Fig 1.

Motor end plate formation in culture. For in vitro visualization, muscle progenitor cells in this Figure are expressing enhanced green fluorescent protein (EGFP: green), although tissue-engineered muscle-polymer implants used for implantation were created with unmodified primary muscle stem cells (MSCs). A) When primary cultures were passaged to prevent myotube (MT) differentiation (as described in Primary Muscle Progenitor Cell Cultures section) and incubated with acetylcholine, agrin, and neuregulin, α-bungarotoxin staining depicted expression of acetylcholine receptors (AChRs) on individual MSC surface (gray). B) To induce multinucleated MT formation (as described for MT group, ie, group with MT-derived muscle–PCL [poly(D,L-lactide-co-ε-caprolactone)] constructs), we incubated cells in nutrient-poor microenvironment (10% horse serum) that induced myoblasts to fuse with one another (arrowheads demarcating contact points between fusing myoblasts). In MEE group (group with motor end plate–expressing muscle–PCL constructs), cells were incubated in MT medium with acetylcholine, agrin, and neuregulin (as described in Methods) to induce AChR formation. Note that whereas AChRs formed on the MSC surface (in A), developing MTs formed mature motor end plates (arrow).