RESUMEN
Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100ß, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.
Asunto(s)
Astrocitos/fisiología , Ingeniería Celular/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Animales , Astrocitos/trasplante , Supervivencia Celular/fisiología , Células Cultivadas , Desoxirribonucleasas , Dependovirus/genética , Fibroblastos/fisiología , Genes Reporteros , Vectores Genéticos , Sustancia Gris/citología , Sustancia Gris/fisiología , Sustancia Gris/cirugía , Proteínas Fluorescentes Verdes/genética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Ratones , Regiones Promotoras Genéticas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/fisiología , Médula Espinal/cirugía , Dedos de ZincRESUMEN
ALS is a neurodegenerative disease with a prevalence rate of up to 7.4/100,000 and the overall risk of developing ALS over a lifetime is 1:400. Most patients die from respiratory failure following a course of progressive weakness. To date, only one traditional pharmaceutical agent-riluzole, has been shown to afford a benefit on survival but numerous pharmaceutical interventions have been studied in preclinical models of ALS without subsequent translation to patient efficacy. Despite the relative selectivity of motor neuron cell death, animal and tissue culture models of familial ALS suggest that non-neuronal cells significantly contribute to neuronal dysfunction and death. Early efforts to transplant stem cells had focused on motor neuron replacement. More practically for this aggressive neurodegenerative disease, recent studies, preclinical efforts, and early clinical trials have focused on the transplantation of neural stem cells, mesenchymal stem cells, or glial progenitors. Using transgenic mouse or rat models of ALS, a number of studies have shown neuroprotection through a variety of different mechanisms that have included neurotrophic factor secretion, glutamate transporter regulation, and modulation of neuroinflammation, among others. However, given that cell replacement could involve a number of biologically relevant factors, identifying the key pathway(s) that may contribute to neuroprotection remains a challenge. Nevertheless, given the abundant data supporting the interplay between non-neuronal cell types and motor neuron disease propagation, the replacement of disease-carrying host cells by normal cells may be sufficient to confer neuroprotection. Key preclinical issues that currently are being addressed include the most appropriate methods and routes for delivery of cells to disease-relevant regions of the neuraxis, cell survival and migration, and tracking the cells following transplantation. Central to the initial development of stem cell transplantation into patients with ALS is the demonstration that transplanted cells lack tumorigenicity and have the appropriate biodistribution to ensure the safety of ALS patients receiving these therapies. Here, we review preclinical and clinical studies focusing on the transplantation of neural and glial progenitor cells as a promising neuroprotective therapy for ALS. The rationale for stem cell transplantation for neuroprotection, proof-of-concept animal studies, and current challenges facing translation of these therapies to the clinic is presented. Lastly, we discuss advancements on the horizon including induced pluripotent stem cell technology and developments for cellular tracking and detection post-transplantation. With the safe completion of the first-in-human Phase I clinical trial for intraspinal stem cell transplantation for ALS in the United States, the time is ripe for stem cell therapies to be translated to the clinic and excitingly, evaluated for neuroprotection for ALS. This article is part of a Special Issue entitled SI: Neuroprotection.
Asunto(s)
Esclerosis Amiotrófica Lateral/cirugía , Neuroglía/fisiología , Neuronas/fisiología , Trasplante de Células Madre/métodos , Animales , HumanosRESUMEN
Although Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease, basic research studies have highlighted that astrocytes contribute to the disease process. Therefore, strategies which replace the diseased astrocyte population with healthy astrocytes may protect against motor neuron degeneration. Our studies have sought to evaluate astrocyte replacement using glial-restricted progenitors (GRPs), which are lineage-restricted precursors capable of differentiating into astrocytes after transplantation. The goal of our current study was to evaluate how transplantation to the diseased ALS spinal cord versus a healthy, wild-type spinal cord may affect human GRP engraftment and selected gene expression. Human GRPs were transplanted into the spinal cord of either an ALS mouse model or wild-type littermate mice. Mice were sacrificed for analysis at either the onset of disease course or at the endstage of disease. The transplanted GRPs were analyzed by immunohistochemistry and NanoString gene profiling which showed no gross differences in the engraftment or gene expression of the cells. Our data indicate that human glial progenitor engraftment and gene expression is independent of the neurodegenerative ALS spinal cord environment. These findings are of interest given that human GRPs are currently in clinical development for spinal cord transplantation into ALS patients.
Asunto(s)
Esclerosis Amiotrófica Lateral/cirugía , Regulación de la Expresión Génica/fisiología , Neuroglía/fisiología , Neuroglía/trasplante , Trasplante de Células Madre/métodos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Antígenos Nucleares/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadáver , Movimiento Celular , Proliferación Celular/genética , Modelos Animales de Enfermedad , Feto , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Médula Espinal/metabolismo , Médula Espinal/patología , Células Madre , Superóxido Dismutasa/genéticaRESUMEN
The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models.
Asunto(s)
Astrocitos , Diferenciación Celular , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Médula Espinal , Animales , Astrocitos/citología , Astrocitos/metabolismo , Regulación de la Expresión Génica , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Neuroinflammation is one of the most striking hallmarks of amyotrophic lateral sclerosis (ALS). Nuclear factor-kappa B (NF-κB), a master regulator of inflammation, is upregulated in spinal cords of ALS patients and SOD1-G93A mice. In this study, we show that selective NF-κB inhibition in ALS astrocytes is not sufficient to rescue motor neuron (MN) death. However, the localization of NF-κB activity and subsequent deletion of NF-κB signaling in microglia rescued MNs from microglial-mediated death in vitro and extended survival in ALS mice by impairing proinflammatory microglial activation. Conversely, constitutive activation of NF-κB selectively in wild-type microglia induced gliosis and MN death in vitro and in vivo. Taken together, these data provide a mechanism by which microglia induce MN death in ALS and suggest a novel therapeutic target that can be modulated to slow the progression of ALS and possibly other neurodegenerative diseases by which microglial activation plays a role.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Muerte Celular/fisiología , Microglía/citología , Neuronas Motoras/citología , FN-kappa B/metabolismo , Factores de Edad , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Comunicación Celular/fisiología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Microglía/metabolismo , Neuronas Motoras/metabolismo , FN-kappa B/antagonistas & inhibidores , Cultivo Primario de Células , Transducción de Señal/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
The role of glia as a contributing factor to motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) is becoming increasingly appreciated. However, most studies implicating astrocytes have focused solely on models of ALS caused by superoxide dismutase 1 (SOD1) mutations. The goal of our study was to determine whether astrocytes contribute to wild-type MN death in the case of ALS caused by mutations in tar-DNA binding protein 43 (TDP-43). Since it is currently unknown how TDP-43 mutations cause disease, we derived astrocytes for study from both gain and loss of function mouse models of TDP-43. Astrocytes overexpressing mutant TDP-43(A315T) as well as astrocytes lacking TDP-43 were morphologically indistinguishable from wild-type astrocytes in vitro. Furthermore, astrocytes with these TDP-43 alterations did not cause the death of wild-type MNs in co-culture. To investigate the in vivo effects of TDP-43 alterations in astrocytes, glial-restricted precursors were transplanted to the wild-type rat spinal cord where they differentiated into astrocytes and interacted with host MNs. Astrocytes with TDP-43 alterations did not cause host wild-type MN damage although they were capable of engrafting and interacting with host MNs with the same efficiency as wild-type astrocytes. These data indicate that astrocytes do not adopt the same toxic phenotype as mutant SOD1 astrocytes when TDP-43 is mutated or expression levels are modified. Our study reinforces the heterogeneity in ALS disease mechanisms and highlights the potential for future screening subsets of ALS patients prior to treatment with cell type-directed therapies.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Astrocitos/metabolismo , Proteínas de Unión al ADN/genética , Neuronas Motoras/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Supervivencia Celular , Técnicas de Cocultivo , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Transgénicos , Ratas , Ratas Sprague-DawleyRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.