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A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity
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High content analysis identifies unique morphological features of reprogrammed cardiomyocytes.
Matthew D Sutcliffe1, Philip M Tan1, Antonio Fernandez-Perez2
1Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, 22908, USA.
Scientific Reports
|January 21, 2018
Summary
Direct reprogramming of fibroblasts into cardiomyocytes shows promise for cardiac repair. New image analysis methods reveal variability in induced cells, suggesting current assessments may be insufficient for predicting functionality.
Area of Science:
- Cardiovascular Biology
- Regenerative Medicine
- Biomedical Imaging
Background:
- Direct reprogramming offers a potential strategy for cardiac regeneration by converting fibroblasts into cardiomyocytes.
- Current methods for assessing reprogramming efficiency often rely on transcriptional signatures, which may not fully capture cellular maturity or functionality.
- Optimizing reprogramming protocols necessitates objective, scalable methods to evaluate cardiomyocyte phenotype.
Purpose of the Study:
- To develop and validate automated image analysis techniques for quantifying cardiomyocyte morphology and sarcomere structure.
- To assess the phenotypic variability of induced cardiac-like myocytes (iCLMs) generated through direct reprogramming.
- To compare the characteristics of iCLMs with neonatal mouse cardiomyocytes.
Main Methods:
- Automated segmentation of reprogrammed cardiomyocytes from immunofluorescence images.
- Analysis of cell morphology, including size and shape.
- Quantification of sarcomere structure using Haralick texture features via SarcOmere Texture Analysis (SOTA).
Main Results:
- Induced cardiac-like myocytes (iCLMs) exhibited significant variability in cardiomyocyte marker expression and morphology.
- iCLMs displayed less organized sarcomere structure and reduced sarcomere length compared to neonatal mouse cardiomyocytes.
- Automated image analysis revealed phenotypic heterogeneity not typically observed in vivo.
Conclusions:
- Traditional assessments of cardiomyocyte reprogramming based solely on marker protein induction may be inadequate for predicting functional outcomes.
- Automated image analysis, including SOTA, provides objective metrics for evaluating cellular phenotype and improving reprogramming efficiency.
- These advanced imaging techniques offer a more systematic approach to advancing cardiac regeneration strategies beyond transcriptome profiling.
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