Biology subjects

Monaghan, M. G.

Publications and source records attributed to Monaghan, M. G..

5 recordsLinked to original sources

bioRxiv2024

Synergistic generation of cardiac resident-like macrophages and cardiomyocyte maturation in tissue engineered platforms

Cardiovascular disease stands as the leading cause of death globally, claiming approximately 19million lives in 2020. On the contrary, the development of cardiovascular drugs is experiencing a decline, largely due to the bottleneck in understanding the pathophysiology of various heart diseases and assessing the effects of drugs on healthy human hearts. The development of induced pluripotent stem cell (iPSC) technology and the availability of cardiac cell types in vitro, has resulted in a surge in efforts to fabricate human cardiac models for disease modelling and drug discovery applications. Although numerous attempts evidence successful fabrication of 3 dimensional (3D) engineered heart tissues, the innate immune cell population of the myocardium - particularly cardiac macrophages, was until recently, overlooke. With increasing appreciation of the interactions between cardiomyocytes and macrophages in the myocardium, in this work, isogenic populations of cardiac resident-like macrophages and cardiomyocytes were generated using iPSCs, to understand the interactions between the two cell types in both 2D and 3D settings, and subjected to electric stimulation. After characterizing iPSC-derived macrophages (iMacs) and iPSC-derived cardiomyocytes (iCMs) in depth, the conditioning of iMacs to align to a cardiac resident macrophage-like phenotype in the presence of iCMs in 2D culture was explored. In co-culture with iCMs, iMacs upregulated known genes expressed by cardiac resident macrophages. Additionally, in co-culture with iMacs, iCMs displayed an elongated morphology, improved calcium function and an increase in known maturation genes such as the ratio between MYH7 and MYH6 as well as SERCA2. In a 2D setting, iMacs showed the ability to electrically couple with iCMs and facilitate synchronous beating in iCM cultures. The 2D characterisation was translated into an engineered cardiac tissue model, wherein, improvement in tissue characteristics in the presence of iMacs was demonstrated in terms of increased cell alignment, enhanced cardiomyocyte elongation, physiologically relevant beat rates and improved tissue compaction. Taken together, these findings may open new avenues to use iMacs in engineered cardiac tissue models, not only as an innate immune cell source, but also as a support cell type to improve cardiomyocyte function and maturation.

bioengineering↗

bioRxiv2023

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Human mesenchymal stem cells (hMSCs) fuel discrete biosynthetic pathways to multiply and differentiate into specific cell lineages; with undifferentiated hMSCs showing reliance on glycolysis. hMSCs differentiating towards an osteogenic phenotype rely on oxidative phosphorylation as an energy source. Here, the metabolic profile of hMSCs was profiled during osteogenic differentiation over 14 days using a non-invasive live-cell imaging platform- two-photon fluorescence lifetime imaging microscopy (2P-FLIM) which images and measures NADH fluorescence. During osteogenesis, we observe a higher dependence on oxidative phosphorylation for cellular energy; concomitant with an increased reliance on anabolic pathways. We validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time-points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further during osteogenic differentiation. An indirect method of promoting glutaminolysis was explored so as to not impact cellular differentiation. As Lactate has been shown to promote glutamine uptake via c-Myc activation triggering expression of glutamine transmembrane transporters and glutaminase 1; we chose to increase extracellular lactate concentrations to drive increased glutaminolysis rates leading to higher levels of mineral deposition and osteogenic gene expression. Lactate supplementation of osteogenic medium also promoted upregulation of lactate metabolism and increased the expression of transmembrane cellular lactate transporters. Higher rates of lactate dehydrogenase gene expression coupled with higher NADH fluorescence intensity demonstrate a conversion of lactate to pyruvate. During this conversion, NADH is formed by the reverse enzymatic reaction of lactate dehydrogenase resulting in increased NADH fluorescence intensity. In order to evaluate the importance of glutaminolysis and lactate metabolism in osteogenic differentiation, these metabolic pathways were shut down using BPTES and -CHC respectively which led to reduced hMSC mineralisation. In summary, we demonstrate that hMSCs osteogenic differentiation has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture. Osteogenic differentiation was demonstrated to be directly dependent on OxPhos and on glutaminolysis and validated using biochemical assays. Furthermore, extracellular lactate is an essential metabolite to ensure osteogenic differentiation as a metabolic fuel and signalling molecule to promote glutaminolysis. These findings have significant impact in generating potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite driven osteogenesis.

bioengineering↗

bioRxiv2022

A guide to the manufacture of sustainable, ready to use in vitro platforms for the electric-field pacing of cellularised 3D porous scaffolds

Electrical activity is a key feature of most native tissues, with the most notable examples being the nervous and the cardiac systems. Modern medicine has moved towards the mimicking and regenerations of such systems both with in vitro models and therapies. Although researchers have now an increased repertoire of cell types and bio-physical cues to generate increasingly complex in vitro models, the inclusion of novel biomaterials in such systems has been negligible, with most approaches relying on scaffold-free self-assembling strategies. However, the rapid development of functional biomaterials and fabrication technologies - such as electroconductive scaffolds - warrants consideration and inclusion of materials, with recent evidence supporting the benefit of incorporating electrically active materials and their influence on the maturation of cardiac cells and tissues. In order to be manipulated in bioreactor systems, scaffold-based in vitro models require bespoke rig and bioreactors that vary from those commonly used for scaffold-free systems. In this work, we detail methods to rapid prototype an electrical pacing bioreactor and R3S - a Rig for Stimulation of Sponge-like Scaffolds. As a proof of concept and validation we demonstrate that these systems are compatible with isotropic and anisotropic porous scaffolds composed of collagen or poly(3,4-ethylene dioxythiophene):polystyrene sulfonate (PEDOT:PSS). External pacing of C3H10 cells on anisotropic porous scaffolds led to a metabolic increase and enhanced cell alignment. This setup has been designed for pacing and simultaneously live tracking of in vitro models. This platform has wide suitability for the study of electrical pacing of cellularized scaffolds in 3D in vitro cultures.

bioengineering↗

bioRxiv2022

Non-Invasive classification of macrophage polarisation by 2P-FLIM and machine learning

1.In this study, fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence is applied as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages were obtained from human blood-circulating monocytes, polarised using established treatments, and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Fluorescence lifetime imaging microscopy (FLIM) quantified variations in NAD(P)H-derived fluorescent lifetimes in large field-of-view images of individual polarised macrophages also challenged, in real-time with small molecule perturbations of metabolism during imaging. We uncover FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) which strongly stratifies the phenotype of polarised human macrophages. This stratification and parameters emanating from a FLIM approach, served as the basis for machine learning models. Applying a random forest model, identified three strongly governing FLIM parameters, achieving a ROC AUC value of 0.944 when classifying human macrophages.

bioengineering↗

bioRxiv2020

Intracellular label-free detection of mesenchymal stem cell metabolism within a perivascular niche-on-a-chip

The stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overall fate of stem cells within it. Mesenchymal stem cells (MSCs), in particular, experience influential microenvironmental conditions, which induce specific metabolic profiles that affect processes such as cell differentiation and dysregulation of the immunomodulatory funtion. Reports focusing specifically on the metabolic status of MSCs under the effect of pathophysiological stimuli - in terms of flow velocities, shear stresses or oxygen tension - do not model heterogenous gradients, highlighting the need of more advanced models reproducing the metabolic niche. Organ-on-a-chip technology offers the most advanced tools for stem cell niche modelling thus allowing for controlled dynamic culture conditions while profiling tunable oxygen tension gradients. However, current systems for live cell detection of metabolic activity inside microfluidic devices require the integration of microsensors that allow for extracellular measurments only, giving innacurate and indirect information about the metabolic state of cells. Here, we present a metabolic toolbox coupling a miniatuirzed in vitro system for human-MSCs dynamic culture, that mimics microenvironmental conditions of the perivascular niche, with high-resolution imaging of intracellular metabolism. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we monitor the spatial metabolic machinery and correlate it with experimentally validated intracellular oxygen concentration after designing the oxygen tension decay along the fluidic chamber by in silico models prediction. Our platform allows for the subjection of a metabolic profile to MSCs, mimicking the physiological niche in space and time, and its real-time monitoring representing a functional tool for modelling perivascular niches, relevant diseases and metabolic-related uptake of pharmaceuticals.

bioengineering↗