Versene edta

BG01V/hOG (Invitrogen – R7799-105) and feeder-free H9 (WiCell Research Institute – Lot WB007–WA09) hESC lines were cultured according to supplier protocols. BG01V/hOG hESCs were expanded on a feeder layer of mitomycin C-inactivated (10 µg/ml, Invitrogen) mouse embryonic fibroblasts (MEFs, Millipore) in hESC medium (DMEM/F12 with 2 mM GLUTAMAX (Invitrogen), 20% (v/v) knockout serum replacement (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 55 µM β-mercaptoethanol (Gibco), 4 ng/ml recombinant human bFGF (Inivitrogen), and 50 µg/ml hygromycin B (Invitrogen)). hESC colonies were passaged by cutting them into uniform pieces (StemPro EZPassage tool, Invitrogen). Following initial expansion, BG01V/hOG hESC were switched to matrigel (Growth Factor Reduced Matrigel, BD Bioscences) and expanded in chemically defined mTeSR1 medium (mTeSR1 Basal Medium with mTeSR1 5X supplement, Stem Cell Technologies). BG01V/hOG hESC were passaged five times to eliminate MEF from the culture. H9 hESC are cultured under a feeder-free condition. Their expansion protocol was the same as that for the feeder-free culture of the BG01V/hOG hESC. H9 colonies were passaged using 0.2 g/L Versene (EDTA) (Lonza). BG01V/hOG and H9 hESC colonies were incubated with Accutase (Innovative Cell Technologies) for 5 min at 37°C to form single cell suspensions.

Human induced pluripotent stem cells (hiPSCs) were a foreskin fibroblast-derived cell line iPS(foreskin)-3 (purchased from WiCell Research Institute, Madison, WI– cat# WB0002) and cultured in chemically defined stem cell medium (mTeSR1 basal medium with mTeSR1 supplement, Stem Cell Technologies, Ontario, Canada) on a Matrigel matrix (BD Biosciences, San Jose, CA). iPSC colonies were passaged using Versene (EDTA) (Lonza, Allendale, NJ) for 8 minutes at room temperature.s

Two control hiPSC lines, WT3 (Ad4) [1 (link)] and WT4 (MJN1), were cultured in mTeSR^™1 (StemCell Technologies, 05850) supplemented with penicillin/streptomycin (P/S) (Gibco, 15140–122) on 6-well plates (Corning, NY) pre-coated with reduced growth-factor Matrigel (BD, 354230). Cell culture medium was changed every day and hiPSCs were passaged every 4–5 days using 0.02% Versene EDTA (Lonza BE17-711E) at a ratio of 1:6. Cells were maintained in a humidified environment at 37 °C with 5% CO₂.

Cells were disassociated using Versene (EDTA) (Lonza), washed with PBS and fixed in paraformaldehyde (PFA; 2% final concentration in PBS) at 37 °C for 10 minutes. After washing with PBS, the cells were permeabilised with pre-chilled methanol (−18 °C) and incubated at 4 °C for 30 minutes, followed by a washing step. 0.2–0.5 × 10⁶ cells were resuspended in a total volume of 200 μl PBS containing 1% BSA and incubated with appropriate amounts of anti-CD44-BV421 (Cat.N.562890, BD Biosciences; 1:300 dilution), anti-TRA-1-60-FITC (Cat. N. FCMAB115F, Merck Millipore; 1:100 dilution) and Phospho-p38 MAPK (Cat. N. 4552, Cell Signaling Technology; 1:50 dilution; Mouse mAb Ig1 Isotype Control Alexa Fluor 647 Conjugate Cat. N.4843, Cell Signaling) monoclonal antibodies for 1 hour on a shaker, plate in the dark at room temperature. Finally, samples were washed using BD FACS™ Lyse Wash Assistant (BD Biosciences) and immediately analysed on a flow cytometer. FACS analysis was performed using BD FACS Canto II flow cytometer with FACSDiva software (BD Biosciences). A minimum of 20,000 events were recorded for each sample. Fluorescence minus One control (for each antibody) was used to gate the subpopulations.
Alkaline Phosphatase detection was performed with Alkaline Phosphotase detection kit (SCR004, Millipore) according to manufacture instructions.

One hESC line (H9, WiCell) one neonatal iPSC line (Neo1, male 38, 39) and one adult iPSC line (AD3, from a 37‐year‐old female 40) were adapted from MEF‐supported to feeder‐free culture conditions on 6 well plates coated with growth factor‐reduced Matrigel basement membrane matrix (GFRM; Corning, New York) in TeSR1 (Stem Cell Technologies, Vancouver, Canada) defined medium. Cells were fed daily and passaged every 5 days at a ratio of up to 1:10 using 0.02% versene EDTA (Lonza, Basel, Switzerland). Cells were then differentiated by initiating EB formation, followed by long‐term culture (up to 150 days). EBs were generated either by mechanical, enzymatic, or dissociation–reaggregation approaches (Fig. 1). Each method was tested in the presence or absence of ROCK inhibitor (Y‐27632, Chemdea, NJ) for the first 48 hours of differentiation, during the early phase of EB formation. All cultures were tested in parallel under either stationary (static, “St”) or shaking (“Sh”) conditions throughout differentiation. Shaking cultures were achieved by placing cells on an orbital shaker (30 rpm, Stuart) housed inside the incubator for the entire period of differentiation, or in the case of EBs generated via the dissociation–reaggregation method, from day 12 onward. Biological replicates were performed in triplicate (n ≥ 3) for all experimental conditions tested.

A human adipose tissue-derived mesenchymal stem cell line (ADSCs; Donor 26508, #000034977, Poietics, Lonza, Basel, Switzerland) was maintained at 37°C, in 90% humidified air with 5% CO₂. SGM consisted of high-glucose (4.5 g/L) DMEM with ultra-glutamine (BioWhittaker, Lonza, Basel, Switzerland), 1% penicillin/streptomycin (BioWhittaker, Lonza, Basel, Switzerland), and 10% FBS (Biochrom, Berlin, Germany). For culture expansion, adherent ADSCs were liberated with 0.5% trypsin (200 mg/L Versene EDTA, #BE171616, Lonza, Belgium) and sub-cultured at a seeding density of 5000 cells/cm². For cryopreservation, ADSCs were stored in liquid N₂ in SGM containing 10% DMSO (Merck, Darmstadt, Germany). All experiments were performed in triplicate on ADSCs between passages five and seven.