Biology subjects

Orlova, V.

Publications and source records attributed to Orlova, V..

4 recordsLinked to original sources

bioRxiv2026

Mapping the regulatory wiring of endothelial cell differentiation reveals a critical role for Hippo mechanosignaling

At each step of development, stem cells encounter a branching decision to differentiate into one type of cell or another. This decision is executed through the action of competing gene programs that specify different cellular identities. Consequently, creating a comprehensive map of the gene programs that drive each branching step of human cell differentiation remains a long-sought goal of developmental and stem cell biology. Here, we introduce a framework combining genome-wide CRISPR screens, single-cell readouts, and gene program inference to systematically discover and characterize regulators of cell differentiation. This framework includes a method for CRISPR perturbations paired with single-cell RNA-seq called circular capture Perturb-seq (CC-Perturb-seq), which improves gRNA detection from split-pool combinatorial indexing protocols and enables perturbing many candidate regulators to map their effects on gene programs. We applied this framework to map how human pluripotent stem cells differentiate into arterial endothelial cells (ECs), and identified 174 regulators that promote or inhibit EC differentiation. By tracking gene programs across three stages of differentiation, we resolved when and how regulators divert cells toward alternative fates. This map revealed an unexpected requirement for the Hippo mechanosignaling pathway in EC specification. During lateral mesoderm differentiation, Hippo mechanosignaling was required to activate ETV2, the master transcriptional regulator of endothelial identity. Inhibiting Hippo blocked ETV2 even in the presence of VEGF and diverted progenitors to a mesenchymal fate in vitro. Likewise, Hippo signaling was required for maximal etv2 expression in developing zebrafish embryos. Conversely, pharmacologically activating the Hippo pathway increased the purity of both arterial and venous EC differentiation in vitro. Our framework thus identifies novel regulators of endothelial cell differentiation, improves stem-cell differentiation protocols via manipulation of mechanosignaling, and provides a generalizable platform for mapping the regulatory wiring of human development.

genomics↗

bioRxiv2025

Cytokine-induced memory-like responses in endothelial cells link chronic inflammation to vascular disease risk

Chronic inflammation plays a central role in the progression of both infectious and vascular diseases, yet its impact on endothelial cells (ECs), which form the interface between blood and tissue, remains poorly understood. Given their constant exposure to inflammatory cytokines such as TNF- and IFN-{gamma}, we set out to investigate how cytokine induced inflammation shapes EC function at the molecular level. Using primary human umbilical vein endothelial cells (HUVECs), we modeled repeated cytokine exposure to simulate a chronically inflamed microenvironment. Transcriptomic and epigenetic profiling revealed that ECs respond to this chronic stimulation with durable transcriptional and chromatin changes. These responses included phenotypes resembling immune cell priming, training, and tolerance, which are commonly associated with innate immune memory, a phenomenon whereby innate immune cells mount altered response following previous stimulation. Although we did not observe classical trained immunity pathways, several genes known to mediate immune training, including TLR2, IL1B, and HDAC9, exhibited persistent activation following TNF- re-exposure. IFN-{gamma} stimulation uniquely induced sustained expression and chromatin accessibility at MHC class II loci, suggesting cytokine-specific modes of reprogramming. Functionally, re-stimulated ECs exhibited enhanced monocyte adhesion in a 3D vessel-on-chip model, highlighting the relevance of these molecular changes to vascular inflammation. Moreover, the regulatory regions altered by cytokine exposure were enriched for disease-associated SNPs, particularly those linked to COVID-19, sepsis, and cardiovascular disorders. In summary, these findings reveal that repeated exposure to cytokines as seen in chronic inflammation can induce memory-like responses in ECs and suggest that endothelial reprogramming may contribute to vascular dysfunction.

molecular biology↗

bioRxiv2025

A simplified co-culture reveals altered cardiotoxic responses to doxorubicin in hPSC-derived cardiomyocytes in the presence of endothelial cells

Cardiotoxicity is a significant challenge in cancer therapies, particularly with doxorubicin, a widely used anthracycline known for its broad anti-cancer spectrum but life-threatening cardiac side effects. There is a critical need for more predictive in vitro models to understand doxorubicin-induced cardiotoxicity and patient-specific drug responses. In this study, we used human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFBs) and endothelial cells (hPSC-ECs) to investigate the cardiotoxic effects of doxorubicin in two-dimensional mono-and multi-cell type cultures. By mimicking the cumulative dose effect seen in patients through repeated doxorubicin treatments and using a machine learning-based in silico image analysis tool, we could precisely quantify caspase 3/7 activity as an early toxicity marker and identify hPSC-CMs in multi-cell type cultures. This innovative approach allowed continuous monitoring of apoptosis from phase-contrast images, revealing that hPSC-ECs showed higher sensitivity to doxorubicin than isogenic hPSC-CMs or hPSC-cFBs and significantly enhanced cardiomyocyte toxicity in co-culture. In contrast, dermal fibroblasts differentiated from the same hPSC line showed no toxic response under the same treatment regimen. These results challenge the conventional focus on cardiomyocytes as the target of drug-induced cardiac damage. Our findings not only highlight the complex interplay among different cardiac cell types in mediating the toxic effects of doxorubicin, but also demonstrate the potential of AI-enabled tools to advance personalized drug screening and safety assessments.

pharmacology and toxicology↗

bioRxiv2023

Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

The non-clinical assessment of CAR-T cells demands innovative models that are capable of predicting safety and efficacy in the clinical setting. Here, we present a novel solid tumor-on-chip model that allows CAR-T cell perfusion and integrates the vasculature and tumor lesions to recapitulate key events of CAR-T cell performance including extravasation, tumor infiltration and cytokine release. We assessed CAR-T cells targeting the ROR1 antigen against tumor aggregates that were derived from a breast cancer cell line and primary breast cancer organoids. The data show the temporal kinetic of ROR1 CAR-T cell migration and expansion, lytic activity and cytokine production over the course of 8 days, and reveal a correlation between anti-tumor efficacy and ROR1 antigen density on tumor cells. CAR-modified T cells extravasated faster, infiltrated tumor lesions stronger, persisted longer and in higher numbers than non-CAR modified T cells. Intriguingly, we detected cytokine release levels and kinetics typically observed in patients who developed cytokine release syndrome, and administered dasatinib as a pharmacologic OFF switch to control this inflammatory response. The data illustrate the ability of this tumor-on-chip platform to assess parameters associated withherapeutic outcome and the potential to aid in patient stratification and monitoring of CAR-T cell therapy.

bioengineering↗