RESUMO
BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.
Assuntos
Cardiomiopatia Dilatada , Feminino , Gravidez , Animais , Humanos , Cardiomiopatia Dilatada/genética , Peixe-Zebra , Volume Sistólico , Função Ventricular Esquerda , Centrossomo/metabolismo , Miócitos CardíacosRESUMO
Chondrogenesis of human mesenchymal stem cells (hMSCs) is an important biological process in many applications including cartilage tissue engineering. We investigated the glucose uptake characteristics of aggregates of hMSCs undergoing chondrogenesis over a 3-week period both experimentally and by using a mathematical model. Initial concentrations of glucose in the medium were varied from 1 to 4.5 g/L to mimic limiting conditions and glucose uptake profiles were obtained. A reaction-diffusion mathematical model was implemented and solved to estimate kinetic parameters. Experimental glucose uptake rates increased with culture time for aggregates treated with higher initial glucose concentrations (3 and 4.5 g/L), whereas they decreased or remained constant for those treated with lower initial glucose concentrations (1 and 2 g/L). Lactate production rate increased by as much as 40% for aggregates treated with higher initial glucose concentrations (2, 3 and 4.5 g/L), whereas it remained constant for those treated with 1 g/L initial glucose concentration. The estimated DNA-normalized maximum glucose uptake rate decreased by a factor of 9 from day 0-2 (12.5 mmol/s/g DNA) to day 6-8 (1.5 mmol/s/g DNA), after which it started to increase. On day 18-20, its value (17.5 mmol/s/g DNA) was about 11 times greater than its lowest value. Further, the extracellular matrix levels of aggregates at day 14 and day 21 correlated with their overall glucose uptake and lactate production. The results suggest that during chondrogenesis, for optimal results, cells require increasing amounts of glucose. Our results also suggest that diffusion limitations play an important role in glucose uptake even in the smaller size aggregate model of chondrogenesis. Further, the results indicate that glucose uptake or lactate production can be a tool for predicting the end quality of tissue during the process of chondrogenesis. The estimated kinetic parameters can be used to model glucose requirements in cartilage tissue engineering applications.
Assuntos
Condrogênese/fisiologia , Glucose/metabolismo , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Células Cultivadas , Humanos , Cinética , Células-Tronco Mesenquimais/citologiaRESUMO
Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor ß containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.
Assuntos
Cartilagem/enzimologia , Condrogênese , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/enzimologia , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidores , Transporte Biológico Ativo , Cartilagem/citologia , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Quinases Associadas a rho/metabolismoRESUMO
Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.
Assuntos
Tomografia Óptica/instrumentação , Tomografia Óptica/métodos , Núcleo Celular/metabolismo , Desenho de Equipamento , Tomografia Computadorizada Quadridimensional/instrumentação , Tomografia Computadorizada Quadridimensional/métodos , Humanos , Células K562/patologia , Mitocôndrias/metabolismo , Reprodutibilidade dos Testes , Análise de Célula ÚnicaRESUMO
Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.