RESUMEN
It has been previously shown that the secretome of Human Umbilical Cord Perivascular Cells (HUCPVCs), known for their mesenchymal like stem cell character, is able to increase the metabolic viability and hippocampal neuronal cell densities. However, due to the different micro-environments of the distinct brain regions it is important to study if neurons isolated from different areas have similar, or opposite, reactions when in the presence of HUCPVCs secretome (in the form of conditioned media-CM). In this work we: 1) studied how cortical and cerebellar neuronal primary cultures behaved when incubated with HUCPVCs CM and 2) characterized the differences between CM collected at two different conditioning time points. Primary cultures of cerebellar and cortical neurons were incubated with HUCPVCs CM (obtained 24 and 96 h after three days of culturing). HUCPVCs CM had a higher impact on the metabolic viability and proliferation of cortical cultures, than the cerebellar ones. Regarding neuronal cell densities it was observed that with 24 h CM condition there were higher number MAP-2 positive cells, a marker for fully differentiated neurons; this was, once again, more evident in cortical cultures. In an attempt to characterize the differences between the two conditioning time points a proteomics approach was followed, based on 2D Gel analysis followed by the identification of selected spots by tandem mass spectrometry. Results revealed important differences in proteins that have been previously related with phenomena such as neurl cell viability, proliferation and differentiation, namely 14-3-3, UCHL1, hsp70 and peroxiredoxin-6. In summary, we demonstrated differences on how neurons isolated from different brain regions react to HUCPVCs secretome and we have identified different proteins (14-3-3 and hsp70) in HUCPVCs CM that may explain the above-referred results.
Asunto(s)
Cerebelo/citología , Corteza Cerebral/citología , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/citología , Proteínas 14-3-3/fisiología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/fisiología , ProteómicaRESUMEN
Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been shown to ameliorate the injured central nervous system (CNS). Although these effects were initially attributed to the putative differentiation of MSCs towards the neural lineage, it is now known that most of them are mediated by the secretome. Up to now most in vitro reports have dealt with the effects of the secretome on neural stem cells and their differentiation. Consequently, there is a lack of information regarding the role of the secretome on the viability and survival of pre-existent matured differentiated cell populations. Moreover, it is also not known how the time points of conditioned media (CM) collection affect such parameters. In the present study, primary cultures of hippocampal neurons and glial cells were incubated with CM obtained from MSCs. To determine how the temporal profiles of CM collection impact on post-natal neurons and glial cells, we collected MSCs CM at 24, 48, 72 and 96 h of conditioning. MTS test revealed that for the hippocampal cultures the incubation with CM increased cell viability for all time points, with significant increases in the percentage of neurons in culture incubated with CM 24 h. For glial cells only the later time point of CM collection (96 h) increased cell viability. Fluorescence microscopy observations also revealed that CM 48 h and 72 h increased astrocytes percentages, while CM 24 h decreased microglial cell and oligodendrocytes values. These results revealed that post-natal neuronal and glial cells respond differently to MSCs CM; moreover, there are specific temporal variations in the composition of the CM of MSCs collected at different time points that trigger different effects on mature neurons and the distinct glial cell populations (astrocytes, oligodendrocytes and microglial cells).
Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Neuroglía/citología , Neuronas/citología , Animales , Células de la Médula Ósea/citología , Células Cultivadas , Hipocampo/citología , Células Madre Mesenquimatosas/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.