Aleksandra Vilotić

Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs).

Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca2+ imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patients stem cells, this biosensor can help to characterize heart diseases such as dystrophies.