Derivation of iPSC-Derived Notochordal Cells

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.