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Human induced pluripotent stem cell-derived astrocytes are differentially activated by multiple sclerosis-associated cytokines

ABSTRACT: Recent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a new serum-free protocol to differentiate human iPSCs into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the thirteen hiPSC lines differentiated. Using this new model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNFα and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This new model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE120411 | GEO | 2018/11/01

REPOSITORIES: GEO

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Embryonic stem cell-derived brainstem and spinal astrocyte-like cells develop a neurotoxic phenotype in vitro following clinically relevant pro-inflammatory stimulation

Project description:Traumatic axonal injury (TAI) in the brainstem carries a particularly poor prognosis. Although the role of neuroinflammation in this process is incompletely characterized, astrogliosis has been shown to coincide with TAI. Moreover, astrogliotic forebrain astrocytes were recently shown to confer a toxic effect on neurons. We sought to assess if ventral brainstem- or rostroventral spinal astrocytes exert similar effects on motor neurons in vitro. We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of embryonic stem cells (ES). ES-astrocyte identity was assessed using RNA-sequencing, immunocytochemistry, and comparison with primary subventricular zone-astrocytes. We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α. In co-cultures with reactive ES-astrocytes and motor neurons, we demonstrated neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions. When exposed to IL-1β and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. This demonstrates that ventral brainstem and rostroventral spinal cord astrocytes can exert neurotoxic effects in vitro. This highlights the importance of neuroinflammation as a secondary injury mechanism following neurotrauma, and presents a possible treatment avenue to improve neuronal survival in particularly vulnerable CNS regions following TAI.

2023-04-02 | GSE213804 | GEO

Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway (ChIP-Seq)

Project description:Neuroinflammation is a common feature of neurodegenerative disorders such as Alzheimer's disease (AD). Neuroinflammation is induced by dysregulated glial activation, and astrocytes, the most abundant glial cells, become reactive upon neuroinflammatory cytokines released from microglia, and actively contribute to neuronal loss. Therefore, blocking reactive astrocyte functions is a viable strategy to manage neurodegenerative disorders. However, factors or therapeutics directly regulating astrocyte subtype remain unexplored. Here, we identified transcription factor NF-E2-related factor 2 (Nrf2) as a therapeutic target in neurotoxic reactive astrocytes upon neuroinflammation. We found that the absence of Nrf2 promoted the activation of reactive astrocytes in the brain tissue samples obtained from AD model 5xFAD mice, whereas enhanced Nrf2 expression blocked the induction of reactive astrocyte gene expression by counteracting NF-kB subunit p65 recruitment. Neuroinflammatory astrocytes robustly upregulated genes associated with type I interferon and the antigen-presenting pathway, which were suppressed by Nrf2 pathway activation. Moreover, impaired cognitive behaviors observed in AD mice were rescued upon ALGERNON2 treatment, which potentiated Nrf2 pathway and reduced the induction of neurotoxic reactive astrocytes. Thus, we highlight the potential of astrocyte-targeting therapy by promoting the Nrf2 pathway signaling for neuroinflammation-triggered neurodegeneration.

2023-08-07 | GSE237423 | GEO

Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway ( nascent RNA-Seq )

2023-08-07 | GSE237422 | GEO

Functional astrocytes differentiated from hiPSCs

Project description:Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. Using this method, we generated hiPSC-derived astrocyte populations (hiPSC-astrocytes) from 42 NPC lines (derived from 30 individuals) with an average of ~90% S100β-positive cells. Transcriptomic analysis demonstrated that the hiPSC-astrocytes are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our novel protocol is a reproducible, straightforward (single media) and rapid (<30 days) method to generate homogenous populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.

2017-08-18 | GSE97904 | GEO

Isolation of ferret astrocytes reveals their morphological, transcriptional, and functional differences from mouse astrocytes

Project description:To identify differently expressed genes between ferret and mouse astrocytes, we performed RNA-seq studies on primary ferret and mouse astrocytes cultured in serum free medium. We isolated primary astrocytes from mixed glial cultures which are prepared from the cortices of ferrets and mice at postnatal day 1. Each biological replicate was generated from 2 individuals of the same species. We detected substantial changes in the expression of genes associated with cell proliferation and migration, and identified ferret astrocyte-specific genes.

2022-09-20 | GSE201199 | GEO

Expression analysis of astrocytes isolated from the rat forebrain

Project description:Comparison of expression data of rat forebrain astrocytes from P1, P7 acutely isolated by immunopanning or cultured with astrocytes prepared by McCarthy and de Vellis' (1980) method. Elucidating the genes induced by serum in immunopannedrat astrocytes. Three biological replicates for each sample were done. MD-astrocytes were prepared as described in McCarthy and de Vellis 1980 and harvested for mRNA after 7DIV. IP-astrocytes were isolated from P1 or P7 Sprague Dawley rats and processed for RNA immediately (IP-astrocytes P1/P7), or cultured for 7 days in HBEGF before harvesting (Cult. IP-astrocytes P1/P7). For the serum studies, we plated IP-astrocytes P7 in MD-astrocyte media containing 10% fetal calf serum immediately after isolation and cultured them for 7 days. After 7 days, the cultures were either processed for total RNA or washed 3x with dPBS and astrocyte base media with HBEGF was added. The cells were cultured for an additional 7 days and then processed for RNA. We isolated total RNA with the QIAshredder and Qiagen RNeasy Mini Kit. We used the 3’IVT Express kit for preparation of the RNA and the Rat Genome 230 2.0 Array chip (Affymetrix, Santa Clara). RT-PCR was used to elucidate the level of contamination in each cell sample.

2011-09-05 | E-GEOD-26066 | biostudies-arrayexpress

Rat Astrocyte-Neuron Co-cultures versus purified co-cultured Astrocytes versus Astrocyte alone

Project description:Profiling of transcriptional changes in rat astrocytes when co-cultured with neurons: comparison of astrocytes cultured alone with astrocytes co-cultured with mouse hippocampal neurons. Co-cultured astrocytes are isolated using cold jet, a novel tool for these neuron-glia cultures. Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that at least part of the involved astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-induced astrocyte genes in vitro, we tested the effectiveness of the ‘cold jet’, a new method for separation of neurons from co-cultured astrocytes. The cold jet method is performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis revealed changes in expression of a large number of mRNAs and biological processes, including novel findings. Thus, cold jet is an efficient method to separate astrocytes from neurons in co-culture, and in this study reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

2016-11-09 | GSE52481 | GEO

Traumatic brain injury results in unique microglial and astrocyte transcriptomes enriched for type I interferon response

Project description:In this study, we performed bulk RNA sequencing of concurrently isolated microglia and astrocytes to evaluate their cell-specific transcriptional responses to TBI 7 days post-injury. Our findings revealed sustained, cell-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute timepoint. The microglia and astrocyte transcriptional response to TBI was unique from other neurodegenerative and neuroinflammatory diseases that have been described. In addition, microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation.

2021-06-01 | GSE167459 | GEO

CROP-seq of hiPSC-derived astrocytes (scRNA-Seq)

Project description:In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states with altered functions. Certain inflammatory insults induce reactive astrocyte states that lose homeostatic functions and gain neurotoxicity, and likely contribute to neuroinflammatory and neurodegenerative diseases. However, the cellular pathways controlling these states are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-kB activation drove two distinct inflammatory reactive states dependent on STAT3 and IRF1, respectively. Furthermore, these states corresponded with those observed in other experimental contexts, including in vivo, and their markers were upregulated in the human brain in Alzheimer's disease and ischemic-hypoxic encephalopathy. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

2021-08-24 | GSE182308 | GEO

CROP-seq of hiPSC-derived astrocytes (bulk RNA-Seq)

2021-08-24 | GSE182307 | GEO

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