RESUMEN
Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.
Asunto(s)
Lesiones Encefálicas/cirugía , Potenciales Evocados Somatosensoriales/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Sinapsis/fisiología , Potenciales de Acción , Vías Aferentes/fisiología , Animales , Encéfalo/citología , Encéfalo/ultraestructura , Lesiones Encefálicas/etiología , Línea Celular Transformada , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Modelos Animales de Enfermedad , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Neuronas/fisiología , Neuronas/ultraestructura , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Accidente Cerebrovascular/complicaciones , Sinapsis/ultraestructura , Núcleos Talámicos Ventrales/citologíaRESUMEN
Stroke is a leading cause of disability and currently lacks effective therapy enabling long-term functional recovery. Ischemic brain injury causes local inflammation, which involves both activated resident microglia and infiltrating immune cells, including monocytes. Monocyte-derived macrophages (MDMs) exhibit a high degree of functional plasticity. Here, we determined the role of MDMs in long-term spontaneous functional recovery after middle cerebral artery occlusion in mice. Analyses by flow cytometry and immunocytochemistry revealed that monocytes home to the stroke-injured hemisphere., and that infiltration peaks 3 d after stroke. At day 7, half of the infiltrating MDMs exhibited a bias toward a proinflammatory phenotype and the other half toward an anti-inflammatory phenotype, but during the subsequent 2 weeks, MDMs with an anti-inflammatory phenotype dominated. Blocking monocyte recruitment using the anti-CCR2 antibody MC-21 during the first week after stroke abolished long-term behavioral recovery, as determined in corridor and staircase tests, and drastically decreased tissue expression of anti-inflammatory genes, including TGFß, CD163, and Ym1. Our results show that spontaneously recruited monocytes to the injured brain early after the insult contribute to long-term functional recovery after stroke. SIGNIFICANCE STATEMENT: For decades, any involvement of circulating immune cells in CNS repair was completely denied. Only over the past few years has involvement of monocyte-derived macrophages (MDMs) in CNS repair received appreciation. We show here, for the first time, that MDMs recruited to the injured brain early after ischemic stroke contribute to long-term spontaneous functional recovery through inflammation-resolving activity. Our data raise the possibility that inadequate recruitment of MDMs to the brain after stroke underlies the incomplete functional recovery seen in patients and that boosting homing of MDMs with an anti-inflammatory bias to the injured brain tissue may be a new therapeutic approach to promote long-term improvement after stroke.
Asunto(s)
Macrófagos , Monocitos , Recuperación de la Función , Accidente Cerebrovascular/fisiopatología , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Antígenos de Diferenciación Mielomonocítica/genética , Conducta Animal/efectos de los fármacos , Quimera , Lateralidad Funcional , Infarto de la Arteria Cerebral Media/fisiopatología , Inflamación/patología , Lectinas/biosíntesis , Lectinas/genética , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/patología , Plasticidad Neuronal/fisiología , Desempeño Psicomotor/efectos de los fármacos , Receptores CCR2/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Recuperación de la Función/efectos de los fármacos , Accidente Cerebrovascular/patología , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , beta-N-Acetilhexosaminidasas/biosíntesis , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.
Asunto(s)
Corteza Cerebral/citología , Células Madre Pluripotentes Inducidas/fisiología , Infarto de la Arteria Cerebral Media/cirugía , Neuronas/fisiología , Recuperación de la Función/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/trasplante , Modelos Animales de Enfermedad , Estimulación Eléctrica , Glutaminasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Infarto de la Arteria Cerebral Media/patología , Neuronas/clasificación , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Desnudas , Ratas Sprague-DawleyRESUMEN
Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain.
Asunto(s)
Isquemia Encefálica/patología , Encéfalo/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Células-Madre Neurales/fisiología , Accidente Cerebrovascular/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antimetabolitos , Bromodesoxiuridina , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Electroporación , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Infarto de la Arteria Cerebral Media/patología , Masculino , Proteínas de la Membrana , Ratones , Ratones Noqueados , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Recuperación de la Función , Retroviridae/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/fisiología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/fisiología , Factores de Transcripción/metabolismo , Transfección/métodosRESUMEN
Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells survive long-term, differentiate to functional neurons, and induce recovery in the stroke-injured brain are unclear. We have transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into the stroke-damaged mouse and rat striatum or cortex. Recovery of forepaw movements was observed already at 1 week after transplantation. Improvement was most likely not due to neuronal replacement but was associated with increased vascular endothelial growth factor levels, probably enhancing endogenous plasticity. Transplanted cells stopped proliferating, could survive without forming tumors for at least 4 months, and differentiated to morphologically mature neurons of different subtypes. Neurons in intrastriatal grafts sent axonal projections to the globus pallidus. Grafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons. Our study provides the first evidence that transplantation of human iPSC-derived cells is a safe and efficient approach to promote recovery after stroke and can be used to supply the injured brain with new neurons for replacement.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Neuronas/citología , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/cirugía , Anciano , Animales , Encéfalo/citología , Encéfalo/patología , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Ratones , RatasRESUMEN
Ischemic stroke, caused by middle cerebral artery occlusion, leads to long-lasting formation of new striatal neurons from neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ) of adult rodents. Concomitantly with this neurogenic response, SVZ exhibits activation of resident microglia and infiltrating monocytes. Here we show that depletion of circulating monocytes, using the anti-CCR2 antibody MC-21 during the first week after stroke, enhances striatal neurogenesis at one week post-insult, most likely by increasing short-term survival of the newly formed neuroblasts in the SVZ and adjacent striatum. Blocking monocyte recruitment did not alter the volume of the ischemic lesion but gave rise to reduced astrocyte activation in SVZ and adjacent striatum, which could contribute to the improved neuroblast survival. A similar decrease of astrocyte activation was found in and around human induced pluripotent stem cell (iPSC)-derived NSPCs transplanted into striatum at one week after stroke in monocyte-depleted mice. However, there was no effect on neurogenesis in the graft as determined 8weeks after implantation. Our findings demonstrate, for the first time, that a specific cellular component of the early inflammatory reaction in SVZ and adjacent striatum following stroke, i.e., infiltrating monocytes, compromises the short-term neurogenic response neurogenesis from endogenous NSPCs.
Asunto(s)
Encéfalo/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Monocitos/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Accidente Cerebrovascular/terapia , Factores de Edad , Animales , Encéfalo/citología , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/trasplante , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/patologíaRESUMEN
BACKGROUND: Intracerebral transplantation of human induced pluripotent stem cells (iPSCs) can ameliorate behavioral deficits in animal models of stroke. How the ischemic lesion affects the survival of the transplanted cells, their proliferation, migration, differentiation, and function is only partly understood. METHODS: Here we have assessed the influence of the stroke-induced injury on grafts of human skin iPSCs-derived long-term neuroepithelial-like stem cells using transplantation into the rostral migratory stream (RMS), adjacent to the neurogenic subventricular zone, in adult rats as a model system. RESULTS: We show that the occurrence of an ischemic lesion, induced by middle cerebral artery occlusion, in the striatum close to the transplant does not alter the survival, proliferation, or generation of neuroblasts or mature neurons or astrocytes from the grafted progenitors. In contrast, the migration and axonal projection patterns of the transplanted cells are markedly influenced. In the intact brain, the grafted cells send many fibers to the main olfactory bulb through the RMS and a few of them migrate in the same direction, reaching the first one third of this pathway. In the stroke-injured brain, on the other hand, the grafted cells only migrate toward the ischemic lesion and virtually no axonal outgrowth is observed in the RMS. CONCLUSIONS: Our findings indicate that signals released from the stroke-injured area regulate the migration of and fiber outgrowth from grafted human skin-derived neural progenitors and overcome the influence on these cellular properties exerted by the neurogenic area/RMS in the intact brain.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Células-Madre Neurales/trasplante , Neurogénesis , Accidente Cerebrovascular/terapia , Animales , Astrocitos/metabolismo , Axones/metabolismo , Encéfalo/patología , Diferenciación Celular/genética , Humanos , Infarto de la Arteria Cerebral Media , Células-Madre Neurales/inmunología , Neuronas/inmunología , Neuronas/patología , Ratas , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/patologíaRESUMEN
Ischemic stroke is often associated with loss of cortical neurons leading to various neurological deficits. A cell replacement based on stem cell transplantation to repair the damaged brain requires the generation of specific neuronal subtypes. Recently, induced pluripotent stem cells have been used to generate various subtypes of neurons in vitro for transplantation in stroke-damaged brains. However, whether these cells can be primed as neuronal precursors to become cortical projection neurons by means of biomaterials releasing differentiation factors is not known. Here, we report that microspheres of biodegradable poly(ester-amide) composed of adipic acid, L-phenyl-alanine and 1,4-butanediol, loaded with differentiation factors, can be used to fate human induced pluripotent stem cell-derived long-term expandable neuroepithelial-like stem cells to cortical projection neurons. The three factors, Wnt3A, BMP4 and cyclopamine, were released from loaded microspheres over at least one month following biphasic dynamic time course, promoting cortical differentiation of the cells in vitro. Microspheres did not evoke significant inflammatory response after transplantation into intact rodent brain. Our study shows the potential of biodegradable polymer microspheres to promote neuronal differentiation by continuous release of factors, thereby creating the appropriate microenvironment. This new strategy may improve the efficacy of stem cell-based therapeutic approaches.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Implantes Absorbibles , Animales , Materiales Biocompatibles/química , Proteína Morfogenética Ósea 4/administración & dosificación , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Sistemas de Liberación de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/trasplante , Ensayo de Materiales , Microesferas , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/trasplante , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Poliésteres/química , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/terapia , Alcaloides de Veratrum/administración & dosificación , Proteína Wnt3A/administración & dosificaciónRESUMEN
Ischemic stroke affecting the adult brain causes increased progenitor proliferation in the subventricular zone (SVZ) and generation of neuroblasts, which migrate into the damaged striatum and differentiate to mature neurons. Meteorin (METRN), a newly discovered neurotrophic factor, is highly expressed in neural progenitor cells and immature neurons during development, suggesting that it may be involved in neurogenesis. Here, we show that METRN promotes migration of neuroblasts from SVZ explants of postnatal rats and stroke-subjected adult rats via a chemokinetic mechanism, and reduces N-methyl-D-asparate-induced apoptotic cell death in SVZ cells in vitro. Stroke induced by middle cerebral artery occlusion upregulates the expression of endogenous METRN in cells with neuronal phenotype in striatum. Recombinant METRN infused into the stroke-damaged brain stimulates cell proliferation in SVZ, promotes neuroblast migration, and increases the number of immature and mature neurons in the ischemic striatum. Our findings identify METRN as a new factor promoting neurogenesis both in vitro and in vivo by multiple mechanisms. Further work will be needed to translate METRN's actions on endogenous neurogenesis into improved recovery after stroke.