Triple‐negative breast cancer (TNBC) represents the most aggressive breast tumor subtype. However, the molecular determinants responsible for the metastatic TNBC phenotype are only partially understood. We here show that expression of the mitochondrial calcium uniporter (MCU), the selective channel responsible for mitochondrial Ca2+ uptake, correlates with tumor size and lymph node infiltration, suggesting that mitochondrial Ca2+ uptake might be instrumental for tumor growth and metastatic formation. Accordingly, MCU downregulation hampered cell motility and invasiveness and reduced tumor growth, lymph node infiltration, and lung metastasis in TNBC xenografts. In MCU‐silenced cells, production of mitochondrial reactive oxygen species (mROS) is blunted and expression of the hypoxia‐inducible factor‐1α (HIF‐1α) is reduced, suggesting a signaling role for mROS and HIF‐1α, downstream of mitochondrial Ca2+. Finally, in breast cancer mRNA samples, a positive correlation of MCU expression with HIF‐1α signaling route is present. Our results indicate that MCU plays a central role in TNBC growth and metastasis formation and suggest that mitochondrial Ca2+ uptake is a potential novel therapeutic target for clinical intervention.

a b s t r a c tHuman pluripotent stem cells provide new tools for developmental and pharmacological studies as well as for regenerative medicine applications. Calcium homeostasis and ligand-dependent calcium signaling are key components of major cellular responses, including cell proliferation, differentiation or apoptosis. Interestingly, these phenomena have not been characterized in detail as yet in pluripotent human cell sates. Here we review the methods applicable for studying both short-and long-term calcium responses, focusing on the expression of fluorescent calcium indicator proteins and imaging methods as applied in pluripotent human stem cells. We discuss the potential regulatory pathways involving calcium responses in hPS cells and compare these to the implicated pathways in mouse PS cells. A recent development in the stem cell field is the recognition of so called "naïve" states, resembling the earliest potential forms of stem cells during development, as well as the "fuzzy" stem cells, which may be alternative forms of pluripotent cell types, therefore we also discuss the potential role of calcium homeostasis in these PS cell types.

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

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NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzymebinding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM). These protocols allow differences in metabolism to be detected between cell types and altered physiological and pathological states.

Human induced pluripotent stem cells (hiPSCs) and their differentiated derivatives became a new, promising source for in vitro screening techniques. Cell lines derived from healthy individuals can be applied for drug safety testing, while patient-derived cells provide a platform to model diseases in vitro and can be used as a tool for personalized medicine including specific drug efficacy testing, identification of new pharmacological targets as well as for tailoring pharmacological therapies. Efficient differentiation protocols yielding cardiomyocytes or endothelial cells derived from iPSCs have been developed recently.Phenotypic characterization and gene expression profiling of these derivatives can reveal clues for developmental and pathological questions. Moreover, functional analysis and cell-based assays using automated fluorescence imaging platform and high content analysis characterize cell type specific profiles of hiPSC-derived cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC) at the cellular and subcellular levels. This can be utilized in a platform which can provide multiple endpoint profiles of candidate compounds.

Aims Hippo signalling is an evolutionarily conserved pathway that controls organ size by regulating apoptosis, cell proliferation, and stem cell self‐renewal. Recently, the pathway has been shown to exert powerful growth regulatory activity in cardiomyocytes. However, the functional role of this stress‐related and cell death‐related pathway in the human heart and cardiomyocytes is not known. In this study, we investigated the role of the transcriptional co‐activators of Hippo signalling, YAP and TAZ, in human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) in response to cardiotoxic agents and investigated the effects of modulating the pathway on cardiomyocyte function and survival. Methods and results RNA‐sequencing analysis of human heart samples with doxorubicin‐induced end‐stage heart failure and healthy controls showed that YAP and ERBB2 (HER2) as upstream regulators of differentially expressed genes correlated with doxorubicin treatment. Thus, we tested the effects of doxorubicin on hiPSC‐CMs in vitro. Using an automated high‐content screen of 96 clinically relevant antineoplastic and cardiotherapeutic drugs, we showed that doxorubicin induced the highest activation of YAP/TAZ nuclear translocation in both hiPSC‐CMs and control MCF7 breast cancer cells. The overexpression of YAP rescued doxorubicin‐induced cell loss in hiPSC‐CMs by inhibiting apoptosis and inducing proliferation. In contrast, silencing of YAP and TAZ by siRNAs resulted in elevated mitochondrial membrane potential loss in response to doxorubicin. hiPSC‐CM calcium transients did not change in response to YAP/TAZ silencing. Conclusions Our results suggest that Hippo signalling is involved in clinical anthracycline‐induced cardiomyopathy. Modelling with hiPSC‐CMs in vitro showed similar responses to doxorubicin as adult cardiomyocytes and revealed a potential cardioprotective effect of YAP in doxorubicin‐induced cardiotoxicity.

Human induced pluripotent stem cells (hiPSCs) and their differentiated derivatives are new, promising models for studying disease-related phenotypes in vitro. DiGeorge syndrome is caused by the deletion of the 22q11.2 chromosome region on one allele and the hemizygous presence of the affected genes is not sufficient for the healthy phenotype. DGCR8 gene encoding a key component of the microprocessor complex essential for microRNA biogenesis, located in this region. We generated iPSCs from peripheral blood mononuclear cells of members of a family where the disease is present in three generations. The manifestations of the disease differ between family members as grandfather and mother have milder symptoms including minimal facial dysmorphia and hypocalcaemia, while progeny had severe symptoms including pulmonary atresia, ventricular and atrial septal defect and hypoparathyroidism. MLPA genetic assay shows that mother and progeny lack the same 3 Mb long chromosome region and karyotype analysis showed normal chromosomal arrangement in both cases. The unaffected father has no microdeletion but karyotype analysis revealed a translocation between chromosomes 6 and 12. Reprogramming of cells was performed by the expression of four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) via Sendai virus vector transduction. Generated iPSCs were characterised by mRNA and protein expressions of pluripotency markers Nanog (ΔCt father: 0.034, mother: 0.069, progeny: 0.049, p>0.05) and Oct4 (ΔCt father: 0.48, mother: 0.50, progeny: 0.39, p>0.05). FACS revealed >90% of the cells were positive for SSEA4 cell surface pluripotency marker. mRNA levels of DGCR8 were decreased to 41% in mother (n=3, p<0.05) and to 26% in progeny (n=3, p<0.01) compared to father reflecting the genetic background. The hiPSCs were differentiated into endothelial cells and cardiomyocytes to compare phenotypes of disease-affected and control cells. During endothelial differentiation, expression of mesodermal markers Mesp1 and Brachyury was increased at day5 (n=3, p<0.0001) and CD31 (n=3, p<0.05). VE-cadherin and Angiopoietin2 (n=3, p<0.0001) endothelial markers showed increased expression on day 19. CD31/NRP1-double positive arterial like endothelial cells were characterized by immunocytochemistry, tube formation and ac-LDL assays. Beating cardiac clusters showed significantly decreased frequency (control: 76.3±0.9/min, progeny: 57.7±4.9/min, p<0.0001) and higher beating rhythm irregularity index (control: 0.03±0.04, progeny: 0.09±0.12, p<0.5) in case of progeny (n=108) compared to healthy hiPSC line XCL1 (n=10) on day 18 of cardiac differentiation. Our study can provide insights to the development of disease and may serve as human in vitro model for design of new drug targets in complex multiorgan disorder. Further comparative analyses on cellular morphology, viability, transcriptomics, proteomics and functionality are warranted.