Fig. 4. Development and morphology of CMCs, ETCs, and SMCs in CBs.

A Composed bright-field image (18 images) of a CB at day 13 after aggregation. Rhythmically contracting areas are highlighted in magenta. Insets: left, CMC derived from CVPCs; right, primary CMCs from a 2-day-old mouse heart. B CMCs start to contract as single cells or as small aggregates of 1–6 cells. Inset: magnification of a small aggregate of CMCs. Note the typical gray cytoplasm and bright nuclei of CMCs. C Single contracting CMCs proliferate and form patches composed of dozens of cells. D Elongated bundles of discrete CMCs. E Branching networks of CMC bundles. F Indirect immunofluorescence staining of large arrays of CMCs at day 16 with anti-cTnT (green). G Single CMC at day 22 stained for cTnT (red); the arrow indicates myofibrils with a sarcomeric substructure. Nuclei stained with DAPI (blue). H Connexin 43 staining (red) indicating cell-cell interaction between 2 adjacent CMCs. cTnT (green); nuclei (blue). ETCs at day 11 stained with CD31 antibody (red) (I) and with anti-vWF antibody (green) (J). K Epithelial cluster of ETCs at day 17. L Dark-field image of cord-like networks of ETCs forming vascular-like strings at day 36. M SMCs start to form at day 13 and occasionally contract very slowly once they are in a parallel alignment. Brackets indicate elongated nuclei typical for SMCs. N Elongating SMCs stained with anti-SMA specific antibodies (red). Nuclei (blue). O Large clusters of SMCs aligned in vessel-like structures contracted occasionally between days 20 and 25, stained with SMA-specific antibodies (green). Nuclei (blue). B–E, K, L Phase-contrast images. Scale bars = 1 mm (A), 50 μm (B, D, E, M, N), 100 μm (C, F, K, O), 10 μm (G, H), 20 μm (I, J), and 200 μm (L).