Papel de la ruta mTOR sobre el envejecimiento de las células madre

[Resumen] Las células madre mesenquimales (CMMs) tienen una amplia capacidad de auto-renovación, un amplio potencial de diferenciación y propiedades inmunosupresoras. Una desventaja en las aplicaciones clínicas es su limitada viabilidad cuando existe un microambiente adverso. Las vesículas extracelulares (VEs), tales como los exosomas y microvesículas, son liberadas por las células en el medio, participando en la interacción entre ellas, facilitando la presentación de antígenos, la señalización trans a las células vecinas y la transferencia de ARN y proteínas. Se seleccionaron CMMs procedentes de médula ósea de individuos juveniles y adultos de ratas Wistar y sus VEs fueron aisladas, y caracterizadas por citometría de flujo. Se trataron las células con las VEs obtenidas de las diferentes poblaciones, juvenil y adulta. El objetivo fue determinar si estas VEs modificaban la ruta de mTOR de las CMM donde se introducían, demostrando así su capacidad comunicativa. Se realizaron estudios proteómicos mediante Western Blot y génicos mediante qPCR-RT para determinar el nivel de expresión de los genes y proteínas de la ruta mTOR. Además, se intentó evaluar el papel de RICTOR en dicha ruta mediante la inhibición del miR-188-3p a través de VEs en una población juvenil de CMMs. Las VEs internalizan en las CMMs independientemente de la edad y producen cambios en la expresión de genes y proteínas relacionados con el envejecimiento en la ruta de mTOR. La transfección de las CMMs juveniles con el miR-188-3p reveló que sus VEs modificaban la senescencia de las CMMs adultas tratadas con las mismas. Nuevos experimentos deben ser realizados para demostrar que el microRNA está actuando sobre el RICTOR.[Resumo] As células nai mesenquimais (CNMs) teñen unha amplia capacidade de auto-renovación, un amplio potencial de diferenciación e propiedades inmunosupresoras. Unha desventaxe nas aplicacións clínicas é a súa limitada viabilidade cando existe un microambiente adverso. As vesículas extracelulares (VEs), tales coma os exosomas e microvesículas, son liberadas polas células no medio, participando na interacción entre elas, facilitando a presentación de antíxenos, a sinalización trans ás células veciñas e a transferencia de ARN e proteínas. Seleccionáronse CNMs procedentes de médula ósea de individuos xuveníis e adultos de ratas Wistar e as súas VEs foron illadas, e caracterizadas por citometría de fluxo. Tratáronse as células coas VEs obtidas das diferentes poboacións, xuvenil e adulta. O obxetivo foi determinar se estas VEs modificaban a ruta de mTOR das CNM onde se introducían, demostrando así a súa capacidade comunicativa. Realizáronse estudos proteómicos, a partir destas CNMs modificadas, estudios proteómicos mediante Western Blot e xénicos mediante qPCR-RT para determinar o nivel de expresión dos xenes e proteínas da ruta mTOR. Ademais, intentouse avaliar o papel do RICTOR na ruta mediante a inhibición do miR-188-3p a través de VEs nunha poboación xuvenil de CNMs. As VEs internalizan nas CNMs independentemente da idade e producen cambios na expresión de xenes e proteínas relacionados có avellantamento na ruta de mTOR. A transfección das CNMs xuveníis có miR-188-3p revelou que as súas VEs modificaban a senescencia das CNMs adultas tratadas coas mesmas. Novos experimentos deben ser realizados para demostrar que o microRNA está actuando sobre o RICTOR.[Abstract] Mesenchymal stem cells (MSCs) have a broad capacity for self-renewal, a broad potential for differentiation and immunosuppressive properties. A disadvantage in clinical applications is their limited viability when there is an adverse microenvironment. Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by the cells in the medium, participating in the interaction between them, improving their antigen retrieval, trans- signaling to neighboring cells and the transfer of RNA and proteins. Bone marrow MSCs from young and adult Wistar rats were selected and their EVs were isolated, and characterized by flow cytometry. The cells were treated with the EVs obtained from the different populations, young and adult. The objective was to determine if these EVs modified the mTOR pathway, involved in senescence,of the CMM where they were introduced, thus demonstrating their communicative capacity. From these modified CMMs, proteomic and genetic studies were carried out by Western Blot and qPCR-RT respectively were done. In addition, an attempt was made to evaluate the role of RICTOR in this route by inhibition miR-188-3p through VEs in a juvenile population of CMMs. The VEs internalize in the CMMs regardless of age and produce changes in the expression of genes and proteins related to aging in the mTOR pathway. The transfection of the young CMMs with the miR-188-3p revealed that their Evs modified the senescence of the adult CMM treated with them. New experiments must be performed to show that miRNA is working through the RICTOR.Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en Fundamentos de investigación biomédica. Curso 2017/2018

Effect of Aging on Behaviour of Mesenchymal Stem Cells

[Abstract] Organs whose source is the mesoderm lineage contain a subpopulation of stem cells that are able to differentiate among mesodermal derivatives (chondrocytes, osteocytes, adipocytes). This subpopulation of adult stem cells, called “mesenchymal stem cells” or “mesenchymal stromal cells (MSCs)”, contributes directly to the homeostatic maintenance of their organs; hence, their senescence could be very deleterious for human bodily functions. MSCs are easily isolated and amenable their expansion in vitro because of the research demanding to test them in many diverse clinical indications. All of these works are shown by the rapidly expanding literature that includes many in vivo animal models. We do not have an in-depth understanding of mechanisms that induce cellular senescence, and to further clarify the consequences of the senescence process in MSCs, some hints may be derived from the study of cellular behaviour in vivo and in vitro, autophagy, mitochondrial stress and exosomal activity. In this particular work, we decided to review these biological features in the literature on MSC senescence over the last three years.Xunta de Galicia; ED481B 2017/11

High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome

[Abstract] Hutchinson–Gilford progeria syndrome (HGPS) is a deadly childhood disorder, which is considered a very rare disease. It is caused by an autosomal dominant mutation on the LMNA gene, and it is characterized by accelerated aging. Human cell lines from HGPS patients and healthy parental controls were studied in parallel using next-generation sequencing (NGS) to unravel new non-previously altered molecular pathways. Nine hundred and eleven transcripts were differentially expressed when comparing healthy versus HGPS cell lines from a total of 21,872 transcripts; ITPR1, ITPR3, CACNA2D1, and CAMK2N1 stood out among them due to their links with calcium signaling, and these were validated by Western blot analysis. It was observed that the basal concentration of intracellular Ca2+ was statistically higher in HGPS cell lines compared to healthy ones. The relationship between genes involved in Ca2+ signaling and mitochondria-associated membranes (MAM) was demonstrated through cytosolic calcium handling by means of an automated fluorescent plate reading system (FlexStation 3, Molecular Devices), and apoptosis and mitochondrial ROS production were examined by means of flow cytometry analysis. Altogether, our data suggest that the Ca2+ signaling pathway is altered in HGPS at least in part due to the overproduction of reactive oxygen species (ROS). Our results unravel a new therapeutic window for the treatment of this rare disease and open new strategies to study pathologies involving both accelerated and healthy aging.Xunta de Galicia; ED481D-2021-020This work was funded by the Spanish National Health Institute Carlos III (PI20/00497) awarded to M.C.A. Furthermore, J.A.F.-L. is funded by the Xunta de Galicia Fellowship (ED481D-2021-020

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells

[Abstract] Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used smallmolecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.Instituto de Salud Carlos III; PI20/00497Xunta de Galicia; ED481B 2017/11

Influence of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Vitro and their Role in Ageing

[Abstract] Introduction: This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. Methods: MSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing. Results: Incubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining. Conclusions: MSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.JAF-L is the recipient of a postdoctoral fellowship funded by Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Spain

Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA

[Abstract] Osteoarthritis (OA) is closely linked to the increase in the number of senescent cells in joint tissues, and the senescence-associated secretory phenotype (SASP) is implicated in cartilage degradation. In the last decade, extracellular vesicles (EV) in combination with the use of miRNAs to modify post-transcriptional expressions of multiple genes have shown their utility in new therapies to treat inflammatory diseases. This work delves into the anti-inflammatory effect of extracellular vesicles derived from mesenchymal stem cells (MSC) previously modified to inhibit the expression of miR-21. We compare the efficacy of two treatments, MSC with their miR-21 inhibited through lentiviral transfection and their EV, against inflammation in a new OA animal model. The modified MSC and their EV were intraperitoneally injected in an OA animal model twice. One month after treatment, we checked which therapy was the most effective to reduce inflammation compared with animals untreated. Treated OA model sera were analyzed for cytokines and chemokines. Subsequently, different organs were analyzed to validate the results obtained. EV were the most effective treatment to reduce chemokines and cytokines in serum of OA animals as well as SASP, in their organs checked by proteomic and genomic techniques, compared with MSC alone in a statistically significant way. In conclusion, MSC-miR-21--derived EV showed a higher therapeutic potential in comparison with MSCs-miR-21-. They ameliorate the systemic inflammation through inactivation of ERK1/2 pathway in OA in vivo model. Workflow of the realization of the animal model of OA by injecting cells into the joint cavity of the left knee of the animals, which produces an increase in serum cytokines and chemokines in the animals in addition to the increase in SASP and markers of inflammation. Inhibition of miR-21 in MSCs, from the stroma of the human umbilical cord, by lentivirus and extraction of their EVs by ultracentrifugation. Finally, application of MSC therapy with its miR-21 inhibited or its EVs produces a decrease in serum cytokines and chemokines in the treated animals, in addition to an increase in SASP and markers of inflammation. The cell-free therapy being the one that produces a greater decrease in the parameters studied.Xunta de Galicia; ED481D-2021-020Instituto de Salud Carlos III; PI20/0049

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC

Action Mechanisms of Small Extracellular Vesicles in Inflammaging [Review]

This article belongs to the Special Issue Extracellular Vesicles Research in Inflamm-Aging[Abstract] The accumulation process of proinflammatory components in the body due to aging influences intercellular communication and is known as inflammaging. This biological mechanism relates the development of inflammation to the aging process. Recently, it has been reported that small extracellular vesicles (sEVs) are mediators in the transmission of paracrine senescence involved in inflammatory aging. For this reason, their components, as well as mechanisms of action of sEVs, are relevant to develop a new therapy called senodrugs (senolytics and senomorphic) that regulates the intercellular communication of inflammaging. In this review, we include the most recent and relevant studies on the role of sEVs in the inflammatory aging process and in age-related diseases such as cancer and type 2 diabetes.J.F.L. was funded by Xunta de Galicia, Grant Number ED481D-2021-020. M.C.A. received a grant from the Spanish National Health Institute Carlos III (PI20/00497)Xunta de Galicia; ED481D-2021-02

Proteomic study of mesenchymal stem cells derived exosomes modified using miR

Poster.-- Annual Meeting ISEV (International Society Extracellular Vesicles) 2020, 20-22 JulyThe project we are working on is tomodify the immunogenic profile of human CMMs from the umbilical cord stroma through its stabletransfection with anti-miR-21-5p, and therefore of the exosomes that these cells generate, for use in free-celltherapy to treat inflammatory processProteoRed Concept Test Project GrantPeer reviewe

Mesenchymal Stem Cell-Derived Extracellular Vesicle Isolation and Their Protein Cargo Characterization

10 pages, 2 figuresIn the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVsPeer reviewe