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

## TL;DR

This paper introduces a new method to measure and analyze skeletal muscle cell behavior and quality using a semi-automated workflow for image analysis.

## Contribution

The novel contribution is a validated semi-automated workflow for quantifying migration and fusion behavior in skeletal muscle cells.

## Key findings

- Culture media significantly affect cell fusion dynamics in skeletal muscle cells.
- Optogenetic stimulation increases acetylcholine receptors in myonuclei.
- Primary mouse cells show greater myoblast persistence and earlier cell fusion compared to hiPSC-SMCs.

## Abstract

In vitro 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 in vitro 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.