Mtesrtm1

Patient-derived AD hiPSCs sourced from a male patient with an APP gene mutation causing fAD (ADAPP) and hiPSCs from an age-matched healthy female (HN1) were cultured in mTeSR^TM1 (STEMCELL Technologies cat no. 85850) on cell culture plates coated with Corning®Matrigel®. The hiPSCs were then passaged onto 6-well plates coated with 50 µg/mL of poly-L-ornithine (PLO, cat no. P4957, Sigma, St. Louis, MO, USA) and 10 µg/mL of laminin (cat no. L2020, Sigma, St. Louis, MO, USA). Neural induction was performed using a monolayer culture protocol and a STEMdiff™ SMADi Neural Induction Kit (STEMCELL Technologies cat no. 08581). Briefly, after the hiPSCs were plated as single cells onto 6-well plates, they were cultured for seven days at 37 °C and 5% CO₂ with daily media changes performed using STEMdiff™ Neural Induction Medium + SMADI. On day seven the cells were again passaged onto 6-well plates and daily media changes were performed with STEMdiff™ Neural Induction Medium + SMADI. This continued until passage three on day twenty-one when the cells were ready for expansion.

Cells were grown at 37 °C in a humidified environment under hypoxic conditions (5% O₂, 5% CO₂, and 90% N₂). Cells were cultured in mTeSR^TM1 (Stemcell Technologies, Cologne, Germany) on Matrigel^TM-coated 6-well plates (VWR, Darmstadt, Germany; 0.083 mg/well) in 1 mL Knockout^TM DMEM (Thermo Fisher Scientific, Bremen, Germany). For passaging, cells were treated with 1 mg/mL collagenase IV (Thermo Fisher Scientific) in Knockout DMEM for 5–7 min at 37 °C, followed by rinsing once in Knockout DMEM, and subsequently mechanical dissociation and seeding at a 1:10–1:100 split ratio with daily medium replacement.

Three different human episomal hiPSC cell lines were used for this study. One of the lines was a donation from Columbia Stem Cell Initiative [23 (link)], which was derived from dermal fibroblasts from a heathy male. Commercially purchased lines from Gibco (Cat. No. A18945) and ATCC (Cat No. ACS-1024) were derived from the CD34 + cord blood cells from a healthy female and the CD34 + bone marrow cells from a healthy male, respectively. hiPSCs were cultured in mTeSRTM1 (StemCell Technologies, Cat. No. 85,850) media. For regular maintenance, cells were sub-cultured every 3 to 4 days in Geltrex-coated 6-well plates. The growth chamber was maintained at 37°C with 5% CO2. Regular testing for mycoplasma contamination was performed.

GENEA002 and GENEA016 cell lines were obtained from Genea Biocells Ltd. (Sydney, Australia) and were previously described in [19 (link),20 (link)]. The ATCC-BXS0116 hiPSC line was used in this study (ATCC catalog number ACS-1030). The H9 cell line was obtained from the WiCell institute.
GENEA016, GENEA002, H9 hESC and ATCC-hiPSC were cultured on Matrigel^® (Corning) coated plates in mTeSR^TM1 (StemCell Technologies), Vancouver, BC, Canada supplemented with 0.5% penicillin–streptomycin (ThermoFisher Scientific, Waltham, MA, USA). Cells were grown in a 37 °C incubator with 10% O₂ and 5% CO₂ and passaged every 3–4 days as needed.

iPSCs (WTC with dCas9-KRAB, AICS90) and hESCs (H1 line, WAe001-A) were maintained under feeder-free conditions on Matrigel-coated plates (#354277, BD Biosciences) in mTeSR^TM1, (#05871/05852, StemCell Technologies Inc.). iPSCs were routinely passaged with acctuase (# 07920, StemCell Technologies Inc.), while hESCs were routinely passaged with collagenase (#07909, StemCell Technologies Inc.). Cells were routinely tested for mycoplasma using the MycoAlert Mycoplasma Detection Kit from Lonza (LT07-118).

For induction of hepatocytes, liver progenitor cells were collected and seeded onto collagen type I-coated plates at 2 × 10⁴ cells/cm². The cells were cultured for 2 days in SHM medium and the medium was supplemented with 1 μM Dexamethasone (Dex) (Sigma) and 20 ng/ml human Oncostatin M (PeproTech) for 6 days. For induction of biliary epithelial cells, the liver progenitor cells were isolated and co-cultured with pre-inoculated low density mouse embryonic fibroblasts (passage 2). The mouse embryonic fibroblasts were isolated from E13.5 C57BL/6 J mouse embryos and cultured in DMEM high-glucose media (Gibco) supplemented with 10% FBS (Ausbian). The medium was replaced with mTeSRTM1 (STEMCELL Technologies) containing YAC (mTeSR1+YAC), and on day 6, the medium was replaced with mTeSR1+YAC supplemented with 2% Matrigel (Corning) for another 6 days. All media were changed every other day.