Differentiation of human and murine induced pluripotent stem cells to microglia-like cells [human]
Ontology highlight
ABSTRACT: Microglia are myeloid-lineage inflammatory cells that activate innate and adaptive immune responses within the central nervous system (CNS). They also have important roles in CNS development and homeostasis and can modulate the course of a variety of CNS diseases. We report here the sequential differentiation of murine induced pluripotent stem cells (iPSC) into hematopoietic progenitor-like cells and then into cells with a phenotype and gene expression profile resembling that of primary brain-isolated microglia. Functionally, the iPSC-derived microglia (iPS-MG) produce inflammatory cytokines and reactive oxygen species, demonstrate phagocytic activity and migrate to areas of pathology within the brain following intracranial injection. The ability to readily generate iPS-MG in vitro will facilitate the study of normal and disease-specific microglia and be useful for the development of microglia-based treatments for CNS diseases.
Project description:Microglia are the resident inflammatory cells of the central nervous system (CNS) and have important roles in development, homeostasis and a variety of neurologic and psychiatric diseases. Difficulties in procuring human microglia have limited their study and hampered the clinical translation of microglia-based treatments shown to be effective in animal disease models. Here, we report the differentiation of human induced pluripotent stem cells (iPSC) into microglia-like cells by exposure to defined factors and co-culture with astrocytes. These iPSC-derived microglia (iPS-MG) have the phenotype, gene expression profile and functional properties of brain-isolated microglia. Murine iPS-MG generated using a similar protocol have equivalent efficacy to primary brain-isolated microglia in the treatment of murine syngeneic intracranial malignant gliomas. The ability to generate human microglia facilitates the further study of this important CNS cell type and raises the possibility of their use in personalized medicine applications.
Project description:Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglia responses are believed to worsen CNS diseases, nevertheless their impact during the neuroinflammatory processes remains largely obscure. Here, using a combination of multicolor flow cytometry and single-cell RNA sequencing, we comprehensively profiled microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we showed that microglia isolated from LPS-injected mice displayed a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identified distinct microglia activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglia heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases.
Project description:The role of microglia and infiltrating monocytes in experimental autoimmune encephalomyelitis (EAE) pathogenesis has been controversial. To gain insight into their respective roles, we developed a method for differentiating between microglia and infiltrating monocytes in the CNS using CD44. We used this system to monitor changes in cell number, activation status, and gene expression by RNA sequencing (RNA-Seq) over the course of disease. This in vivo characterization and RNA-Seq dataset improves our understanding of myeloid cell biology in the brain under inflammatory conditions and may lead to strategies to identify therapies for inflammatory pathways active in neuroinflammatory diseases. Pooled samples from 10 mice were analyzed for both microglia and monocytes at distinct timepoints post EAE inducation. Peritoneal macrophages were isolated and analyzed for five samples, and also in a pool.
Project description:iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Ab-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Ab-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically-modified microglia.
Project description:iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Ab-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Ab-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically-modified microglia.
Project description:MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Here, we identified microglia as the main cellular source of miR-146a among mouse CNS resident cells. We further characterized the phenotype of miR-146a KO microglia cells during in vivo demyelination induced by cuprizone (CPZ) and found reduced number of CD11c+ microglia in the KO compared to WT mice. Microglia were also isolated from the brain, and the proteome was analyzed by liquid chromatography mass spectrometry.
Project description:Combining proteomics and systems biology analyses, we demonstrated that neonatal microglial cells derived from two different CNS locations (cortex and spinal cord) displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited great variability in terms of both cellular and small extracellular vesicles (sEVs) protein contents and levels. Bioinformatics data analysis showed that the cortical microglia had anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia was rather involved in inflammatory response process. Of interest, while both sEVs microglia sources enhanced growth of DRGs axons, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed through neurite outgrowth assays in DRGs cell line and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays indicated that the microglia localized at different CNS regions can ensure different biological functions. Together, these works indicate that neonatal microglia locations regulate their physiological and pathological functional fates, and could explain the high prevalence of brain vs. spinal cord glioma in adults.
Project description:In the brain the cells that control inflammation are called a type of white blood cell called microglia. Microglia are located throughout the brain and spinal cord and account for 10–15% of all cells found within the brain. As the resident white blood cells, they are the main active immune defence in the central nervous system (CNS). Microglia are part of an important class of cells known as macrophages that have two main states: M1 and M2. M1 cells are pro- inflammatory, leading to more inflammation, while M2 are anti-inflammatory, and drive wound healing. In this study, we will collect primary microglia from surgical biospies of 100 individuals.
This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Several viruses can infect the mammalian nervous system and induce neurological dysfunction. Adoptive immunotherapy (AI) is an approach that involves administration of antiviral T cells and has shown promise in clinical studies for the treatment of peripheral virus infections in humans such as cytomegalovirus, Epstein-Barr virus, and adenovirus, among others. Clearance of neurotropic infections, on the other hand, is particularly challenging because the central nervous system (CNS) is relatively intolerant of immunopathological reactions. Therefore, it is essential to develop and mechanistically understand therapies that noncytopathically eradicate pathogens from the CNS. Here, we used mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV) to demonstrate that therapeutic antiviral T cells can completely purge the persistently infected brain without causing blood brain barrier breakdown or tissue damage. Mechanistically, this is accomplished through a tailored release of chemoattractants that recruit antiviral T cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Upon arrival, T cells enlisted the support of nearly all brain resident myeloid cells (microglia) by converting them into CD11c+ antigen-presenting cells (APCs) – a cell population also found in the brain of a human immunodeficiency virus infected patient. Two-photon imaging studies revealed that antiviral CD8+ and CD4+ T cells interacted directly with CD11c+ microglia and induced STAT1 signaling, but did not initiate programmed cell death. We propose that noncytopathic CNS viral clearance can be achieved by therapeutic antiviral T cells reliant on restricted chemoattractant production and interactions with apoptosis-resistant microglia. 6 Mouse Microglia-sorted Brain Samples: 3 (-) AI, 3 (+) AI.
Project description:Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the central nervous system, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell (PSC) lines.