Mtesr media

H9 hESCs were maintained under feeder-free conditions in mTeSR media (STEMCELL Technologies). Media was changed every day. When cell density reached 70%–80% confluence, colonies were dissociated using accutase (STEMCELL Technologies) and plated onto Matrigel (BD Biosciences)-coated plates at a 1:6 dilution. During passaging, the media was supplemented with 2 μM thiazovivin overnight. For hESC-iN formation, dissociated single cells were plated at a density of ∼2.5 × 10⁵ cells per 35 mm² well. Lentivirus infections (with an additional EGFP-expressing virus) and transgene induction were performed similarly to as described for the fibroblast-iN production, using N3 media. Puromycin selection continued from day 2–6 postinfection, with media changes every other day. On day 7, cells were dissociated into single cells using PBS-EDTA (0.5 mM) and seeded onto mouse glia. The next day, media was replaced with Neurobasal media (Neurobasal [Invitrogen], L-glut [Invitrogen], B27 [Invitrogen], penicillin/streptomycin [Invitrogen], doxycycline [2 μg/ml], BDNF [10 ng/ml] [PeproTech], GDNF [20 ng/ml] [PeproTech], and Ara-C). Media was half-exchanged every 3–4 days.

Sex chromosomal mosaicism in starting fibroblasts (partial 45,X recorded in Coriell Biorepository) was confirmed by interphase DNA FISH using sex centromere (DXZ1/DYZ3) probes (WiCell). Fibroblasts were reprogrammed to hiPSCs using the CytoTune iPSC 2.0 Sendai Reprogramming kit (Thermo Fisher Scientific) and maintained by weekly mechanical passaging, as described (26 (link)). Prior to BAP differentiation, hiPSCs were transitioned to mTeSR media (Stem Cell Technologies), grown on extracellular matrix (Geltrex, Thermo Fisher), and passaged weekly with ethylenediaminetetraacetic acid. Standard IF was performed for pluripotency markers and H3K27me3, as well as FISH and qRT-PCR for XIST (SI Appendix, SI Methods include details).

All human pluripotent stem cell studies were carried out in accordance with consent from the University of Queensland’s Institutional Human Research Ethics approval (HREC#: 2015001434). hiPSCs were maintained in mTeSR media (Stem Cell Technologies, Cat.#05850). Unless otherwise specified, cardiomyocyte directed differentiation using a monolayer platform was performed with a modified protocol based on previous reports(Burridge et al., 2014 (link)). On day −1 of differentiation, hPSCs were dissociated using 0.5% EDTA, plated into vitronectin coated plates at a density of 1.8 × 10⁵ cells/cm², and cultured overnight in mTeSR media. Differentiation was induced on day 0 by first washing with PBS, then changing the culture media to RPMI (ThermoFisher, Cat.#11875119) containing 3μM CHIR99021 (Stem Cell Technologies, Cat.#72054), 500μg/mL BSA (Sigma Aldrich, Cat.#A9418), and 213μg/mL ascorbic acid (Sigma Aldrich, Cat.#A8960). After 3 days of culture, the media was replaced with RPMI containing 500μg/mL BSA, 213μg/mL ascorbic acid, and 5μM Xav-939 (Stem Cell Technologies, Cat.#72674). On day 5, the media was exchanged for RPMI containing 500μg/mL BSA, and 213μg/mL ascorbic acid without supplemental cytokines. From day 7 onwards, the cultures were fed every 2 days with RPMI plus 1x B27 supplement plus insulin (Life Technologies Australia, Cat.#17504001).