Relesr

iPSCs were cultivated on Matrigel hESC-qualified matrix (Corning, Corning, NY, USA, 354277) coated dishes with daily mTeSR1^TM medium changes (Stemcell Technologies, Vancouver, BC, Canada, 85850) in a cell incubator at 37 °C and 5% CO₂. When 70% confluence was reached, the iPSCs were split into a 1:4 ratio using ReleSR^TM, following the instructions of the manufacturer (Stemcell Technologies, Canada, 100-0484).

All iPSCs cell lines were maintained on hESC-qualified Matrigel (Corning®, NY, USA) in mTeSR^TM1 medium (STEM CELL Technologies, Vancouver, BC, Canada) and passaged using mechanical dissociation at the beginning of the establishment of the cell line. After 7 to 10 passages, cells where passaged using ReLeSR^TM (STEM CELL Technologies) and maintained until the passage 40 for the experiments.

Two iPSC cell lines were used in this study. As described in our previous studies, KSCBi005, a human-induced pluripotent stem cell line from the National Stem Cell Bank of Korea (KSCB, Chung-Ju, Republic of Korea), and PSEN1-mutated patient-derived CS40iFAD-nxx (RRID:CVCL_YX94) iPSCs from the Cedars Sinai Medical Center iPSC Core Facility (CSMC, Los Angeles, CA, USA) were used to generate wild-type and AD brain organoids, respectively. These iPSCs were plated and cultured according to their manufacturers’ cell culture manuals. In short, KSCBi005 cells were cultured on fibronectin (Corning, Corning, NY, USA)-coated plates using Essential 8 medium (Gibco, Grand Island, NY, USA), and CS40iFAD-nxx cells were cultured on GFR-Matrigel (Corning, NY, USA)-coated plates in mTeSR^TM Plus medium (STEMCELL Technologies, Cambridge, MA, USA). Each cell line was passaged every 4–5 days, depending on the confluency of the cells, using ReLeSR^TM (STEMCELL Technologies, Cambridge, MA, USA).

Human iPSCs, including P72, R72, and p53 null cells, were established and maintained as previously reported [17 (link)]. Human fibroblasts were reprogrammed using episomal vectors to express Oct4, Sox2, Klf4, Lin28, and L-myc using the Neon Transfection System (Invitrogen, Carlsbad, CA, USA). Human iPSCs were cultured in mTeSR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) on a Matrigel-coated cell culture dish or feeder layer. ReLeSR^TM (STEMCELL Technologies) was used to passage hiPSCs.

Wild-type BJ fibroblast-derived iPSCs (BJ-iPS), patient-derived MELAS iPSCs and corrected MELAS isogenic control iPSCs (c-MELAS) were maintained feeder-free on Matrigel-coated plates in StemMACS^TM iPS-Brew media (Miltenyi Biotec). Routine passaging of iPSCs was performed using ReLeSR^TM (STEMCELL Technologies) at 1:6 split ratio every week. Pluripotent stem cells were differentiated towards spinal motor neurons following established protocol published previously^{3 (link)}. To generate spinal organoids, iPSCs colonies were first dissociated into single cells and seeded at 30,000 cells per well in a 96-well low attachment round-bottom plate. At day 10 of differentiation, embryoid bodies were then encapsulated in 15 μl Matrigel droplets before maturing them in spinner flask at day 14. Organoids were routinely harvested at days 21, 28, 25 and 42 for downstream analysis.
Deep sequencing of the entire mtDNA genome was performed for MELAS and c-MELAS iPSCs previously^{1 (link)} to confirm the heteroplasmic levels of the A3243G mutation. No other deleterious mutations were found. Subsequently, heteroplasmy was determined for MELAS and c-MELAS iPSCs every 5–6 passages using the restriction fragment length polymorphism (RFLP) method as described previously^{1 (link)}.

Human ES cell line H1 cells and stromal cell line OP9 cells were used in this study. Both cell lines were purchased from ATCC. H1 cells were cultured in mTeSRTM1 (Stemcell #85850) medium using Corning^® Matrigel^® hESC‐Qualified Matrix (StemCell #07181) as the surface coating matrix. The medium was changed daily. Cells were passaged using ReLeSRTM (StemCell #05872) every 4–5 days. OP9 cells were cultured in α‐MEM (Gibco #12571) with 20% FBS (BI #04002), passaged every 2–3 days. Undifferentiated H1 cells were seeded onto irradiated OP9 cells and cultured in hESCs maintenance medium.²⁸, ²⁹ Three days later, medium was replaced by haematopoiesis‐inducing medium.²⁸, ²⁹ H1 cells were co‐cultured with OP9 cells for 15 days. On Day 15, the cells were stained with PE‐conjugated CD34 and APC‐conjugated CD71 at 4℃ in the dark. The CD34⁺CD71⁻ hematopoietic stem cells were sorted using BD FACSAria fusion Cell Sorter.

Human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs)—were maintained in TeSRTM-E8TM medium (STEMCELL Technologies). For passaging hPSCs, ReLeSRTM (STEMCELL Technologies) was used according to the manufacturer's instructions.