cardiomyocytes to understand the complex pathogenic mechanisms of HCM.

Resource details
HCM is a genetic disorder characterized by left ventricular hypertrophy.HCM is predominantly caused by mutations in genes encoding sarcomere or sarcomere-associated proteins (Lan et al., 2013;Marian & Braunwald, 2017;Wu et al., 2019).Thin filaments of the sarcomeres are composed of tropomyosin, troponin and actin (van der Velden & Stienen, 2019).The TNNI3 gene encodes cardiac troponin I (cTnI), a subunit of the troponin complex.Notably, mutations in TNNI3 have been reported in 2%-7% of HCM cases (Mogensen et al., 2004).
In this report, we generated three iPSC lines SCVIi017-A, SCVIi018-A, and SCVIi019-A from three HCM patients each carrying distinct mutation in TNNI3.Peripheral blood mononuclear cells (PBMCs) collected from these patients were reprogrammed into iPSCs using Sendai virus carrying reprogramming factors OCT4, SOX2, KLF4, and c-MYC.
All of the three iPSC lines showed typical iPSC morphology (Fig. 1A).High expression levels of pluripotency markers were confirmed by immunofluorescence staining and reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Fig. 1B and  1C).Genetic testing confirmed c.433C > T, c.610C > T and c.235C > T mutations in TNNI3 of SCVIi017-A, SCVIi018-A, and SCVIi019-A, respectively (Fig. 1D).Neither reprogramming nor long-term maintenance compromised the karyotype integrity of these iPSC lines (Fig. 1E).All of the three iPSC lines demonstrated full potential to generate three lineages by expressing endoderm (Sox17 and Foxa2), mesoderm (Brachyury and Tbx6), and ectoderm (Otx2 and Pax6) markers (Fig. 1F).While trace amount of Sendai virus was detectable at early passages of iPSCs, it was absent at passages 24-27 (Fig. 1G).All iPSC clones were tested negative for mycoplasma (Supplementary Fig. 1).A set of 16 polymorphic short tandem repeats (STR) analysis confirmed the identicalness of the three iPSC lines to the patients' PBMCs (data archived) (Table 1).

Reprogramming
PBMCs were isolated and collected by gradient centrifugation from the peripheral blood of patients.PBMCs were isolated by Percoll separation (GE Healthcare) and purified by washing with DPBS buffer (Thermo Fisher Scientific).After replating, PBMCs were cultured in PBMC medium containing complete StemPro-34 medium (Thermo Fisher Scientific) supplemented with 100 ng/mL SCF (Peprotech), 100 ng/mL FLT3 (Thermo Fisher Scientific), 20 ng/mL IL-3 (Peprotech), 20 ng/mL IL-6 (Thermo Fisher Scientific), and 20 ng/mL EPO (Thermo Fisher Scientific).PBMCs were reprogrammed to iPSCs by the CytoTune®-iPSC Sendai Reprogramming Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.Briefly, transduced PBMCs were resuspended and plated.The StemPro™−34 medium was refreshed every two days.At day 7, the medium was changed to fresh StemMACS™ iPS-Brew XF medium (Miltenyi Biotechnology).Medium was refreshed every other day until day 10-15 post-infection when colonies were ready to be picked.Picked colonies were further expanded and frozen down for downstream applications.

Cell culture
iPSCs were cultured in StemMACS™ iPS-Brew XF medium in 6-well plates coated with Matrigel (Corning) at a dilution of 1:400 in a humidified incubator at 37 °C with 5% CO 2 .Medium was changed every other day.iPSCs were passaged at a ratio of 1:6 to 1:12.Y-27632 (10 μM), a potent inhibitor of ROCK1 (Selleck Chemicals), was added in the medium during the first 24 h of cell replating to improve cell survival and attachment.

Immunofluorescence staining
iPSCs at passages 15-20 and iPSC derivatives were fixed with 4% paraformaldehyde for 15 min at room temperature (RT), permeabilized with 0.3% Triton X-100 (Sigma) for 10 min at RT, and blocked with 3% bovine serum albumin (BSA, Sigma) for 30 min at RT. Then cells were incubated with primary antibodies overnight at 4 °C and fluorescence-conjugated secondary antibodies for 60 min at RT. Cell nuclei were counter stained with Hoechst 33342 (Thermo Fisher Scientific) for 5 min at RT. Images were captured using an inverted fluorescence microscope.The antibody information and dilution ratios are listed in Table 2.

Trilineage differentiation potential assay
iPSCs at passages 15-20 were differentiated using the STEMdiff™ trilineage differentiation kit (Stemcell Technologies) according to the manufacturer's instructions.Differentiations were assessed by the expressions of classical lineage markers in each germ layer.

RT-qPCR
Total RNA was extracted by miRNeasy Micro Kit (Qiagen).RT-qPCR was performed by iScript™ Reverse Transcription Supermix (Bio-rad) according to the manufacturer's instructions.iPSCs at passages 15-20 were used for the detection of pluripotency markers.iPSCs at passages 24-27, as well as early passage (P9), were used for the detection of Sendai virus genome.

Karyotyping
A total of 2 × 10 6 iPSCs were collected from each line between passages 11-15 and analyzed using the KaryoStat™ assay (Thermo Fisher Scientific).

Short tandem repeat (STR) analysis
Genomic DNAs of PBMCs and iPSCs at passages 15-20 were isolated by QuickExtract™ DNA Extraction Solution (Lucigen).STR analysis was performed using a CLA IdentiFiler™ Direct PCR Amplification Kit (Thermo Fisher Scientific).Capillary electrophoresis was performed on ABI3130xl by the Stanford Protein Nucleic Acid (PAN) Facility.

Mycoplasma detection
Mycoplasma detection was performed by a MycoAlert™ Detection Kit (Lonza) according to the manufacturer's instructions.

DNA sequencing
Genomic DNA was isolated from iPSCs at passages 15-20 using the QuickExtract™ DNA Extraction Solution (Lucigen) and amplified by PCR.Information of the designed primers was listed in Table 2. Purified PCR products were subjected to Sanger sequencing.The presence of TNNI3 mutations was identified by aligning the Sanger sequencing data with wildtype TNNI3 sequence using SnapGene software.

Fig
Fig.1 panel A

Table 1
Characterization and validation.

Table 2
Reagents details.RRID Requirement for antibodies: use http://antibodyregistry.org/ to retrieve RRID for antibodies and include ID in table as shown in examples.
Stem Cell Res.Author manuscript; available in PMC 2023 May 12.