Figure 3. Foxa2+ CM cells can be identified and characterized during mESC differentiation.
(a) Schematic of mESC in vitro differentiation protocols to the cardiovascular and endoderm lineages. Mesoderm cells are generated through addition of Bmp4 and Activin A, whereas endoderm cells are generated through addition of high levels of Activin A. CM is specified to the cardiomyocyte lineage through addition of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and XAV. (b,c) Fluorescence-activated cell sorting (FACS) strategies for the isolation of Foxa2+ and Foxa2− CM (green and teal gates, respectively), Kdrhigh haematopoietic mesoderm (purple gate) or Foxa2+ endoderm (red gate) at day 5 of differentiation. (d) Chord plot showing a selection of genes upregulated in Foxa2+ CM (over Foxa2− CM) present in the represented enriched GO terms. Outer ring shows log2 fold change (left, key at upper right) or GO term grouping (right, key below). Chords connect gene names with GO term groups. (e,f) IF analysis of differentiated cardiomyocytes generated from Foxa2+ (e) or Foxa2− (f) CM showing expression of cTnT and Mlc2v. Images show typical cells generated in each condition. (g) Quantification of experiments in (e/f) (n=3 differentiations, error bars reflect s.e.m.).