Fig. 5

Functional validation of iMG microglial identity. A-F. Phagocytic activity in iMG. A-A”. Microscopic images showing iMG (C1-iPSC) phagocytosing Flour 555-labeled beta-amyloid (A”), which is inhibited by cytochalasin D (A). B. Flow cytometric analysis of Flour 555-labeled beta-amyloid treated iMG derived from C1-iPSC line. Red histogram shows the control sample (untreated), and light blue histogram shows the sample exposed to Flour 555-labeled beta-amyloid. C. Flow cytometry of fluorescent beta-amyloid treated iMG differentiated from three different iPSC lines. Control: cytochalasin D treatment. (D-D”) iMG phagocytosing pHrodo-red labeled myelin with (D) and without cytochalasin D (D”). E-F. iMG engulfing pHrodo-red labeled human synaptosomes, confirmed by microscopy (E) and flow cytometry (F). Red histogram shows the control sample and light blue histogram shows the sample exposed to pHrodo labeled synaptosomes but not cytochalasin D. A-F n = 3. A, A’’, D, D’’, E: CU-iPSC. C: all three iPSC lines. Scale bars: 10 μm in A-A”, D-D” and E. G. iMG response to LPS challenge by releasing cytokines and chemokines. The volcano plot shows the means of three independent experiments of LPS or vehicle-only treatment for 12 h. A two-sided paired t-test for each cytokine/chemokine was applied to assess if LPS treatment had an overall impact on cytokine/chemokine secretion (P < 0.05). H. A23187 and ATP evoke Ca²⁺ transients in iMG, as depicted by live imaging of Fluo-4 labeled iMG following addition of either compound. Arrows highlight Ca²⁺driven signal accumulation in iMG over time. I-J. Image-derived fluorescence intensity signal measurements over time post A23187 (I) or ATP (J) treatment. Recordings began 60 s after baseline measurements and continued for 600 s. Supplementary Figure S5D shows the recording up to 1800 s. Error bars in I and J express the standard error of means of three independent experiments. The full registry of Ca²⁺ dynamics in video format can be found in the supplementary material. K. iMG migrate toward IL-34 cytokine, as demonstrated by fluorometric InnoCyte™ cell migration assay results, represented as relative green, fluorescent intensity (GFI). Neuron: SH-SY5Y neuronal cell line. Error bars represent the standard deviation of three independent experiments (iMG vehicle-only vs. iMG IL-34; Student’s t-test p = 0.0013). Abbreviations: Aß amyloid beta; AF555 AlexaFluor555, a fluorochrome; APC allophycocyanine, a fluorochrome; THP-1 a monocytic cell line; iMG induce pluripotent stem cell derived microglia