Metabolomics Profiling of iPSCs

The generation of the PWB and control iPSC lines from skin biopsies are detailed in our recent study [16 (link)]; these cell lines were maintained and propagated under feeder-free conditions using an Essential 8 Medium on a geltrex-coated plate (ThermoFisher, Waltham, MA, USA). The following iPSC lines were used in this study: PWB_4221_3, PWB_4221_6, PWB_3921_9, PWB_3921_16, control_52521_8, and control_52521_9 [16 (link)]. There were experimental duplications for PWB_4221_3, PWB_3921_9, and control_52521_8, resulting in a total of 9 samples. The iPSCs (~10⁶ cells) were dissociated using StemPro Accutase (ThermoFisher, Waltham, MA, USA); cell pellets were collected and stored at −80 °C until processing. Samples were thawed with the addition of 300 µL of 80% methanol. Samples were vortexed for 60 s, sonicated for 30 min at 4 °C, and incubated at −20 °C for one hour. The samples were then subjected to centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatant (200 µL) was mixed with 5 µL of L-o-Chlorophenylalanine (0.14 mg/mL) for liquid chromatography–mass spectrometry (LC–MS) analysis.