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Biomedical data are becoming increasingly multimodal and thereby capture the underlying complex relationships among biological processes. Deep learning (DL)-based data fusion strategies are a popular approach for modeling these nonlinear relationships. Therefore, we review the current state-of-the-art of such methods and propose a detailed taxonomy that facilitates more informed choices of fusion strategies for biomedical applications, as well as research on novel methods. By doing so, we find that deep fusion strategies often outperform unimodal and shallow approaches. Additionally, the proposed subcategories of fusion strategies show different advantages and drawbacks. The review of current methods has shown that, especially for intermediate fusion strategies, joint representation learning is the preferred approach as it effectively models the complex interactions of different levels of biological organization. Finally, we note that gradual fusion, based on prior biological knowledge or on search strategies, is a promising future research path. Similarly, utilizing transfer learning might overcome sample size limitations of multimodal data sets. As these data sets become increasingly available, multimodal DL approaches present the opportunity to train holistic models that can learn the complex regulatory dynamics behind health and disease.
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Aprendizado ProfundoRESUMO
Revolutionary advances in AI and deep learning in recent years have resulted in an upsurge of papers exploring applications within the biomedical field. Within stem cell research, promising results have been reported from analyses of microscopy images to, that is, distinguish between pluripotent stem cells and differentiated cell types derived from stem cells. In this work, we investigated the possibility of using a deep learning model to predict the differentiation stage of pluripotent stem cells undergoing differentiation toward hepatocytes, based on morphological features of cell cultures. We were able to achieve close to perfect classification of images from early and late time points during differentiation, and this aligned very well with the experimental validation of cell identity and function. Our results suggest that deep learning models can distinguish between different cell morphologies, and provide alternative means of semi-automated functional characterization of stem cell cultures.
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Inteligência Artificial , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular , Hepatócitos/metabolismo , Técnicas de Cultura de Células/métodosRESUMO
The different chambers of the human heart demonstrate regional physiological traits and may be differentially affected during pathological remodeling, resulting in heart failure. Few previous studies, however, have characterized the different chambers at a transcriptomic level. We, therefore, conducted whole tissue RNA sequencing and gene set enrichment analysis of biopsies collected from the four chambers of adult failing (n = 8) and nonfailing (n = 11) human hearts. Atria and ventricles demonstrated distinct transcriptional patterns. When compared with nonfailing ventricles, the transcriptional pattern of nonfailing atria was enriched for many gene sets associated with cardiogenesis, the immune system and bone morphogenetic protein (BMP), transforming growth factor-ß (TGF-ß), MAPK/JNK, and Wnt signaling. Differences between failing and nonfailing hearts were also determined. The transcriptional pattern of failing atria was distinct compared with that of nonfailing atria and enriched for gene sets associated with the innate and adaptive immune system, TGF-ß/SMAD signaling, and changes in endothelial, smooth muscle cell, and cardiomyocyte physiology. Failing ventricles were also enriched for gene sets associated with the immune system. Based on the transcriptomic patterns, upstream regulators associated with heart failure were identified. These included many immune response factors predicted to be similarly activated for all chambers of failing hearts. In summary, the heart chambers demonstrate distinct transcriptional patterns that differ between failing and nonfailing hearts. Immune system signaling may be a hallmark of all four heart chambers in failing hearts and could constitute a novel therapeutic target.NEW & NOTEWORTHY The transcriptomic patterns of the four heart chambers were characterized in failing and nonfailing human hearts. Both nonfailing atria had distinct transcriptomic patterns characterized by cardiogenesis, the immune system and BMP/TGF-ß, MAPK/JNK, and Wnt signaling. Failing atria and ventricles were enriched for gene sets associated with the innate and adaptive immune system. Key upstream regulators associated with heart failure were identified, including activated immune response elements, which may constitute novel therapeutic targets.
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Insuficiência Cardíaca , Transcriptoma , Adulto , Humanos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Átrios do Coração/metabolismo , Perfilação da Expressão Gênica , Fator de Crescimento Transformador beta/metabolismo , Miocárdio/metabolismoRESUMO
The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, many cell lines and primary cells do not recreate physiological restriction of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low paracellular permeability. Given the possibility to generate large amounts of human iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. iPSC-derived brain endothelial cells form tight cell layers in vitro and their barrier properties can be enhanced through coculture with other cell types of the BBB. Currently, many different models of the BBB using iPSC-derived cells are under evaluation to study BBB formation, maintenance, disruption, drug transport and diseases affecting the BBB. This review summarizes important functions of the BBB and current efforts to create iPSC-derived BBB models in both static and dynamic conditions. In addition, it highlights key model requirements and remaining challenges for human iPSC-derived BBB models in vitro.
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Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Células Endoteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Barreira Hematoencefálica/patologia , Técnicas de Cocultura/métodos , HumanosRESUMO
Transcriptional studies of the human heart provide insight into physiological and pathophysiological mechanisms, essential for understanding the fundamental mechanisms of normal cardiac function and how they are altered by disease. To improve the understanding of why men and women may respond differently to the same therapeutic treatment it is crucial to learn more about sex-specific transcriptional differences. In this study the transcriptome of right atrium and left ventricle was compared across sex and regional location. Paired biopsies from five male and five female patients undergoing aortic valve replacement or coronary artery bypass grafting were included. Gene expression analysis identified 620 differentially expressed transcripts in atrial and ventricular tissue in men and 471 differentially expressed transcripts in women. In total 339 of these transcripts overlapped across sex but notably, 281 were unique in the male tissue and 162 in the female tissue, displaying marked sex differences in the transcriptional machinery. The transcriptional activity was significantly higher in atrias than in ventricles as 70% of the differentially expressed genes were upregulated in the atrial tissue. Furthermore, pathway- and functional annotation analyses performed on the differentially expressed genes showed enrichment for a more heterogeneous composition of biological processes in atrial compared with the ventricular tissue, and a dominance of differentially expressed genes associated with infection disease was observed. The results reported here provide increased insights about transcriptional differences between the cardiac atrium and ventricle but also reveal transcriptional differences in the human heart that can be attributed to sex.
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Perfilação da Expressão Gênica , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Fatores Sexuais , Transcrição Gênica , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/cirurgia , Biópsia , Análise por Conglomerados , Ponte de Artéria Coronária , Feminino , Regulação da Expressão Gênica , Implante de Prótese de Valva Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , TranscriptomaRESUMO
There is a strong anticipated future for human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far, their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics with multiple functional assays. The transcriptomics analysis revealed that 86% of the genes were expressed at similar levels in hiPS-HEP as in human primary hepatocytes (hphep). Adult characteristics of the hiPS-HEP were confirmed by the presence of important hepatocyte features, e.g., Albumin secretion and expression of major drug metabolizing genes. Normal energy metabolism is crucial for modeling metabolic diseases, and both transcriptomics data and functional assays showed that hiPS-HEP were similar to hphep regarding uptake of glucose, low-density lipoproteins (LDL), and fatty acids. Importantly, the inflammatory state of the hiPS-HEP was low under standard conditions, but in response to lipid accumulation and ER stress the inflammation marker tumor necrosis factor α (TNFα) was upregulated. Furthermore, hiPS-HEP could be co-cultured with primary hepatic stellate cells both in 2D and in 3D spheroids, paving the way for using these co-cultures for modeling non-alcoholic steatohepatitis (NASH). Taken together, hiPS-HEP have the potential to serve as an in vitro model for metabolic diseases. Furthermore, differently expressed genes identified in this study can serve as targets for future improvements of the hiPS-HEP.
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Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Metabólicas/metabolismo , Transcriptoma , Idoso , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Estresse do Retículo Endoplasmático , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lipoproteínas LDL/metabolismo , Masculino , Doenças Metabólicas/genética , Pessoa de Meia-Idade , Cultura Primária de Células/métodos , Esferoides Celulares/citologia , Esferoides Celulares/metabolismoRESUMO
Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation. Stem Cells 2018;36:1816-12.
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Barreira Hematoencefálica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma/fisiologia , Diferenciação Celular , HumanosRESUMO
Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.
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Fígado/citologia , Fígado/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Transcriptoma/genéticaRESUMO
Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed®, and Cellartis®, and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis® medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis® medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis® medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure.
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Enzimas/metabolismo , Fígado/metabolismo , Técnicas de Cultura de Órgãos/métodos , Trifosfato de Adenosina/metabolismo , Albuminas/biossíntese , Proteínas de Transporte/metabolismo , Meios de Cultura , Fibrose/genética , Regulação da Expressão Gênica , Humanos , Inativação Metabólica , Fígado/efeitos dos fármacos , Fígado/patologia , Estresse Oxidativo/genética , Xenobióticos/metabolismo , Xenobióticos/farmacocinéticaRESUMO
Regenerative therapies hold great potential to change the treatment paradigm for cardiac diseases. Human cardiac progenitor cells can be used for drug discovery in this area and also provide a renewable source of cardiomyocytes. However, a better understanding of their characteristics is critical for interpreting data obtained from drug screening using these cells. In the present study, we performed global transcriptional analysis of two important sources of cardiac progenitors, i.e., patient epicardium-derived cells (EPDCs) and cardiac progenitor cells (CPCs) derived from human induced pluripotent stem cells. In addition, we also compared the gene expression profiles of these cells when they were cultured under normoxic and hypoxic conditions. We identified 3,289 mRNAs that were differentially expressed between EPDCs and CPCs. Gene ontology annotation and pathway enrichment analyses further revealed possible unique functions of these two cell populations. Notably, the impact of hypoxia vs normoxia on gene expression was modest and only a few genes (e.g., AK4, ALDOC, BNIP3P1, PGK1, and SLC2A1) were upregulated in EPDCs and CPCs after the cells were exposed to low oxygen for 24 h. Finally, we also performed a focused analysis of the gene expression patterns of a predefined set of 92 paracrine factors. We identified 30 of these genes as differentially expressed, and 29 were expressed at higher levels in EPDCs compared with CPCs. Taken together, the results of the present study advance our understanding of the transcriptional programs in EPDCs and CPCs and highlights important differences and similarities between these cell populations.
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Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Pericárdio/citologia , Biomarcadores/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Análise por Conglomerados , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Anotação de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxigênio/farmacologia , Comunicação Parácrina/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/genéticaRESUMO
Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.
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Diferenciação Celular/genética , Genes , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Padrões de ReferênciaRESUMO
BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories. METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified. RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC. CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.
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Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/metabolismo , RNA/genética , Fatores de Transcrição/genética , Transcriptoma , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/citologia , Fatores de Transcrição/biossínteseRESUMO
Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Hepatócitos/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Células-Tronco Pluripotentes/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Fígado Gorduroso/induzido quimicamente , Células Hep G2 , Humanos , Lipidoses/induzido quimicamente , Fígado/efeitos dos fármacosRESUMO
Vascular diseases are the largest cause of death globally and impose a major global burden on healthcare. The gold standard for treating vascular diseases is the transplantation of autologous veins, if applicable. Alternative treatments still suffer from shortcomings, including low patency, lack of growth potential, the need for repeated intervention, and a substantial risk of developing infections. The use of a vascular ECM scaffold reconditioned with the patient's own cells has shown successful results in preclinical and clinical studies. In this study, we have compared the proteomes of personalized tissue-engineered veins of humans and pigs. By applying tandem mass tag (TMT) labeling LC/MS-MS, we have investigated the proteome of decellularized (DC) veins from humans and pigs and reconditioned (RC) DC veins produced through perfusion with the patient's whole blood in STEEN solution, applying the same technology as used in the preclinical studies. The results revealed high similarity between the proteomes of human and pig DC and RC veins, including the ECM texture after decellularization and reconditioning. In addition, functional enrichment analysis showed similarities in signaling pathways and biological processes involved in the immune system response. Furthermore, the classification of proteins involved in immune response activity that were detected in human and pig RC veins revealed proteins that evoke immunogenic responses, which may lead to graft rejection, thrombosis, and inflammation. However, the results from this study imply the initiation of wound healing rather than an immunogenic response, as both systems share the same processes, and no immunogenic response was reported in the preclinical and clinical studies. Finally, our study assessed the application of STEEN solution in tissue engineering and identified proteins that may be useful for the prediction of successful transplantations.
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Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs.
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BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.
Assuntos
Diabetes Mellitus Experimental , Pé Diabético , Humanos , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 1 de Crescimento de Fibroblastos , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Modelos Animais de DoençasRESUMO
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery.
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Acetil-CoA Carboxilase , Diferenciação Celular , Miócitos Cardíacos , Células-Tronco Pluripotentes , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Acetil-CoA Carboxilase/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , AnimaisRESUMO
Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Infarto do Miocárdio , Miócitos Cardíacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/transplante , Humanos , Animais , Camundongos , Infarto do Miocárdio/terapia , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Fibroblastos/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/metabolismo , Modelos Animais de Doenças , Neovascularização Fisiológica , Células CultivadasRESUMO
Stem cell niches have been thoroughly investigated in tissue with high regenerative capacity but not in tissues where cell turnover is slow, such as the human heart. The left AtrioVentricular junction (AVj), the base of the mitral valve, has previously been proposed as a niche region for cardiac progenitors in the adult human heart. In the present study, we explore the right side of the human heart, the base of the tricuspid valve, to investigate the potential of this region as a progenitor niche. Paired biopsies from explanted human hearts were collected from multi-organ donors (N = 12). The lateral side of the AVj, right atria (RA), and right ventricle (RV) were compared for the expression of stem cell niche-related biomarkers using RNA sequencing. Gene expression data indicated upregulation of genes related to embryonic development and extracellular matrix (ECM) composition in the proposed niche region, that is, the AVj. In addition, immunohistochemistry showed high expression of the fetal cardiac markers MDR1, SSEA4, and WT1 within the same region. Nuclear expression of HIF1α was detected suggesting hypoxia. Rare cells were found with the co-staining of the proliferation marker PCNA and Ki67 with cardiomyocyte nuclei marker PCM1 and cardiac Troponin T (cTnT), indicating proliferation of small cardiomyocytes. WT1+/cTnT+ and SSEA4+/cTnT+ cells were also found, suggesting cardiomyocyte-specific progenitors. The expression of the stem cell markers gradually decreased with distance from the tricuspid valve. No expression of these markers was observed in the RV tissue. In summary, the base of the tricuspid valve is an ECM-rich region containing cells with expression of several stem cell niche-associated markers. Co-expression of stem cell markers with cTnT indicates cardiomyocyte-specific progenitors. We previously reported similar data from the base of the mitral valve and thus propose that human adult cardiomyocyte progenitors reside around both atrioventricular valves.
Assuntos
Nicho de Células-Tronco , Valva Tricúspide , Adulto , Humanos , Valva Tricúspide/patologia , Miócitos Cardíacos/metabolismo , Ventrículos do Coração , Biomarcadores/metabolismoRESUMO
Tissue engineering is a promising methodology to produce advanced therapy medicinal products (ATMPs). We have developed personalized tissue engineered veins (P-TEV) as an alternative to autologous or synthetic vascular grafts utilized in reconstructive vein surgery. Our hypothesis is that individualization through reconditioning of a decellularized allogenic graft with autologous blood will prime the tissue for efficient recellularization, protect the graft from thrombosis, and decrease the risk of rejection. In this study, P-TEVs were transplanted to vena cava in pig, and the analysis of three veins after six months, six veins after 12 months and one vein after 14 months showed that all P-TEVs were fully patent, and the tissue was well recellularized and revascularized. To confirm that the ATMP product had the expected characteristics one year after transplantation, gene expression profiling of cells from P-TEV and native vena cava were analyzed and compared by qPCR and sequencing. The qPCR and bioinformatics analysis confirmed that the cells from the P-TEV were highly similar to the native cells, and we therefore conclude that P-TEV is functional and safe in large animals and have high potential for use as a clinical transplant graft.