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

Estudio del potencial terapéutico de los trasplantes de precursores GABAérgicos en modelos animales de epilepsia

[Introducción]: La Medicina Regenerativa o Terapia Celular utiliza células madre (CM) como agente terapéutico para dar respuesta a enfermedades crónico-degenerativas en las que los fármacos o la cirugía se han mostrado ineficaces. En el caso de las neuropatologías, la Terapia Celular permite el reemplazo de neuronas que degeneran progresivamente, o bien reparar circuitos neuronales dañados. Resultados previos de nuestro grupo, demuestran que CM neurales fetales derivadas de una región del cerebro en desarrollo conocida como eminencia ganglionar media (MGE) se diferen-cian hacia interneuronas GABAérgicas tras ser implantadas en el cerebro de ratones normales. Estos progenitores GABAérgicos son capaces de: 1) Migrar largas distancias, abarcando am-plias zonas del cerebro. 2) Diferenciar, adquiriendo morfologías y marcadores moleculares ca-racterísticos de interneuronas GABAérgicas. 3) Ser funcionales electrofisiológicamente, mos-trando potenciales de acción y propiedades de membrana característicos de interneuronas GABAérgicas maduras. 4) Integrarse plenamente en los circuitos corticales existentes, ya que son capaces de modificar específicamente la actividad inhibitoria cortical en las regiones en donde se implantan. En la actualidad no se conoce ningún tipo celular que haya sido capaz de lograr semejante in-tegración y funcionalidad al ser trasplantado en el cerebro. Estas propiedades les hace ideales para su utilización terapéutica en el tratamiento de la epilepsia, ya que podrían corregir las alte-raciones del sistema GABAérgico, una de sus principales causas, y paliar las crisis epilépticas al incrementar selectivamente la inhibición local. Por ello, el Proyecto de Tesis tiene como objetivo demostrar la capacidad antiepiléptica de las precursores GABAérgicos de la MGE mediante su trasplante en varios modelos animales de epilepsia. Pretendemos estudiar la distribución, supervivencia, diferenciación y funcionalidad de las células derivadas del trasplante en un ambiente epileptogénico. Así mismo, analizaremos, mediante whole cell voltage/current clamp y electroencefalogramas cómo las células de MGE trasplantadas son capaces de modificar la actividad cortical y recuperar los perfiles normales de actividad electrofisiológica en los animales epilépticos. Ello nos permitirá establecer las bases para una posible aplicación clínica de éstas células y proporcionar en el futuro una alternativa terapéutica para el tratamiento de la epilepsia.[Objetivos]: El Proyecto de Tesis que se presenta tiene como objetivo principal demostrar que los pre-cursores neuronales fetales derivados de la eminencia media ganglionar pueden ser útiles para el tratamiento de la epilepsia mediante su trasplante en el hipocampo. Para ello, nos marcamos los siguientes objetivos: - Comprobar la capacidad terapéutica de los precursores neuronales procedentes de la MGE mediante trasplantes en la corteza y el hipocampo de los siguientes modelos animales de epilepsia. a. Modelo inducido por Pilocarpina en ratón b. Modelo de supresión local específica de interneuronas mediante la administra-ción de un análogo de la Sustancia P conjugado al neurotóxico Saporina. - Estudiar la diferenciación de las células trasplantadas en el cerebro epiléptico mediante análisis inmunohistológico de neurotransmisores y marcadores específicos de interneu-ronas. - Determinar el grado de recuperación de los animales epilépticos trasplantados, compro-bando su comportamiento y efecto sobre las crisis epilépticas, así como la electrofisio-logía (whole-cell current-clamp) de las células trasplantadas y su capacidad de modular la actividad inhibitoria y excitatoria del hipocampo.[Material y Métodos]: -Obtención de los modelos de epilepsia. -Modelo inducido por Pilocarpina. -Modelo de ablación específica de interneuronas mediante SSP-Saporina. -Disección y disociación de precursores neuronales de la MGE. -Trasplantes estereotáxicos de precursores de la MGE en los modelos animales de epilepsia. -En los ratones adultos. -En los ratones neonatos. -Estudios electrofisiológicos. -Mediante la tecnica del patch-clamp whole-cell current clamp. -Patrones de disparo, IPSCs y efecto sobre excitación. -Inmunocitoquímica e inmunohistoquímica con marcadores específicos de los diferentes subtipos de interneuronas. -Tinciones de Timm y Nissl. -Pruebas de comportamiento. -Campo Abierto (CA). -Laberinto en Cruz Levantada (LCL). -Test de repulsión Electrica (TRE).Peer Reviewe

Bone marrow-derived cell therapy in combination with lipoic acid is effective therapeutic approach for functional recovery after traumatic brain injury

Póster presentado al 9th World Congress on Brain Injury, celebrado en Edimburgo (Escocia) del 21 al 25 de marzo de 2012Peer Reviewe

Efficient Differentiation of Human Embryonic Stem Cells into Functional Cerebellar-Like Cells

12 páginas, 6 figuras.-- et al.The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum-related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell differentiation into cerebellar-like cells such as granule neurons, Purkinje cells, interneuron, and glial cells. Neurons derived using our protocol showed a T-shaped polarity phenotype and express similar markers to the developed human cerebellum. Electrophysiological measurements confirmed functional electrical properties compatible with these cells. In vivo implantation of differentiated human embryonic stem cells transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular and the Purkinje cell layers and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in the development of cerebellum can be efficiently recapitulated in vitro, which enables the design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases.This work was supported by funds for research in the field of regenerative medicine from the Regional Government Health Department (Generalitat Valenciana) and the Instituto Carlos III belonging to the Spanish Ministry of Health and Consumer Affairs, and through grants from the Spanish Ministry of Science and Innovation (SAF2007-63193) and La Marató (TV3 070330).Peer reviewe

Effect of neuronal precursor cells derived from medial ganglionic eminence in an acute epileptic seizure model

5 páginas, 1 figura, 1 tabla.-- Special Issue: 10th Workshop on the Neurobiology of Epilepsy (WONOEP): Novel Therapeutic Approaches to Epileptogenesis.-- et al.Most of the γ-aminobutyric acid (GABA)ergic interneurons in the cerebral cortex originate from restricted regions of the ventral telencephalon known as the caudal and medial ganglionic eminence (MGE) and from the preoptic area. It is well established that dysfunction of GABAergic interneurons can lead to epilepsy. During the last decade new approaches to prevent, reduce, or reverse the epileptic condition have been studied, including cell-based therapy from different sources. Recent studies have shown that transplanted neuronal precursor cells derived from MGE have the ability to migrate, differentiate into inhibitory GABAergic interneurons, and integrate into cortical and hippocampal networks, modifying the inhibitory tone in the host brain. Therefore, transplantation of neuronal precursors derived from MGE into the postnatal central nervous system (CNS) could modify the neuronal circuitry in neurologic diseases in which inhibitory synaptic function is altered, such as in epilepsy. Here, we evaluated the seizure susceptibility of mice transplanted with MGE-derived cells in the maximum electroconvulsive shock (MES) model and we review some data from different studies using GABAergic precursor or GABA-releasing cell grafts in animal models of seizure and epilepsy.This work was supported by FAPESP, FAPERJ, and CNPq.Peer reviewe

GABAergic precursors grafts in animal models of epilepsy

Trabajo presentado en II EMBO Workshop on Cortical Interneurons in Health and Disease, celebrado en Costa d’en Blanes, Mallorca (España), del 24 al 27 de junio de 2012Introduction: Refractory epilepsy is present in about 30% of TLE patients, despite carefully optimized drug treatment. These patients have no treatment other than major resective surgery. In the last years, cell-based therapies have emerged as a promising alternative. Recently, we have demonstrated that MGE-derived precursors transplanted into the normal adult and neonatal telencephalon migrate, differentiate and incorporate as fully functional GABAergic interneurons. Moreover, grafting in a mouse model of hyperactivity, generated by partial elimination of GABAergic interneurons, replaced the deficit in interneurons and restored the normal levels of inhibition in the ablated hippocampus. To further analyze the use of these precursors as an antiepileptic therapy for refractory TLE epilepsy, we have performed transplants in the pilocarpine mouse model of epilepsy. Results: Intrahippocampal bilateral transplants of GFP+ cells from the MGE were performed 5 days after pilocarpine induction of status epilepticus. We monitorized the control and transplanted mice daily from 2 weeks to 4 months after the transplant to asses the possible antiepileptic effects. We observed a significant delay in the latency time to develop the spontaneous recurrent seizures (SRS) in the transplanted group. In addition, the percentage of transplanted mice that fully developed the SRS was significantly reduced with respect their controls and this small group suffered the SRS four times less frequently. Immohistochemical analysis reveled that GFP+ cells spread widely through CA and DG areas. They differentiated into normal interneuron subtypes, with mature morphology and expressing specific markers such as parvalbumin, somatostatin, NP-Y, and calretinin. Interestingly, we observed a protective effect on the hippocampal sclerosis, correlated with reduced levels of cell death. Grafted cells did not affect the sprouting of mossy fibers, although we cannot discard a direct interaction with the grafted cells. In fact, patch clamp analysis of sIPSC in the grafted area confirmed a modulation of the inhibitory synaptic function. Conclusion: MGE-derived precursor grafts into the hippocampus protects against the development of TLE and elicit a reduction in hippocampal sclerosis. This cell-based therapy could be extremely beneficial for the treatment of refractory epilepsy.Financial Support: Ministerio de Ciencia y Tecnología (SAF 2009- 07746)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

Lipoic acid and bone marrow derived cells therapy induce angiogenesis and cell proliferation after focal brain injury

Introduction: Traumatic brain injury is a main cause of disability and death in developed countries, above all among children and adolescents. The intrinsic inability of the central nervous system to efficiently repair traumatic injuries renders transplantation of bone marrow-derived cells (BMDC) a promising approach towards repair of brain lesions. On the other hand, many studies have reported the beneficial effect of Lipoic acid (LA), a potent antioxidant promoting cell survival, angiogenesis and neuroregeneration. Methods: In this study, the cortex of adult mice was cryo-injured in order to mimic local traumatic brain injury. Vehicle or freshly prepared BMDC were grafted in the cerebral penumbra area 24 hours after unilateral local injury alone or combined with intra-peritoneal LA administration as a new regenerative strategy. Results: Differences were found in the process of cell proliferation, angiogenesis and glial scar formation after local injury depending of the applied treatment, either LA or BMDC alone or in combination. Conclusion: The data presented here suggest that transplantation of BMDC is a good alternative and valid strategy to treat a focal brain injury when LA could not be prescribed due to its non-desired secondary effectsPeer Reviewe