Patient recruitment and ethics committee
A blood sample was collected from the F97 patient, after obtaining written informed consent, following the ethical guidelines outlined in European and National regulations (law 12/2005) of the Lisbon Academic Medical Center Biobank (Biobanco-iMM) and receiving approval from the Ethics Committee of Lisbon Academic Medical Center (Ref. nº 468/20, February 23, 2020).
Generation and maintenance of iPSC lines
PBMCs were isolated from the whole blood using Ficoll-Paque and preserved in 20% DMSO in FBS, following the standardised procedure of Biobanco-iMM. Before the reprogramming phase, thawed PBMCs were cultured for 4 days in StemPro™-34 medium (Gibco, 10639011) supplemented with specific cytokines (IL-3, IL-6, FLT-3, and SCF - Prepotech/Tebu-bio, 200.03, 200.06, 300 − 19, 300-07). Subsequently, 5x106 cells were transduced using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, A16517) with the following Multiplicity of infection (MOI) for each virus: KOS MOI = 5, hc-Myc MOI = 5 and hKlf4 MOI = 3. On the third day post-transduction, cells were reseeded into Matrigel®-coated 6-well culture plates and transitioned to mTeSR™Plus Medium (StemCell Technologies, 100–0276) in the following days. Colonies exhibiting characteristic stem cell morphology were individually picked and transferred to new Matrigel®-coated dishes in mTeSR™Plus. Continuous passaging was performed until the absence of SeV expression was confirmed by qRT-PCR (passage 14 for clone 1F97 and passage 11 for clone 5F97). iPSCs were maintained at 37°C in a humidified 5% CO2, 20% O2 incubator and passaged 1:3 − 1:6 every 3–4 days when colonies covered approximately 80% of the culture dish surface area, utilising 0.5mM EDTA dissociation buffer (Thermo Fisher Scientific, 15575020). Further characterization analyses of the two iPSC clones were performed between passages 11 and 20, after SeV clearance.
Targeted sequencing
Genomic DNA from iPSCs was extracted using the conventional phenol:chloroform:isoamyl alcohol method. Following extraction, the DNA underwent PCR amplification with primers for the region of interest of the MYBPC3 gene (Table 2) and subjected to Sanger sequencing, by StabVida Lda (Lisbon, Portugal).
G-banding karyotyping
For G-banding karyotyping, iPSCs in an exponential growth phase were arrested in metaphase by exposure to colcemid (10 µg/ml; Thermo Fisher Scientific, 15212012) for 5 hours at 37°C. Subsequently, cells were harvested using Accutase (STEMCELL Technologies, 07922), treated with a hypotonic potassium chloride solution for 30 minutes at 37°C, and fixed with a 1:3 (v/v) acetic acid:methanol solution. Karyotype analysis was completed by Genomed SA (Lisbon, Portugal).
Trilineage differentiation assays
The trilineage differentiation potential of iPSCs into endoderm, mesoderm, and ectoderm was evaluated using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies, 05230), according to the manufacturer’s instructions. Assessment of lineage-specific markers was carried out through both immunofluorescence (IF) and quantitative real time PCR (qRT-PCR) (Table 2).
Quantitative real time (qRT)-PCR
Total RNA was isolated using NZYol (NZYTech®, MB18501) and DNase I treatment (Roche®, 04716728001). cDNA was synthesised using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche®, 5081963001) and amplified by qRT-PCR with specific primers for the targeted genes (Table 2), using the Universal SYBR Green Supermix (Bio-Rad, 1725274). All PCR reactions were performed in triplicate for 40 cycles using the ViiA™7 RT-PCR Systems (Applied BioSystems). The mRNA expression levels are depicted as the fold change of the target gene normalised against GAPDH (for SeV clearance) or U6 (for differentiation markers) housekeeping genes (2–ΔCt).
Immunofluorescence assays
Coverslip-seeded cells underwent fixation with 3.7% paraformaldehyde (PFA) in PBS, for 10 minutes at room temperature (RT) and subsequent permeabilization with 0.5% Triton X-100 in PBS, for 10 minutes at RT. Following this, cells were blocked with 5% foetal bovine serum (FBS, Life Technologies, A5256701) in PBS, for 30 minutes at RT. Cells were then incubated overnight at 4°C with the specified primary antibodies (Table 2); the following day, incubation with the corresponding secondary antibodies diluted in 1% FBS in PBS (Table 2), was performed for 1 hour at RT and nuclei were then counterstained with DAPI (0.2 mg/ml; Cat# D9542, Sigma). Images were acquired using a Zeiss LSM 710 Confocal Laser Point-Scanning Microscope.
Flow cytometry
iPSCs, dissociated into single cells, were fixed in 2% PFA, and stored at 4°C. For intracellular staining, cells were permeabilized with 0.1% Saponin (Sigma, SAE0073), for 15 minutes at RT, and incubated with primary antibodies (OCT4 or SOX2) (Table 2) for 1 hour at RT, after washing. 1% FBS/1xPBS was used to wash cells, twice, before further incubation for 45 minutes, in the dark, with the goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Thermo Fisher Scientific, A-11001). For surface staining, cells were resuspended in conjugated antibodies (SSEA4 or TRA1-60) (Table 2), diluted in 3% FBS/1xPBS, for 30 minutes at RT. Before analysis on the FACSCalibur™ flow cytometer (Becton Dickinson), all cells were resuspended in 1xPBS. In each experimental sample, at least 10,000 events were recorded within the defined gate based on side scatter (SSC) and forward scatter (FSC). Results were analysed using FlowJo software.
Short tandem repeat (STR) analysis
To assess the clonality of each generated cell line, genomic DNA isolated from both iPSCs and the respective PBMCs, using phenol:chloroform:isoamyl alcohol extraction, was sent to Genomed SA (Lisbon, Portugal). There, STR DNA analysis was performed using the AmpFLSTR® Identifiler® Plus PCR Amplification Kit, which amplifies STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA), along with a gender-determining marker, Amelogenin (AMEL).
Mycoplasma detection
iPSC cultures were checked for mycoplasma contamination with the qPCR Mycoplasma Test (Mycoplasmacheck, Eurofins Genomics), according to the manufacturer’s instructions.