The muscularis medium maintains mature smooth muscle cells, ICC, neurons and glial cells.

<p>(<b>A</b>) Immunofluorescence of MHC, c-Kit, β-tubulin III, and GFAP in the serum or muscularis media at d7 and d28. Nuclei (DAPI, blue); F-actin (phalloidin, green). Scale bars, 200 μm. (<b>B</b>-<b>E</b>) Details of cells in the muscularis medium at day 28. (<b>B</b>) Filament bundles in contractile smooth muscle cells (arrow). (<b>C</b>) Multipolar ICC network (left) and dipolar ICC (right, day 21). (<b>D</b>) Ganglia-like neural aggregates (white arrow), thick neurite bundles (black arrow) and neural fibers (white asterisk) are present in the muscularis medium, top left, scale bar, 100 μm; neurites extend over 2,000 μm (left bottom, arrows, scale bars, 500 μm); and different types of neurons (middle and right). (<b>E</b>) Four different types of glial cells (arrows). (<b>F</b>) Close associations of smooth muscle cells, neurons and glial cells (arrows). (<b>G</b>) Serosal mesothelial cells (cytokeratin, red) in the muscularis medium at day 28 (left, middle) and in muscle strips (right). Scale bars, 100 μm.