RESUMO
LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity.
Assuntos
Pan troglodytes/genética , Retroelementos , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoplasma/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Processamento Pós-Transcricional do RNA , RNA Antissenso/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/metabolismo , Alinhamento de SequênciaRESUMO
Neurogenesis in the adult hippocampus declines with age, a process that has been implicated in cognitive and emotional impairments. However, the mechanisms underlying this decline have remained elusive. Here, we show that the age-dependent downregulation of lamin B1, one of the nuclear lamins in adult neural stem/progenitor cells (ANSPCs), underlies age-related alterations in adult hippocampal neurogenesis. Our results indicate that higher levels of lamin B1 in ANSPCs safeguard against premature differentiation and regulate the maintenance of ANSPCs. However, the level of lamin B1 in ANSPCs declines during aging. Precocious loss of lamin B1 in ANSPCs transiently promotes neurogenesis but eventually depletes it. Furthermore, the reduction of lamin B1 in ANSPCs recapitulates age-related anxiety-like behavior in mice. Our results indicate that the decline in lamin B1 underlies stem cell aging and impacts the homeostasis of adult neurogenesis and mood regulation.
Assuntos
Envelhecimento/metabolismo , Ansiedade/genética , Regulação para Baixo , Hipocampo/citologia , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Envelhecimento/genética , Animais , Diferenciação Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , RatosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Differentiation of multipotent stem cells occurs through the highly coordinated control of gene expression. Repressor element 1 (RE1) silencing transcription factor (REST), a master transcriptional regulator in neuronal stem cells, restricts neuronal gene expression. REST activity is context-dependent and is modified by its cofactors, such as Ctdsp2. In this issue of Genes & Development, Dill and colleagues (pp. 25-30) report on the microRNA-mediated regulation of neural differentiation. Interestingly, this microRNA is post-transcriptionally regulated and modulates expression of its host gene, ctdsp2.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas de Peixe-Zebra/genética , AnimaisRESUMO
Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.
Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Pan paniscus/genética , Pan troglodytes/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Proteínas Argonautas/metabolismo , Linhagem Celular , Forma Celular , Citidina Desaminase/metabolismo , Evolução Molecular , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Camundongos Nus , Antígenos de Histocompatibilidade Menor , Pan paniscus/metabolismo , Pan troglodytes/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de RNA , Especificidade da EspécieRESUMO
Researchers have long sought to understand the genetic basis of the cognitive differences between primates, with particular focus on the human brain. Although all mutational types have worked in concert with evolutionary forces to generate the current human brain, in this review we will explore the impact of mobile elements, specifically non-LTR retrotransposons. Non-LTR retrotransposons have contributed coding and regulatory sequences to the genome throughout evolution. During primate evolution there have been multiple waves of LINE retrotransposition as well as the birth of new mobile elements such as the SINEs Alu and SVA and we will explore what kinds of impacts these may have had on the evolving human brain.
Assuntos
Evolução Biológica , Encéfalo/fisiologia , Genoma , Primatas/fisiologia , Retroelementos/genética , Animais , Evolução Molecular , Primatas/genéticaRESUMO
RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non-cell-autonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non-cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non-cell-autonomously via SCG2. SIGNIFICANCE STATEMENT: Our results reveal that REST regulates differentiation and maturation of neural progenitor cells in vitro by orchestrating both cell-intrinsic and non-cell-autonomous factors and that Scg2 is a major secretory target of REST with a differentiation-enhancing activity in a paracrine manner. In vivo, REST depletion causes accelerated differentiation of newborn neurons at the expense of spine defects, suggesting a potential role for REST in the timing of the maturation of granule neurons.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Proteínas Repressoras/fisiologia , Secretogranina II/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Ratos WistarRESUMO
Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.
Assuntos
Diferenciação Celular , Linhagem da Célula , Reprogramação Celular , Endotélio Vascular/citologia , Fibroblastos/citologia , Miócitos de Músculo Liso/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.
Assuntos
Drosophila/metabolismo , MicroRNAs/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Animais , Proteínas Argonautas , Linhagem Celular , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Iniciação em Eucariotos , Inativação Gênica , Genes de Insetos , MicroRNAs/genética , Modelos Biológicos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Complexo de Inativação Induzido por RNA/genéticaRESUMO
Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.
Assuntos
Células-Tronco Neurais/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Regiões 5' não Traduzidas/genética , Animais , DNA/metabolismo , Feminino , Inativação Gênica , Células HEK293 , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Ratos , Receptores Citoplasmáticos e Nucleares/deficiênciaRESUMO
Changes in chromatin state contribute to the switch in gene expression programs that characterizes the transition of dividing neural stem cells toward a neuronal fate. In this issue of Neuron, Lessard et al. show that this process is regulated by specific cofactor exchanges within the SWI/SNF chromatin remodeling complex.
Assuntos
Diferenciação Celular/fisiologia , Cromatina/metabolismo , Neurônios/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Neurônios/fisiologia , Transcrição Gênica/fisiologiaRESUMO
RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Transcrição Gênica , Proteínas Argonautas , Centrômero/genética , Centrômero/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Polimerase II/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Virais/metabolismoRESUMO
Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here we show that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Genes Reporter/genética , Humanos , MicroRNAs/genética , Complexos Multiproteicos , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Ribonuclease III/genéticaRESUMO
RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.
Assuntos
Nuclease do Micrococo/isolamento & purificação , Nuclease do Micrococo/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , Complexo de Inativação Induzido por RNA/química , Animais , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Drosophila melanogaster/enzimologia , Substâncias Macromoleculares , Nuclease do Micrococo/química , Estrutura Terciária de Proteína , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
In recent years, sequence-specific gene silencing has been an area of increasing focus, both because of its interesting biology and because of its power as an experimental tool. A growing understanding of one such phenomenon, RNA interference (RNAi), has provided clues that many homology-dependent gene-silencing mechanisms share a common trigger, double-stranded RNA. Recent findings that RNAi and related pathways are involved not only in the response to exogenous pathogenic and endogenous parasitic nucleic acids but also in basic cellular processes, such as gene regulation and heterochromatin formation, have further fueled interest in this rapidly expanding field.
Assuntos
Inativação Gênica , Interferência de RNA , Animais , Endorribonucleases/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease IIIRESUMO
Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.
Assuntos
Neurônios/citologia , Pan paniscus/fisiologia , Pan troglodytes/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular/genética , Dendritos/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Especificidade da EspécieRESUMO
microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs(f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.
Assuntos
Drosophila melanogaster/fisiologia , Células Germinativas/fisiologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Masculino , Dados de Sequência Molecular , RNA Helicases/metabolismo , Interferência de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismoRESUMO
Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.
Assuntos
Evolução Molecular , Genoma Humano , Hominidae/genética , Animais , Mapeamento de Sequências Contíguas , Variação Genética , Humanos , Anotação de Sequência Molecular , Análise de Sequência de DNARESUMO
Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1ß or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1ß. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.