For Research Use Only. Not for Use in Diagnostic Procedures.
Imaging and Analysis of Stem Cells and Stem Cell-Derived Cardiomyocytes, Dr James Smith, University of Nottingham, Nottingham
In the Denning Laboratory within the Department of Stem Cell Biology, the PerkinElmer Operetta system is used in stem-cell analysis and for the investigation of early differentiation markers and fully differentiated populations. A collaborative project is currently ongoing to develop metrics and quality standards for the scale-up of human induced pluripotent stem cells (iPSCs), which involves staining for specific early differentiation markers at specific time points and quantification of these.
A differentiation protocol is routinely used to turn iPSCs into cardiomyocytes. Firstly, new developed tissue is dissociated and stained for cardiac-specific markers and the percentage purity of the differentiated populations is analysed. A difference is clearly visible between the nuclei of the positive and negative populations within the differentiated cells, with the positive nuclei appearing much smaller and rounder.
Research is ongoing to mature cardiomyocytes to make them more physiologically relevant, e.g. for use in drug screens and modelling of specific cardiac diseases. The Operetta is used by the researchers to track the characteristics of cardiomyocytes from human pluripotent stem cells and compare them with those of adult cardiomyocytes. The two are very different, with the former demonstrating a disorganised sarcomeric structure, while the adult cells show a highly organized and aligned structure.
Multinucleation of cardiomyocytes is associated with maturation and diseases such as hypertrophy. Specific mutations are introduced into stem cells using Cas9/clustered regularly interspaced short palindromic repeats (CRISPR) technology. These are differentiated into cardiomyocytes and a range of parameters including multinucleation are tracked. It is possible to determine the number of mono-, bi- and multinucleated cardiomyocytes and relate these data to specific mutations and drug-screening applications. In disease modelling, cardiomyocyte action potential is measured using calcium fluorescence, with the PerkinElmer Envision multimode plate reader used to monitor calcium kinetics.