The Myotube Analyzer: how to assess myogenic features in muscle stem cells

Abstract

Background The analysis of in vitro cultures of human adult muscle stem cells obtained from biopsies delineates the potential of skeletal muscles and may help to understand altered muscle morphology in patients. In these analyses, the fusion index is a commonly used quantitative metric to assess the myogenic potency of the muscle stem cells. Since the fusion index only partly describes myogenic potency, we developed the Myotube Analyzer tool, which combines the definition of the fusion index with extra features of myonuclei and myotubes obtained from satellite cell cultures. Results The software contains image adjustment and mask editing functions for preprocessing and semi-automatic segmentation, while other functions can be used to determine the features of nuclei and myotubes. The fusion index and a set of five novel parameters were tested for reliability and validity in a comparison between satellite cell cultures from children with cerebral palsy and typically developing children. These novel parameters quantified extra nucleus and myotube properties and can be used to describe nucleus clustering and myotube shape. Two analyzers who were trained in cell culture defined all parameters using the Myotube Analyzer app. Out of the six parameters, five had good reliability reflected by good intra-class correlation coefficients (> 0.75). Children with cerebral palsy were significantly different from the typically developing children (p < 0.05) for five parameters, and for three of the six parameters, these differences exceeded the minimal detectable differences. Conclusions The Myotube Analyzer can be used for the analysis of fixed differentiated myoblast cultures with nuclear and MyHC staining. The app can calculate the fusion index, an already existing parameter, but also provides multiple new parameters to comprehensively describe myogenic potential in its output. The raw data used to determine these parameters are also available in the output. The parameters calculated by the tool can be used to detect differences between cultures from children with cerebral palsy and typically developing children. Since the program is open source, users can customize it to fit their own analysis requirements.

Generation of Mature <em>Toxoplasma gondii</em> Bradyzoites in Human Immortalized Myogenic KD3 Cells

“…Optional: Confirm cell fusion by appropriate staining protocols (e.g., immunofluorescence staining for myosin heavy chain and nuclei) and calculate myogenic index ( Noë et al, 2022 ).…”

Hypoxia promotes proliferation and inhibits myogenesis in broiler satellite cells

“…Cell cycle status of SC was classified by counting nuclei expressing Pax7- or MyoD-positive or both using ImageJ (imagej.nih.gov). Fusion index, myotube coverage, and number of myotubes in MF20 images were measured using Myotube Analyzer, using guidelines previously described ( Noë et al, 2022 ). The fusion index was determined by counting the number of myonuclei within myosin heavy chain-positive myotubes divided by the total number of myonuclei.…”

Morphological and Molecular Responses of Lateolabrax maculatus Skeletal Muscle Cells to Different Temperatures

“…Images were taken under a fluorescence microscope (ECHO, Chicago, America). NFI was calculated manually as a ratio of the number of nuclei inside myotubes to the number of total nuclei after myotube formation at 21 °C, 25 °C, and 28 °C, as previously described [ 93 ]. Three microscopic vision fields of each well were randomly selected to calculate the myotube NFI, and the experiment was repeated three times.…”