Estudio de la fusión celular en respuesta a diversas condiciones patológicas

La hipótesis de la fusión celular considera que una célula derivada de la médula ósea se fusiona con un precursor local o con una célula madura, transfiriendo su material genético y mezclando sus citoplasmas. La fusión celular supone un cambio conceptual en los campos de la terapia celular y de la genética, ya que las células de la médula, al fusionarse, aportarían material genético nuevo que podría permitir el rescate de una célula en proceso de degeneración, o corregir un defecto genético de la misma. Por tanto, la fusión celular posee un gran potencial terapéutico. Sin embargo, el papel de la fusión celular en el tratamiento de sintomatologías del sistema nervioso esta aun en fase de estudio. La fusión celular tiene como principal diana en el cerebro a las neuronas de Purkinje. La fusión celular en el cerebelo, al igual que ocurre en el hígado, podría revertir procesos degenerativos y rescatar mutaciones recesivas que afecten a las neuronas de Purkinje, como es el caso de algunas Ataxia. Por ello, en este trabajo hemos estudiado la fusión celular producida tras un trasplante de médula ósea en animales que presentan ataxia causada por la degeneración de neuronas de Purkinje. También hemos observado que, pese a la ausencia de eventos de fusión celular, se producen mejoras funcionales en el comportamiento de dichos animales. Por otra parte, la isquemia cerebral es la tercera causa de muerte en los países industrializados y constituye la principal causa de discapacidad en el adulto. Aunque se desconoce si el mecanismo de fusión celular también participa activamente en la recuperación y generación de tejido vascular tras la isquemia cerebral. Por ello en este trabajo hemos estudiado la fusión celular producida tras un trasplante de médula ósea bajo condiciones normales y tras un evento de isquemia cerebral. También hemos logrado caracterizar que los pericitos son el tipo celular que presenta mecanismos de fusión celular tanto en condiciones fisiológicas como tras un proceso isquémico. Por último, el proceso de fusión celular no se ha estudiado en detalle dentro del propio sistema hematopoyético, pese a que una de las parejas en los eventos de fusión es de este origen. Por ello hemos investigado en profundidad la presencia de eventos de fusión celular dentro de este sistema y comprobado que un proceso inflamatorio puede tener consecuencias en los niveles de fusión celular dentro del sistema hematopoyético

Transplant of GABAergic Precursors Restores Hippocampal Inhibitory Function in a Mouse Model of Seizure Susceptibility

16 páginas, 8 figuras.-- Licencia Creative Commons Reconocimiento-No comercial.Defects in GABAergic function can cause epilepsy. In the last years, cell-based therapies have attempted to correct these defects with disparate success on animal models of epilepsy. Recently, we demonstrated that medial ganglionic eminence (MGE)-derived cells grafted into the neonatal normal brain migrate and differentiate into functional mature GABAergic interneurons. These cells are able to modulate the local level of GABA-mediated synaptic inhibition, which suggests their suitability for cell-based therapies. However, it is unclear whether they can integrate in the host circuitry and rescue the loss of inhibition in pathological conditions. Thus, as proof of principle, we grafted MGE-derived cells into a mouse model of seizure susceptibility caused by specific elimination of GABAergic interneuron subpopulations in the mouse hippocampus after injection of the neurotoxic saporin conjugated to substance P (SSP-Sap). This ablation was associated with significant decrease in inhibitory postsynaptic currents (IPSC) on CA1 pyramidal cells and increased seizure susceptibility induced by pentylenetetrazol (PTZ). Grafting of GFP+ MGE-derived cells in SSP-Sap-treated mice repopulates the hippocampal ablated zone with cells expressing molecular markers of mature interneurons. Interestingly, IPSC kinetics on CA1 pyramidal cells of ablated hippocampus significantly increased after transplantation, reaching levels similar to the normal mice. More importantly, this was associated with reduction in seizure severity and decrease in postseizure mortality induced by PTZ. Our data show that MGE-derived cells fulfill most of the requirements for an appropriate cell-based therapy, and indicate their suitability for neurological conditions where a modulation of synaptic inhibition is needed, such as epilepsy.This work was supported by grants from Spanish Ministry of Science and Innovation (SAF 07/61880 and FIS 07/0079), and the Regenerative Medicine Programme from CIPF. M.E.C. and I.Z. were recipients of Miguel Servet contract from Carlos III Institute (Spanish Ministry of Science and Innovation) and Ph.D. fellowship from Generalitat Valenciana, respectively.Peer reviewe

Analysis of the Ush2a Gene in Medaka Fish (Oryzias latipes)

Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndrome is clinically classified into three types: USH1, USH2 and USH3. USH2 accounts for well over one-half of all Usher cases and mutations in the USH2A gene are responsible for the majority of USH2 cases, but also for atypical Usher syndrome and recessive non-syndromic RP. Because medaka fish (Oryzias latypes) is an attractive model organism for genetic-based studies in biomedical research, we investigated the expression and function of the USH2A ortholog in this teleost species. Ol-Ush2a encodes a protein of 5.445 aa codons, containing the same motif arrangement as the human USH2A. Ol-Ush2a is expressed during early stages of medaka fish development and persists into adulthood. Temporal Ol-Ush2a expression analysis using whole mount in situ hybridization (WMISH) on embryos at different embryonic stages showed restricted expression to otoliths and retina, suggesting that Ol-Ush2a might play a conserved role in the development and/or maintenance of retinal photoreceptors and cochlear hair cells. Knockdown of Ol-Ush2a in medaka fish caused embryonic developmental defects (small eyes and heads, otolith malformations and shortened bodies with curved tails) resulting in late embryo lethality. These embryonic defects, observed in our study and in other ciliary disorders, are associated with defective cell movement specifically implicated in left-right (LR) axis determination and planar cell polarity (PCP)

Estudio del proceso de fusión celular bajo diferentes condiciones patológicas

Tesis presentada para optar al grado de Doctor por la Universidad de Valencia, Facultad de Ciencias Biológicas-Departamento de Biología Celular y Parasitología: Programa de Neurociencias Básicas y Aplicadas y que ha sido realizada en las siguientes instituciones: - Hospital General Universitario de Valencia; - Centro de Investigación Príncipe Felipe (CIPF) de Valencia; - Universidad de Salamanca; - CABIMER de Sevilla.La hipótesis de la fusión celular considera que una célula derivada de la médula ósea se fusiona con un precursor local o con una célula madura, transfiriendo su material genético y mezclando sus citoplasmas. La fusión celular supone un cambio conceptual en los campos de la terapia celular y de la genética, ya que las células de la médula, al fusionarse, aportarían material genético nuevo que podría permitir el rescate de una célula en proceso de degeneración, o corregir un defecto genético de la misma. Por tanto, la fusión celular posee un gran potencial terapéutico. Sin embargo, el papel de la fusión celular en el tratamiento de sintomatologías del sistema nervioso esta aun en fase de estudio. La fusión celular tiene como principal diana en el cerebro a las neuronas de Purkinje. La fusión celular en el cerebelo, al igual que ocurre en el hígado, podría revertir procesos degenerativos y rescatar mutaciones recesivas que afecten a las neuronas de Purkinje, como es el caso de algunas Ataxia. Por ello, en este trabajo hemos estudiado la fusión celular producida tras un trasplante de médula ósea en animales que presentan ataxia causada por la degeneración de neuronas de Purkinje. También hemos observado que, pese a la ausencia de eventos de fusión celular, se producen mejoras funcionales en el comportamiento de dichos animales. Por otra parte, la isquemia cerebral es la tercera causa de muerte en los países industrializados y constituye la principal causa de discapacidad en el adulto. Aunque se desconoce si el mecanismo de fusión celular también participa activamente en la recuperación y generación de tejido vascular tras la isquemia cerebral. Por ello en este trabajo hemos estudiado la fusión celular producida tras un trasplante de médula ósea bajo condiciones normales y tras un evento de isquemia cerebral. También hemos logrado caracterizar que los pericitos son el tipo celular que presenta mecanismos de fusión celular tanto en condiciones fisiológicas como tras un proceso isquémico. Por último, el proceso de fusión celular no se ha estudiado en detalle dentro del propio sistema hematopoyético, pese a que una de las parejas en los eventos de fusión es de este origen. Por ello hemos investigado en profundidad la presencia de eventos de fusión celular dentro de este sistema y comprobado que un proceso inflamatorio puede tener consecuencias en los niveles de fusión celular dentro del sistema hematopoyético.Marina Piquer ha disfrutado varias becas asociadas a proyectos de investigación financiados por la Fundación Mutua Madrileña del Automovilismo, CIPF (Instituto de Salud Carlos III) y la Fundación Alicia Koplowitz. La presente investigación obtuvo de forma competitiva fondos para su financiación de las instituciones anteriormente mencionadas, además del Ministerio de Investigación y Ciencia.Peer Reviewe

Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System Through Different Mechanisms of Plasticity

14 páginas, 6 figuras, 3 tablas.-- et al.Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.This work was supported by the Ministerio de Ciencia e Innovación (BFU2010-18284), the Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas), the Junta de Castilla y León, “MMA,” “Samuel Solórzano Barruso,” and the “Alicia Koplowitz” Foundations, and Centre for Regenerative Medicine and Cell Therapy of Castilla y León.Peer reviewe

Cell fusion contributes to pericyte formation after stroke

6 páginas, 2 figuras.Recent reports have shown that bone marrow-derived cells (BMDCs) contribute to the formation of vasculature after stroke. However, the mechanism by which mural cells are formed from BMDC remains elusive. Here, we provide direct evidence that the cell fusion process contributes to the formation of pericytes after stroke. We generated mouse bone marrow chimeras using a cre/lox system that allows the detection of fusion events by X-gal staining. In these mice, we detected X-gal-positive cells that expressed vimentin and desmin, specific markers of mature murine pericytes. Electron microscopy confirmed that fused cells possessed basal lamina and characteristics of pericytes. Furthermore, induction of stroke increased significantly the presence of fused cells in the ischemic area. These cells expressed markers of developing pericytes such as NG2. We conclude that cell fusion participates actively in the generation of vascular tissue through pericyte formation under normal as well as pathologic conditions.This work was supported by grants from Spanish Ministry of Health (FIS 04/2744) and Regenerative Medicine Programme from the CIPF. M.P-G and IZ were recipients of PhD fellowships from CIPF and Generalitat Valenciana, respectively.Peer reviewe

Enhanced Hematovascular Contribution of SCL 3′ Enhancer Expressing Fetal Liver Cells Uncovers Their Potential to Integrate in Extramedullary Adult Niches

13 páginas, 6 figuras, 1 tabla.-- et al.Fetal liver (FL) hematopoietic progenitors have superior blood engraftment competence compared with adult bone marrow (BM), however less is known about FL in vivo vascular capacity. Here we show in transplantation assays that FL cells possess enhanced vascular endothelial potential compared with adult bone marrow. We generated high-level hematopoietic chimeras using donor cells from mice transgenic for the stem cell leukaemia 3′ enhancer human placental alkaline phosphatase (SCL3′Enh-PLAP) reporter construct, active in vascular endothelium, and blood progenitor and stem cells. Long-term lineage tracing analysis revealed PLAP+ vascular-like patches in FL-derived chimeras, whereas adult BM-derived chimeras presented only rare and scattered PLAP+ cells. PLAP+ vascular-like patches were formed following transplantation into both newborn and adult recipient mice, although their frequency was reduced in adult recipients. Confocal analysis of multiple labeled tissues revealed that whereas most liver and heart PLAP+ vascular patch-associated cells were endothelial, PLAP+ vascular patches in the kidney contained endothelial, hematopoietic, and putative hemangioblastic cells. Moreover, fluorescence-activated cell sorting assays showed that only FL PLAPbright+ donor cells can generate PLAP+ vascular patches upon transplantation. Taken together, these data demonstrate superior vascular contribution potential of FL cells, and not only provide new insights into the developmental pathways controlling endothelial development but also may prove informative when addressing the mechanisms involved in vascular regeneration and hemangiogenic recovery in a clinical context.This work was supported by the Spanish Ministry of Education and Science Grant SAF64679, Junta de Andalucia Grants PAI-BIO-295 and PAIDI-CTS1614, CONSOLIDER INGENIO 2010 Grant, the Leukemia Research Fund, fellowship CONACYT-179065 to A.M.G.-O., fellowship I3P-CSIC to C.Q., and fellowship UPO to A.C. A.M.G.-O. and A.C. contributed equally to this work.Peer reviewe

Non Coding RNAs as Regulators of Wnt/β-Catenin and Hippo Pathways in Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy histologically characterized by the replacement of myocardium by fibrofatty infiltration, cardiomyocyte loss, and inflammation. ACM has been defined as a desmosomal disease because most of the mutations causing the disease are located in genes encoding desmosomal proteins. Interestingly, the instable structures of these intercellular junctions in this disease are closely related to a perturbed Wnt/β-catenin pathway. Imbalance in the Wnt/β-catenin signaling and also in the crosslinked Hippo pathway leads to the transcription of proadipogenic and profibrotic genes. Aiming to shed light on the mechanisms by which Wnt/β-catenin and Hippo pathways modulate the progression of the pathological ACM phenotype, the study of non-coding RNAs (ncRNAs) has emerged as a potential source of actionable targets. ncRNAs comprise a wide range of RNA species (short, large, linear, circular) which are able to finely tune gene expression and determine the final phenotype. Some share recognition sites, thus referred to as competing endogenous RNAs (ceRNAs), and ensure a coordinating action. Recent cancer research studies regarding the key role of ceRNAs in Wnt/β-catenin and Hippo pathways modulation pave the way to better understanding the molecular mechanisms underlying ACM

Bone marrow transplantation improves motor activity in a mouse model of ataxia

Ataxias are locomotor disorders that can have an origin both neural and muscular, although both impairments are related. Unfortunately, ataxia has no cure, and the current therapies are aimed at motor re‐education or muscular reinforcement. Nevertheless, cell therapy is becoming a promising approach to deal with incurable neural diseases, including neuromuscular ataxias. Here, we have used a model of ataxia, the Purkinje Cell Degeneration (PCD) mutant mouse, to study the effect of healthy (wild‐type) bone marrow transplantation on the restoration of defective mobility. Bone marrow transplants (from both mutant and healthy donors) were performed in wild‐type and PCD mice. Then, a wide battery of behavioural tests was employed to determine possible motor amelioration in mutants. Finally, cerebellum, spinal cord, and muscle were analysed to study the integration of the transplant‐derived cells and the origin of the behavioural changes. Our results demonstrated that the transplant of wild‐type bone marrow restores the mobility of PCD mice, increasing their capabilities of movement (52–100% of recovery), exploration (20–71% of recovery), speed (35% of recovery), and motor coordination (25% of recovery). Surprisingly, our results showed that bone marrow transplant notably improves the skeletal muscle structure, which is severely damaged in the mutants, rather than ameliorating the central nervous system. Although a multimodal effect of the transplant is not discarded, muscular improvements appear to be the basis of this motor recovery. Furthermore, the results from our study indicate that bone marrow stem cell therapy can be a safe and effective alternative for dealing with movement disorders such as ataxiasThis work was supported by the Fondo de Investigaciones Sanitarias(FIS 04/2744), the Ministerio de Ciencia e Innovación (BFU2010‐18284), the Ministerio de Economía y Competitividad (SAF2013‐41175R), the Generalitat Valenciana (ACOMP06/131), the Junta deCastilla y León, the Junta de Andalucía (PI‐0736‐2010), the Centre forRegenerative Medicine and Cell Therapy of Castilla y León, the SamuelSolórzano Foundation, the FOLTRA Foundation, the Mututa Madrileña(MMA) Foundation, the Colectivo Ataxias en Movimiento Foundation,the Alicia Koplowitz Foundation, and the University of SalamancaPeer reviewe