RESUMO
Human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC CMs) may be used in regenerative medicine for individualized tissue transplants in the future. For application in patients, the generated CMs have to be highly pure and well characterized. In order to overcome the prevalent scarcity of CM-specific markers, we quantitatively assessed cell-surface-exposed sialo-glycoproteins and N-glycans of hiPSCs, CM progenitors, and CMs. Applying a combination of metabolic labeling and specific sialo-glycoprotein capture, we could highly enrich and quantify membrane proteins during cardiomyogenic differentiation. Among them we identified a number of novel, putative biomarkers for hiPSC CMs. Analysis of the N-glycome by capillary gel electrophoresis revealed three novel structures comprising ß1,3-linked galactose, α2,6-linked sialic acid and complex fucosylation; these were highly specific for hiPSCs. Bisecting GlcNAc structures strongly increased during differentiation, and we propose that they are characteristic of early, immature CMs.
Assuntos
Membrana Celular/química , Glicômica/métodos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , Polissacarídeos/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sequência de Carboidratos , Diferenciação Celular , Membrana Celular/metabolismo , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Fucose/química , Fucose/metabolismo , Galactose/química , Galactose/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Polissacarídeos/metabolismo , Receptor EphA7/genética , Receptor EphA7/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Coloração e Rotulagem/métodosRESUMO
Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8-96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing.
Assuntos
Envelhecimento/urina , Modelos Biológicos , Peptídeos/urina , Proteoma/metabolismo , Proteômica , Animais , Eletrocromatografia Capilar , Feminino , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos KnockoutRESUMO
Based on demographic trends, the societies in many developed countries are facing an increasing number and proportion of people over the age of 65. The raise in elderly populations along with improved health-care will be concomitant with an increased prevalence of ageing-associated chronic conditions like cardiovascular, renal, and respiratory diseases, arthritis, dementia, and diabetes mellitus. This is expected to pose unprecedented challenges both for individuals and societies and their health care systems. An ultimate goal of ageing research is therefore the understanding of physiological ageing and the achievement of 'healthy' ageing by decreasing age-related pathologies. However, on a molecular level, ageing is a complex multi-mechanistic process whose contributing factors may vary individually, partly overlap with pathological alterations, and are often poorly understood. Proteome analysis potentially allows modelling of these multifactorial processes. This review summarises recent proteomic research on age-related changes identified in animal models and human studies. We combined this information with pathway analysis to identify molecular mechanisms associated with ageing. We identified some molecular pathways that are affected in most or even all organs and others that are organ-specific. However, appropriately powered studies are needed to confirm these findings based in in silico evaluation.