Figure 1.

Neural crest differentiation protocol and cartilage organoids

(A) Neural crest stem cell (NCSC) differentiation protocol. hESCs were differentiated using the stepwise replacement of knockout serum replacement (KSR) media with N2 for 5 days, followed by the addition of NCSC specification media containing BDNF, FGF8, SHH, and ROCKi. Small molecule inhibitors were added to guide differentiation (see STAR Methods). NCSCs and organoid-producing cells were maintained in N2 media containing FGF2 and EGF.

(B) Representative light microscopy images of cell morphology throughout differentiation and after prolonged culture. Day = number of days after the original differentiation protocol. Organoids continued to spontaneously form in cell culture dishes containing differentiated NCSCs. Three images at day 68 show highly plastic morphology of cells as they migrate individually and begin to assemble into an organoid.

(C) Histology and stain of human ear tissue (top) and representative organoid (bottom). H&E, hematoxylin and eosin. Saffranin O and Toluidine Blue stain cartilage ECM components red-orange and purple, respectively; Weigert’s resorcin-fuchsin stains elastic fibers violet. (N = 5). Scale bar = 50 μm.