Nucleofection technology

The derivation of iPSC-derived notochordal cells (iNCs) from iPSCs was performed using our 3-step protocol. During Step 1, the iPSCs were differentiated into Primitive Streak Mesoderm (PSM) cells via a 3-day exposure to 5µM GSK3 inhibitor (Millipore, Billerica, MA) in a manner similarly used in previously published studies 88 (link), 89 . The media was replaced every 24 hours supplemented with fresh 5µM GSK3 reconstituted in Dimethyl sulfoxide (DMSO). During Step 2, the GSK3i-treated cells were transfected using commercially available Nucleofection technology (Lonza, Basel, Switzerland) with human Brachyury-encoding pCMV6-AC-GFP vector plasmid (OriGene, Rockville, MD) and cultured for 2 days in Advanced-RPMI medium. The transfection efficiency was validated using flow cytometry to GFP+ cells, and transfection efficiency over 70% was considered successful. During Step 3, the cells were encapsulated in TF hydrogel as previously described 52 (link), grown in hypoxic conditions (2% O₂) for maturation in vitro, and harvested for RNA isolation and gene expression analysis or for vibro-sectioning and immunofluorescence staining.

Adherent cells (MCF7, HeLa, HepG2, HCT-116 and HCT-116 p53^−/−) were cultured in Dulbecco modified essential medium (DMEM) media. Suspension cells (K562, KG-1, HEL and U937) were cultured in RPMI 1640 media. Both culture media were supplemented with 10% (v/v) Fetal Bovine Serum, 1% (v/v) L- Glutamine, penicillin-streptomycin, and 1% (v/v) sodium pyruvate. Cells were cultured under humidified condition at 37°C in a 5% CO incubator. The miRNA transfections and negative control pBabe transfections were performed using nucleofection technology according to the manufacturer's instructions (Lonza). The miRNA sequence is listed in Supplementary Table S1.

Human glioblastoma U251 MG (U251) and U87 MG (U87) cell lines were purchased from CLS Cell Lines Service (Germany) and monitored for correct genetic profile via microsatellite analyses according to a protocol described earlier (Peickert et al. 2012 (link)). LN-229 glioblastoma cells were from Prof. Marta Miączyńska from the International Institute of Molecular and Cell Biology in Warsaw, Poland. Cells were cultured at 37 °C and 5 % CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with GlutaMAX-1, 10 % fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco Live Technologies, USA). For most experiments, a formulated amino acid-free DMEM (Sigma-Aldrich, USA) was used. In order to prepare arginine- (-Arg) or lysine-free (-Lys) conditions, the medium was supplemented with all the amino acids except of arginine or lysine, respectively. A complete (control) medium was obtained by supplementation with all the amino acids (0.4 mM for arginine, and 0.8 mM for lysine and leucine). -Arg, -Lys and control media were supplemented with 5 % dialyzed FBS (Sigma-Aldrich, USA), lacking small molecules such as amino acids. U251 cells cultivated in the control medium were transfected with pmaxGFP plasmid using Nucleofection™ technology (Lonza Group Ltd., Switzerland).