Fig 2: Surface marker expression changes in PSC-derived neural crest development. (a) Flow cytometric characterization of surface markers across four different stages of NCR differentiation (Passages 0, 3, 6 and 9) shows the temporal changes in surface marker expression as the cells progress along NCR differentiation. Error bars represent arithmetic mean ± SD (n = 3; CD90high at P9 n = 2). (b) Figure depicts the arbitrary threshold (indicated by red gate line) set and consistently applied across the various passages of NCR differentiation to identify CD29dhigh, CD44high, CD73high and CD90high cells. (c) Heatmap representation of CD antigen levels (percentage of expression) in relation to established intracellular NCR and EMT protein expression (lines of squares indicating passage no. at P0, P3, P6, P9; also see Fig. 2e and Suppl. Fig. 2), illustrating a critical transition stage around P3 and association of CD markers with either NSC (left, top) or post-EMT (right, bottom) phenotypes. (d) Time-dependent correlation of percentage of CD57 versus CD51/CD61 expression underlining a gradual decrease of HNK1 accompanied by a gain of CD51/61 expression when focusing on P3 to P6 as a critical transition period (left panel), with the clearest drop occuring between P3 to P4 (right panel). (e) Spearman correlation matrix of flow cytometric expression data across NCR differentiation (percentage positivity at P0, P3, P4, P5, P6, P9) highlights the largest negative correlation of surface antigen expression of CD57 vs. CD51/61 (–0.89; see Suppl. Table 1) over the course of NCR EMT