Generating NGLY1-deficiency hPSC Models

WA09-C3, -C4, and -C6 human embryonic stem cells (hESCs) were generated through CRISPR-Cas9-mediated editing of the NGLY1 gene in WA09 hESCs followed by single-cell cloning [16 (link)]. Human-induced pluripotent stem cells (hiPSCs) were established from NGLY1-deficiency patients’ fibroblasts (GM25990 and GM26607; Coriell Institute for Medical Research) using CytoTune Sendai Reprogramming Kit (Thermo Fisher Scientific). Except using TeSR-E8 medium (Stemcell Technologies) and 2 mM EDTA passaging solution (Thermo Fisher Scientific), we generally followed the reported method [31 (link)] to culture undifferentiated hPSCs in a feeder cell-free condition. All the hPSCs were routinely subcultured when cell density reached 80% to 90%. The passage numbers of the hESCs and hiPSCs used in our studies spanned across 65–95 and 35–65, respectively. Additional information relevant to the cells was summarized in Table S1. The experiments using hPSCs were performed in compliance with the guidelines and approval of the institutional biosafety committee and embryonic stem cell research oversight committee. All cells were periodically tested using the MycoAlert mycoplasma detection kit (Lonza) and free of mycoplasma.