Biology subjects

Orlova, V. V.

Publications and source records attributed to Orlova, V. V..

8 recordsLinked to original sources

bioRxiv2026

Dissecting human fetal cardiac repair using cardioids

Human cardiac injury responses are governed by dynamic interacting processes that are difficult to resolve. Unlike adults, fetal mammalian hearts regenerate through coordinated remodeling and proliferation supported by a pro-regenerative immune environment, extracellular matrix (ECM), and immature cardiomyocytes, including trabecular subtypes. Here, we establish a modular human cardioid injury platform to dissect these interactions. We show that anti-inflammatory macrophages selectively migrate to the injury, clear debris, and promote ECM remodeling, whereas inflammatory macrophages suppress cardiomyocyte proliferation. Synergistic FGF2-NRG1 signaling induces trabecular identity and morphology in a hyaluronan-dependent manner, conferring enhanced injury repair, characterized by cytoskeletal remodeling and cardiomyocyte proliferation mediated by YAP and WNT signaling. Exogenous YAP, but not WNT, is sufficient to promote repair in non-trabecular cardioids. These findings uncover coordinated immune-ECM-cardiomyocyte interactions governing human fetal regenerative competence and mechanistically resolve remodeling and proliferative components of cardiac repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/735236v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e647a2org.highwire.dtl.DTLVardef@16911f8org.highwire.dtl.DTLVardef@11ad14eorg.highwire.dtl.DTLVardef@1a35d94_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Vessel-on-Chip Model Of The Microcirculation In Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAA) are pathological dilations of the abdominal aorta. To date, surgical intervention is the only option for managing large AAAs, with no pharmacological therapies to prevent growth of small aneurysms. A current limitation in investigating further pharmacological avenues is the translatability of results from animal models, or from patient trials that are limited by co-morbidities and disease severity. To bridge this knowledge gap, we created a novel, patient-specific vessel-on-chip (VoC) model of the microcirculation in AAA (AAA-VoC). We found that co-culture of both C (control)-VSMCs and AAA-patient derived VSMCs with healthy, hiPSC-derived ECs generate lumenized and perfusable microvascular networks. We show that AAA-VoCs are characterized by an enlarged average vascular diameter. We furthermore found that AAA-VSMCs show phenotypical deviations from C- VSMCs after 7 days in co-culture such as increased number and surface area, indicative of a preserved pathological phenotype in our in vitro model. Lastly, we demonstrate that AAA-VoCs showed an increased level of pro-inflammatory cytokine expression over C-VoCs and displayed an impaired endothelial barrier function, resulting in vascular leakage. With this study, we show that AAA-VSMCs affect microvascular networks formed by healthy hiPSC-ECs and that a AAA phenotype is preserved in 3D co-culture, making this model valuable for future studies investigating treatments for AAA.

cell biology↗

bioRxiv2025

Controlled Induction of ALK1 in ACVRL1-null hiPSC-derived Endothelial Cells Provides Insight into Organ-Restricted AVMs in HHT

Genetic alterations in activin receptor-like kinase 1 (ACVRL1, ALK1) are linked to hereditary hemorrhagic telangiectasia (HHT), a condition characterized by hemorrhages, arteriovenous malformations (AVMs) and endothelial cell (EC) dysfunction. Haploinsufficiency is considered the central disease-driving mechanism, but it remains unclear why vascular lesions are tissue-restricted. To investigate whether ACVRL1 gene dosage in human ECs plays a role in organ-specific susceptibility, we developed a tunable human in vitro model by deleting ACVRL1 in human induced pluripotent stem cells (hiPSCs), and then reintroduced wild-type ACVRL1 under control of a doxycycline-inducible promoter. This enabled temporal, dose-dependent induction of ALK1 expression in ECs, allowing definition of threshold levels required to restore endothelial function and support vascular homeostasis. ALK1-deficient hiPSC-ECs displayed disrupted SMAD1/5 signaling, hyperproliferation, altered tip/stalk cell specification, and dysregulated transcriptional programs. Even low-level reinduction of ALK1 was sufficient to restore endothelial function, indicating threshold-dependence for ALK1 in vascular homeostasis. These data indicated that one mechanism underlying tissue-restricted prevalence of AVMs in vascular beds with low basal ACVRL1 expression, such as the liver, is a minimal requirement for ALK1 in sustaining EC functionality. This tunable human platform offers a powerful tool for dissecting HHT pathobiology and a platform for identifying strategies to restore ALK1 signaling therapeutically in affected tissues.

molecular biology↗

bioRxiv2024

Engineered microvasculature using maskless photolithography and on-chip hydrogel patterning: a facile approach

In vitro models of human microvasculature are increasingly used to understand blood vessel diseases and to support drug development. Most engineered models, however, are slow and labor-intensive to produce. Here, we used a single commercial digital micromirror device (DMD)-based setup for maskless photolithography to both fabricate microfluidic chips and pattern the inside of these chips with gelatin methacrylate (GelMA) hydrogels. These hydrogel scaffolds had tunable stiffness, could be generated rapidly and were suitable for forming perfusable microvasculature from human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs). When cultured in narrow channels, the hiPSC-ECs adopted a tubular morphology that was similar to capillaries in vivo, but they followed the square channel geometry in wider channels. Compartmentalization of the chips allowed co-culture of hiPSC-ECs with hiPSC-derived astrocytes, thereby increasing model complexity. Furthermore, valve-like structures could be patterned inside the channels, mimicking functional vascular valves, holding promise for thrombosis and lymphatic vasculature research.

bioengineering↗

bioRxiv2022

Tissue microenvironment dictates the state of human induced pluripotent stem cell-derived endothelial cells of distinct developmental origin in 3D cardiac microtissues

Each tissue and organ in the body has its own type of vasculature. Here we demonstrate that organotypic vasculature for the heart can be recreated in a three-dimensional cardiac microtissue (MT) model composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), cardiac fibroblasts (CFs) and endothelial cells (ECs). ECs in cardiac MTs upregulated expression of markers enriched in human intramyocardial ECs (iECs), such as CD36, CLDN5, APLNR, NOTCH4, IGFBP3, ARHGAP18, which were previously identified in the single-cell RNA-seq dataset from the human fetal heart (6.5-7 weeks post coitum). We further show that the local microenvironment largely dictates the organ-specific identity of hiPSC-derived ECs: we compared ECs of different developmental origins derived from two distinct mesoderm subtypes (cardiac and paraxial mesoderm) and found that independent of whether the ECs were cardiac or paraxial mesoderm derived, they acquired similar identities upon integration into cardiac microtissues. This was confirmed by single-cell RNA-seq. Overall, the results indicated that whilst the initial gene profile of ECs was dictated by developmental origin, this could be modified by the local tissue environment such that the original identity was lost and the organotypic identity acquired through local environmental signals. This developmental "plasticity" in ECs has implications for multiple pathological and disease states.

developmental biology↗

bioRxiv2022

ETV2 upregulation marks the specification of early cardiomyocytes and endothelial cells during co-differentiation

The ability to differentiate human induced pluripotent stem cells (hiPSCs) efficiently into defined cardiac lineages, such as cardiomyocytes and cardiac endothelial cells, is crucial to study human heart development and model cardiovascular diseases in vitro. The mechanisms underlying the specification of these cell types during human development are not well-understood which limits fine-tuning and broader application of cardiac model systems. Here, we used the expression of ETV2, a master regulator of hematoendothelial specification in mice, to identify functionally distinct subpopulations during the co-differentiation of endothelial cells and cardiomyocytes from hiPSCs. Targeted analysis of single-cell RNA sequencing data revealed differential ETV2 dynamics in the two lineages. A newly created fluorescent reporter line allowed us to identify early lineage-predisposed states and show that a transient ETV2-high state initiates the specification of endothelial cells. We further demonstrated, unexpectedly, that functional cardiomyocytes can originate from progenitors expressing ETV2 at a low level. Our study thus sheds light on the in vitro differentiation dynamics of two important cardiac lineages. SIGNIFICANCE STATEMENTIn vitro differentiation of cardiac cell types is of great importance for understanding heart development, disease modeling and future regenerative medicine. Currently, underlying molecular mechanisms are incompletely understood, which limits the efficiency and fine-tuning of present differentiation protocols. Here, we investigated the master regulator ETV2 and showed that its upregulation marks the specification of two cardiac cell types during co-differentiation. Using single-cell RNA-seq and a new fluorescent reporter line we identified lineage-predisposed subpopulations in the ETV2+ cells. We thus resolved ETV2 dynamics at the single-cell level in the context of in vitro human cardiac differentiation.

developmental biology↗

bioRxiv2022

Pressure-driven fluidic system to control, multiplex and recirculate cell culture medium for Organs-on-Chips

Organ-on-Chip (OoC) devices are increasingly used to mimic the tissue microenvironment of cells in intact organs. This includes microchannels to mimic, for example, fluidic flow through vessels. Present methods for controlling microfluidic flow in these systems rely on gravity, rocker systems or external pressure pumps. For many purposes, pressure pumps give the most consistent flow profiles but they are not well-suited for high throughput as might be required for testing drug responses. Here, we describe a method which allows multiplexing of microfluidic channels in OoC devices plus the accompanying custom software necessary to run the system. Moreover, we show the approach is also suited to for recirculation of culture medium, an essential cost consideration when expensive culture reagents are used that are not "spent" through uptake by the cells during transient unidirectional flow.

bioengineering↗

bioRxiv2017

Versatile open software to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo

Contraction of muscle reflects its physiological state. Methods to quantify contraction are often complex, expensive and tailored to specific models or recording conditions, or require specialist knowledge for data extraction. Here we describe an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of contractility in (i) single cardiomyocytes from primary adult heart and human pluripotent stem cells, (ii) multicellular 2D-cardiomyocyte cultures, 3D engineered heart tissues and cardiac organoids/microtissues in vitro and (iii) intact hearts of zebrafish and humans in vivo. Good correlation was found with conventional measures of contraction in each system. Thus, using a single method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell- and animal models and human echocardiograms.

physiology↗