Skeletal muscle derived cells (SMDC) hold tremendous potential for replenishing dysfunctional muscle associated with incontinence. Autologous SMDC-based therapy for faecal incontinence (FI) is reported to be safe, but the level of efficacy reported in previous studies suggests that heterogeneity in the proposed therapy may contribute to inconsistent clinical outcomes. 

Improved methods for patient stratification to identify patients who are (un)likely to respond well to regenerative therapies are advocated to ensure the potential benefits of cell-based regenerative medicine can be fully exploited. Predicting the therapeutic potency of SMDC in vitro prior to implantation will facilitate development of successful therapeutics in regenerative medicine and reduce implementation costs. 

Here, we report on the development of a novel SMDC profiling tool to examine populations of cells in vitro derived from different donors. The primary aim of the study was to evaluate the applicability of multi-parametric imaging-based phenotypic characterization to distinguish myogenic potency of SMDC. Heterogeneity in the formation of myotubes from different donors was correlated with cell shape descriptors.

Commercially available human SMDC (Cook MyoSite) consisting of non-differentiated primary human muscle cells were derived from 14 individual human donors. Image acquisition of SMDC growth was performed using a CytoSMART Lux2 microscope. Images were acquired every 5 minutes over a period of 72 hours. CellProfiler software was used to create an image analysis pipeline for analysis of different cell shape characteristics during 2 hours of tracking at 12- and 24-hours post-culture. The Konstanz Information Miner (KNIME; open-source data analytics) software was used to calculate the mean and SD of the characteristic for all the objects identified in one image. These data were then used for statistical correlation with the fusion index calculated from myotube formation assays established in the same cultures after 5 days of subsequent incubation in differentiation medium. The fusion index was calculated as the ratio of nuclei number in myocytes with 2 or more nuclei divided by the total number of nuclei. Correlation studies used the Spearman test with r < -0.5 or > +0.5 indicating correlation.

Several early cell shape characteristics were found to negatively correlate with the fusion index. Images collected at 12 hours after initiating culture revealed that 5 cell shape characteristics negatively correlated with the fusion index, including the total area occupied by cells (r=-0.815, p=0.001), area shape (r=-0.631; p=0.028), bounding box area (r=-0.576; p=0.049), minimum ferret diameter (r=-0.589; p=0.044) and minor axis length (r=-0.614; p=0.034).  Images collected at 24 hours after initiating revealed 8 cell shape characteristics that negatively correlated with the fusion index. These included: total area occupied by cells (r=-0.686; p=0.007), area shape (r=-0.709; p=0.005), bounding box area (r=-0.563; p=0.036), compactness (r=-0.534; p=0.049), equivalent diameter (r=-0.576; p=0.031), minimum ferret diameter (r=-0.620; p=0.018), minor axis length (r=-0.590; p=0.026) and perimeter (r=-0.654; p=0.011). There was a high correlation between all these characteristics at 24 hours of imaging after initiating culture.

Results indicate that monitoring of cell shape during the early stages of bioprocessing using real-time imaging could be used to predict cellular competency necessary for differentiation and myofibre formation in vivo.

Further studies could establish whether selection of either patients or cell populations on this basis have a higher probability of yielding better outcomes in cell-based therapy for incontinence.

Continence 2S2 (2022) 100466
DOI: 10.1016/j.cont.2022.100466