Biology subjects

Fernandez, M. C.

Publications and source records attributed to Fernandez, M. C..

5 recordsLinked to original sources

bioRxiv2025

Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have become an invaluable tool for disease modelling and drug testing. However, while many etiologies of heart failure involve defects in excitation-contraction coupling, mitochondrial energetics or both, iPSC-CM are limited by the developmental immaturity of these processes. Here, we report a combinatorial strategy to enhance the maturation of human iPSC-CM by integrating three-dimensional (3D) spheroid culture conditions with a defined hormone- and fatty acid-enriched maturation medium (MM). A comprehensive analysis of structural, electrophysiological and Ca2+ handling parameters was performed to evaluate cellular and functional maturation. The iPSC-CM generated under these conditions (3D_MM) exhibit many phenotypic characteristics that resemble those of isolated adult human CM, including (i) a rod-shaped morphology, (ii) cardiac ultrastructural features such as aligned myofilaments, unidirectional organized sarcomeres, and the presence of transverse (t)-tubules, (iii) refined action potential (AP) parameters and Ca2+ handling, and (iv) {beta}-adrenergic responsiveness and a positive force-frequency relationship. Compared with long-term (LT) monolayer cultures of 90 days or the individual cues (3D or MM alone), the 3D_MM protocol achieves mostly superior or at the least non-inferior maturation effects. This systematic investigation further demonstrates that while 3D culturing or MM alone improved specific aspects of maturation, only their synergistic combination produced a comprehensive enhancement of key CM processes, such as excitation-contraction coupling and mitochondrial energetics.

cell biology↗

bioRxiv2023

3D structure of fibroblasts and macrophages in the healthy and cryo-ablated heart

IntroductionCardiac non-myocytes (NM) play important roles in heart development, homeostasis, and lesion repair. To assess the relevance of different NM populations for cardiac (patho)physiology, a quantitative assessment of their abundance and structure in the different heart chambers is an essential prerequisite. We here present an experimental approach to determine the distribution, dimensions, and 3D morphology of fibroblasts (FB) and macrophages (M{Phi}) in healthy and pathologically remodelled hearts. Methods and resultsWe used Cre-loxP recombination to selectively target channelrhopsin-2 (ChR2)-eYFP to either FB or M{Phi} in healthy and cryo-ablated mouse hearts. Hearts were optically cleared using X-CLARITY and membrane-bound eYFP fluorescence was recorded by confocal microscopy. The resulting image stacks were segmented to generate 3D reconstructions of labelled cell populations in near native tissue. In doing so, we show that FB and M{Phi} have similar surface areas, volumes and morphologies, but that FB occupy larger fractional volumes than M{Phi} in all chambers of healthy murine hearts. Furthermore, M{Phi} appear primarily as single cells, whereas FB form extended networks of interconnected cells. In left-ventricular tissue following cryo-ablation, we observed large disordered networks of FB in the scar area with an increased volume occupied by FB both in the scar and remotely. In cryo-ablated ventricles, M{Phi} form comparatively small, but dense networks in the scar without changing their abundance in remote myocardium. ConclusionsOur study assesses the 3D distribution and structure of fluorescently labelled FB and M{Phi} in healthy and lesioned murine hearts. Based on 3D reconstructions of FB and M{Phi} networks, we quantified the surface areas and volumes of individual non-myocytes in the different chambers of the heart and in ventricular scar tissue, thus providing important quantitative data serving as basis for computational modelling of non-myocyte contributions to cardiac structure and physiology.

cell biology↗

bioRxiv2023

Age-related structural and functional changes of the intracardiac nervous system

BackgroundAlthough aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. Methods and ResultsLower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20-24 months) compared to young mice (2-3 months). In old mice, CSPG4 was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. ConclusionsOn the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/568538v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11dbccdorg.highwire.dtl.DTLVardef@1563d75org.highwire.dtl.DTLVardef@dcda58org.highwire.dtl.DTLVardef@182e598_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Global and single-cell proteomics view of the co-evolution between neural progenitors and breast cancer cells in a co-culture model

Tumor neurogenesis, a process by which new nerves invade tumors, is a growing area of interest in cancer research. Nerve presence has been linked to aggressive features of various solid tumors, including breast and prostate cancer. A recent study suggested that the tumor microenvironment may influence cancer progression through recruitment of neural progenitor cells from the central nervous system. However, the presence of neural progenitors in human breast tumors has not been reported. Here, we investigate the presence of Doublecortin (DCX) and Neurofilament-Light (NFL) co-expressing (DCX+/NFL+) cells in patient breast cancer tissue using Imaging Mass Cytometry. To map the interaction between breast cancer cells and neural progenitor cells further, we created an in vitro model mimicking breast cancer innervation, and characterized using mass spectrometry-based proteomics on the two cell types as they co- evolved in co-culture. Our results indicate stromal presence of DCX+/NFL+ cells in breast tumor tissue from a cohort of 107 patient cases, and that neural interaction contribute to drive a more aggressive breast cancer phenotype in our co-culture models. Our results support that neural involvement plays an active role in breast cancer and warrants further studies on the interaction between nervous system and breast cancer progression.

cancer biology↗

bioRxiv2020

Postglacial migration across a large dispersal barrier outpaces regional expansion from glacial refugia: evidence from two conifers in the Pacific Northwest.

Understanding how climate refugia and migration over great distances have facilitated species survival during periods of past climate change is crucial for evaluating contemporary threats to biodiversity. In addition to tracking a changing climate, extant species must face complex, anthropogenically fragmented landscapes. The dominant conifer species in the mesic temperate forests of the Pacific Northwest are split by the arid rain-shadow of the Cascade Range into coastal and interior distributions, with continued debate over the origins of the interior populations. If the Last Glacial Maximum extirpated populations in the interior then postglacial migration across the arid divide would have been necessary to create the current distribution, whereas interior refugial persistence could have locally repopulated the disjunction. These alternative scenarios have significant implications for the postglacial development of the Pacific Northwest mesic forests and the impact of dispersal barriers during periods of climate change. Here we use genotyping-by-sequencing (ddRADseq) and phylogeographical modeling to show that the postglacial expansion of both mountain hemlock and western redcedar consisted largely of long-distance spread inland in the direction of dominant winds, with limited expansion from an interior redcedar refugium. Our results for these two key mesic conifers, along with fossil pollen data, address the longstanding question on the development of the Pacific Northwest mesic forests and contrast with many recent studies emphasizing the role of cryptic refugia in colonizing modern species ranges. Statement of SignificanceUnderstanding whether habitat fragmentation hinders range shifts as species track a changing climate presents a pressing challenge for biologists. Species with disjunct distributions provide a natural laboratory for studying the effects of fragmentation during past periods of climate change. We find that dispersal across a 50-200-km inhospitable barrier characterized the expansion of two conifer species since the last ice age. The importance of migration, and minimal contribution of more local glacial refugia, contrasts with many recent studies emphasizing the role of microrefugia in populating modern species distributions. Our results address a longstanding question on the development of the disjunct mesic conifer forests of the Pacific Northwest and offer new insights into the spatiotemporal patterns of refugial populations and postglacial vegetation development previously unresolved despite decades of paleoecological studies.

plant biology↗