Quantitative measurement of morphometric indicators of skeletal muscle cell behaviour and quality

<i>In vitro</i> culturing of effective human-induced pluripotent stem cell-derived skeletal muscle cells (hiPSC-SMCs) has proven to be challenging. Progress is hindered by the limited range of metrics applied to assess experimental success. We present a semi-automated workflow for segmenting, tracking and quantifying migration and fusion behaviour in live and static images of myoblast and myotube cells. Workflow outputs are validated against manually labelled images and the metrics applied to images from case studies of <i>in vitro</i> cultures of primary mouse muscle cells under varying culture media conditions, mouse primary cells undergoing optogenetic stimulation and hiPSC-SMC. We show culture media-dependent differences in cell fusion dynamics and increased acetylcholine receptors in myonuclei under optogenetic stimulation. We show that myoblasts have greater persistence and proliferation in primary mouse cells than hiPSC, and cell-cell fusion occurred earlier but at a steadier rate in primary mouse cells.