The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands—or even millions—of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.

The unfolded protein response (UPR) pathway senses unfolded proteins and regulates proteostasis and cell fate through activity of the transcription factors ATF4, ATF6, and XBP1 within a complex network of three main branches. Here, we investigated contributions of the three branches to UPR activity in single cells using microscopy-based quantification and dynamic modeling. BAC-GFP HepG2 reporter cell lines were exposed to tunicamycin, and activation of various UPR components was monitored for 24 h. We constructed a dynamic model to describe the adaptive UPR signaling network, for which incorporation of all three branches was required to match the data. Our calibrated model suggested that ATF6 shapes the early dynamics of pro-apoptotic CHOP. We confirmed this hypothesis by measurements beyond 24 h, by perturbing single siRNA knockdowns and by ATF6 measurements. Overall, our work indicates that ATF6 is an important regulator of CHOP, which in turn regulates cell fate decisions.

Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.

Mitochondria are the main bioenergetic organelles of cells. Exposure to chemicals targeting mitochondria therefore generally results in the development of toxicity. The cellular response to perturbations in cellular energy production is a balance between adaptation, by reorganisation and organelle biogenesis, and sacrifice, in the form of cell death. In homeostatic conditions, aerobic mitochondrial energy production requires the maintenance of a mitochondrial membrane potential (MMP). Chemicals can perturb this MMP, and the extent of this perturbation depends both on the pharmacokinetics of the chemicals and on downstream MMP dynamics. Here we obtain a quantitative understanding of mitochondrial adaptation upon exposure to various mitochondrial respiration inhibitors by applying mathematical modeling to partially published high-content imaging time-lapse confocal imaging data, focusing on MMP dynamics in HepG2 cells over a period of 24 h. The MMP was perturbed using a set of 24 compounds, either acting as uncoupler or as mitochondrial complex inhibitor targeting complex I, II, III or V. To characterize the effect of chemical exposure on MMP dynamics, we adapted an existing differential equation model and fitted this model to the observed MMP dynamics. Complex III inhibitor data were better described by the model than complex I data. Incorporation of pharmacokinetic decay into the model was required to obtain a proper fit for the uncoupler FCCP. Furthermore, oligomycin (complex V inhibitor) model fits were improved by either combining pharmacokinetic (PK) decay and ion leakage or a concentration-dependent decay. Subsequent mass spectrometry measurements showed that FCCP had a significant decay in its PK profile as predicted by the model. Moreover, the measured oligomycin PK profile exhibited only a limited decay at high concentration, whereas at low concentrations the compound remained below the detection limit within cells. This is consistent with the hypothesis that oligomycin exhibits a concentration-dependent decay, yet awaits further experimental verification with more sensitive detection methods. Overall, we show that there is a complex interplay between PK and MMP dynamics within mitochondria and that data-driven modeling is a powerful combination to unravel such complexity.

Introduction: Drug-induced liver injury (DILI) is a significant threat to human health and a major problem in drug development. It is hard to predict due to its idiosyncratic nature and which does not show up in animal trials. Hepatic adaptive stress response pathway activation is generally observed in drug-induced liver injury. Dynamical pathway modeling has the potential to foresee adverse effects of drugs before they go in trial. Ordinary differential equation modeling can offer mechanistic insight, and allows us to study the dynamical behavior of stress pathways involved in DILI. Areas covered: This review provides an overview on the progress of the dynamical modeling of stress and death pathways pertinent to DILI, i.e. pathways relevant for oxidative stress, inflammatory stress, DNA damage, unfolded proteins, heat shock and apoptosis. We also discuss the required steps for applying such modeling to the liver. Expert opinion: Despite the strong progress made since the turn of the century, models of stress pathways have only rarely been specifically applied to describe pathway dynamics for DILI. We argue that with minor changes, in some cases only to parameter values, many of these models can be repurposed for application in DILI research. Combining both dynamical models with in vitro testing might offer novel screening methods for the harmful side-effects of drugs.