Biology subjects

Mummery, C. L.

Publications and source records attributed to Mummery, C. L..

13 recordsLinked to original sources

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↗

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↗

bioRxiv2025

Gene syntax defines supercoiling-mediated transcriptional feedback

Gene syntax--the order and arrangement of genes and their regulatory elements--shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

synthetic biology↗

bioRxiv2024

STRAIGHT-IN Dual: a platform for dual, single-copyintegrations of DNA payloads and gene circuits into human induced pluripotent stem cells

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influence transgene silencing, demonstrating the impact these design features have on reporter gene expression and forward programming of hiPSCs into neurons, motor neurons, and endothelial cells. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of different transcription factors within the same cell. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

synthetic 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↗

bioRxiv2023

Gain of chromosome region 1q31.3 in human iPSCs confers growth advantage and alters contraction in derivative cardiomyocytes

hPSCs can acquire chromosomal aberrations such as copy number variations during prolonged maintenance in vitro, conferring growth advantages. However, the effect of these culture-acquired mutations on the phenotypes of the differentiated hPSCs is largely unstudied. Here, we identified mosaicism in a hPSC line in which some cells showed a gain of chromosome 1q31.3. We subcloned the wild-type and variant hPSCs and could maintain both as stable lines. While both variant and wildtype lines differentiated efficiently to cardiomyocytes (hPSC-CMs), molecular analysis revealed the variant hPSC-CMs had increased expression of TNNT2, a gene encoding one of the major sarcomere proteins mediating cardiomyocyte contractility and located within the gained chromosome 1q region. Moreover, the variant hPSC-CMs showed altered contraction kinetics. Together these results highlight the importance of careful monitoring of chromosome aberrations in hPSC lines as these could have confounding effects in various applications such as disease modelling and drug discovery.

cell biology↗

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↗

bioRxiv2021

STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) by: (i) inserting various combinations of fluorescent reporters into hiPSCs to assess excitation-contraction coupling cascade in derivative cardiomyocytes, and; (ii) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorder into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically-matched panels of hiPSC lines efficiently and cost-effectively.

cell biology↗

bioRxiv2019

Isogenic sets of hiPSC-CMs harboring KCNH2 mutations capture location-related phenotypic differences

AimsLong QT syndrome type 2 (LQT2) is caused by mutations in the gene KCNH2, encoding the hERG ion channel. Clinically, mild and severe phenotypes are associated with this cardiac channelopathy, complicating efforts to predict patient risk. The location of the mutation within KCNH2 contributes to this variable disease manifestation. Here we determined whether such phenotypic differences could be detected in cardiomyocytes derived from isogenic human induced pluripotent stem cells (hiPSCs) genetically edited to harbour a range of KCNH2 mutations. Methods and ResultsThe hiPSC lines heterozygous for missense mutations either within the pore or tail region of the ion channel were generated using CRISPR-Cas9 editing and subsequently differentiated to cardiomyocytes (hiPSC-CMs) for functional assessment. Electrophysiological analysis confirmed the mutations prolonged the action potentials and field potentials of the hiPSC-CMs, with differences detected between the pore and tail region mutations when measured as paced 2D monolayers. This was also reflected in the cytosolic Ca2+ transients and contraction kinetics of the different lines. Pharmacological blocking of the hERG channel in the hiPSC-CMs also revealed that mutations in the pore-loop region conferred a greater susceptibility to arrhythmic events. ConclusionThese findings establish that subtle phenotypic differences related to the location of the KCNH2 mutation in LQT2 patients are reflected in hiPSC-CMs under genetically controlled conditions. Moreover, the results validate hiPSC-CMs as a strong candidate for evaluating the underlying severity of individual KCNH2 mutations in humans which could ultimately facilitate patient risk stratification. Translational perspectiveClinical management of patients diagnosed with cardiac channelopathy diseases such as LQT2 is complicated by the variable disease phenotypes observed among mutation carriers, creating challenges for diagnosis, risk stratification and treatment. The genotype of the patient contributes to this clinical heterogeneity, with the influence of the mutations location within KCNH2 on a patients risk of a cardiac event being an example. Here we demonstrate that under stringently controlled genetic and experimental conditions, hiPSC-CMs are able to reflect these subtle genotype-phenotype differences, thereby providing new opportunities to stratify and potentially lessen sudden cardiac death risk amongst KCNH2 mutation carriers.

cell biology↗

bioRxiv2019

Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology, and drug screening. All these applications require large quantities of well-characterised and standardised batches of hiPSC-CMs. Cryopreservation of hiPSC-CMs without affecting their biochemical or biophysical phenotype is essential for facilitating this, but ideally requires the cells being unchanged by the freeze-thaw procedure. We therefore compared the in vitro functional and molecular characteristics of fresh and cryopreserved hiPSC-CMs generated from two independent hiPSC lines. While the frozen hiPSC-CMs exhibited poorer replating than their freshly-derived counterparts, there was no difference in the proportion of cardiomyocytes retrieved from the mixed population when this was factored in. Interestingly, cryopreserved hiPSC-CMs from one line exhibited longer action potential durations. These results provide evidence that cryopreservation does not compromise the in vitro molecular, physiological and mechanical properties of hiPSC-CMs, though can lead to an enrichment in ventricular myocytes. It also validates this procedure for storing hiPSC-CMs, thereby allowing the same batch of hiPSC-CMs to be used for multiple applications and evaluations.

cell biology↗

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↗