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Pro-inflammatory cytokine stimulation of human cortical spheroids containing neuroectoderm-derived and mesoderm-derived cell types

ABSTRACT: Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)functioning. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. In this study, we developed a five-months culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) comprising not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations. We then set out to explore the effects of stimulating our hCSs for three time periods with the cytokines TNFα and IL-1β. We use transcriptome-wide RNA-sequencing (RNA-seq) analysis to show that the major process induced by TNFα as well as by IL-1β is neuroinflammation. Central in this process are endothelial cells, microglia and astrocytes, and activation of the NFκB and STAT pathways. In addition to this equiva-lent impact of TNFα and IL-1β, we find that each of the two cytokines has specific and stimulation-time-dependent effects on hCS gene expression, and that IL-1β exhibits a faster self-inhibitory feedback response to dampen neuroinflammation than TNFα. Thus, our protocol provides an inducible 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200779 | GEO | 2022/05/02

REPOSITORIES: GEO

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Project description:Purpose: In the early phases of T1D development exposure of β-cells to IFNγ, IL-1β, and TNFα is thought to induce β-cell dysfunction and the expression of chemokines and cytokines. These changes are mediated in part by post-translational histone modifications within cis-regulatory regions. The purpose of this study was to determine if prevention of these epigenetic changes can alter cytokine induced gene expression changes. Methods: Pancreatic islets were isolated from both male and female CD1 mice by collagenase digestion and were picked by hand. Hand-picked islets were cultured for four days with 1.6ng/ml IFNγ, 0.25ng/ml IL-1β and 0.16ng/ml TNFα (cytokine) or were left untreated (nocytokine). For RNA-seq islets were collected and processed for NGS using the Illumina TruSeq library prep kit. Chromatin immunoprecipitation (ChIP) was performed using 3μg of anti-H3K4me1 (Abcam) with islets from at least 10 adult ICR mice (8-10 weeks old) exposed to 1.6ng/ml IFNγ, 0.25ng/ml Il-1β and 0.16ng/ml TNFα for four days. DNA from 4 separate ChIP experiments was pooled for library construction and sequencing using the Illumina platform. Results: Comparison of gene expression levels between untreated and cytokine-treated islets resulted in the identification of 205 genes that showed an IFNγ, Il-1β, and TNFα-induced chromatin state change (from repressed or inactive to H3K4me1 enriched, i.e. the de novo regions), or increased H3K4me1 enrichment, that were associated with genes with up-regulated expression in response to IFNγ, Il-1β, and TNFα, and 111 genes with reduced H3K4me1 and down-regulated expression in response to IFNγ, Il-1β, and TNFα. Conclusions: Our study confirms that exposure of islets to pro-inflammatory cytokines induces the expression of chemokines and that these changes are the result of a switch from an inactive to an active chromatin state. Further we show that disruption of the trithorax group complex inhibits the upregulation of pro-inflammatory gene expression.

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Pro-inflammatory cytokine stimulation of human cortical spheroids containing neuroectoderm-derived and mesoderm-derived cell types

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