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