iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

[1]  R. Faull,et al.  Isolation of highly enriched primary human microglia for functional studies , 2016, Scientific Reports.

[2]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[3]  H. Weiner,et al.  Differential roles of microglia and monocytes in the inflamed central nervous system , 2014, The Journal of experimental medicine.

[4]  H. Neumann,et al.  Sensing the neuronal glycocalyx by glial sialic acid binding immunoglobulin-like lectins , 2014, Neuroscience.

[5]  S. Hickman,et al.  Microglial Dysfunction and Defective β-Amyloid Clearance Pathways in Aging Alzheimer's Disease Mice , 2008, The Journal of Neuroscience.

[6]  J. Grutzendler,et al.  CX3CR1 in Microglia Regulates Brain Amyloid Deposition through Selective Protofibrillar Amyloid-β Phagocytosis , 2010, The Journal of Neuroscience.

[7]  M. Giustetto,et al.  Synaptic Pruning by Microglia Is Necessary for Normal Brain Development , 2011, Science.

[8]  P. Rezaie,et al.  The origin and cell lineage of microglia—New concepts , 2007, Brain Research Reviews.

[9]  I. Amit,et al.  Microglia development follows a stepwise program to regulate brain homeostasis , 2016, Science.

[10]  R. Nitsch,et al.  Astrocyte‐released cytokines induce ramification and outward K+ channel expression in microglia via distinct signalling pathways , 2001, The European journal of neuroscience.

[11]  A. Stalder,et al.  Invasion of Hematopoietic Cells into the Brain of Amyloid Precursor Protein Transgenic Mice , 2005, The Journal of Neuroscience.

[12]  G. Keller,et al.  Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells , 2014, Nature Biotechnology.

[13]  H. Kettenmann,et al.  Physiology of microglia. , 2011, Physiological reviews.

[14]  Janna H. Neltner,et al.  Disease-related microglia heterogeneity in the hippocampus of Alzheimer’s disease, dementia with Lewy bodies, and hippocampal sclerosis of aging , 2015, Acta Neuropathologica Communications.

[15]  D. Maric,et al.  Differentiation of human and murine induced pluripotent stem cells to microglia-like cells , 2017, Nature Neuroscience.

[16]  N. Perrimon,et al.  Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms. , 2014, Human molecular genetics.

[17]  L. Tan,et al.  Role of pro-inflammatory cytokines released from microglia in Alzheimer's disease. , 2015, Annals of translational medicine.

[18]  Tom Michoel,et al.  Microglial brain region-dependent diversity and selective regional sensitivities to ageing , 2015, Nature Neuroscience.

[19]  H. Neumann,et al.  Alleviation of Neurotoxicity by Microglial Human Siglec-11 , 2010, The Journal of Neuroscience.

[20]  W. Gan,et al.  The P2Y12 receptor regulates microglial activation by extracellular nucleotides , 2006, Nature Neuroscience.

[21]  H. Neumann,et al.  Neuronal ‘On’ and ‘Off’ signals control microglia , 2007, Trends in Neurosciences.

[22]  F. Ginhoux,et al.  Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia. , 2012, Immunity.

[23]  Peter Riederer,et al.  Interleukin-1β and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer's and de novo Parkinson's disease patients , 1995, Neuroscience Letters.

[24]  Ben A. Barres,et al.  Complement and microglia mediate early synapse loss in Alzheimer mouse models , 2016, Science.

[25]  A. Mildner,et al.  P2Y12 receptor is expressed on human microglia under physiological conditions throughout development and is sensitive to neuroinflammatory diseases , 2017, Glia.

[26]  D. Underhill,et al.  C9orf72 is required for proper macrophage and microglial function in mice , 2016, Science.

[27]  I. Amit,et al.  Host microbiota constantly control maturation and function of microglia in the CNS , 2015, Nature Neuroscience.

[28]  M. Prinz,et al.  Factors regulating microglia activation , 2013, Front. Cell. Neurosci..

[29]  Tatsuya Tsukahara,et al.  A Family of non-GPCR Chemosensors Defines an Alternative Logic for Mammalian Olfaction , 2016, Cell.

[30]  A. Aguzzi,et al.  Microglia: Scapegoat, Saboteur, or Something Else? , 2013, Science.

[31]  B. Barres,et al.  The complement system: an unexpected role in synaptic pruning during development and disease. , 2012, Annual review of neuroscience.

[32]  B. Winblad,et al.  Up-regulation of the inflammatory cytokines IFN-γ and IL-12 and down-regulation of IL-4 in cerebral cortex regions of APPSWE transgenic mice , 2002, Journal of Neuroimmunology.

[33]  Michelle K. Cahill,et al.  Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation , 2016, Cell.

[34]  Keith A. Johnson,et al.  CD33 Alzheimer’s disease locus: Altered monocyte function and amyloid biology , 2013, Nature Neuroscience.

[35]  G. Keller,et al.  Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. , 2007, Blood.

[36]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[37]  P Riederer,et al.  Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer's and de novo Parkinson's disease patients. , 1995, Neuroscience letters.

[38]  S. Gygi,et al.  Identification of a Unique TGF-β Dependent Molecular and Functional Signature in Microglia , 2013, Nature Neuroscience.

[39]  F. C. Bennett,et al.  New tools for studying microglia in the mouse and human CNS , 2016, Proceedings of the National Academy of Sciences.

[40]  G. Paxinos,et al.  ABCA7 Mediates Phagocytic Clearance of Amyloid-β in the Brain. , 2016, Journal of Alzheimer's disease : JAD.

[41]  B. Miller,et al.  Rare TREM2 variants associated with Alzheimer’s disease display reduced cell surface expression , 2016, Acta Neuropathologica Communications.

[42]  M. Diamond,et al.  IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia , 2012, Nature Immunology.

[43]  A. Palucka,et al.  Development and function of human innate immune cells in a humanized mouse model , 2014, Nature Biotechnology.

[44]  Alison M. Goate,et al.  Alzheimer’s Disease Risk Polymorphisms Regulate Gene Expression in the ZCWPW1 and the CELF1 Loci , 2016, PloS one.

[45]  J. Antel,et al.  Isolating, culturing, and polarizing primary human adult and fetal microglia. , 2013, Methods in molecular biology.

[46]  I. Amit,et al.  Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment , 2014, Cell.

[47]  V. Mathura,et al.  Inflammatory cytokine levels correlate with amyloid load in transgenic mouse models of Alzheimer's disease , 2005, Journal of Neuroinflammation.

[48]  T. Möller,et al.  Central nervous system myeloid cells as drug targets: current status and translational challenges , 2015, Nature Reviews Drug Discovery.

[49]  Madeline A. Lancaster,et al.  Cerebral organoids model human brain development and microcephaly , 2013, Nature.

[50]  J. Hardy,et al.  Microglial genes regulating neuroinflammation in the progression of Alzheimer's disease , 2016, Current Opinion in Neurobiology.

[51]  B. Stevens,et al.  New insights on the role of microglia in synaptic pruning in health and disease , 2016, Current Opinion in Neurobiology.

[52]  U. Sengupta,et al.  Identification of oligomers at early stages of tau aggregation in Alzheimer's disease , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[53]  K. Lunetta,et al.  Association of TREM2 variants with Alzheimer's disease in African-Americans: For the Alzheimer's Disease Genetics Consortium (ADGC) , 2013, Alzheimer's & Dementia.

[54]  T. Maniatis,et al.  An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex , 2014, The Journal of Neuroscience.

[55]  J. Pollard,et al.  A Lineage of Myeloid Cells Independent of Myb and Hematopoietic Stem Cells , 2012, Science.

[56]  J. Koenigsknecht-Talboo,et al.  Microglial Phagocytosis Induced by Fibrillar β-Amyloid and IgGs Are Differentially Regulated by Proinflammatory Cytokines , 2005, The Journal of Neuroscience.

[57]  Jennifer Luebke,et al.  Depletion of microglia and inhibition of exosome synthesis halt tau propagation , 2015, Nature Neuroscience.

[58]  Lino C. Gonzalez,et al.  TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia , 2016, Neuron.

[59]  F. Müller,et al.  Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease , 2009, Proceedings of the National Academy of Sciences.

[60]  D. Liebermann,et al.  Interferon regulatory factor 1 is a myeloid differentiation primary response gene induced by interleukin 6 and leukemia inhibitory factor: role in growth inhibition. , 1991, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[61]  Steffen Jung,et al.  TGF‐β signaling through SMAD2/3 induces the quiescent microglial phenotype within the CNS environment , 2012, Glia.

[62]  R. Ransohoff,et al.  Heterogeneity of CNS myeloid cells and their roles in neurodegeneration , 2011, Nature Neuroscience.

[63]  Li-Huei Tsai,et al.  Efficient derivation of microglia-like cells from human pluripotent stem cells , 2016, Nature Medicine.

[64]  C. Glass,et al.  Molecular control of activation and priming in macrophages , 2015, Nature Immunology.

[65]  F. Gage,et al.  Mutant Huntingtin Promotes Autonomous Microglia Activation via Myeloid Lineage-determining Factors Fold Difference from the Mean , 2022 .

[66]  Andrew D. Rouillard,et al.  Enrichr: a comprehensive gene set enrichment analysis web server 2016 update , 2016, Nucleic Acids Res..

[67]  F. Geissmann,et al.  The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C− monocytes , 2011, Nature Immunology.

[68]  C. Cotman,et al.  Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[69]  Mark D. Robinson,et al.  edgeR: a Bioconductor package for differential expression analysis of digital gene expression data , 2009, Bioinform..

[70]  Toshiro K. Ohsumi,et al.  The Microglial Sensome Revealed by Direct RNA Sequencing , 2013, Nature Neuroscience.

[71]  A. Lakatos,et al.  The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function , 2016, Proceedings of the National Academy of Sciences.

[72]  K. Gylys,et al.  Quantitative characterization of crude synaptosomal fraction (P‐2) components by flow cytometry , 2000, Journal of neuroscience research.

[73]  Marco Prinz,et al.  Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease , 2014, Nature Reviews Neuroscience.

[74]  Stephen J. Smith,et al.  Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways , 2013, Nature.

[75]  F. Ginhoux,et al.  Targeting innate immunity for neurodegenerative disorders of the central nervous system , 2016, Journal of neurochemistry.

[76]  F. Ginhoux,et al.  Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages , 2010, Science.

[77]  B. Finsen,et al.  Accelerated microglial pathology is associated with Aβ plaques in mouse models of Alzheimer’s disease , 2014, Aging cell.

[78]  F. Rosenbauer,et al.  Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways , 2013, Nature Neuroscience.

[79]  P. Séguéla,et al.  P2Y12 expression and function in alternatively activated human microglia , 2015, Neurology: Neuroimmunology & Neuroinflammation.

[80]  P. Rezaie,et al.  Colonisation of the developing human brain and spinal cord by microglia: a review , 1999, Microscopy research and technique.

[81]  Avi Ma'ayan,et al.  Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool , 2013, BMC Bioinformatics.

[82]  M. Guillot-Sestier,et al.  Innate immunity in Alzheimer's disease: a complex affair. , 2013, CNS & neurological disorders drug targets.

[83]  M. A. Ajmone-Cat,et al.  TGF‐β and LPS modulate ADP‐induced migration of microglial cells through P2Y1 and P2Y12 receptor expression , 2010, Journal of neurochemistry.