Generation of two induced pluripotent stem cell lines from patients with cardiac amyloidosis carrying heterozygous transthyretin (TTR) mutation

Specific mutations in the TTR gene are responsible for the development of variant (hereditary) ATTR amyloidosis. Here, we generated two human induced pluripotent stem cell (iPSC) lines from patients diagnosed with Transthyretin Cardiac Amyloidosis (ATTR-CM) carrying heterozygous mutation in the TTR gene (i.e., p.Val30Met). The patient-derived iPSC lines showed expression of high levels of pluripotency markers, trilineage differentiation capacity, and normal karyotype. The generation of these iPSC lines represents a great tool for modeling patient-specific amyloidosis in vitro, allowing the investigation of the pathological mechanisms related to the disease in different cell types and tissues.


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Transthyretin Amyloidosis (ATTR) is a severe systemic and fatal disease caused by a misfolding and aggregation of the normal, non-mutated transthyretin protein (wild-type ATTR) or by a heterozygous mutation in the TTR gene (hereditary/variant ATTR) that promotes misfolding and aggregation.Usually, the clinical presentation of the disease includes polyneuropathy (ATTR-PN) and/or cardiomyopathy (ATTR-CM) caused by the deposition of these amyloid aggregates and fibrils into the target organs.Over 130 mutations in the TTR gene have been identified, most of them being pathogenic (Adams et al., 2019;Ruberg and Berk, 2012).Here, we described the generation of two iPSC lines derived from a 65 year-old Caucasian male patient (SCVIi066-A) and a 63 year-old Caucasian male patient (SCVIi067-A) diagnosed with transthyretin cardiac amyloidosis due to a heterozygous mutation in the TTR gene (c.148G>Aencoding p.Val30Met; pathogenic variant).The mutation Val30Met (also known as V50M) is one of the most common mutations in the TTR gene and is endemic in some countries (e.g., Portugal and Sweden), presenting with clinical manifestations related to ATTR-PN with or without ATTR-CM (Adams et al., 2019).Cardiac manifestations of TTR-CA include a restrictive cardiomyopathy phenotype, reduced stroke volume, compromised cardiac output, atrial fibrillation, diastolic dysfunction, and cardiac fibrosis (Griffin et al., 2021).Therefore, the cell reprogramming approach provides an unlimited source for generating a plethora of cell types affected by the Transthyretin Amyloid disease, such as hepatocytes (the main source of TTR production), cardiomyocytes (target tissue of ATTR deposition) and peripheral nerves (target tissue of ATTR deposition), allowing in vitro disease modeling and drug screening assays (Giadone et al., 2018).
Reprogramming of a patient's peripheral blood mononuclear cells (PBMCs) to iPSCs was performed using Sendai virus containing the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) (see Material and Methods).iPSC clones (SCVIi066-A and SCVIi067-A) showed typical morphology (Fig. 1A) and normal karyotype (Fig. 1B).Immunofluorescence staining showed the expression of pluripotency markers OCT3/4, NANOG and SOX2 at the protein level (Fig. 1C).Quantitative analysis of gene expression of NANOG and SOX2 was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Fig. 1D-E, respectively).Both genes presented mRNA levels as high as a control iPSC (i.e., healthy non-mutated cells) and significantly higher than iPSC-derived cardiomyocytes (iPSC-CMs) generated from the control line.Furthermore, expression of the non-integrative Sendai virus (SEV) was absent from both lines at passage 16 (Fig. 1F).The generated iPSC lines were able to successfully differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) (Fig. 1G).The presence of heterozygous mutation (c.148G>A) was confirmed by Sanger sequencing and was absent in the control cell line (Fig. 1H).SCVIi066-A and SCVIi067-A lines were mycoplasma-negative (Supplementary Fig. 1A).Short tandem repeat (STR) analysis confirmed that both lines demonstrated overlapping profiles with corresponding somatic donor cells (submitted in archive with journal).

Trilineage differentiation
Differentiation capacity into cells of the three germ layers was performed using the STEMdiff ™ Definitive Endoderm Differentiation Kit (STEMCELL ™ Technologies #05110) for endoderm differentiation.Ectoderm differentiation was induced with the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems #SC027B).Mesoderm differentiation was induced using RPMI media supplemented with 827-Minus Insulin (Gibco #11875-085 and #A18956-01) for 48 h.

Immunofluorescent staining
For qualitative analysis of pluripotency and trilineage differentiation, cells were fixed in 4 % paraformaldehyde (PFA) at room temperature (RT) for 10 min.Afterwards, cells were permeabilized using 50 μg/mL digitonin (Sigma Aldrich #D141) for 10 min, followed by a blocking step using a solution of 1 % of Bovine Serum Albumin (BSA) for 30 min at RT. Cells were incubated with primary antibodies (Table 2) overnight at 4 °C.Cells were washed three times and incubated for 30 min at RT with secondary antibodies (Table 2).Nuclei were stained with Molecular Probes NucBlue ™ (Thermo Fisher Scientific #R37606) for 10 min at RT.

Short tandem repeat (STR) analysis
Genomic DNA (gDNA) was isolated from iPSCs (passage number 20) and PBMCs using the DNeasy Blood & Tissue Kit (Qiagen #69504).STR analysis was performed using CLA Identifiler ™ Direct PCR Amplification Kit (Thermo Fisher Scientific #A44661).Capillary electrophoresis was performed on ABI3130xl by the Stanford Protein Nucleic Acid (PAN) Facility.

Karyotyping
Approximately 2 × 10 6 cells were collected from the iPSC lines at passage number 11 and analyzed using the KaryoStat ™ assay (Thermo Fisher Scientific).

Sanger sequencing
PCR primers were designed to detect TTR mutations (Table 2) and used to amplify the genomic region of the gDNA using the KOD One PCR Master Mix (DiagnoCine, #KMM-101).The PCR reaction was performed using the following conditions: 94 °C 1 min, 94 °C 30 s; 62 °C 15 s, 68 °C 1 min for 35 cycles / 68 °C 5 min.Sanger sequencing was submitted to and performed by Azenta.
Stem Cell Res.Author manuscript; available in PMC 2024 January 27.