RESUMEN
OBJECTIVE: Glycogen storage disease type 1a (GSD Ia) is a rare inherited metabolic disorder caused by mutations in the glucose-6-phosphatase (G6PC1) gene. When untreated, GSD Ia leads to severe fasting-induced hypoglycemia. Although current intensive dietary management aims to prevent hypoglycemia, patients still experience hypoglycemic events. Poor glycemic control in GSD Ia is associated with hypertriglyceridemia, hepatocellular adenoma and carcinoma, and also with an increased bleeding tendency of unknown origin. METHODS: To evaluate the effect of glycemic control on leukocyte levels and coagulation in GSD Ia, we employed hepatocyte-specific G6pc1 deficient (L-G6pc-/-) mice under fed or fasted conditions, to match good or poor glycemic control in GSD Ia, respectively. RESULTS: We found that fasting-induced hypoglycemia in L-G6pc-/- mice decreased blood leukocytes, specifically proinflammatory Ly6Chi monocytes, compared to controls. Refeeding reversed this decrease. The decrease in Ly6Chi monocytes was accompanied by an increase in plasma corticosterone levels and was prevented by the glucocorticoid receptor antagonist mifepristone. Further, fasting-induced hypoglycemia in L-G6pc-/- mice prolonged bleeding time in the tail vein bleeding assay, with reversal by refeeding. This could not be explained by changes in coagulation factors V, VII, or VIII, or von Willebrand factor. While the prothrombin and activated partial thromboplastin time as well as total platelet counts were not affected by fasting-induced hypoglycemia in L-G6pc-/- mice, ADP-induced platelet aggregation was disturbed. CONCLUSIONS: These studies reveal a relationship between fasting-induced hypoglycemia, decreased blood monocytes, and disturbed platelet aggregation in L-G6pc-/- mice. While disturbed platelet aggregation likely accounts for the bleeding phenotype in GSD Ia, elevated plasma corticosterone decreases the levels of proinflammatory monocytes. These studies highlight the necessity of maintaining good glycemic control in GSD Ia.
Asunto(s)
Ayuno , Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Hepatocitos/metabolismo , Hipoglucemia/metabolismo , Monocitos/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Hepatocitos/patología , Hipoglucemia/patología , Hielo , Masculino , Ratones Noqueados , Ratones Transgénicos , Monocitos/patología , Agregación PlaquetariaRESUMEN
Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana/metabolismo , Netrina-1/metabolismo , Nicho de Células Madre , Animales , Arteriolas/metabolismo , Diferenciación Celular , Proliferación Celular , Senescencia Celular , Eliminación de Gen , Trasplante de Células Madre Hematopoyéticas , Ratones Mutantes , Ratones Transgénicos , Transducción de SeñalRESUMEN
Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.
Asunto(s)
Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células/genética , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Marcaje Isotópico , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/trasplante , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone area-forming cell assay; CAFC). These assays measure the potential of a test cell population retrospectively, i.e., at the time its activity is evident when the stem cell itself is often not detectable anymore. Although the in vitro LTC-IC and CAFC assays have been shown to correlate with in vivo activity, in vivo transplantation assays, where it can be shown that cells possess the ability to indefinitely repopulate all blood lineages, are the ultimate proof for HSC activity. Nevertheless, these in vitro assays provide an excellent method to screen for stem cell activity of a putative stem cell population or for screening the effect of a certain treatment on HSCs.