Kardiomiociti diferencirani iz ljudskih pluripotentnih stanica kao eksperimentalni model za prekondicioniranje potaknuto anesteticima [Cardiomyocytes differentiated from human pluripotent stem cells as an experimental model for anesthetic-induced preconditioning ]

The lack of human myocardium for research purposes represents a great problem in studying pathogenesis of human cardiac disease. The doctoral dissertation is designed to test whether cardiomyocytes differentiated from human pluripotent stem cells can be used for studying anesthetic preconditioning (APC), a cardioprotective strategy. Human embryonic stem cell (hESC) lines, H1 and H9, and human induced pluripotent stem cell (hiPSC) lines, C2a and C6a were differentiated into cardiomyocytes by directed differentiation. All experiments with hESCs were conducted at the Medical College of Wisconsin using commercially available lines and their use was approved by the local Ethical Committee. hiPSC were obtained from the laboratory of Dr. S.A Duncan, MCW. Cardiac differentiation of hESC and hiPSC lines was accompanied with the occurrence of spontaneous contractions in cell clusters. Cardiac enrichment was greater than 65% in beating clusters of all cell lines. Living differentiated cardiomyocytes were labelled with a genetic construct, MLC 2v-EGFP, delivered by a lentiviral vector. Immunostaining for sarcomeric proteins, sarcomeric-α actinin and cardiac troponin T revealed that sarcomeres in hESC- and hiPSC-derived cardiomyocytes exhibited a proper striated pattern. The time course of expression of genes involved at various stages of cardiomyogenesis was analyzed by qPCR in all hESC and hiPSC lines. It revealed that the loss of expression of pluripotency and later mesodermal gene was accompanied by the increase in expression of genes of early phase of cardiac differentiation. Genes characteristic for terminal cardiac differentiation were expressed during the second week of cardiomyogenesis. APC of hESC- and hiPSC-derived cardiomyocytes induced a delay in opening of the mitochondrial permeability transition pore (mPTP) in stressed cells, which is a critical mediator of protection by APC. Increase in the survival of preconditioned cardiomyocytes correlated with the delay in mPTP opening in hESC-derived cardiomyocytes. Isoflurane activated characteristic mediators of preconditioning in hESC-derived cardiomyocytes. It induced opening of mitoKATP channels and moderately increased production of reactive oxygen species by inhibiting respiratory chain. In conclusion, functional human pluripotent stem cell-derived cardiomyocytes can be efficiently differentiated in vitro. These cells exhibit competent responses to APC and therefore could be used as a reliable experimental models for studying APC