Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2–dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised [Ca2+]i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric γ-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric γ-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan.

Telomerase is a ribonucleoprotein that counteracts telomere shortening and can immortalise human cells. There is also evidence for a telomere-independent survival function of telomerase. However, its mechanism is not understood. We show here that TERT, the catalytic subunit of human telomerase, protects human fibroblasts against oxidative stress. While TERT maintains telomere length under standard conditions, telomeres under increased stress shorten as fast as in cells without active telomerase. This is because TERT is reversibly excluded from the nucleus under stress in a dose- and time-dependent manner. Extranuclear telomerase colocalises with mitochondria. In TERT-overexpressing cells, mtDNA is protected, mitochondrial membrane potential is increased and mitochondrial superoxide production and cell peroxide levels are decreased, all indicating improved mitochondrial function and diminished retrograde response. We propose protection of mitochondria under mild stress as a novel function of TERT.

SummaryMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

Key points• Within cells, mitochondria oxidize carbohydrates, and fatty and amino acids to use the released energy to form ATP, and in the process, they also generate reactive oxygen species. Their maximal rates are linked to the magnitude of the mitochondrial membrane potential.• Here we derive a model of fluorescent potentiometric probe dynamics, and on these principleswe introduce an absolute quantitative method for assaying mitochondrial membrane potential in millivolts in individual cultured cells.• This is the first micro-scale method to enable measurement of differences in mitochondrial membrane potential between cells with different properties, e.g. size, mitochondrial density and plasma membrane potential, including cases when plasma membrane potential fluctuates.• Mitochondrial membrane potential in cultured rat cortical neurons is −139 mV at rest. In response to electrical stimulation of the cells, it is regulated between −108 mV and −158 mV by concerted increases in energy demand and metabolic activation. Abstract Mitochondrial membrane potential ( M ) is a central intermediate in oxidative energy metabolism. AlthoughM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential ( P ) and the M probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of M in millivolts as they vary in time in individual cells in monolayer culture. The P -dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute P and M in time from the probe fluorescence intensities, taking into account their slow, P -dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high-and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, M is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca 2+ -dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.