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Browsing by Subject "cardiac differentiation"

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  • Tepsell, Juhani (2018)
    During and after myocardial infarction, millions to a billion cells die off. Scar tissue formed by fibroblasts replaces the injured myocardium during recovery. While the newly formed tissue is durable and prevents rupture of the heart, it doesn´t contribute to pump function. Depending on the extent of cardiomyocyte loss, the remaining functional myocardium get strained. Adult mammalian heart has inadequate capacity to regenerate after such injury. In case of sustained substantial increase in workload, the compensatory mechanisms turn into pathological processes including excessive fibrosis and myocyte apoptosis. The progressive decline of hearts contractile function results in heart failure (HF). Current drug treatments for managing HF aim to prevent progression of the disease and relieve symptoms. ACE inhibitors, beta blockers and diuretics are effective along with healthy lifestyle. No practical treatments are available to restore cardiac function yet. Human myocardium normally regenerates, but only 1% or less of myocytes get replaced yearly. Heart’s resident stem/progenitor cells (CPCs) likely play a role in the turnover. The aim of this study was to develop a screening method to identify small molecules that possibly promote differentiation of cardiac progenitor cells to cardiomyocytes. Cell population differentiated from mouse embryonic stem cells (mESCs) was used as a model for CPCs. Directed differentiation protocol of mESCs used here promotes commitment to cells of cardiac mesoderm, part of which will further differentiate to cardiac progenitors. The resulting population at day 6 is heterogenous but many of these cells are progenitors that turn into cardiomyocytes (CMs) by day 8. 10 000 cells per well are plated on 384 well plates at day 5. Test compounds are added at day 6 and removed day 8 for effect in progenitors and day 7-9 for effect in early cardiomyocytes. 0,1% DMSO is used as vehicle and Wnt pathway inhibitor XAV939 as positive control. The effects are quantified with plate reader on day 9. E14 derived mESC reporter line was used. Myl2v-eGFP + SMyHC3-RFP double reporter line allows the specific identification of ventricular CMs with green fluorescence and atrial CMs with red fluorescence. Plate reader measures the total fluorescence of the wells at 485/520nm on day 9, which is used as a readout for ventricular CMs. The fluorescence intensity depends on the amount of GFP+ cells but also on the level of Myl2v expression. Atrial CMs could be quantified similarly but the population doesn´t contain enough RFP+ cells. The assay was shown to reliably point out ‘hits’ that have a strong effect. Any compounds that only produce a moderate effect could be a false negative, however. The effect on cardiac progenitors could likely be increased by simply adding the compounds earlier on day 5. Variability of key reagents causes the main technical troubles through unpredictably affecting cytokine concentrations which decreases the amount of cardiac progenitors. Partially similar screening assays are being used by the big pharma where they cryopreserve progenitors in bulk for later use, thus simplifying and speeding up their method. Same approach could be adopted.