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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

Reactive astrocytes with inflammatory signature in CCM disease in hypoxia and normoxia conditions

Project description:The CCM endothelium is hypersensitive to angiogenesis and can induce a hypoxic program associated with changes in angiogenesis, inflammation, and endothelial-cell metabolism under normoxic conditions. However, the role of active drivers of angiogenesis as CCM disease modifiers in human disease remains unclear. To assess whether CCM reactive astrocytes with neuroinflammatory capacity respond to hypoxia-induced CCM exacerbation, we employed the astrocyte-specific (Aldh1l1-EGFP/Rpl10a) Translational Ribosome Affinity Purification (TRAP) system in combination with a CCM mouse model (Pdcd10BECKO;Aldh1l1-EGFP/Rpl10a). TRAP protocol was performed using Slco1c1-iCreERT2;Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a mice to isolate ribosomes from astrocytes as previously described. Astrocyte-TRAP mRNAs were from cerebral tissue of mice age P50 exposed to hypoxia or normoxia conditions.

2024-09-23 | GSE236162 | GEO

Assessment and Evaluation of Contemporary Approaches for Astrocyte Differentiation from hiPSCs: A Modeling Paradigm for Alzheimer's Disease

Project description:Background: Astrocytes have recently gained attention as key players in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. Numerous differentiation protocols have been developed to study human astrocytes in vitro. However, the properties of the resulting glia are inconsistent, making it difficult to select an appropriate method for a given research question. Therefore, we compared three approaches for the generation of iPSC-derived astrocytes. We performed a detailed analysis using a widely used long serum-free (LSFP) and short serum-free (SSFP) protocol, as well as a TUSP protocol using serum for a limited time of differentiation. Results: We used RNA sequencing and immunochemistry to characterize the cultures. Astrocytes generated by the LSFP and SSFP methods differed significantly in their characteristics from those generated by the TUSP method using serum. The TUSP astrocytes had a less neuronal pattern, showed a higher degree of extracellular matrix formation, and were more mature. The short-term presence of FBS in the medium facilitated the induction of astroglia characteristics but did not result in reactive astrocytes. Data from cell-type deconvolution analysis applied to bulk transcriptomes from the cultures assessed their similarity to primary and fetal human astrocytes. Conclusions: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol for solving specific research tasks or drug discovery studies with iPSC-derived astrocytes.

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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.

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

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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

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

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2022-09-20 | GSE201199 | GEO

Expression analysis of astrocytes isolated from the rat forebrain

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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

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