Human insulin solution

MCF7 and 293T/LentiX cells were cultivated in DMEM (+GlutaMAX, Thermo Scientific) supplemented with 10% FBS (Sigma) and 1% penicillin–streptomycin (Sigma). T47D culture medium was additionally supplemented with 1 µl/ml human insulin solution (Sigma). Cell lines were tested for mycoplasma contamination by PCR. MCF7, T47D and MCF10A cells were a kind gift from Almut Schulze (University of Würzburg, Germany) and they were authenticated using STR profiling.
Transfections were carried out using Lipofectamine 3000 reagent (Thermo Scientific) or polyethylenimine (PEI, Sigma) with Opti-MEM reduced serum medium (Thermo Scientific). For siRNA transfections, cells were transfected using the Lipofectamine RNAiMAX reagent (Thermo Scientific). siRNAs were purchased from Dharmacon and are listed in Supplementary Table 4.

Reductase activity was assessed by an insulin precipitation assay (Bardwell et al., 1991 (link); Kpadeh et al., 2013 (link)) using human insulin solution (Sigma). Reactions (triplicate) were carried out in 200 μl of 100 mM sodium phosphate buffer, pH 7.0, 131 μM insulin, 1 mM dithiothreitol (DTT), 2 mM EDTA, and 10 μM HP0231m or EcDsbA; reaction mixtures were incubated in a 96-well plate format at room temperature in a Sunrise^TM (Tecan) plate reader. Reactions were started by adding DTT to a final concentration of 1 mM. The changes in the absorbance (A₆₅₀) as a function of time were measured (Collet et al., 2003 (link); Kpadeh et al., 2013 (link)). The results are presented as the average of three independent experiments.

hPSCs were detached with 2 mg/mL collagenase, type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA), for 30 minutes at 37°C, followed by centrifugation at 300 g for 5 minutes, and the suspension was transferred to a fresh bacteriological petri dish. For NCSC generation, EBs were cultured for 4 days in DMEM/F12 containing 20% knockout serum replacement, 1% non‐essential amino acids and 55 μmol/L β‐mercaptoethanol (all from Invitrogen) supplemented with 10 μmol/L SB431542 (Tocris Bioscience, Bristol, UK) and 0.5‐5 μmol/L DMH1 (or DM) (Merck Millipore, Burlington, MA, USA). During differentiation, the medium was changed daily. On day 4, EBs were attached to Matrigel‐coated dishes in NCSC differentiation medium containing 1% N2 supplement (Invitrogen), 20 ng/mL bFGF (CHA Biotech, Pangyo, Korea) and 25 μg/mL human insulin solution (Sigma‐Aldrich) and continued to differentiate for 5 more days with the medium changed every day.

To increase the stiffness of 3D collagen gels, we increased their concentration [34 (link), 35 ]. We utilized the high concentration collagen I solution (Corning 354249) and adjusted the pH using a modification of previously published protocols [37 (link)]. More specifically, we added the desired amount of collagen I so as to get a final collagen concentration of 0.5, 1.0 or 3.0 mg/ml, in a solution containing 10% 10x Minimal Essential Medium, 1% human insulin solution (Sigma-Aldrich), and distilled water. The pH was adjusted to 7.4 by adding 1N NaOH. Cancer cells were added to the collagen solution before it solidified at a concentration of 2.5 x 10⁵ cells/ml [37 (link)] and normal complete culture medium was added on top of the collagen gel containing cells 4h after its solidification. Cells were cultured in 3D collagen gels of 0.5, 1.0 or 3.0 mg/ml for 3 days and were then subjected to gene expression analysis at the mRNA and protein level as specified. In experiments involving RSU-1 silencing, siRNA transfection was performed in traditional 2D culture 1 day prior to embedding cells in the collagen gels. Cells were left to grow in the gels for two more days before being harvested and analyzed for gene expression.

For the enhancement of neural induction properties, MMC-AFSCs and healthy AFSCs were detached by enzymatic treatment, using Accutase (Thermo Fisher Scientific). Cells were plated on laminin-coated plates (Sigma-Aldrich) in a mesenchymal basal medium as described above. The next day, medium was changed to neuronal induction medium, containing DMEM-F12 (Gibco), Neurobasal Medium (Gibco), N-2 Supplement (Gibco), B-27 Supplement (Gibco), human insulin solution (Sigma-Aldrich), L-glutamine, 0.1 mM NEAA (Thermo Fisher Scientific), 2-mercaptoethanol (Thermo Fisher Scientific), basic fibroblast growth factor (bFGF, 20 ng/ml, Lonza), as well as the epithelial growth factor (EGF, 20 ng/ml, Lonza). The medium was refreshed every other day. Cells were cultured for 15 days before processing for immunofluorescence analysis.