CD34+ bulge McSCs exhibit distinct neural crest lineage potential.

(A) Heatmap of scaled and clustered, variance stabilizing transformation (VST) read count values obtained for select genes from RNA-seq analysis of CD34+ and CD34- McSCs. In bulge and SHG McSCs, the melanogenic marker (red) and NCSC marker (blue) are differentially expressed, closely clustered and are statistically significant. (Padjusted < 0.02, Benjamini-Hochberg adjusted p value) (B) RNA-Seq analysis, based on fold change, showed that CD34+GFP+ McSCs (blue; left panel) express higher NCSC markers, whereas CD34-GFP+ McSCs (red; right panel) express higher melanogenic markers. (C) Formation of non-adherent spheroids was studied among CD34+GFP+ (bulge) and CD34-GFP+ (SHG) McSCs when cultured in NCSC medium (top panels). The image in bottom panel depicts retention of GFP expression in spheroids formed by both cell types. The top panel also shows the percentage of cells initially plated that formed spheroids from three independent experiments. (N = 3, standard deviation reported) (D) The size of non-adherent spheroids derived from bulge and SHG McSCs when cultured in NCSC medium as determined at days 2, 4, 6 and 8. (*P ≤ 0.01, **P Value ≤ 0.05 by ANOVA; N = 5). (E) Expression of neural crest lineage markers Gfap, α-Sma, Tuj1, Krt15 among CD34+GFP+ (top panel) and CD34-GFP+ (bottom panel) McSCs following adherent culture in neural crest differentiation medium. Scale bars: 50 μm. (F) Expression of melanocyte lineage marker Tyrp1 among CD34+GFP+ (top panel) and CD34-GFP+ (bottom panel) McSCs following adherent culture in melanocyte differentiation medium. In right two panels, brightfield images show pigmented melanocytes among cultured CD34+GFP+ (top) and CD34-GFP+ (bottom) McSCs. Scale bars: 50 μm. (G) Quantification of neural crest-derived cell and melanocyte marker expression frequency after cells were cultured in either neural crest differentiation (left) or melanocyte differentiation (right) condition. For this experiment, all cells in each well were counted and this graph is a representative of 3 independent experiments.