Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-oligodendrocyte Interactions

doi: 10.3791/61778

Figure Lengend Snippet: Table of materials

Article Snippet: Tempo-iOlogo: Human iPSC-derived OPCs , Tempo BioScience , SKU102.

Techniques:

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative red-green channel overlay images of wild type (WT) (top) and TREM2 knockout (KO) (bottom) induced pluripotent stem cell (iPSC)-microglia loaded with Fluo-4 (green) and Fura-red (red) showing resting cytosolic Ca 2+ before ADP, and Ca 2+ levels 15 s and 5 min after ADP addition. Scale bar = 20 μm. ( B ) Average traces (left panels) showing changes in cytosolic Ca 2+ in response to 2.5 μM ADP in 1 mM Ca 2+ buffer (n = 39–44 cells). Baseline-subtracted peak Ca 2+ response and cytosolic Ca 2+ levels 5 min after ADP shown on the right (n = 250–274 cells, five experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP as in ( A ) and (B ) but in iPSC-microglia expressing the GCaMP6f-tdTomato fusion Ca 2+ probe Salsa6f (n = 41–53 cells, two independent experiments, Mann–Whitney test). Images in ( C ) are overlay of GCaMP6f (green) and tdTomato (red) channel images. Scale bar = 20 μm. ( E ) Ca 2+ responses to 2.5 μM ATP in WT and TREM2 KO iPSC-microglia. Average traces (left panel, n = 63–71 cells) and bar graph summary of peak cytosolic Ca 2+ and Ca 2+ after 5 min (right panel, 165–179 cells, three experiments, Mann–Whitney test). ( F ) Ca 2+ responses to 10 μM UTP. Average traces (45–55 cells) and summary of peak cytosolic Ca 2+ and Ca 2+ after 5 min (175–269 cells, three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by *** for p<0.001, ****p<0.0001 . Figure 1—source data 1. Microglia lacking TREM2 show exaggerated Ca 2+ responses to purinergic stimulation. In this dataset, the results of microglial stimulation with purinergic agonists and validation of Salsa6f isogenic microglia are included.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out, MANN-WHITNEY, Expressing

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative bright field, green (GCaMP6f), red (tdTomato), and green/red channel overlay images of transgenic Salsa6f expressing iPSC-microglia at low (top row) and high (bottom row) cytosolic Ca 2+ levels. Cells were treated with 2 μM thapsigargin (TG) to deplete stores and evoke store-operated Ca 2+ entry (SOCE). Images are shown at the end of TG treatment for low Ca 2+ and at the peak of SOCE for high Ca 2+ . Scale bar = 20 μm. ( B ) Trace of average change in fluorescence intensity of tdTomato (red) and GCamp6f (green) over time. Summary of GCaMP6f and tdTomato intensities before and after invoking SOCE is shown on the right. ( C ) Ratiometric GCaMP6f/ tdTomato signal (green/red or G/R ratio) over time calculated from ( B ). Summary of G/R ratio at low and high cytosolic Ca 2+ ( B, C , n = 19 cells, Mann–Whitney test). ( D ) Immunofluorescence images showing staining for the microglia-specific marker IBA1 in either resting or activated wild type (WT) or Salsa6f-transgenic iPSC-microglia (left). Right panel shows quantification of IBA1 protein expression (n = 4 wells, two independent images per well, t -test). Cells were activated with 100 ng/mL lipopolysaccharide (LPS for 24 hr). ( E ) Microglia cell counts at final day of differentiation (n = 3 wells, t -test). ( F ) Phagocytosis of synaptosomes in WT non-transgenic (open circle) and Salsa6f-expressing (closed circle) iPSC-microglia. Cytochalasin D (gray, 10 µM) used as negative control to inhibit phagocytosis. Live cultures imaged on IncuCyte S3 (n = 4 wells; four images per well). ( G ) Phagocytic load at 24 hr for synaptosomes, beta-amyloid, zymosan A, and S. aureus (n = 4 wells; four images per well; one-way ANOVA with Tukey post-hoc test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns for nonsignificant, ****P<0.0001 .

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Transgenic Assay, Expressing, Fluorescence, MANN-WHITNEY, Immunofluorescence, Staining, Marker, Negative Control

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative trace showing changes in cytosolic Ca 2+ in a single cell to illustrate the scheme for measuring cytosolic Ca 2+ level 5 min after agonist application. ( B ) Bar graph summary of cytosolic Ca 2+ levels in wild type (WT) and TREM2 knockout (KO) induced pluripotent stem cell (iPSC)-microglia 5 min after application of 2.5 μM ADP (blue), 2.5 μM ATP (red), and 10 μM UTP (yellow). N = 165–274 cells pooled from 2 to 3 experiments. One-way ANOVA with multiple comparisons. Data shown as mean ± SEM for the bar graph. p-Values indicated by ****p<0.0001 .

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Schematic highlighting key downstream Ca 2+ signaling events triggered by ADP. Cytosolic Ca 2+ response to ADP is determined by functional expression and activity of P2Y 12 and P2Y 13 receptors, IP 3 receptors, endoplasmic reticulum (ER) store Ca 2+ content, and store-operated Ca 2+ entry (SOCE) regulated by STIM and Orai proteins. ( B ) Representative images (left panel) showing overlay of Fluo-4 (green) and Fura-red (red) channels in wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. Average trace showing Ca 2+ response to ADP in Ca 2+ -free buffer (middle panel, 64–83 cells). Quantification of peak signal (right panel, n = 264–289 cells, four experiments, Mann–Whitney test). ( C–E ) Dose–response curves showing baseline-subtracted peak Ca 2+ responses to ADP in Ca 2+ -free buffer ( C ), percent of ‘responding’ cells ( D ), and peak Ca 2+ responses only in ‘responding’ cells ( E ). N = 84–474 WT cells and 70–468 TREM2 KO cells, 2–5 experiments. ( F ) RNA normalized read counts of P2Y 12 and P2Y 13 receptor expression from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4, adjusted p-values from DESeq2). ( G ) Representative histogram (left panel) showing plasma membrane (PM) expression of P2Y 12 receptor in WT and TREM2 KO microglia. Cells were stained with BV421-labeled anti-human P2Y 12 receptor antibody. Isotype control is shown as dashed line. Right panel shows summary of median fluorescence intensity (MFI) of P2Y 12 receptor-labeled cells (n = 10 samples each, Student’s t -test). ( H ) Ca 2+ traces (left panel) showing response to 1 μM ADP in Ca 2+ -free buffer after 30 min pretreatment with a combination of P2Y 12 receptor antagonist PSB 0739 (10 μM) and P2Y 13 receptor antagonist MRS 2211 (10 μM). Summary of the peak Ca 2+ response (right panel, n = 40–79 cells, two experiments, Mann–Whitney test). Data are mean ± SEM. p-Values indicated by ****p<0.0001. Figure 2—source data 1. Higher sensitivity of TREM2 knockout (KO) microglia to ADP is driven by increased purinergic receptor expression. In this dataset, the results of ADP stimulation in 0 Ca 2+ , dose curve of ADP in wild type (WT) and TREM2 KO, P2Y receptor expression, expression of key calcium signaling proteins, and inhibition of P2Y receptors are included.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Functional Assay, Expressing, Activity Assay, MANN-WHITNEY, RNA Sequencing Assay, Staining, Labeling, Fluorescence, Knock-Out, Inhibition

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative green (GCaMP6f) and red (tdTomato) channel overlay images of wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. ( B ) Average trace (left panel) showing Ca 2+ response to 100 nM ADP in Ca 2+ -free buffer. Quantification of peak signal (right panel, n = 46–75 cells, two experiments, Mann–Whitney test). ( C ) Comparison of peak cytosolic Ca 2+ in response to ADP (2.5 μM ADP) in 1 mM Ca 2+ or Ca 2+ -free buffer (n = 38–96 cells , ordinary one-way ANOVA with multiple comparisons). ( D ) Volcano plot of differentially expressed genes from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4). Genes for IP3R, STIM1, and ORAI1 are highlighted. ( E ) RNA normalized read counts for IP 3 receptor type 2 (ITPR2), PMCA1 (ATP2B1), SERCA2 (ATP2A2), SERCA3 (ATP2A3), STIM1, and ORAI1 in WT and TREM2 KO iPSC-microglia. Isoforms expressed lower than 10 reads in any sample are not considered expressed and are not shown. Relative expression of P2Y 12 and P2Y 13 receptors is shown for comparison of the relative fold change between WT and TREM2 KO cells. ( F, G ) Peak Ca 2+ response in Ca 2+ -free buffer after treatment with 1 or 10 μM ADP in the presence of P2Y 12 receptor antagonist PSB 0739 ( F ) or P2Y 13 receptor antagonist MRS 2211 ( G ), respectively. Cells were pretreated with 10 μM of PSB 0739 or 10 μM MRS 2211 for 30 min before imaging. (72–128 cells, F ; 83–117 cells, G ; representative of three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p<0.01, ***p<0.001, ****p<0.0001 .

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: MANN-WHITNEY, RNA Sequencing Assay, Expressing, Imaging

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Average trace showing SOCE triggered in TREM2 knockout (KO) microglia via emptying endoplasmic reticulum (ER) Ca 2+ stores with thapsigargin (TG, 2 μM) in Ca 2+ -free buffer followed by readdition of 1 mM Ca 2+ in the absence (control, green trace) or presence (red trace) of the Orai channel inhibitor Synta66. Cells were pretreated with Synta66 (10 μM) for 30 min before experiment. Bar graph summary of the rate of Ca 2+ influx after readdition of 1 mM Ca 2+ (80–126 cells, Mann–Whitney test). ( B ) SOCE evoked by ADP (2.5 μM) in TREM2 KO microglia (green trace) using a similar Ca 2+ addback protocol. Red trace shows the effect of Synta66 on ADP-evoked SOCE. Right panel summarizes the rate of ADP-triggered Ca 2+ influx after readdition of 1 mM Ca 2+ (n = 125–154 cells, two experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP in TREM2 KO iPSC-microglia pretreated with 2-APB (50 μM) or Gd 3+ (5 μM) to block SOCE. Average traces ( C ), baseline-subtracted initial peak Ca 2+ responses to ADP ( D , left panel), and baseline-subtracted Ca 2+ after 5 min of ADP addition ( D , right panel) are shown (n = 41–74 cells, ordinary one-way ANOVA with multiple comparisons). ( E, F ) Role of Orai1 in TG- and ADP-evoked SOCE in iPSC-microglia. ( E ) Comparison of TG-evoked SOCE in WT and Orai1 KO cell showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–54 cells, 3–4 experiments, Mann–Whitney test). ( F ) ADP-evoked SOCE in WT and Orai1 KO showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–53 cells, 3–4 experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, ****p<0.0001 .

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out, MANN-WHITNEY, Blocking Assay

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Thapsigargin (TG) pulse experiment to measure residual ER Ca 2+ pool in cells after initial treatment with ADP (1 μM) and subsequent treatment with TG (2 μM). Imaging was done in Ca 2+ -free buffer to prevent Ca 2+ influx across the plasma membrane (PM). Average trace (left panel), peak ADP Ca 2+ response (middle panel), and extent of TG-induced ER store release measured as area under the curve (AUC, right panel) (n = 81–108 cells, Mann–Whitney test). ( B ) Control experiment comparing the ER-Ca 2+ pool in WT and TREM2 KO microglia after store depletion with TG and without any pretreatment with ADP (n = 29–63 cells, Mann–Whitney test). ( C, D ) Relationship between ADP-induced store release and store-operated Ca 2+ entry (SOCE) in induced pluripotent stem cell (iPSC)-microglia. ( C ) Representative single-cell trace of Ca 2+ signal in response to ADP in 1 mM extracellular Ca 2+ buffer showing the scheme for measuring ER store release as the initial Ca 2+ peak and SOCE as cytosolic Ca 2+ level 5 min after ADP application. ( D ) Scatter plot showing correlation of initial ADP-induced Ca 2+ response (store release) and cytoplasmic Ca 2+ after 5 min (SOCE) in WT (gray) and KO (green) cells (n = 866–935 cells from multiple imaging runs with a range of ADP doses; in μM: 0.001, 0.1, 0.5, 1, 2, 2.5, 5, 10; comparison of slopes between WT and TREM2 KO: p=0.7631; extra sum of squares F -test). ( E, F ) Comparison of cytosolic Ca 2+ clearance indicative of PMCA pump activity in WT and TREM2 KO microglia. SOCE was invoked and rate of Ca 2+ decline was measured after addition of 0 mM Ca 2+ . ( E ) Average trace showing invoking SOCE with 2 μM TG (left panel). Right panel shows the drop in cytosolic Ca 2+ following addition of Ca 2+ -free solution as highlighted (pink) in the SOCE trace. ( F ) Summary of rate of Ca 2+ decline after addition of 0 mM Ca 2+ (n = 8 imaging fields, 142–175 total cells, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p<0.01 .

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Imaging, MANN-WHITNEY, Activity Assay

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Average trace showing closure of scratch wound produced with IncuCyte S3 WoundMaker. Induced pluripotent stem cell (iPSC)-microglia imaged every 30 min after scratch wound with or without ADP stimulation (n = 4 wells; two images per well). ( B ) Representative image of WT iPSC-microglia motility 30 min after ADP exposure with cell tracks overlain (left). Pseudocolored images (center) across time: 0 min (red), 4 min (orange), 8 min (yellow), 12 min (green), 16 min (cyan), 20 min (blue), 24 min (purple), and 28 min (magenta). Scale bar = 100 μm. White boxes zoomed in at right to demonstrate motile (top) and nonmotile (bottom) cells. ( C ) Representative color images (top left) and displacement vectors (bottom left) of WT iPSC-microglia at baseline (no ADP, gray) and 30 min after 2.5 μM ADP treatment (red). Summary of mean speed (µm/min), Displacement over 10 min (μm/10 min) and track straightness (track length/track displacement) (414–602 cells, two experiments). ( D ) Representative images, displacement vectors, and quantification of WT iPSC-microglia motility for 20 min following ADP addition. Cells were pretreated with vehicle (gray), MRS 2211 (10 μM, gold), or PBS 0739 (10 μM, blue) (180–187 cells, two experiments). ( E ) Representative images, displacement vectors, and quantification of WT iPSC-microglia motility after ADP in 1 mM Ca 2+ (light gray) or Ca 2+ -free buffer (dark gray) (401–602 cells, three experiments). ( F ) Representative images (left) and process extension (right) of iPSC-microglia (cytoplasmic GFP, gray) before or 30 min after ADP addition. Cells were pretreated with vehicle (gray), MRS 2211 (10 μM, gold), or PBS 0739 (10 μM, blue) (52–163 cells, 3–4 experiments). ( C–F ) One-way ANOVA with Tukey post hoc test. Data shown as mean ± SEM ( A, F ) and as violin plots with mean, 25th and 75th percentile ( C–E ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Figure 4—source data 1. Nondirectional ADP exposure increases wild type (WT) microglial speed and process extension. In this dataset, the results of motility experiments and process extension in WT cells are included.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Produced

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative images of a cell (Cell 1) from a time-lapse experiment showing increased branching and extension of processes in GFP-expressing WT iPSC-microglia, at times indicated following addition of 2.5 μM ADP. Bright-field DIC images (top row) and GFP images (bottom row) are shown. ( B ) Another example of a cell (Cell 2) showing process extension in the same imaging field. ( C ) A motile cell (Cell 3) in the same imaging field is shown for comparison. Note the lack of displacement in cells that extend their process and lack of significant process extension in a highly motile cell. Scale bar: 15 μM.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Expressing, Imaging

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A–E ) Motility of wild type (WT) (gray) and TREM2 KO (green) iPSC-microglia over 20 min following ADP addition in 1 mM Ca 2+ -containing buffer. ( A ) Plots of track displacement in μm centered from point of origin at (0,0). ( B ) Mean squared displacement (MSD) vs. time. Mean cell track speeds ( C ), total track displacement in 10 min interval ( D ), and track straightness ( E ) for 130–327 cells, seven experiments, Student’s t -test. ( F–J ) Same as ( A–F ) but in Ca 2+ -free medium (125–279 cells, two experiments, Student’s t -test). ( K ) Representative images of GFP-expressing WT (top) and TREM2 KO (bottom) iPSC-microglia, before and 30 min after 2.5 μM ADP addition. ( L ) Quantification of total number of branches per cell before and after ADP treatment (left) and paired dot plots showing fold change in branch number from pre-ADP levels (right). Each data point represents an imaging field in the paired plots. ( M ) Total process length before and after ADP treatment displayed as raw values per cell (left) and as fold change from baseline conditions per imaging field (right). For ( L ) and ( M ). n = 151–158 cells, WT; 133–167 cells, KO; 9–10 imaging fields, 3–4 experiments. One-way ANOVA with multiple comparisons for single-cell data, two-tailed paired t -test for the paired plots. Data shown as mean ± SEM ( B, G, L, M ) and as violin plots with mean, 25th and 75th percentile ( C– E, H–J ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, and ****p<0.0001. Figure 5—source data 1. ADP-driven process extension and cell displacement are increased in TREM2 knockout (KO) induced pluripotent stem cell (iPSC)-microglia. In this dataset, the results of motility experiments and process extension in wild type (WT) and TREM2 KO cells, as well as baseline motility and directional persistence, are included.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Expressing, Imaging, Two Tailed Test, Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Summary of microglial mean speeds, displacement over 10 min, and track straightness in open-field migration in the absence of any purinergic stimulation (Student’s t -test). ( B ) Flower plots show similar displacement from origin for WT (left) and TREM2 KO (right) cells. ( C ) Directional autocorrelation calculated via DiPer Excel Macro. Due to lack of directional gradient, directional autocorrelation of motility vectors is expected to drop quickly. Time constants for best-fit single-exponential curves are indicated, consistent with increased straightness for TREM2 KO cells treated with ADP. ( D ) Directional autocorrelation of WT (gray) and TREM2 KO (green) iPSC-microglia at baseline (open circles) or after ADP addition (filled circles). Mean autocorrelation values in the first 5 min (left panel, one-way ANOVA) and time (min) until autocorrelation reaches zero (right panel). Data shown as mean ± SEM for the bar graph in ( D ), and as violin plots with mean, 25th and 75th percentile in ( A ). p-Values indicated by ns, nonsignificant, *p<0.05, and ****p<0.0001.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Migration

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: Branching and process extension in WT and TREM2 KO induced pluripotent stem cell (iPSC)-microglia 30 min after addition of ADP in 1 mM ( A, B ) or 0 mM extracellular Ca 2+ buffer ( C, D ). ( A ) Data displayed as paired plots showing average branch number per cell in an imaging field (top row) and normalized to pre-ADP values for each imaging field (middle row). Bottom row shows fold change in branching after ADP treatment for WT (gray) and KO (green) iPSC-microglia. ( B ) Changes in process length in the same dataset as ( A ). n = 151–158 cells, WT; 133–167 cells, KO; 9–10 imaging fields, 3–4 experiments. ( C, D ) Same analysis as ( A, B ) but with ADP in Ca 2+ -free buffer. n = 137–143 cells, eight imaging fields, 2–3 experiments. ( A–D ) p-Values calculated by two-tailed paired Student’s t -test for the paired plots and unpaired t -test when comparing fold change in WT and KO cells. Data shown as paired plots and as mean ± SEM for the bar graphs. p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Imaging, Two Tailed Test

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Schematic of traditional store-operated Ca 2+ entry (SOCE) pathway with store refilling (left) and protocol for sustaining cytoplasmic Ca 2+ to ‘low’ and ‘high’ levels with 0.2 and 2 mM extracellular Ca 2+ and using thapsigargin (TG) to inhibit store refilling (right). ( B ) Average SOCE traces in wild type (WT) Salsa6f induced pluripotent stem cell (iPSC)-microglia showing changes in cytoplasmic Ca 2+ after addition of either 0.2 or 2 mM extracellular Ca 2+ (n = 78–110 cells). ( C ) Average change in cytoplasmic Ca 2+ levels in WT and TREM2 KO microglia over 25 min after SOCE activation. ( D ) Comparison of Ca 2+ levels and microglia motility in WT (top) and TREM2 KO (bottom) microglia. Cytosolic Ca 2+ levels indicated by instantaneous single-cell G/R ratio (n = 74–158 cells). Mean of instantaneous speeds, track displacement, and track straightness calculated as before in and . Yellow (0.2 mM Ca, TG), green (2 mM Ca, TG). Student’s t -test ****p<0.0001; **p=0.0062; *p=0.432; ns > 0.9999. ( E ) Correlation of instantaneous Ca 2+ and instantaneous speed in WT and KO cells. Red line denotes 10 μm/s (cells above this threshold considered ‘fast moving’). For WT: p<0.0001; r = –0.1316; number pairs = 5850. For KO: p<0.0001; r = –0.1433; number pairs = 6,063 (Spearman’s correlation). ( F ) Mean speed of cells binned by instantaneous G/R Ca 2+ ratio (one-way ANOVA ****p<0.0001). Each data point is calculated for a bin increment of 0.5 G/R ratio. ( G ) Percentage of fast-moving cells quantified as a function of G/R Ca 2+ ratio. X-axis G/R ratios binned in increments of 0.5 as in ( F ). In ( E–G ), n = 78–100 cells. Data shown as mean ± SEM ( B, F ) and as violin plots with mean, 25th and 75th percentile ( D ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, and ****p<0.0001. Figure 6—source data 1. Cytosolic Ca 2+ levels tune microglial motility in TREM2 knockout (KO) cells. In this dataset, the results showing the effect of calcium levels on motility in TREM2 wild type (WT) and KO cells are included.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Activation Assay, Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: Scratch closure over 24 hr in WT (gray) and TREM2 KO (green) induced pluripotent stem cell (iPSC)-microglia with (filled symbols) or without (empty symbols) pre-stimulation of iPSC-microglia with ADP (10 μM, 30 min). N = 2 wells, two images per well. Data shown as mean ± SEM.

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques:

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet:

Article Snippet: Cell line (human) , WT GFP-expressing iPSC-microglia , Coriell , AICS-0036;RRID : CVCL_JM19 , iPSC-line with GFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: CRISPR, Knock-Out, Expressing, Construct, Transfection, Plasmid Preparation, Cell Culture, Stem Cell Culture, Recombinant, Selection, Blocking Assay, Clone Assay, Incubation, Microscopy, Chemotaxis Assay, Software, Cell Tracking Assay

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative red-green channel overlay images of wild type (WT) (top) and TREM2 knockout (KO) (bottom) induced pluripotent stem cell (iPSC)-microglia loaded with Fluo-4 (green) and Fura-red (red) showing resting cytosolic Ca 2+ before ADP, and Ca 2+ levels 15 s and 5 min after ADP addition. Scale bar = 20 μm. ( B ) Average traces (left panels) showing changes in cytosolic Ca 2+ in response to 2.5 μM ADP in 1 mM Ca 2+ buffer (n = 39–44 cells). Baseline-subtracted peak Ca 2+ response and cytosolic Ca 2+ levels 5 min after ADP shown on the right (n = 250–274 cells, five experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP as in ( A ) and (B ) but in iPSC-microglia expressing the GCaMP6f-tdTomato fusion Ca 2+ probe Salsa6f (n = 41–53 cells, two independent experiments, Mann–Whitney test). Images in ( C ) are overlay of GCaMP6f (green) and tdTomato (red) channel images. Scale bar = 20 μm. ( E ) Ca 2+ responses to 2.5 μM ATP in WT and TREM2 KO iPSC-microglia. Average traces (left panel, n = 63–71 cells) and bar graph summary of peak cytosolic Ca 2+ and Ca 2+ after 5 min (right panel, 165–179 cells, three experiments, Mann–Whitney test). ( F ) Ca 2+ responses to 10 μM UTP. Average traces (45–55 cells) and summary of peak cytosolic Ca 2+ and Ca 2+ after 5 min (175–269 cells, three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by *** for p<0.001, ****p<0.0001 . Figure 1—source data 1. Microglia lacking TREM2 show exaggerated Ca 2+ responses to purinergic stimulation. In this dataset, the results of microglial stimulation with purinergic agonists and validation of Salsa6f isogenic microglia are included.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out, MANN-WHITNEY, Expressing

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative bright field, green (GCaMP6f), red (tdTomato), and green/red channel overlay images of transgenic Salsa6f expressing iPSC-microglia at low (top row) and high (bottom row) cytosolic Ca 2+ levels. Cells were treated with 2 μM thapsigargin (TG) to deplete stores and evoke store-operated Ca 2+ entry (SOCE). Images are shown at the end of TG treatment for low Ca 2+ and at the peak of SOCE for high Ca 2+ . Scale bar = 20 μm. ( B ) Trace of average change in fluorescence intensity of tdTomato (red) and GCamp6f (green) over time. Summary of GCaMP6f and tdTomato intensities before and after invoking SOCE is shown on the right. ( C ) Ratiometric GCaMP6f/ tdTomato signal (green/red or G/R ratio) over time calculated from ( B ). Summary of G/R ratio at low and high cytosolic Ca 2+ ( B, C , n = 19 cells, Mann–Whitney test). ( D ) Immunofluorescence images showing staining for the microglia-specific marker IBA1 in either resting or activated wild type (WT) or Salsa6f-transgenic iPSC-microglia (left). Right panel shows quantification of IBA1 protein expression (n = 4 wells, two independent images per well, t -test). Cells were activated with 100 ng/mL lipopolysaccharide (LPS for 24 hr). ( E ) Microglia cell counts at final day of differentiation (n = 3 wells, t -test). ( F ) Phagocytosis of synaptosomes in WT non-transgenic (open circle) and Salsa6f-expressing (closed circle) iPSC-microglia. Cytochalasin D (gray, 10 µM) used as negative control to inhibit phagocytosis. Live cultures imaged on IncuCyte S3 (n = 4 wells; four images per well). ( G ) Phagocytic load at 24 hr for synaptosomes, beta-amyloid, zymosan A, and S. aureus (n = 4 wells; four images per well; one-way ANOVA with Tukey post-hoc test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns for nonsignificant, ****P<0.0001 .

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Transgenic Assay, Expressing, Fluorescence, MANN-WHITNEY, Immunofluorescence, Staining, Marker, Negative Control

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative trace showing changes in cytosolic Ca 2+ in a single cell to illustrate the scheme for measuring cytosolic Ca 2+ level 5 min after agonist application. ( B ) Bar graph summary of cytosolic Ca 2+ levels in wild type (WT) and TREM2 knockout (KO) induced pluripotent stem cell (iPSC)-microglia 5 min after application of 2.5 μM ADP (blue), 2.5 μM ATP (red), and 10 μM UTP (yellow). N = 165–274 cells pooled from 2 to 3 experiments. One-way ANOVA with multiple comparisons. Data shown as mean ± SEM for the bar graph. p-Values indicated by ****p<0.0001 .

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Schematic highlighting key downstream Ca 2+ signaling events triggered by ADP. Cytosolic Ca 2+ response to ADP is determined by functional expression and activity of P2Y 12 and P2Y 13 receptors, IP 3 receptors, endoplasmic reticulum (ER) store Ca 2+ content, and store-operated Ca 2+ entry (SOCE) regulated by STIM and Orai proteins. ( B ) Representative images (left panel) showing overlay of Fluo-4 (green) and Fura-red (red) channels in wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. Average trace showing Ca 2+ response to ADP in Ca 2+ -free buffer (middle panel, 64–83 cells). Quantification of peak signal (right panel, n = 264–289 cells, four experiments, Mann–Whitney test). ( C–E ) Dose–response curves showing baseline-subtracted peak Ca 2+ responses to ADP in Ca 2+ -free buffer ( C ), percent of ‘responding’ cells ( D ), and peak Ca 2+ responses only in ‘responding’ cells ( E ). N = 84–474 WT cells and 70–468 TREM2 KO cells, 2–5 experiments. ( F ) RNA normalized read counts of P2Y 12 and P2Y 13 receptor expression from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4, adjusted p-values from DESeq2). ( G ) Representative histogram (left panel) showing plasma membrane (PM) expression of P2Y 12 receptor in WT and TREM2 KO microglia. Cells were stained with BV421-labeled anti-human P2Y 12 receptor antibody. Isotype control is shown as dashed line. Right panel shows summary of median fluorescence intensity (MFI) of P2Y 12 receptor-labeled cells (n = 10 samples each, Student’s t -test). ( H ) Ca 2+ traces (left panel) showing response to 1 μM ADP in Ca 2+ -free buffer after 30 min pretreatment with a combination of P2Y 12 receptor antagonist PSB 0739 (10 μM) and P2Y 13 receptor antagonist MRS 2211 (10 μM). Summary of the peak Ca 2+ response (right panel, n = 40–79 cells, two experiments, Mann–Whitney test). Data are mean ± SEM. p-Values indicated by ****p<0.0001. Figure 2—source data 1. Higher sensitivity of TREM2 knockout (KO) microglia to ADP is driven by increased purinergic receptor expression. In this dataset, the results of ADP stimulation in 0 Ca 2+ , dose curve of ADP in wild type (WT) and TREM2 KO, P2Y receptor expression, expression of key calcium signaling proteins, and inhibition of P2Y receptors are included.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Functional Assay, Expressing, Activity Assay, MANN-WHITNEY, RNA Sequencing Assay, Staining, Labeling, Fluorescence, Knock-Out, Inhibition

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative green (GCaMP6f) and red (tdTomato) channel overlay images of wild type (WT) (top) and TREM2 KO (bottom) induced pluripotent stem cell (iPSC)-microglia before and peak Ca 2+ response after ADP addition in Ca 2+ -free buffer. Scale bar = 20 μm. ( B ) Average trace (left panel) showing Ca 2+ response to 100 nM ADP in Ca 2+ -free buffer. Quantification of peak signal (right panel, n = 46–75 cells, two experiments, Mann–Whitney test). ( C ) Comparison of peak cytosolic Ca 2+ in response to ADP (2.5 μM ADP) in 1 mM Ca 2+ or Ca 2+ -free buffer (n = 38–96 cells , ordinary one-way ANOVA with multiple comparisons). ( D ) Volcano plot of differentially expressed genes from bulk RNA-sequencing of WT and TREM2 KO iPSC-microglia (n = 4). Genes for IP3R, STIM1, and ORAI1 are highlighted. ( E ) RNA normalized read counts for IP 3 receptor type 2 (ITPR2), PMCA1 (ATP2B1), SERCA2 (ATP2A2), SERCA3 (ATP2A3), STIM1, and ORAI1 in WT and TREM2 KO iPSC-microglia. Isoforms expressed lower than 10 reads in any sample are not considered expressed and are not shown. Relative expression of P2Y 12 and P2Y 13 receptors is shown for comparison of the relative fold change between WT and TREM2 KO cells. ( F, G ) Peak Ca 2+ response in Ca 2+ -free buffer after treatment with 1 or 10 μM ADP in the presence of P2Y 12 receptor antagonist PSB 0739 ( F ) or P2Y 13 receptor antagonist MRS 2211 ( G ), respectively. Cells were pretreated with 10 μM of PSB 0739 or 10 μM MRS 2211 for 30 min before imaging. (72–128 cells, F ; 83–117 cells, G ; representative of three experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p<0.01, ***p<0.001, ****p<0.0001 .

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: MANN-WHITNEY, RNA Sequencing Assay, Expressing, Imaging

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Average trace showing SOCE triggered in TREM2 knockout (KO) microglia via emptying endoplasmic reticulum (ER) Ca 2+ stores with thapsigargin (TG, 2 μM) in Ca 2+ -free buffer followed by readdition of 1 mM Ca 2+ in the absence (control, green trace) or presence (red trace) of the Orai channel inhibitor Synta66. Cells were pretreated with Synta66 (10 μM) for 30 min before experiment. Bar graph summary of the rate of Ca 2+ influx after readdition of 1 mM Ca 2+ (80–126 cells, Mann–Whitney test). ( B ) SOCE evoked by ADP (2.5 μM) in TREM2 KO microglia (green trace) using a similar Ca 2+ addback protocol. Red trace shows the effect of Synta66 on ADP-evoked SOCE. Right panel summarizes the rate of ADP-triggered Ca 2+ influx after readdition of 1 mM Ca 2+ (n = 125–154 cells, two experiments, Mann–Whitney test). ( C, D ) Cytosolic Ca 2+ response to ADP in TREM2 KO iPSC-microglia pretreated with 2-APB (50 μM) or Gd 3+ (5 μM) to block SOCE. Average traces ( C ), baseline-subtracted initial peak Ca 2+ responses to ADP ( D , left panel), and baseline-subtracted Ca 2+ after 5 min of ADP addition ( D , right panel) are shown (n = 41–74 cells, ordinary one-way ANOVA with multiple comparisons). ( E, F ) Role of Orai1 in TG- and ADP-evoked SOCE in iPSC-microglia. ( E ) Comparison of TG-evoked SOCE in WT and Orai1 KO cell showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–54 cells, 3–4 experiments, Mann–Whitney test). ( F ) ADP-evoked SOCE in WT and Orai1 KO showing average traces (left panel) and summary of SOCE rate (right panel; n = 42–53 cells, 3–4 experiments, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, ****p<0.0001 .

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Knock-Out, MANN-WHITNEY, Blocking Assay

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Thapsigargin (TG) pulse experiment to measure residual ER Ca 2+ pool in cells after initial treatment with ADP (1 μM) and subsequent treatment with TG (2 μM). Imaging was done in Ca 2+ -free buffer to prevent Ca 2+ influx across the plasma membrane (PM). Average trace (left panel), peak ADP Ca 2+ response (middle panel), and extent of TG-induced ER store release measured as area under the curve (AUC, right panel) (n = 81–108 cells, Mann–Whitney test). ( B ) Control experiment comparing the ER-Ca 2+ pool in WT and TREM2 KO microglia after store depletion with TG and without any pretreatment with ADP (n = 29–63 cells, Mann–Whitney test). ( C, D ) Relationship between ADP-induced store release and store-operated Ca 2+ entry (SOCE) in induced pluripotent stem cell (iPSC)-microglia. ( C ) Representative single-cell trace of Ca 2+ signal in response to ADP in 1 mM extracellular Ca 2+ buffer showing the scheme for measuring ER store release as the initial Ca 2+ peak and SOCE as cytosolic Ca 2+ level 5 min after ADP application. ( D ) Scatter plot showing correlation of initial ADP-induced Ca 2+ response (store release) and cytoplasmic Ca 2+ after 5 min (SOCE) in WT (gray) and KO (green) cells (n = 866–935 cells from multiple imaging runs with a range of ADP doses; in μM: 0.001, 0.1, 0.5, 1, 2, 2.5, 5, 10; comparison of slopes between WT and TREM2 KO: p=0.7631; extra sum of squares F -test). ( E, F ) Comparison of cytosolic Ca 2+ clearance indicative of PMCA pump activity in WT and TREM2 KO microglia. SOCE was invoked and rate of Ca 2+ decline was measured after addition of 0 mM Ca 2+ . ( E ) Average trace showing invoking SOCE with 2 μM TG (left panel). Right panel shows the drop in cytosolic Ca 2+ following addition of Ca 2+ -free solution as highlighted (pink) in the SOCE trace. ( F ) Summary of rate of Ca 2+ decline after addition of 0 mM Ca 2+ (n = 8 imaging fields, 142–175 total cells, Mann–Whitney test). Data shown as mean ± SEM for traces and bar graphs. p-Values indicated by ns, nonsignificant, **p<0.01 .

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Imaging, MANN-WHITNEY, Activity Assay

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Average trace showing closure of scratch wound produced with IncuCyte S3 WoundMaker. Induced pluripotent stem cell (iPSC)-microglia imaged every 30 min after scratch wound with or without ADP stimulation (n = 4 wells; two images per well). ( B ) Representative image of WT iPSC-microglia motility 30 min after ADP exposure with cell tracks overlain (left). Pseudocolored images (center) across time: 0 min (red), 4 min (orange), 8 min (yellow), 12 min (green), 16 min (cyan), 20 min (blue), 24 min (purple), and 28 min (magenta). Scale bar = 100 μm. White boxes zoomed in at right to demonstrate motile (top) and nonmotile (bottom) cells. ( C ) Representative color images (top left) and displacement vectors (bottom left) of WT iPSC-microglia at baseline (no ADP, gray) and 30 min after 2.5 μM ADP treatment (red). Summary of mean speed (µm/min), Displacement over 10 min (μm/10 min) and track straightness (track length/track displacement) (414–602 cells, two experiments). ( D ) Representative images, displacement vectors, and quantification of WT iPSC-microglia motility for 20 min following ADP addition. Cells were pretreated with vehicle (gray), MRS 2211 (10 μM, gold), or PBS 0739 (10 μM, blue) (180–187 cells, two experiments). ( E ) Representative images, displacement vectors, and quantification of WT iPSC-microglia motility after ADP in 1 mM Ca 2+ (light gray) or Ca 2+ -free buffer (dark gray) (401–602 cells, three experiments). ( F ) Representative images (left) and process extension (right) of iPSC-microglia (cytoplasmic GFP, gray) before or 30 min after ADP addition. Cells were pretreated with vehicle (gray), MRS 2211 (10 μM, gold), or PBS 0739 (10 μM, blue) (52–163 cells, 3–4 experiments). ( C–F ) One-way ANOVA with Tukey post hoc test. Data shown as mean ± SEM ( A, F ) and as violin plots with mean, 25th and 75th percentile ( C–E ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Figure 4—source data 1. Nondirectional ADP exposure increases wild type (WT) microglial speed and process extension. In this dataset, the results of motility experiments and process extension in WT cells are included.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Produced

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Representative images of a cell (Cell 1) from a time-lapse experiment showing increased branching and extension of processes in GFP-expressing WT iPSC-microglia, at times indicated following addition of 2.5 μM ADP. Bright-field DIC images (top row) and GFP images (bottom row) are shown. ( B ) Another example of a cell (Cell 2) showing process extension in the same imaging field. ( C ) A motile cell (Cell 3) in the same imaging field is shown for comparison. Note the lack of displacement in cells that extend their process and lack of significant process extension in a highly motile cell. Scale bar: 15 μM.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Expressing, Imaging

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A–E ) Motility of wild type (WT) (gray) and TREM2 KO (green) iPSC-microglia over 20 min following ADP addition in 1 mM Ca 2+ -containing buffer. ( A ) Plots of track displacement in μm centered from point of origin at (0,0). ( B ) Mean squared displacement (MSD) vs. time. Mean cell track speeds ( C ), total track displacement in 10 min interval ( D ), and track straightness ( E ) for 130–327 cells, seven experiments, Student’s t -test. ( F–J ) Same as ( A–F ) but in Ca 2+ -free medium (125–279 cells, two experiments, Student’s t -test). ( K ) Representative images of GFP-expressing WT (top) and TREM2 KO (bottom) iPSC-microglia, before and 30 min after 2.5 μM ADP addition. ( L ) Quantification of total number of branches per cell before and after ADP treatment (left) and paired dot plots showing fold change in branch number from pre-ADP levels (right). Each data point represents an imaging field in the paired plots. ( M ) Total process length before and after ADP treatment displayed as raw values per cell (left) and as fold change from baseline conditions per imaging field (right). For ( L ) and ( M ). n = 151–158 cells, WT; 133–167 cells, KO; 9–10 imaging fields, 3–4 experiments. One-way ANOVA with multiple comparisons for single-cell data, two-tailed paired t -test for the paired plots. Data shown as mean ± SEM ( B, G, L, M ) and as violin plots with mean, 25th and 75th percentile ( C– E, H–J ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, and ****p<0.0001. Figure 5—source data 1. ADP-driven process extension and cell displacement are increased in TREM2 knockout (KO) induced pluripotent stem cell (iPSC)-microglia. In this dataset, the results of motility experiments and process extension in wild type (WT) and TREM2 KO cells, as well as baseline motility and directional persistence, are included.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Expressing, Imaging, Two Tailed Test, Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Summary of microglial mean speeds, displacement over 10 min, and track straightness in open-field migration in the absence of any purinergic stimulation (Student’s t -test). ( B ) Flower plots show similar displacement from origin for WT (left) and TREM2 KO (right) cells. ( C ) Directional autocorrelation calculated via DiPer Excel Macro. Due to lack of directional gradient, directional autocorrelation of motility vectors is expected to drop quickly. Time constants for best-fit single-exponential curves are indicated, consistent with increased straightness for TREM2 KO cells treated with ADP. ( D ) Directional autocorrelation of WT (gray) and TREM2 KO (green) iPSC-microglia at baseline (open circles) or after ADP addition (filled circles). Mean autocorrelation values in the first 5 min (left panel, one-way ANOVA) and time (min) until autocorrelation reaches zero (right panel). Data shown as mean ± SEM for the bar graph in ( D ), and as violin plots with mean, 25th and 75th percentile in ( A ). p-Values indicated by ns, nonsignificant, *p<0.05, and ****p<0.0001.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Migration

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: Branching and process extension in WT and TREM2 KO induced pluripotent stem cell (iPSC)-microglia 30 min after addition of ADP in 1 mM ( A, B ) or 0 mM extracellular Ca 2+ buffer ( C, D ). ( A ) Data displayed as paired plots showing average branch number per cell in an imaging field (top row) and normalized to pre-ADP values for each imaging field (middle row). Bottom row shows fold change in branching after ADP treatment for WT (gray) and KO (green) iPSC-microglia. ( B ) Changes in process length in the same dataset as ( A ). n = 151–158 cells, WT; 133–167 cells, KO; 9–10 imaging fields, 3–4 experiments. ( C, D ) Same analysis as ( A, B ) but with ADP in Ca 2+ -free buffer. n = 137–143 cells, eight imaging fields, 2–3 experiments. ( A–D ) p-Values calculated by two-tailed paired Student’s t -test for the paired plots and unpaired t -test when comparing fold change in WT and KO cells. Data shown as paired plots and as mean ± SEM for the bar graphs. p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Imaging, Two Tailed Test

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: ( A ) Schematic of traditional store-operated Ca 2+ entry (SOCE) pathway with store refilling (left) and protocol for sustaining cytoplasmic Ca 2+ to ‘low’ and ‘high’ levels with 0.2 and 2 mM extracellular Ca 2+ and using thapsigargin (TG) to inhibit store refilling (right). ( B ) Average SOCE traces in wild type (WT) Salsa6f induced pluripotent stem cell (iPSC)-microglia showing changes in cytoplasmic Ca 2+ after addition of either 0.2 or 2 mM extracellular Ca 2+ (n = 78–110 cells). ( C ) Average change in cytoplasmic Ca 2+ levels in WT and TREM2 KO microglia over 25 min after SOCE activation. ( D ) Comparison of Ca 2+ levels and microglia motility in WT (top) and TREM2 KO (bottom) microglia. Cytosolic Ca 2+ levels indicated by instantaneous single-cell G/R ratio (n = 74–158 cells). Mean of instantaneous speeds, track displacement, and track straightness calculated as before in and . Yellow (0.2 mM Ca, TG), green (2 mM Ca, TG). Student’s t -test ****p<0.0001; **p=0.0062; *p=0.432; ns > 0.9999. ( E ) Correlation of instantaneous Ca 2+ and instantaneous speed in WT and KO cells. Red line denotes 10 μm/s (cells above this threshold considered ‘fast moving’). For WT: p<0.0001; r = –0.1316; number pairs = 5850. For KO: p<0.0001; r = –0.1433; number pairs = 6,063 (Spearman’s correlation). ( F ) Mean speed of cells binned by instantaneous G/R Ca 2+ ratio (one-way ANOVA ****p<0.0001). Each data point is calculated for a bin increment of 0.5 G/R ratio. ( G ) Percentage of fast-moving cells quantified as a function of G/R Ca 2+ ratio. X-axis G/R ratios binned in increments of 0.5 as in ( F ). In ( E–G ), n = 78–100 cells. Data shown as mean ± SEM ( B, F ) and as violin plots with mean, 25th and 75th percentile ( D ). p-Values indicated by ns, nonsignificant, *p<0.05, **p<0.01, and ****p<0.0001. Figure 6—source data 1. Cytosolic Ca 2+ levels tune microglial motility in TREM2 knockout (KO) cells. In this dataset, the results showing the effect of calcium levels on motility in TREM2 wild type (WT) and KO cells are included.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: Activation Assay, Knock-Out

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet: Scratch closure over 24 hr in WT (gray) and TREM2 KO (green) induced pluripotent stem cell (iPSC)-microglia with (filled symbols) or without (empty symbols) pre-stimulation of iPSC-microglia with ADP (10 μM, 30 min). N = 2 wells, two images per well. Data shown as mean ± SEM.

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques:

Journal: eLife

Article Title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

doi: 10.7554/eLife.73021

Figure Lengend Snippet:

Article Snippet: Cell line (human) , WT RFP-expressing iPSC-microglia , Coriell , AICS-0031-035;RRID : CVCL_LK44 , iPSC-line with RFP tagged to αtubulinOriginally developed by Dr. Bruce Conklin.

Techniques: CRISPR, Knock-Out, Expressing, Construct, Transfection, Plasmid Preparation, Cell Culture, Stem Cell Culture, Recombinant, Selection, Blocking Assay, Clone Assay, Incubation, Microscopy, Chemotaxis Assay, Software, Cell Tracking Assay

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia incorporate into sliced cerebral organoids (A) Representative confocal images of a day 144 organoid with neurons (MAP2 + ), astrocytes (GFAP + ), and microglia (IBA1 + ). Scale bars, 250 μm. (B) Representative confocal images of day 144 organoids 14 days after the addition of HIV-1 JRFL-infected or mock-infected microglia; (B1) shows a high-magnification image of HIV-1-infected microglia from (B). Scale bars, 250 μm or 20 μm as indicated. (C) High-magnification image of a single HIV-1-infected microglial cell within a sliced organoid. Scale bars, 20 μm. (D) RT-qPCR of gag/pol mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) ELISA analysis of secreted p24 in culture supernatant from HIV-1 JRFL-infected or mock-infected microglia organoid day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗ p < 0.01; data are presented as the mean ± SEM. (F) RT-qPCR analysis of ISG15 , RSAD2 , IFITM3 , MX1 , IFI44 , and IFI27 mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗ p < 0.05; data are presented as the mean ± SEM.

Article Snippet: Recent advances in stem cell technologies have opened up new possibilities for modeling and studying HAND, particularly through the development of human iPSC-derived neurons, astrocytes, and microglia capable of mimicking complex in vivo neuroimmune interactions when grown in three dimensional cerebral organoid cultures., , Cerebral organoids offer a powerful and physiologically relevant platform to study complex neuroimmune interactions, but traditional organoid differentiation protocols do not produce microglia.

Techniques: Infection, Quantitative RT-PCR, One-tailed Test, Enzyme-linked Immunosorbent Assay