Figure S5.
Inflammatory response and Astrostim cell intracellular signaling mechanisms via chemogenetic activation of human astrocytes. (A) Subset of reactive astrocyte markers significantly upregulated in day-12 Astrostim cells after 2-h (left) and 2-d (right) CNO treatment compared to controls (n = 3 each), as detected by RNAseq and shown as log2FoldChange. Identified genes were (i) restricted or (ii) not restricted to those also upregulated in iAstros compared with NPCs, with IEGs omitted; see Data S4. (B) Complete connectome map (CNet plot) of top 5 KEGG pathways significantly upregulated in 2-h CNO treatment of Astrostim cells (see Fig. 4 C). Pathways are shown as yellow hubs (left), with associated genes listed as geneIDs (right) and colorized according to FoldChange. (C) Top KEGG pathways upregulated in 2-d CNO treatment compared with 2-h CNO treatment. Numbers indicate total DEG counts concerning these pathways, for Padj ≤ 0.0001 and −log10(Padj) > 2. (D) Quantification of conditioned media using a Quantibody Human Cytokine Array revealed that CCL2 (MCP-1) protein was consistently secreted upon CNO treatment from Astrostim cells in monoculture (2-d CNO; n = 3) as well as in coculture spheres with OptoNeurons (6-d CNO; n = 2). (E) 2-h (left) and 2-d (right) CNO treatment resulted in upregulation of CCL2 expression in Astrostim cell monocultures, as quantified by qPCR (n = 4 each). (F) 6-d CNO treatment resulted in significant upregulation of CCL2 expression within cocultures spheres of Astrostim cells and OptoNeurons, as quantified by qPCR (n = 4 each). (G) Pretreatment with 10 µM of the IκB inhibitor ACHP for 1 h significantly decreased nuclear NF-κB signal in Astrostim cells, compared with Astrostim cells without ACHP pretreatment (n = 3 groups each, with five images per group). Scale: 100 µm. (H) Chronic (15 d) treatment with 100 nM recombinant human CCL2 in BP+GF did not significantly alter neuronal firing rates on MEAs compared with non–CCL2 treated controls (n = 8–10). Results are shown as mean ± SEM. In H, dots represent individual electrodes. For A, significance was determined using the DESeq2 R package. For E–G, significance was determined using two-tailed unpaired t tests, with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (see asterisks overlaid on heatmap).

Inflammatory response and Astrostim cell intracellular signaling mechanisms via chemogenetic activation of human astrocytes. (A) Subset of reactive astrocyte markers significantly upregulated in day-12 Astrostim cells after 2-h (left) and 2-d (right) CNO treatment compared to controls (n = 3 each), as detected by RNAseq and shown as log2FoldChange. Identified genes were (i) restricted or (ii) not restricted to those also upregulated in iAstros compared with NPCs, with IEGs omitted; see Data S4. (B) Complete connectome map (CNet plot) of top 5 KEGG pathways significantly upregulated in 2-h CNO treatment of Astrostim cells (see Fig. 4 C). Pathways are shown as yellow hubs (left), with associated genes listed as geneIDs (right) and colorized according to FoldChange. (C) Top KEGG pathways upregulated in 2-d CNO treatment compared with 2-h CNO treatment. Numbers indicate total DEG counts concerning these pathways, for Padj ≤ 0.0001 and −log10(Padj) > 2. (D) Quantification of conditioned media using a Quantibody Human Cytokine Array revealed that CCL2 (MCP-1) protein was consistently secreted upon CNO treatment from Astrostim cells in monoculture (2-d CNO; n = 3) as well as in coculture spheres with OptoNeurons (6-d CNO; n = 2). (E) 2-h (left) and 2-d (right) CNO treatment resulted in upregulation of CCL2 expression in Astrostim cell monocultures, as quantified by qPCR (n = 4 each). (F) 6-d CNO treatment resulted in significant upregulation of CCL2 expression within cocultures spheres of Astrostim cells and OptoNeurons, as quantified by qPCR (n = 4 each). (G) Pretreatment with 10 µM of the IκB inhibitor ACHP for 1 h significantly decreased nuclear NF-κB signal in Astrostim cells, compared with Astrostim cells without ACHP pretreatment (n = 3 groups each, with five images per group). Scale: 100 µm. (H) Chronic (15 d) treatment with 100 nM recombinant human CCL2 in BP+GF did not significantly alter neuronal firing rates on MEAs compared with non–CCL2 treated controls (n = 8–10). Results are shown as mean ± SEM. In H, dots represent individual electrodes. For A, significance was determined using the DESeq2 R package. For E–G, significance was determined using two-tailed unpaired t tests, with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (see asterisks overlaid on heatmap).

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