Journal: Molecular Neurodegeneration

Article Title: An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

doi: 10.1186/s13024-024-00723-x

Figure Lengend Snippet: Pedigree of the proband’s family, MRI images, and genotyping data. A Pedigree of the proband’s family (prepared via https://cegat.com/ ). Shading indicates carriers of the c.2350G > A (p.V784M) CSF1R variant with ALSP diagnosis. Symbol with a dot indicates a carrier without clinical manifestations of ALSP. B - E Brain MRI of subject II.3. B Sagittal FLAIR T2-weighted MR image showing thinning and hyperintense signal of the anterior portion of the body of the corpus callosum (red circle). C - D Axial T2-weighted MR images showing the presence of bilateral multifocal and confluent lesions in the frontal lobes (red circles) and multifocal lesions in the left fontal and parietal lobe with areas of restricted diffusion in the diffusion weighted image ( E , red circles). F – H Brain MRI of a healthy control, matched by sex and age to subject II.3. F Sagittal T1-weighted image showing normal size and signal of the brain parenchyma, specifically of the corpus callosum. G - H Axial T2-weighted images showing no atrophy, normal ventricular size and normal signal at the level of the cerebral white matter. (I) Chromatogram of PBMC-derived DNA showing heterozygous c.2350G > A CSF1R variant (shown here as position 190)

Article Snippet: Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour.

Techniques: Variant Assay, Biomarker Discovery, Diffusion-based Assay, Control, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

doi: 10.1186/s13024-024-00723-x

Figure Lengend Snippet: Derivation of microglia-like cells from the ALSP-CSF1R patient with a c.2350G > A (p.V784M) CSF1R variant. A Phase contrast images of ALSP-CSF1R iPSC line differentiated into iMGL using the 2.0 or 2.9 protocol. Scale bar = 150 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m . B Viable cell yield per well of a 6-well plate assessed by trypan blue exclusion assay following 2.0 and 2.9 differentiation of the ALSP-CSF1R iPSCs into iMGL. n = 7 differentiation batches. C Viable cell yield per well of a 6-well plate assessed by trypan blue exclusion assay following side-by-side differentiation of healthy control lines ( n = 14 differentiation batches from 6 lines) and the ALSP-CSF1R line ( n = 14 differentiation batches) using the 2.9 protocol. A t-test was performed, * p < 0.05. (D) Quantification of IBA1- and PU.1-immunopositivity. n = 4 healthy control lines and 4 batches of a single ALSP-CSF1R line, differentiated side-by-side using the 2.9 protocol. E qRT-PCR assessment of microglia marker expression on day 0, 14 and 28 of microglial differentiation. n = 4 healthy control lines and 4 batches of a single ALSP-CSF1R line, differentiated side-by-side using the 2.9 protocol. Two-way ANOVA were performed, followed by Sidak’s post hoc tests. *** p < 0.001. F Flow cytometry assessment of P2RY12, CX3CR1 and MerTK cell surface expression. T-tests were performed. n = 4 healthy control lines and 4 batches of a single ALSP-CSF1R line, differentiated side-by-side using the 2.9 protocol. ** p < 0.01. G Flow cytometry assessment of CSF1R cell surface expression. A t-test was performed. n = 4 healthy control lines and 4 batches of a single ALSP-CSF1R line, differentiated side-by-side using the 2.9 protocol. * p < 0.05. H Western blot assessment of CSF1R and its tyrosine 723-phosphorylated form, and GAPDH. A t-test was performed. n = 5 healthy control lines and 5 batches of a single ALSP-CSF1R line, differentiated side-by-side using the 2.9 protocol. ** p < 0.01

Article Snippet: Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour.

Techniques: Variant Assay, Trypan Blue Exclusion Assay, Control, Quantitative RT-PCR, Marker, Expressing, Flow Cytometry, Western Blot

Journal: Molecular Neurodegeneration

Article Title: An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

doi: 10.1186/s13024-024-00723-x

Figure Lengend Snippet: Functional phenotype of iMGL derived from the ALSP-CSF1R patient with a c.2350G > A (p.V784M) CSF1R variant. All iMGL were generated using the 2.9 protocol. Quantification ( A ) and images ( B ) of iMGL migratory activity toward ADP assessed by Boyden chamber assay. Cells were concomitantly treated or not with PSB0739. Kruskal–Wallis tests followed by Dunn’s multiple comparison tests were performed. n = 6 healthy control lines and 6 batches of a single ALSP patient line, differentiated side-by-side. White = Hoechst 33342, scale bar = 75 μm in ( B ). Quantification of green fluorescence intensity per cell ( C ) and representative images ( D ) of iMGL exposed to vehicle or pHrodo.™ Green-labelled myelin, opsonized red blood cells (IgG-RBC) or E. coli for three hours and then counterstained with Hoescht 33342. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05. Scale bar = 250 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( D ). Quantification of mean fluorescence intensities (MFI; E ) and representative images ( F ) of LAMP1, CD68 and IBA1 immunostaining of iMGL. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, ** p < 0.01, *** p < 0.001. Scale bar = 100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m in ( F ). G Cytokine secretion assessed in cell supernatants following a 24-h treatment with vehicle, LPS (100 ng/mL) or Pam 3 CSK 4 (100 ng/mL). A two-way ANOVA was performed, followed by Tukey’s post hoc test. n = 4 healthy control lines and 4 batches of a single ALSP patient line, differentiated side-by-side, *** p < 0.001, ns = non-significant. H qRT-PCR performed after three hours of Pam 3 CSK 4 (100 ng/mL) vs. vehicle treatment. T-tests were performed. n = 3 healthy control lines and 3 batches of a single ALSP patient line, differentiated side-by-side, * p < 0.05

Article Snippet: Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour.

Techniques: Functional Assay, Derivative Assay, Variant Assay, Generated, Activity Assay, Boyden Chamber Assay, Comparison, Control, Fluorescence, Immunostaining, Quantitative RT-PCR

Journal: Molecular Neurodegeneration

Article Title: An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders

doi: 10.1186/s13024-024-00723-x

Figure Lengend Snippet: Effect of CSF1R loss of function on microglial phenotype. A - C CSF1R WT/WT and CSF1R WT/KO iPSCs were differentiated into iMGL side-by-side. A Viable cell yield per well of a 6-well plate assessed by trypan blue exclusion assay following 2.0 and 2.9 differentiation. A Kruskal–Wallis test was performed, followed by Dunn’s post hoc test. n = 4 differentiation batches. B Phase contrast images of iMGL 2.9. Red triangles show presence of round or dysmorphic floating cells. Scale bar = 150 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m . C Flow cytometry assessment of cell surface marker expression. Kruskal–Wallis tests were performed, followed by Dunn’s post hoc test. n = 4 differentiation batches using the 2.9 protocol, * p < 0.05, ** p < 0.01 vs. unstained control. MFI = median fluorescence intensity, a.u. = arbitrary unit. D - G CSF1R WT/REV and CSF1R WT/E633K iPSCs were differentiated into iMGL side-by-side. D Viable cell yield per well of a 6-well plate assessed by trypan blue exclusion assay following 2.0 and 2.9 differentiation. A Kruskal–Wallis test was performed, followed by Dunn’s post hoc test. n = 4 differentiation batches. E Phase contrast images of iMGL 2.9. Scale bar = 150 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu m$$\end{document} μ m . F qRT-PCR assessment of microglia markers. T-tests were performed. n = 5 differentiation batches using the 2.9 protocol, * p < 0.05 vs CSF1R WT/REV iMGL. G Flow cytometry assessment of cell surface marker expression. Mann–Whitney tests were performed. n = 5 differentiation batches using the 2.9 protocol * p < 0.05, ** p < 0.01. MFI = median fluorescence intensity. H - I Healthy control, mature iMGL were generated following the 2.9 protocol. H Microglia marker expression assessed by qRT-PCR in iMGL three days after siCON or siCSF1R transfection. T-tests were performed. n = 3 lines, * p < 0.05 vs siCON. I Microglia marker expression assessed by qRT-PCR in iMGL following a 3-day treatment with PLX3397 (1 μM, every other day). T-tests were performed. n = 4 lines, * p < 0.05 vs vehicle treatment. J - L eGFP and either WT or V784M CSF1R were stably co-expressed in ALSP-CSF1R iHPCs using lentiviruses and cells were differentiated into iMGL following the 2.9 protocol. J Merged phase contrast and green fluorescence images of ALSP-CSF1R iMGL. Scale bar = 150 μm. (K) Viable cell yield per well of a 6-well plate assessed by trypan blue exclusion assay. A t-test was performed. n = 4 differentiation batches. L Flow cytometry assessment of eGFP signal and microglia marker expression. MFI = median fluorescence intensity. n = 1 differentiation batch

Article Snippet: Membranes were immunoblotted for CSF1R (1:250; #MAB3291, R&D Systems) and its phosphorylated form (Y723; 1:500; #3155, Cell Signaling), nuclear factor kappa B (NF-κB; 1:500; #8282, Cell Signaling) and its phosphorylated form (S536; 1:500; #3033, Cell Signaling), caspase-1 (1:500; #ab179515, Abcam), IL-1β (1:500; #12,242, Cell Signaling), NLR family pyrin domain containing 3 (NLRP3; 1:500; #15101S, Cell Signaling) and GAPDH (1:5000; G8795, Sigma Aldrich) overnight at 4 °C, and then with horse radish peroxidase-linked secondary antibodies (1:10,000; Jackson Laboratory) for one hour.

Techniques: Trypan Blue Exclusion Assay, Flow Cytometry, Marker, Expressing, Control, Fluorescence, Quantitative RT-PCR, MANN-WHITNEY, Generated, Transfection, Stable Transfection