Browsing by Subject "coculture"

Human induced pluripotent stem cells (hiPSC) can be propagated in a long-term culture and further differentiated into many cell types, including cardiomyocytes (CM) and endothelial cells (EC). Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are promising tools in cardiac research, since they retain the original genotype of the individual donor and thus enable the use of patient- and disease specific cells. Crucial for the optimal use of hiPSC-CMs in experiments are methods for assessing cardiomyocyte phenotype. Contraction is a prominent feature for CMs, and it is essential that contraction can be quantified accurately. Reliable quantification is relevant when hiPSC-CMs are used for studying disease phenotypes, cardiac safety pharmacology, genotype-phenotype correlations, cardiac disease mechanisms and cardiac function over time. In this thesis project, contractile behavior of hiPSC-CMs was analyzed using video microscopy and online tool MUSCLEMOTION. Contraction parameters were obtained from hiPSC-CMs derived from patients with hypoplastic left heart syndrome (HLHS) and healthy controls on multiple timepoints during differentiation. In addition, contraction was analyzed in iPSC-CMs cocultured with induced pluripotent stem cell derived endothelial cells (iPSC-ECs), since it has been suggested that ECs can promote morphological and functional maturation of CMs in culture. Contraction duration (CD), time to peak (TTP), relaxation time (RT) and contraction amplitude (CA) was compared between different timepoints as well as between CMs cocultured with ECs and CMs cultured alone. Compared to control cell lines, HLHS patient hiPSC-CMs exhibited longer CD, TTP and RT as well as higher CA values. This difference was present in most of the timepoints, suggesting slower contractile kinetics in HLHS patient iPSC-CMs compared to control iPSC-CMs. Significant changes were also observed in contraction parameters when comparing hiPSC-CMs in coculture and monoculture. Contraction parameters of coculture iPSC-CMs changed in a relatively consistent manner over time, increasing or decreasing throughout the monitoring period whereas in hiPSC-CM monoculture there was more variation between timepoints. This project and results support the use of modern methods in detailed functional characterization of hiPSC-derived cells. In addition, it highlights the potential of coculture in disease modeling and the fact that hiPSC-CMs express variation in phenotypes. However, experiments should be repeated, and additional methods should be used in order to further validate the results and conclusions.