Igf 1

LCLs were cultured in upright non-adherent T25 tissue culture flasks (Nunc) at 37 °C and 5% CO₂. LCL media consisted of RPMI 1640 (Thermo Fisher, Waltham, MA, USA), supplemented with 1× GlutaMAX™ (Thermo Fisher), and 20% fetal bovine serum (Thermo Fisher). Erythroblasts were enriched in mixed PBL cultures following a previously published protocol [33 (link)]. Briefly, PBLs were cultured in erythroblast media containing StemSpan™ SFEM (Stem Cell Technologies, Vancouver, BC, Canada), 50 ng/mL recombinant human stem cell factor (R&D Systems, Minneapolis, MN, USA), 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL IGF1 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 ng/mL Interleukin 3 (Miltenyi Biotech), 2 U/mL human erythropoietin (R&D Systems), and 10 μg/mL Gentamicin (Thermo Fisher). Magnetic beads labelled with a CD71 antibody were then used to enrich for erythroblasts, following the manufacturer’s instructions (Miltenyi Biotec).

Peripheral blood mononuclear cells (PBMCs) were isolated from a known heterozygous parental carrier of the novel synonymous variant under investigation (Figure 1(k), individual I-2) using Ficoll density centrifugation, and maintenance in PBMC media: Stemspan H3000 (StemCell Technologies) supplemented with 50 ng/ml SCF, 40 ng/ml IGF-1 (Miltenyi), 10 ng/ml IL-3 (ThermoFisher), 2 U/ml EPO (R&D), 50 μg/ml ascorbic acid, and 1 μM dexamethasone (Sigma). Two million PBMCs were nucleofected with episomal reprogramming plasmids (Addgene 27080, 27078 and 27077), which expressed transcription factors L-MYC, LIN28, SOX2, KLF4, and OCT3/4, using the Amaxa-4D nucleofection system, Lonza P3 kit with 1 μg total plasmid amount. Transfected cells were seeded on Geltrex-coated plates containing Essential 8 (E8) medium (Life Technologies) and maintained daily until identifiable hiPSC colonies were observed (2-3 weeks). Suitable hiPSCs were then manually picked and transferred to Geltrex-coated plates for expansion in the E8 medium. The control hiPSC line was derived from normal skin fibroblasts (European Collection of Authenticated Cell Cultures (ECACC)) by the pluripotent stem cell core facility, StemCore (University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia).