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- Modelling left ventricular noncompaction cardiomyopathy in cardioidsPublication . Correia, Cátia Dias; Calado, Sofia; Bragança, JoséCongenital heart defects are the most common deficiency in new-borns. Specifically, a failure during myocardial compaction can lead to excessive trabeculation and intertrabecular recesses seen in left ventricular noncompaction (LVNC). Due to heterogeneity found in patients, more in-depth molecular research, with better disease models, is necessary to identify underlying genetic pathways and pathogenesis. Here, a novel self-assembling human cardioid differentiation protocol was used. Initially, three concentrations of a canonical Wnt pathway activator were tested, and addition of ascorbic acid. A higher concentration led to a full loss of organoids, while ascorbic acid slightly increased the number of organoids discarded but increased the number of beating control-derived organoids. Lastly, with hiPSCs established from LVNC patients, and a healthy relative, three initial seeding densities were tested. We experienced a high degree of heterogeneity in size and morphology, vascularisation formation, number of beating and discarded cardioids. We also analysed organoid circularity at two different stages of differentiation, but no conclusions can be made due to result variability and inconsistency between both cell lines, and/in all seeding conditions presenting no clear pattern. We also experienced a lack of fluorescence signal or a low antibody penetrability after immunofluorescence staining, and increased all incubation periods but still experienced a lack of signal for cardiomyocytes. Then, due to organoid size and density, we observed a low reachable sample depth during fluorescence microscopy. An additional step of ScaleA2 optical clearing was tested, which showed that a 10 day incubation period is crucial for imaging quality and reachable depth improvement. In conclusion, this is a promising but very demanding protocol that has to be thoroughly followed, with many influencing factors. More work is still necessary to improve its efficiency for further studies to determine their cardiac development capacity, and if it can be used to model LVNC in the future.