Mtesrtm1 medium

The human iPSC WTC cells (passage 50 to 60) were cultured in 6-well plates coated with 1% Geltrex (Thermo Fisher Scientific, MA, USA) using mTeSR^TM1 medium (Stem Cell Technologies, Vancouver, Canada) with daily medium replacement. Passage was conducted by incubating cells with ReLeSR ^® reagent (Stem Cell Technologies, Vancouver, Canada) for 4 min at room temperature, following wash with PBS solution, when cells reached 80–90% confluency. Cells were collected by pipetting with culture medium and plated at a 1:3 ratio on new Geltrex-coated 6-well plates, allowing them to grow to 90–100% confluence. Cells were cultured with mTeSR^TM1 medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 10 µM Rho-associated kinase (ROCK) inhibitor Y-27632 (Tocris Bioscience, Bristol, UK) to enhance cell survival. After 24 h, the medium was replaced with mTeSR^TM1 medium without the ROCK inhibitor.

For the dual SMAD and CHIR germ lineage differentiations, 100 cell mixed colonies were generated as previously described, cultured in mTeSR^TM-1 medium (STEMCELL Technologies), and allowed to form patterns for 5 days in pluripotency maintenance conditions. After 5 days, the media was supplemented with either SB 435142 (10μM; Stemgent) and LDN 193189 (0.2μM; Sigma-Aldrich) or CHIR 99021 (12μM; Selleckchem). CHIR was pulsed for 24 hr periods on the first and fourth day of the mesendoderm directed differentiation. Dual SMAD inhibition was kept constant for 6 days by supplementing SB 435142 and LDN 193189 into MTeSR media to direct germ lineage to an ectodermal fate. After 6 days of differentiation, colonies were washed 3X with PBS and fixed for staining with 4% paraformaldehyde (VWR) as previously described.

Calu-3 cells derived from the pleural effusion of a 25 years old patient with a lung adenocarcinoma [48 (link)] and VeroE6 cells, immortalized African green kidney epithelial cells [49 ], (both provided by Prof. Robert Rieben, Department of Biomedical Research (DBMR), University of Bern) were grown in Dulbecco’s modified Eagle Medium (DMEM)–GlutaMAX™, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 mg/mL streptomycin, 100 IU/mL penicillin, 1% (w/v) non-essential amino acids and 15 mM HEPES (all Gibco). HIBCPP (a cell line obtained from a choroid plexus papilloma [47 (link)]) were maintained in DMEM/F12, 10% v/v heat-inactivated FBS (Gibco), 5 µg/mL human insulin (Sigma Aldrich), 4mM L-Glutamine (Gibco) and 100 mg/mL streptomycin, 100 IU/mL penicillin (HIBCPP-10 medium). Human induced pluripotent stem cells (hiPSCs) were generated from erythroblasts reprogrammed through nucleofection with plasmids encoding for OCT4, shRNAp53, SOX2, KLF4, L-Myc, and Lin28 [50 (link)]. In this study 2 iPSC clones per donor were used: LNISi001-A/B, LNISi002-A/B, LNISi003-A/B, LNISi004-A/B, with the following age/sex: 30/female, 50/male, 49/female, 33/female. HiPSCs were maintained on growth factor reduced Matrigel^® (Corning) or growth factor reduced and stem cell certified Cultrex™ (R&D) coated plates in mTeSR^TM1 medium (STEMCELL Technologies) [42 (link), 51 (link)].