Journal: The Journal of Experimental Medicine

Article Title: Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

doi: 10.1084/jem.20220857

Figure Lengend Snippet: Human G795A iPSC-derived microglia exhibit robust viability and minimal alterations to gene expression in response to CSF1R inhibitors. (A) Crystal structure of hCSF1R-PLX3397 and hCSF1R-PLX5622 highlighting G795 residue in the kinase domain. (B) Molecular modeling of G795 variants G795A, G795C, and G795V demonstrating increasing R-group sizes. (C and D) Caspase 3/7 levels imaged over 24 h in iMG cultured in complete medium treated with 0.1% DMSO, 250 nM, 500 nM, or 1 µM PLX3397 (C) and equivalent concentrations of PLX5622 (D). Data captured from four images/well from n = 6 independent wells. Data represented as mean values ± SEM. (E and F) Quantification of cell death by fluorescent Caspase 3/7 Dye for Apoptosis after 24 h in culture with PLX3397 (E) and PLX5622 (F). Data captured from four images/well from n = 6 independent wells. Data represented as mean values ± SEM. P value, one-way ANOVA (P < 0.0001). Tukey’s HSD; ****P < 0.0001. (G–J) Bulk RNA-seq analysis of WT-, G795A-, and G795C-iMG. (G) Principal component analysis using the top 2,000 most variably expressed genes between WT-, G795A-, and G795C-iMG cultured for 24 h with 0.1% DMSO or 250 nM PLX5622 ( n = 4 replicates per line, per condition) revealing that the primary source of variation is the WT response to PLX5622. (H) Linear regression analysis and the coefficient of determination between DMSO-treated G795A or G795C and WT microglia, confirming a high degree of concordance when comparing either the full transcriptome (G795A, R 2 = 0.96–0.99; G795C, R 2 = 0.97–0.98) or a 249-gene microglia signature (G795A, R 2 = 0.97–0.99; G795C, R 2 = 0.97–0.98). (I–K) Volcano plots comparing 24-h treatment with 250 nM PLX5622, revealing significant transcriptomic alterations (Log 2 (FC) ≥ 1; FDR ≤ 0.05) in WT microglia (I), but no significant changes in G795A microglia compared with DMSO-treated cells (J) nor G795C microglia compared with DMSO-treated cells (K).

Article Snippet: PLX3397 (HY-16749), PLX5622 (HY-114153), Edicotinib (JNJ-40346527; JNJ-527; HY-109086), and BLZ945 (Sotuletinib; HY-12768) were obtained from MedChemExpress or Selleckchem and used in vitro at the doses indicated in the text.

Techniques: Derivative Assay, Gene Expression, Residue, Cell Culture, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

doi: 10.1084/jem.20220857

Figure Lengend Snippet: Human G795A and G795C iPSC-derived microglia are resistant to multiple CSF1Ri. (A) Chromatograms of CRISPR-modified isogenic set of CSF1R-G795A, G795C, and G795V human iPSC lines. (B and C) Quantification of iMG cell death by fluorescent Caspase 3/7 Dye for Apoptosis over 24 h in culture with complete medium treated with 0.1% DMSO, 250 nM, 500 nM, or 1 µM Edicotinib (B) and equivalent concentrations of BLZ945 (C). (D) Live (Calcein-AM) versus dead (ethidium homodimer-1) percent quantification of WT and G795A microglia cultured with 0.1% DMSO or 500 nM of PLX3397, PLX5622, Edicotinib, or BLZ945. Data captured from four images/well from n = 6 independent wells using an Incucyte S3 live-cell imaging system. Data represented as percent mean values. (E–G) Percent confluency of iMG normalized to t = 0 h over 24 h in culture with complete medium treated with 0.1% DMSO or 500 nM PLX3397 (E), PLX5622 (F), Edicontinib (G), or BLZ945 (H). Data captured from four images/well from n = 6 independent wells using an Incucyte S3 live-cell imaging system. Data represented as mean values ± SEM. (I and J) Hierarchical clustering and Pearson correlation of normalized RNA counts of the whole transcriptome (I) and a 249-gene microglia signature (J) between WT (black), G795A (green), and G795C (blue) iMG cultured with 0.1% DMSO or 250 nM PLX5622 ( n = 4 replicates per cell line, per condition). Lightest shade of blue in I and J indicates correlations of R = 0.93; darkest shade of blue indicates R = 1.0 correlation.

Article Snippet: PLX3397 (HY-16749), PLX5622 (HY-114153), Edicotinib (JNJ-40346527; JNJ-527; HY-109086), and BLZ945 (Sotuletinib; HY-12768) were obtained from MedChemExpress or Selleckchem and used in vitro at the doses indicated in the text.

Techniques: Derivative Assay, CRISPR, Modification, Cell Culture, Live Cell Imaging

Journal: Stem Cell Research & Therapy

Article Title: Subretinal microglia support donor photoreceptor survival in rd1 mice

doi: 10.1186/s13287-024-04052-0

Figure Lengend Snippet: Microglia depletion and repopulation. A . In rd1 mice with microglia depletion, only dead cell debris was found in the subretinal space. B . After microglia repopulation, surviving graft could be found, and microglial cells were close to donor cells (Separate channels were inserted). C . In wild-type mice, macrophages could still be detected following PLX5622 treatment. Some donor cells survived, while others were engulfed by macrophages. D . After microglia/macrophage repopulation, all donor cells were engulfed

Article Snippet: For depletion of microglia, wild-type ( n = 3)and rd1 mice ( n = 3) were administered dietary PLX5622 (an inhibitor of colony-stimulating factor-1 receptor, 1200 mg/kg feed, M18090601, Moldiets, Biopike, Inc., China), starting from one week before the transplantation to the end of the experiment.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Subretinal microglia support donor photoreceptor survival in rd1 mice

doi: 10.1186/s13287-024-04052-0

Figure Lengend Snippet: RNA-seq analysis of RPE and subretinal cells. A . Comparison between rd1 mice fed with and without PLX5622. The latter showed elevated microglia-related marker genes; B . Cluster analysis of upregulated genes between rd1 mice with and without PLX5622 treatment; C . Cluster analysis of upregulated genes between rd1 and age-matched wild-type mice; D . Comparison of genes in the axonogenesis cluster (GO:0007409) between rd1 and wild-type mice; E . Cluster analysis of upregulated genes between PLX5622-treated rd1 and wild-type mice; F . Comparison of the survival rate of photoreceptor cells in wild-type conditional medium and rd1 conditional medium

Article Snippet: For depletion of microglia, wild-type ( n = 3)and rd1 mice ( n = 3) were administered dietary PLX5622 (an inhibitor of colony-stimulating factor-1 receptor, 1200 mg/kg feed, M18090601, Moldiets, Biopike, Inc., China), starting from one week before the transplantation to the end of the experiment.

Techniques: RNA Sequencing, Comparison, Marker

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation

doi: 10.1177/0271678X241236008

Figure Lengend Snippet: PLX5622 treatment reduced CNS resident and circulating monocyte-derived macrophages in mouse bAVMs. (a) & (d). Representative images of bAVMs collected from mice that received PLX5622 starting 1 week (a) or 8 weeks (d) after model induction. CNS resident macrophages (CNS) are Cx3cr1 + (green) and peripheral monocyte-derived macrophages (Peripheral) are CCR2 + (red). Merged images show some cells expressing both Cx3cr1 + and CCR2 + (yellow). Nuclei were counterstained with DAPI (blue). Scale bar= 50 μm. (b) and (c). Quantifications of CNS resident macrophages (b) and peripheral monocyte-derived macrophages (c) in bAVMs of mice that received PLX5622 starting 1 week after model induction. (e) and (f). Quantifications of CNS resident macrophages (e) and peripheral monocyte-derived macrophages (f) in bAVMs of mice that received PLX5622 8 weeks following model induction. AVM + V: AVM mice treated with vehicle; AVM + P: AVM mice treated with PLX5622; Ang + P: mice with brain angiogenesis and PLX5622 treatment; WT + P: WT mice treated with PLX5622 treatment. N = 5–7.

Article Snippet: PLX5622 (180 mg/kg of body weight/day, Plexxikon Biotech Company, South San Francisco, CA) was incorporated into chow and orally administered for 7 days starting at 1 week or 8 weeks after model induction (Supplementary Fig. 1).

Techniques: Derivative Assay, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation

doi: 10.1177/0271678X241236008

Figure Lengend Snippet: PLX5622 treatment reduced dysplastic vessels in mouse bAVMs. (a) and (d) Representative images of brain sections collected from lectin-perfused mice. Vessels are labeled by Cy5 fluorescence labeled lectin (violet). The pictures under 1 W and 8 W AVM + V and AVM + P are enlarged pictures of the squared areas in the pictures above them showing dysplasia vessels. White arrows indicate dysplastic vessels. Scale bars = 50 μm. (b) and (c) Quantifications of vessel densities (b) and dysplasia index (number of vessels with lumen diameter >15 mm/mm 2 ) (c) for mice treated with PLX5622 starting at 1 week after model induction. (e) and (f) Quantifications of vessel densities (e) and dysplasia index (f) for mice treated with PLX5622 starting at 8 week after model induction. AVM + V: AVM mice treated with vehicle; AVM + P: AVM mice treated with PLX5622; Ang + P: mice with brain angiogenesis and PLX5622 treatment; WT + P: WT mice treated with PLX5622 treatment. 1 W and 8 W: treatment started at 1 and 8 weeks after model induction. N = 6 or 7.

Article Snippet: PLX5622 (180 mg/kg of body weight/day, Plexxikon Biotech Company, South San Francisco, CA) was incorporated into chow and orally administered for 7 days starting at 1 week or 8 weeks after model induction (Supplementary Fig. 1).

Techniques: Labeling, Fluorescence

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation

doi: 10.1177/0271678X241236008

Figure Lengend Snippet: PLX5622 treatment increased claudin 5 expressions in bAVM vessels. (a) Representative images of sections stained with anti-claudin 5 (green) and CD31 (endothelial cells, red) antibodies. Scale bar = 50 μm and (b) Quantification of the ratio of claudin-5 and CD31. AVM + V: AVM mice treated with vehicle; AVM + P: AVM mice treated with PLX5622. N = 6.

Article Snippet: PLX5622 (180 mg/kg of body weight/day, Plexxikon Biotech Company, South San Francisco, CA) was incorporated into chow and orally administered for 7 days starting at 1 week or 8 weeks after model induction (Supplementary Fig. 1).

Techniques: Staining

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation

doi: 10.1177/0271678X241236008

Figure Lengend Snippet: PLX5622 treatment reduced the incidence of severe hemorrhage in bAVMs. (a) Representative images of Prussian blue stained sections. Iron depositions (blue) in bAVMs indicates hemorrhage. Nuclei were counterstained with Fast Red. Scale bar = 1mm. (b) and (c) Frequencies and degrees of hemorrhage in bAVMs treated with PLX5622 starting at 1 week (b) and 8 weeks (c) after bAVM model induction. 1 W and 8 W: treatment started at 1 and 8 weeks after model induction. AVM + V: AVM mice treated with vehicle; AVM + P: AVM mice treated with PLX5622; Ang + P: mice with brain angiogenesis and PLX5622 treatment; WT + P: WT mice treated with PLX5622 treatment. N = 5–6.

Article Snippet: PLX5622 (180 mg/kg of body weight/day, Plexxikon Biotech Company, South San Francisco, CA) was incorporated into chow and orally administered for 7 days starting at 1 week or 8 weeks after model induction (Supplementary Fig. 1).

Techniques: Staining