Enhancing Maturation and Translatability of Human Pluripotent Stem Cell-Derived Cardiomyocytes through a Novel Medium Containing Acetyl-CoA Carboxylase 2 Inhibitor.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery.

Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease.

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/ß-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/ß-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/ß-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

Boolean modeling reveals that cyclic attractors in macrophage polarization serve as reservoirs of states to balance external perturbations from the tumor microenvironment.

Cyclic attractors generated from Boolean models may explain the adaptability of a cell in response to a dynamical complex tumor microenvironment. In contrast to this idea, we postulate that cyclic attractors in certain cases could be a systemic mechanism to face the perturbations coming from the environment. To justify our conjecture, we present a dynamic analysis of a highly curated transcriptional regulatory network of macrophages constrained into a cancer microenvironment. We observed that when M1-associated transcription factors (STAT1 or NF-κB) are perturbed and the microenvironment balances to a hyper-inflammation condition, cycle attractors activate genes whose signals counteract this effect implicated in tissue damage. The same behavior happens when the M2-associated transcription factors are disturbed (STAT3 or STAT6); cycle attractors will prevent a hyper-regulation scenario implicated in providing a suitable environment for tumor growth. Therefore, here we propose that cyclic macrophage phenotypes can serve as a reservoir for balancing the phenotypes when a specific phenotype-based transcription factor is perturbed in the regulatory network of macrophages. We consider that cyclic attractors should not be simply ignored, but it is necessary to carefully evaluate their biological importance. In this work, we suggest one conjecture: the cyclic attractors can serve as a reservoir to balance the inflammatory/regulatory response of the network under external perturbations.

On Deep Landscape Exploration of COVID-19 Patients Cells and Severity Markers.

COVID-19 is a disease with a spectrum of clinical responses ranging from moderate to critical. To study and control its effects, a large number of researchers are focused on two substantial aims. On the one hand, the discovery of diverse biomarkers to classify and potentially anticipate the disease severity of patients. These biomarkers could serve as a medical criterion to prioritize attention to those patients with higher prone to severe responses. On the other hand, understanding how the immune system orchestrates its responses in this spectrum of disease severities is a fundamental issue required to design new and optimized therapeutic strategies. In this work, using single-cell RNAseq of bronchoalveolar lavage fluid of nine patients with COVID-19 and three healthy controls, we contribute to both aspects. First, we presented computational supervised machine-learning models with high accuracy in classifying the disease severity (moderate and severe) in patients with COVID-19 starting from single-cell data from bronchoalveolar lavage fluid. Second, we identified regulatory mechanisms from the heterogeneous cell populations in the lungs microenvironment that correlated with different clinical responses. Given the results, patients with moderate COVID-19 symptoms showed an activation/inactivation profile for their analyzed cells leading to a sequential and innocuous immune response. In comparison, severe patients might be promoting cytotoxic and pro-inflammatory responses in a systemic fashion involving epithelial and immune cells without the possibility to develop viral clearance and immune memory. Consequently, we present an in-depth landscape analysis of how transcriptional factors and pathways from these heterogeneous populations can regulate their expression to promote or restrain an effective immune response directly linked to the patients prognosis.

Transcriptional and Microenvironmental Landscape of Macrophage Transition in Cancer: A Boolean Analysis.

The balance between pro- and anti-inflammatory immune system responses is crucial to face and counteract complex diseases such as cancer. Macrophages are an essential population that contributes to this balance in collusion with the local tumor microenvironment. Cancer cells evade the attack of macrophages by liberating cytokines and enhancing the transition to the M2 phenotype with pro-tumoral functions. Despite this pernicious effect on immune systems, the M1 phenotype still exists in the environment and can eliminate tumor cells by liberating cytokines that recruit and activate the cytotoxic actions of TH1 effector cells. Here, we used a Boolean modeling approach to understand how the tumor microenvironment shapes macrophage behavior to enhance pro-tumoral functions. Our network reconstruction integrates experimental data and public information that let us study the polarization from monocytes to M1, M2a, M2b, M2c, and M2d subphenotypes. To analyze the dynamics of our model, we modeled macrophage polarization in different conditions and perturbations. Notably, our study identified new hybrid cell populations, undescribed before. Based on the in vivo macrophage behavior, we explained the hybrid macrophages' role in the tumor microenvironment. The in silico model allowed us to postulate transcriptional factors that maintain the balance between macrophages with anti- and pro-tumoral functions. In our pursuit to maintain the balance of macrophage phenotypes to eliminate malignant tumor cells, we emulated a theoretical genetically modified macrophage by modifying the activation of NFκB and a loss of function in HIF1-α and discussed their phenotype implications. Overall, our theoretical approach is as a guide to design new experiments for unraveling the principles of the dual host-protective or -harmful antagonistic roles of transitional macrophages in tumor immunoediting and cancer cell fate decisions.

Analysis of Epithelial-Mesenchymal Transition Metabolism Identifies Possible Cancer Biomarkers Useful in Diverse Genetic Backgrounds.

Epithelial-to-mesenchymal transition (EMT) relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation generating mesenchymal-like cells with newly acquired migratory and invasive properties. In cancer cells, EMT leads to drug resistance and metastasis. Moreover, differences in genetic backgrounds, even between patients with the same type of cancer, also determine resistance to some treatments. Metabolic rewiring is essential to induce EMT, hence it is important to identify key metabolic elements for this process, which can be later used to treat cancer cells with different genetic backgrounds. Here we used a mathematical modeling approach to determine which are the metabolic reactions altered after induction of EMT, based on metabolomic and transcriptional data of three non-small cell lung cancer (NSCLC) cell lines. The model suggested that the most affected pathways were the Krebs cycle, amino acid metabolism, and glutathione metabolism. However, glutathione metabolism had many alterations either on the metabolic reactions or at the transcriptional level in the three cell lines. We identified Glutamate-cysteine ligase (GCL), a key enzyme of glutathione synthesis, as an important common feature that is dysregulated after EMT. Analyzing survival data of men with lung cancer, we observed that patients with mutations in GCL catalytic subunit (GCLC) or Glutathione peroxidase 1 (GPX1) genes survived less time than people without mutations on these genes. Besides, patients with low expression of ANPEP, GPX3 and GLS genes also survived less time than those with high expression. Hence, we propose that glutathione metabolism and glutathione itself could be good targets to delay or potentially prevent EMT induction in NSCLC cell lines.

Unveiling functional heterogeneity in breast cancer multicellular tumor spheroids through single-cell RNA-seq.

Heterogeneity is an intrinsic characteristic of cancer. Even in isogenic tumors, cell populations exhibit differential cellular programs that overall supply malignancy and decrease treatment efficiency. In this study, we investigated the functional relationship among cell subtypes and how this interdependency can promote tumor development in a cancer cell line. To do so, we performed single-cell RNA-seq of MCF7 Multicellular Tumor Spheroids as a tumor model. Analysis of single-cell transcriptomes at two-time points of the spheroid growth, allowed us to dissect their functional relationship. As a result, three major robust cellular clusters, with a non-redundant complementary composition, were found. Meanwhile, one cluster promotes proliferation, others mainly activate mechanisms to invade other tissues and serve as a reservoir population conserved over time. Our results provide evidence to see cancer as a systemic unit that has cell populations with task stratification with the ultimate goal of preserving the hallmarks in tumors.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.