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Recent advancement of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technologies has opened the door to next-generation modeling of human cardiac biology and disease. Not only does this alleviate the need for human primary tissue and compensate for the well documented deficiencies of rodent models for cardiovascular disease, but these technologies may lead to the identification of better candidates for clinical development. Unfortunately, most current 2D hiPSC-CM models lack the biochemical, mechanical, and electrical feedback that cardiomyocytes endure in a multi-cellular aligned tissue, which limits their translatability into clinical settings. To address this, we incorporated hiPSC-CMs modeling various genetic dilated cardiomyopathies (DCM) into 3D engineered heart tissues (EHTs). Here, we show these models develop distinguishable contractile defects recapitulating hallmarks of DCM when compared to biologically relevant controls. Moreover, this distinct phenotype is quantitative, reproducible, and demonstrates utility for drug discovery. As this innovative technology continues to develop, EHTs are on the forefront of emerging biomimetic assays that can be used to prevent drug attrition in the late stages of drug development.

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to detect drug-induced toxicity in vitro. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We screened a library of 1280 bioactive compounds and identified those with potential cardiotoxic liabilities in iPSC-CMs using a single-parameter score based on deep learning. Compounds demonstrating cardiotoxicity in iPSC-CMs included DNA intercalators, ion channel blockers, epidermal growth factor receptor, cyclin-dependent kinase, and multi-kinase inhibitors. We also screened a diverse library of molecules with unknown targets and identified chemical frameworks that show cardiotoxic signal in iPSC-CMs. By using this screening approach during target discovery and lead optimization, we can de-risk early-stage drug discovery. We show that the broad applicability of combining deep learning with iPSC technology is an effective way to interrogate cellular phenotypes and identify drugs that may protect against diseased phenotypes and deleterious mutations.

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming. DOI: http://dx.doi.org/10.7554/eLife.03573.001, eLife digest The stem cells found in an embryo are able to develop into any of the cell types found in the body of the animal: an ability called pluripotency. When a cell becomes a specialized cell type, such as a nerve cell or a muscle cell, it loses this ability. However, mature cells can be reprogrammed back to a pluripotent state by artificially introducing certain proteins (known as ‘reprogramming factors’) into the mature cells. A core group of reprogramming factors are known to activate networks of genes that are normally only expressed in stem cells, and by doing so trigger and maintain a pluripotent state. Other proteins help these core factors to regulate these networks of genes. In 2011, researchers discovered that a protein complex called XPC—which is normally involved in DNA repair—also helps two core reprogramming factors to activate an important gene related to pluripotency. Now, Fong et al., including several of the researchers involved in the 2011 work, have identified another unexpected partner for the same two core reprogramming factors. The protein complex, called DKC1, has a number of known functions related to the processing of RNA molecules. This complex has also been linked to a fatal, rare human disorder called dyskeratosis congenita—a condition that affects many parts of the body, including the skin and bone marrow. Fong et al. found that when embryonic stems cells from mice are depleted of the DKC1 complex, the activation of important pluripotency-related genes by two of the core reprogramming factors is markedly reduced. The XPC and DKC1 protein complexes were found to interact in pluripotent cells, and together they can activate a pluripotency-related gene to a greater extent than either can individually. Fong et al. propose that DKC1 binds to XPC, which in turn binds to two of the core reprogramming factors. The DKC1 complex also binds to RNA molecules, and Fong et al. found that when the DKC1 complex binds to certain RNAs it is more able to help reprogramming factors activate pluripotency-related genes. On the other hand, other RNA molecules seem to inhibit the complex's ability to activate these genes. Mutations identified in people with dyskeratosis congenita can prevent the DKC1 complex from binding to a subset of human RNA molecules. Moreover, the activity of stem cells is impaired in people with this developmental condition. As such, one of the next challenges will be to investigate if these mutations and RNA binding could be linked to problems with the activation of genes related to pluripotency in stem cells. DOI: http://dx.doi.org/10.7554/eLife.03573.002