Journal: The Journal of Cell Biology

Article Title: Autophagy gene FIP200 in neural progenitors non–cell autonomously controls differentiation by regulating microglia

doi: 10.1083/jcb.201609093

Figure Lengend Snippet: Suppression of microglia activation and infiltration rescued the defective neurogenesis in 2cKO mice. (A) The percentage of ramified, round, and amoeboid microglia per SVZ section of Ctrl, FIP cKO, 2cKO, and p53 cKO mice treated with vehicle (Veh) or minocycline (Mino; three mice each). (B–D) Number of iNOS + microglia (B), IL-6 + microglia (C), or TNF + microglia (D) per SVZ section in Ctrl, FIP cKO, 2cKO, and p53 cKO mice treated with vehicle (Veh) or minocycline (Mino; mean ± SEM; three mice each). (E–G) Number of DCX + neuroblasts (E) and GFAP + astrocytes (F) per SVZ section or the density of NeuN + neurons per olfactory bulb section (G) in Ctrl, FIP cKO, 2cKO, and p53 cKO mice treated with vehicle or minocycline are shown (mean ± SEM; three mice each). (H) Number of Iba1 + microglia per SVZ section of 2cKO mice treated with vehicle (Veh) or TAK-779 (TAK; mean ± SEM; three mice each). (I–K) Number of DCX + neuroblasts (I) and GFAP + astrocytes (K) per SVZ section or the density of NeuN + neurons per olfactory bulb section (J) in 2cKO mice treated with vehicle (Veh) or TAK-779 (TAK; mean ± SEM; three mice each). (L) Number of Iba1 + microglia per SVZ section in Ctrl, FIP cKO, 2cKO, and p53 cKO mice treated with vehicle (Veh) or PLX3397 (PLX; mean ± SEM; four mice each). (M–O) Number of DCX + neuroblasts (M) and GFAP + astrocytes (O) per SVZ section or the density of NeuN + neurons per olfactory bulb section (N) in Ctrl, FIP cKO, 2cKO, and p53 cKO mice treated with vehicle (Veh) or PLX3397 (PLX) are shown (mean ± SEM; four mice each). **, P < 0.01; ***, P < 0.001.

Article Snippet: PLX3397 (ApexBio) was dissolved in DMSO and then added immediately before using to a solution of 0.5% methyl cellulose (M0512; Sigma-Aldrich) and 1.0% Tween 80 (P1754; Sigma-Aldrich).

Techniques: Activation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Small-molecule agents for cancer immunotherapy

doi: 10.1016/j.apsb.2023.12.010

Figure Lengend Snippet: Small-molecule agents under clinical development in immuno-oncology.

Article Snippet: , CSF1R , Pexidartinib , Daiichi Sankyo , CSF1R, c-KIT, FLT3 , Phase III , NCT04488822.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: Small-molecule agents for cancer immunotherapy

doi: 10.1016/j.apsb.2023.12.010

Figure Lengend Snippet: Small-molecule agents in combinational therapies.

Article Snippet: , CSF1R , Pexidartinib , Daiichi Sankyo , CSF1R, c-KIT, FLT3 , Phase III , NCT04488822.

Techniques: Vaccines, Infection

Journal: bioRxiv

Article Title: Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells

doi: 10.1101/2024.10.11.617794

Figure Lengend Snippet: A : H&E and IHC for indicated markers on day 20-FLOs and fetal liver 18 pcw. Scale bar 50 µm. B : Schematic of CD34+ HPC isolation for hematopoietic characterization from factor-free differentiated FLOs and FLOs with factors GM-CSF, IL34, IL3, and UM171 or UM729 (“FLO + GM/34”) added from day 10. C : Flow cytometry analysis of MACS-CD34 enriched FLO, FLO + GM/34 on days 18-22, and mobilized peripheral blood stem cells (mPBSCs). Gated for viable single cells. The blue population is gated for CD34+ CD43-. The pink population is gated for CD34+ CD43+. Pooled experiment of 40 separate organoid cultures. D : Granulocyte, erythroid, macrophage, and megakaryocyte colony forming units (CFU-GEMM) after two weeks culture in MethoCult SF H4436 (STEMCELL Technologies). FLOs and FLOs + GM/34 on days 22-27 were CD34+ enriched via MACS before plating. Primary CD34+ mPBSCs (2 healthy donors) are shown as controls. The colony numbers were normalized to 10000 plated CD34+ cells assessed via flow cytometry. n = 4 (for FLO, FLO + GM/34) and n = 6 (for mPBSCs); separate cultures are shown as individual data points min to max. Kruskal-Wallis with Dunńs multiple comparison correction was used to assess significance. Corrected p-values shown. E : Schematic of functional hematopoietic differentiation assays of FLOs with or without GM/34 compared to adapted factor-dependent iPSC differentiation protocol of HPCs (iPCS-HPCs). F : Flow cytometry analysis for T-cell markers after four weeks of culture using StemDiff T Cell Kit (STEMCELL Technologies). CD34+ cells were enriched via MACS from FLOs, FLOs + GM/34 on days 18-22 and iPSC-HPCs before plating. Primary human mPBSCs (healthy donor) were used as a control. Gated for viable single cells. Pooled data of n = 40 separate organoid cultures. G : Granulocyte-macrophage colony forming units (CFU-GM) after 2 weeks culture in MethoCult SF H4436 of FLOs and FLOs + GM/34 on days 22-27. Primary CD34+ mPBSCs (2 healthy donors) and iPSC-HPCs are shown as controls. The colony numbers were normalized 10000 plated CD34+ cells assessed via flow cytometry. n = 4 (for FLO, FLO + GM/34 and iPSC-HPCs) and n = 6 (for mPBSCs); separate cultures are shown as individual data points min to max. Kruskal-Wallis with Dunńs multiple comparison correction was used to assess significance. Corrected p-values are shown. H: UMAP feature plots of FLOs + GM/34 scRNA-seq datasets on day 13 displaying expression levels of hematopoietic stem and progenitor (HSPC) signature genes.

Article Snippet: To assess functionality of FLO-derived CSF, CSF-1R inhibitor BLZ945 (Hycultec, 500 nM) was added to the basal medium from day 10.

Techniques: Isolation, Flow Cytometry, Comparison, Functional Assay, Control, Expressing

Journal: bioRxiv

Article Title: Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells

doi: 10.1101/2024.10.11.617794

Figure Lengend Snippet: A: UMAP of integrated scRNA-seq datasets from FLOs +GM/34 on days 10, 13, 16, and 20 with pseudo-time (color-coded) and trajectory analysis using PAGA-Tree (indicated in black arrows, hematopoietic cluster highlighted in magenta dotted lines). Feature gene expression plots for maker genes of HPCs (black), stromal mesenchyme (yellow), endothelium (green), myeloid lineages (magenta), endoderm and hepatoblasts/fetal hepatocytes (red). B: mIF image of day 34 FLO and FLO + GM/34 compared to postnatal liver specimen (24 months) to assess cellular heterogeneity and the emergence of putative neutrophils (MPO). Representative images of n = 4 separate organoid cultures. Scale bar 50 µm. C : UMAP of integrated FLO + GM/34 scRNA-seq datasets (days 10, 13, 16, 20) with a close-up of the hematopoietic cluster. Featuring the gene set expression of HSPC-signature: RUNX1, HOXA9, MECOM, HLF, MLLT3, SPINK2 from Calvanese et al., Nature , 2020; yolk sac-derived myeloid-biased progenitors (YSMPs): CD34, KIT, MYB, AZU1, MPO, MS4A3, LYZ ; YSMP-derived embryonic monocyte lineage (YSMP Mono): CCR2, HLA-DR, CSF1R, MEF2C, CD14; and YSMP-derived embryonic granulocyte lineage (YSMP Granulo): S100P, S100A8, S100A9, S100A12, CEACAM8/ CD66b from Bian et al. . Illustrated with https://github.com/ZornLab/Mona . D : UMAP demonstrating cell clusters (RNA_snn_res.0.8) of integrated FLO + GM/34 scRNA-seq datasets (days 10, 13, 16, 20) with close-up on the hematopoietic cluster. E : Cluster gene expression from integrated FLO + GM/34 dataset (days 10, 13, 16, 20) of markers for YSMP, neutrophilic (neutro) and monocytic (mono) fates per Bian et al. . F : FACS gating strategy to characterize neutrophil granulocytes (CD45+ CD14-CD16+ CD66b+, yellow) and macrophages (CD45+ CD14+ CD163+, magenta) of day 30 FLOs + G/GM/34 (added G-CSF from day 20 to 30 to FLO + GM/34 protocol) compared to fresh adult PBMCs and PBMC-derived granulocytes (Granulo, healthy donor). Gated for viable single cells and CD45+. G : Quantification of CD45+ CD14-CD16+ CD66b+ neutrophil granulocytes (left) and CD45+ CD14+ CD163+ macrophages (right) in FLO + G/GM/34 compared to PBMCs and granulocytes (Granulo). n = 2-3 separate organoid cultures shown as individual data points and mean ± SD. One-way ANOVA with Dunnett multiple comparison test to assess significance. Corrected p-values are shown. H: Representative images of Giemsa-staining after FACS-based isolation for CD45+ CD14-CD16+ CD66b+ neutrophil granulocytes (left) and CD45+ CD14+ CD163+ macrophages (right). Scale bar 50 µm.

Article Snippet: To assess functionality of FLO-derived CSF, CSF-1R inhibitor BLZ945 (Hycultec, 500 nM) was added to the basal medium from day 10.

Techniques: Expressing, Derivative Assay, Comparison, Staining, Isolation

Journal: EMBO Molecular Medicine

Article Title: Aged lipid‐laden microglia display impaired responses to stroke

doi: 10.15252/emmm.202217175

Figure Lengend Snippet: A, B (A) Representative images of the striatum of young female mice with original microglia and repopulated microglia 4 days postischemia ( n = 4 per group). Images show microglial cells immunostained with anti‐P2YR12 (red) in the noninjured contralateral hemisphere, the periphery of ischemia, and the lesion core. Cell nuclei are labeled with DAPI (blue). After repopulation, original and renewed microglia react to ischemia with changes in morphology. (B) Magnification of individual cells marked with squares in (D). Scale bar: (A) 20 μm, (B) 10 μm. C Morphometric analysis of microglial cells showed reduced area and increased solidity and circularity in microglia of the ischemic hemisphere versus the contralateral hemisphere in both groups. However, the increase in circularity at the periphery and core of infarction was attenuated in repopulated versus original microglia (* P = 0.047 in the periphery and * P = 0.033 in the core; Kruskal–Wallis test). Points show individual cells (in the periphery, core, and contralateral regions: n = 233, 96, and 91 cells for the control group, and n = 178, 58, and 109 cells for the renewed group, respectively), and colors indicate different mice ( n = 4 mice per group). Bars show the median with 95% confidence interval. D Renewed microglia derive from brain cells. We generated chimeric mice by bone marrow transplantation from DsRed fluorescent reporter donor male mice to wild‐type recipient male mice ( n = 11). After at least 8 weeks, mice were treated with PLX5622 in the diet ( n = 8) or corresponding control diet ( n = 3). Three weeks later, mice were euthanized ( n = 4) or they were switched to control diet for repopulation for 7 days ( n = 4 per group). The brain was studied via flow cytometry by measuring DsRed + and DsRed − cells in the gate of microglia cells. Absolute number of microglial cells was strongly reduced after microglia depletion (PLX5622 diet; one‐way ANOVA and Holm–Šídák's multiple comparisons test *** P = 0.0002 versus control diet). Microglia numbers recovered after 7 days of repopulation ( &&& P = 0.0002 versus depleted cells). The % of DsRed cells in the microglia gate of mice fed control diet was negligible. However, after microglia depletion (PLX5622 treatment; ANOVA and Holm–Šídák's, *** P < 0.0001 versus control diet) there was a high % of DsRed + cells within the very small population of CD45 low CD11b + cells indicating the presence of a few CSF1R‐independent infiltrating cells. Importantly, the proportion of DsRed + cells was negligible after mice were switched to control diet and the number of microglia increased ( &&& P < 0.0001 versus depleted mice). Values are expressed as the mean ± SEM.

Article Snippet: In rodents, genetic strategies to prevent microglial Csf1r expression or treatment with pharmacological inhibitors of CSF1R cause microglia depletion (Waisman et al , ).

Techniques: Labeling, Control, Generated, Transplantation Assay, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Aged lipid‐laden microglia display impaired responses to stroke

doi: 10.15252/emmm.202217175

Figure Lengend Snippet: Experimental design for microglia depletion and repopulation in old (21–22 month) female mice. We depleted microglia by providing a diet containing the CSF1R antagonist PLX5622 for 3 weeks. Microglia repopulation was induced by switching to control diet for 7 days prior to induction of ischemia ( n = 17). Treatment controls were fed a corresponding control diet ( n = 16). Behavioral testing (including training sessions) was conducted throughout the experiment and mice underwent two MRI studies. Measures were obtained the day prior to ischemia (basal value, time = 0) and at different time points postischemia until day 14. Image created with BioRender. The neuroscore value (mean ± SD) decreased from day 1 to day 14 by 23% in the control group (from 10.8 ± 4.7 to 8.3 ± 4.2; n = 15) and by 46% in the microglia renewed group (from 11.9 ± 6.1 to 6.4 ± 2.8; n = 16). The variability within groups was large and differences between groups were not statistically significant ( P = 0.21, nonparametric Kruskal–Wallis test with a repeated‐measures design). Points are individual values for each mouse, box and whiskers show the median, and bars indicate min. and max. values. One animal of each group died after ischemia and these mice were excluded. The grip test assessing limb strength showed a better performance in the group with repopulated microglia (Two‐way ANOVA and Šídák's multiple comparisons test, * P = 0.047). Values are expressed as % of the corresponding basal value (preischemia) for each mouse. Rotarod test did not show differences between groups. Values are expressed as latency to fall (seconds) from the running wheel at different time points postischemia (higher means better). Laterality was assessed via the cylinder test in a subgroup of mice ( n = 7 renewed, n = 6 control). Positive values in the laterality index (ranging from −1 to +1) indicate impairment of the affected hindlimb, i.e., contralateral to the injured brain hemisphere. There were no differences between groups with the Mann–Whitney test. However, data analysis with the Wilcoxon signed‐rank test showed that the repopulated group was significantly different (* P = 0.016) from a theoretical median of +1 at day 4 postischemia whereas the control group was not ( P = 0.062). The results indicate some transient laterality effect in the control group at day 4 postischemia that was not detected in the repopulated group. Bars represent mean ± SD. The MRI lesion volume, as illustrated in the corresponding false color images below, showed no differences in infarct volume in mice with ( n = 16) or without ( n = 15) microglia renewal. Results correspond to day four postischemia. Representative MRI images were obtained on day four postischemia using T2w MRI and day 14 using a Modified Driven Equilibrium Fourier Transform (MDEFT) MRI sequence (7.0 T BioSpec 70/30, Bruker BioSpin). Values show data for individual mice and/or the mean ± SD.

Article Snippet: In rodents, genetic strategies to prevent microglial Csf1r expression or treatment with pharmacological inhibitors of CSF1R cause microglia depletion (Waisman et al , ).

Techniques: Control, MANN-WHITNEY, Modification, Sequencing