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Microglia Increase Inflammatory Responses in iPSC-Derived Human BrainSpheres

Abreu, Celina Monteiro, Gama, Lucio, Krasemann, Susanne, Chesnut, Megan, Odwin-Dacosta, Shelly, Hogberg, Helena T., Hartung, Thomas, Pamies, David·2018·Frontiers in Microbiology
DOI 10.3389/fmicb.2018.02766PMC PMC6296317
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FIGURE 1 Generation of microglia-containing BS (μBS). Immortalized Human Microglia – SV40 were incorporated to 7 week-differentiated BS by gravity as described in materials and methods. (A) Diagram of μBS generation procedure. (B) Comparison between μBS and BS using different microglia markers (TMEM119, Mertk, Axl) and hematoxylin/eosin (HE). (C) Confocal images of immunohistochemistry for the microglia marker IBA1 (green), the neuronal marker NF200 (red), and nuclear staining (Hoechst 33342, blue) in μBS, 48 h after microglia incorporation. (D) Details of microglia aggregations in μBS. Green arrow indicates microglia cluster and red arrow indicates the BS. Bars represent 100 μm (B), 50 μm (C, upper panel), 20 μm (C, all other panels), and 50 μm (D).
FIGURE 1 Generation of microglia-containing BS (μBS). Immortalized Human Microglia – SV40 were incorporated to 7 week-differentiated BS by gravity as described in materials and methods. (A) Diagram of μBS generation procedure. (B) Comparison between μBS and BS using different microglia markers (TMEM119, Mertk, Axl) and hematoxylin/eosin (HE). (C) Confocal images of immunohistochemistry for the microglia marker IBA1 (green), the neuronal marker NF200 (red), and nuclear staining (Hoechst 33342, blue) in μBS, 48 h after microglia incorporation. (D) Details of microglia aggregations in μBS. Green arrow indicates microglia cluster and red arrow indicates the BS. Bars represent 100 μm (B), 50 μm (C, upper panel), 20 μm (C, all other panels), and 50 μm (D).CC BY · PubMed Central
FIGURE 2 Differences between BS, μBS, and microglia after LPS treatment. The microglia, BS, μBS, were exposed to 20 ng/mL LPS up to 24 h. (A) shows gene expression of IL6, IL10, IL1b, CCL2, and TNFa after LPS treatment on the different models (BS, μBS, and microglia cells). Data are shown as fold change of treated versus untreated up to an hour post treatment (3, 6, 12, and 24 h after treatment). (B) show changes in cell cycle in percentage of the cells in G2 plus S phase in microglia cells, BS and μBS treated or non-treated with LPS. Each dot symbol represent a replicate sample (n+3). (C) shows Annexin V Apoptosis Muse assay. Results show % live cells microglia, BS, ad μBS cells treated (+) or not treated (–) with LPS. Each symbol represent a replicate (n = 3). Statistical analysis in A was performed using Dunnett test on ΔΔCt. The asterisks symbols represents ∗P < 0.05 and ∗∗P < 0.01 (two independent experiments and three biological replicates on each experiment).
FIGURE 2 Differences between BS, μBS, and microglia after LPS treatment. The microglia, BS, μBS, were exposed to 20 ng/mL LPS up to 24 h. (A) shows gene expression of IL6, IL10, IL1b, CCL2, and TNFa after LPS treatment on the different models (BS, μBS, and microglia cells). Data are shown as fold change of treated versus untreated up to an hour post treatment (3, 6, 12, and 24 h after treatment). (B) show changes in cell cycle in percentage of the cells in G2 plus S phase in microglia cells, BS and μBS treated or non-treated with LPS. Each dot symbol represent a replicate sample (n+3). (C) shows Annexin V Apoptosis Muse assay. Results show % live cells microglia, BS, ad μBS cells treated (+) or not treated (–) with LPS. Each symbol represent a replicate (n = 3). Statistical analysis in A was performed using Dunnett test on ΔΔCt. The asterisks symbols represents ∗P < 0.05 and ∗∗P < 0.01 (two independent experiments and three biological replicates on each experiment).CC BY · PubMed Central
FIGURE 3 Virus infection in BrainSpheres. Figure shows virus infection by immunohistochemistry in BrainSpheres with (μBS) and without microglia (BS). Green represents the Flavivirus marker for ZIKV (NS1) and DENV-1, red represents microglia markers IBA1 and NF200. Bars represent 50 μm (lower magnification) and 10 μm (higher magnification).
FIGURE 3 Virus infection in BrainSpheres. Figure shows virus infection by immunohistochemistry in BrainSpheres with (μBS) and without microglia (BS). Green represents the Flavivirus marker for ZIKV (NS1) and DENV-1, red represents microglia markers IBA1 and NF200. Bars represent 50 μm (lower magnification) and 10 μm (higher magnification).CC BY · PubMed Central
FIGURE 4 Effects of Flavivirus infection on 3D human iPSC-derived brain spheres (BrainSpheres, BS) without or with microglia cells (μBS). The microglia cells, BS, and μBS were infected with Dengue, ZIKV-BR, and ZIKV-UG using MOI equal 0.1. (A) shows the growth kinetics of flaviviruses over time for the three models. The viral load (RNA copies/mL) are shown with standard deviation. (B) Changes in cell cycle shown as percentage of cells in G2/S phase in BS, μBS, and microglia treated or non-treated with the different viruses. (C) Annexin V Apoptosis Muse assay results. Results represent % live cells in BS, μBS, and microglia samples after the different flavivirus treatments. (D) Gene expression relative quantification for TNFα, CCL2, IL-1b, and IL-6 over the time. Statistical analysis was performed using the Dunnett’s Test (∗mean equal p < 0.05 and ∗∗p < 0.01) for two independent experiments, three biological replicates on each experiment.
FIGURE 4 Effects of Flavivirus infection on 3D human iPSC-derived brain spheres (BrainSpheres, BS) without or with microglia cells (μBS). The microglia cells, BS, and μBS were infected with Dengue, ZIKV-BR, and ZIKV-UG using MOI equal 0.1. (A) shows the growth kinetics of flaviviruses over time for the three models. The viral load (RNA copies/mL) are shown with standard deviation. (B) Changes in cell cycle shown as percentage of cells in G2/S phase in BS, μBS, and microglia treated or non-treated with the different viruses. (C) Annexin V Apoptosis Muse assay results. Results represent % live cells in BS, μBS, and microglia samples after the different flavivirus treatments. (D) Gene expression relative quantification for TNFα, CCL2, IL-1b, and IL-6 over the time. Statistical analysis was performed using the Dunnett’s Test (∗mean equal p < 0.05 and ∗∗p < 0.01) for two independent experiments, three biological replicates on each experiment.CC BY · PubMed Central

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Metadata

Authors
Abreu, C. M., Gama, L., Krasemann, S., Chesnut, M., Odwin-Dacosta, S., Hogberg, H. T., Hartung, T., & Pamies, D.
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2018-12-04
Volume
9
ISSN
1664-302X
DOI
10.3389/fmicb.2018.02766
PMC
PMC6296317
Cited by
123
References
16
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)
Funders
National Center for Advancing Translational Sciences

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