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Human-relevant tests to predict developmental toxicity are urgently needed. A currently intensively studied approach makes use of differentiating human stem cells to measure chemically-induced deviations of the normal developmental program, as in a recent study based on cardiac differentiation (UKK2). Here, we (i) tested the performance of an assay modeling neuroepithelial differentiation (UKN1), and (ii) explored the benefit of combining assays (UKN1 and UKK2) that model different germ layers. Substance-induced cytotoxicity and genome-wide expression profiles of 23 teratogens and 16 non-teratogens at human-relevant concentrations were generated and used for statistical classification, resulting in accuracies of the UKN1 assay of 87–90%. A comparison to the UKK2 assay (accuracies of 90–92%) showed, in general, a high congruence in compound classification that may be explained by the fact that there was a high overlap of signaling pathways. Finally, the combination of both assays improved the prediction compared to each test alone, and reached accuracies of 92–95%. Although some compounds were misclassified by the individual tests, we conclude that UKN1 and UKK2 can be used for a reliable detection of teratogens in vitro, and that a combined analysis of tests that differentiate hiPSCs into different germ layers and cell types can even further improve the prediction of developmental toxicants.

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca2+]i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca2+-indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP+)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP+-cardiomyocytes and ACTN2-copGFP+-cardiomyocytes, allowing real-time imaging of [Ca2+]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP+-cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.

Personalized regenerative medicine and biomedical research have been galvanized and revolutionized by human pluripotent stem cells in combination with recent advances in genomics, artificial intelligence, and genome engineering. More recently, we have witnessed the unprecedented breakthrough life-saving translation of mRNA-based vaccines for COVID-19 to contain the global pandemic and the investment in billions of US dollars in space exploration projects and the blooming space-tourism industry fueled by the latest reusable space vessels. Now, it is time to examine where the translation of pluripotent stem cell research stands currently, which has been touted for more than the last two decades to cure and treat millions of patients with severe debilitating degenerative diseases and tissue injuries. This review attempts to highlight the accomplishments of pluripotent stem cell research together with cutting-edge genomics and genome editing tools and, also, the promises that have still not been transformed into clinical applications, with cardiovascular research as a case example. This review also brings to our attention the scientific and socioeconomic challenges that need to be effectively addressed to see the full potential of pluripotent stem cells at the clinical bedside.

In vitro differentiation of embryonic stem cells (ESCs) provides a convenient basis for the study of microRNA-based gene regulation that is relevant for early cardiogenic processes. However, to which degree insights gained from in vitro differentiation models can be readily transferred to the in vivo system remains unclear. In this study, we profiled simultaneous genome-wide measurements of mRNAs and microRNAs (miRNAs) of differentiating murine ESCs (mESCs) and integrated putative miRNA-gene interactions to assess miRNA-driven gene regulation. To identify interactions conserved between in vivo and in vitro, we combined our analysis with a recent transcriptomic study of early murine heart development in vivo. We detected over 200 putative miRNA–mRNA interactions with conserved expression patterns that were indicative of gene regulation across the in vitro and in vivo studies. A substantial proportion of candidate interactions have been already linked to cardiogenesis, supporting the validity of our approach. Notably, we also detected miRNAs with expression patterns that closely resembled those of key developmental transcription factors. The approach taken in this study enabled the identification of miRNA interactions in in vitro models with potential relevance for early cardiogenic development. Such comparative approaches will be important for the faithful application of stem cells in cardiovascular research.

The authors wish to make the following corrections to this paper [...]

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

Human-induced pluripotent stem cells (hiPSCs) are discussed as disease modeling for optimization and adaptation of therapy to each individual. However, the fundamental question is still under debate whether stem-cell-based disease modeling and drug discovery are applicable for recapitulating pathological processes under in vivo conditions. Drug treatment and exposure to different chemicals and environmental factors can initiate diseases due to toxicity effects in humans. It is well documented that drug-induced cardiotoxicity accelerates the development of heart failure (HF). Until now, investigations on the understanding of mechanisms involved in HF by anticancer drugs are hindered by limitations of the available cellular models which are relevant for human physiology and by the fact that the clinical manifestation of HF often occurs several years after its initiation. Recently, we identified similar genomic biomarkers as observed by HF after short treatment of hiPSCs-derived cardiomyocytes (hiPSC-CMs) with different antitumor drugs such as anthracyclines and etoposide (ETP). Moreover, we identified common cardiotoxic biological processes and signal transduction pathways which are discussed as being crucial for the survival and function of cardiomyocytes and, therefore, for the development of HF. In the present review, I discuss the applicability of the in vitro cardiotoxicity test systems as modeling for discovering preventive mechanisms/targets against cardiotoxicity and, therefore, for novel HF therapeutic concepts.

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the β-adrenoceptor isoprenalin (ISO), L-type Ca2+ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20–22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC.

Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput “omics” methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy.

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca 2+ ] i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca 2+ -indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP + )-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP + -cardiomyocytes and ACTN2-copGFP + -cardiomyocytes, allowing real-time imaging of [Ca 2+ ] i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP + -cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca 2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.