Materials and Methods

Cell culture and reprogramming

PBMCs were isolated from peripheral blood samples using Ficoll gradient separation (Sigma #GE17–5442-03). The enriched PBMCs were expanded and reprogrammed to hiPSCs according to the instruction of the Erythroid Progenitor Reprogramming Kit (STEMCELL Technologies #05924). Briefly, PBMCs were expanded in Erythroid Expansion Medium for 7 days before reprogramming. Reprogramming was initiated by infecting 2×105 cells with Sendai virus carrying human Yamanaka factors, provided in CytoTune-iPS 2.0 Sendai Reprogramming Kit (Invitrogen #A16517) on Matrigel-coated plate. On day 2, 3 and 5 after infection, 1 ml of ReproTeSR medium was added to cells. On day 7, the culture medium was replaced with fresh ReproTeSR medium, and the medium was refreshed daily until hiPSC colonies appeared.

AFCs were first cultured in AmnioMAX-II Complete Medium (ThermoFisher Scientific #11269016) for 4–5 days to allow cell attachment. Non-attached cells were removed, and the medium was replaced every 3–5 days. AFCs were passaged when reached 70–80 % confluence by using 0.05 % trypsin and seeded at a split ratio of 1:4–1:6. For reprogramming, 2.5 × 104 AFCs were seeded on Matrigel-coated plate 2 days before transduction. Transduction was performed by using the CytoTune-iPS 2.0 Sendai Reprogramming Kit. The medium was replaced with fresh AmnioMAX-II Complete Medium every other day up to 7 days post-infection. On day 7, AFCs were transferred to MatriGel (Corning #354277) – coated plate with TeSR-E7 medium (STEMCELL Technologies #05914) supplemented with 10 μM Y-27632 (ApexBio #501012282). Starting on day 8, the culture medium was replaced with fresh TeSR-E7 medium daily until hiPSC colonies appeared.

hiPSCs were cultured in mTeSR1 (STEMCELL Technologies #85850) on plates coated with MatriGel. mTeSR1 was refreshed daily. 10 μM Y-27632 was added to the medium for 24 h after passage. hiPSCs were routinely passed with Gentle Cell Dissociation Reagent (STEMCELL Technologies #1000485) at 1:3 ratio every 4–6 days. All cells were maintained in a 37 °C humidified incubator with 5 % CO2.

Immunofluorescence staining

Immunofluorescence staining was performed as previously described (Chi et al., 2023). The primary and secondary antibodies used in this study are listed in Table 2.

RT-qPCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen #15596026) according to the manufacturer’s instructions. cDNA synthesis was performed using the iScript Reverse Transcription Supermix (Biorad #1708840). Quantitative PCR (qPCR) was carried out using SYBR PowerUP master mix (Applied Biosystems #4309155) on the StepOne Real-Time PCR System (Applied Biosystems) with gene specific primers (Table 2). The 18S ribosomal RNA gene was used as an internal control.

Trilineage differentiation

Endoderm, ectoderm, and mesoderm differentiation was induced from hiPSCs using STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies #05230). Differentiation was performed at passage 27 (USFi005-A), passage 22 (USFi006-A), and passage 22 (USFi007-A).

Karyotyping

Karyotypic analysis of patient derived hiPSC lines, USFi005-A at passage 26 (p26), USFi006-A at passage 21 (p21), and USFi007-A at passage 21 (p21), was performed using the KaryoStat + Assay (Thermo Fisher Scientific).

Sanger sequencing

Genomic DNA from hiPSCs were prepared as crude lysates by using hot 50 mM NaOH and then neutralized with 1 M Tris-HCl. The genomic region of the KCNH2 gene was amplified by PCR using primers flanking c.1898A > G mutation (Table 2) and the Phusion high-fidelity PCR Kit (New England Biolabs #E0553S). PCR products were sequenced by MCLAB.

STR analysis

Frozen cell pellets (1 × 106 cells) from hiPSCs as well as their respective somatic parental cells (AFCs for USFi005-A, PBMCs for USFi006-A and USFi007-A) were collected. STR analysis was performed using the CellCheck Cell Line Authentication service by IDEXX BioAnalytics.

Mycoplasma detection

Cell culture supernatant was collected by spinning the cell sample at 200×g for 5 min. Mycoplasma contamination was evaluated using the MycoAlert Assay Control Set (Lonza #LT07–518) and the MycoAlert Mycoplasma Detection Kit (Lonza #LT07–418).