Figure 2.

Long-Term Maintenance of Crestospheres

(A) FOXD3 and SOX10 mRNA expression was maintained throughout the 7-week crestosphere culture, initiated from pooled neural folds (n = 6; shown as average values).

(B–D) In situ hybridization (B) and immunostaining (C, D) reveal high and heterogeneous expression of premigratory neural crest markers in 7-week-old crestospheres.

(E) Sphere forming assay from single cells shows maintenance of self-renewal capacity through 7 weeks of crestosphere culture (week 1, n = 10; weeks 2, 3, and 7, n = 6; week 5, n = 12), with a drop in the proliferation rate after 5 weeks (n = 3).

(F) Secondary sphere formation is maintained throughout 7 weeks of crestosphere culture (n = 6).

(G) FOXD3 mRNA-expressing crestospheres formed from single cells during the primary sphere-forming assay.