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Figure 1. Human iPSC-Derived Microglia Exhibit High-Quality Morphology

Cryopreserved Human iPSC-Derived Microglia generated from SCTi003-A iPSCs were thawed and plated onto Matrigel?-coated plates at 1.1 x 10^4 cells/cm? in STEMdiff? Microglia Maturation Kit. Microglia were incubated at 37¡ãC and subsequently imaged on Days 2, 10 (before replating), and 20 (after replating) by brightfield microscopy at 10X magnification. Microglia display a compact morphology with limited ramification due to reduced adherence, as expected in a healthy homeostatic microglia monoculture. iPSC = induced pluripotent stem cell.

Figure 2. Human iPSC-Derived Microglia Exhibit High Purity and Express Characteristic Glial Markers

Cryopreserved Human iPSC-Derived Microglia generated from SCTi003-A iPSCs were thawed and cultured using STEMdiff? Microglia Maturation Kit. Microglia co-expressed CD45 and CD11b and expressed P2RY12 and TREM2 markers, as measured by flow cytometry on Days 0 and 10 post-thaw. The bars show the mean ¡À standard deviation. Technical replicates (n = 1 - 2) were averaged, and each dot in the graph represents an experimental replicate with different manufacturing lots separated by color. iPSC = induced pluripotent stem cell.

Figure 3. Human iPSC-Derived Microglia Display Cytokine Release in Response to Inflammatory Stimuli

Cryopreserved Human iPSC-Derived Microglia generated from SCTi003-A iPSCs were thawed and plated at 100,000 cells per well in a 24-well plate using STEMdiff? Microglia Maturation Kit for 1 day. Microglia were treated with water (control) or stimulated with 100 ng/mL LPS for 24 hours. (A) Brightfield microscopy shows the expected microglia morphology, including small cell bodies and ramified processes, under vehicle control conditions. After LPS treatment, activated microglial morphology with larger cell bodies, hypertrophic ramification, ameboid-like or rod-like shapes is observed. (B) The release of pro-inflammatory cytokines (TNF¦Á and IL-6) was measured by Duoset ELISA with 20X dilution of the LPS-treated supernatants and the undiluted control supernatants. Microglia release cytokines in response to LPS treatment, as expected. Technical replicates (n = 2) were averaged, and each dot in the graph represents an experimental replicate with different manufacturing lots separated by color.*** p ¡Ü 0.001 and **** p ¡Ü 0.0001. LPS = lipopolysaccharide; iPSC = induced pluripotent stem cell.

Figure 4. Human iPSC-Derived Microglia Are Capable of Phagocytosis

Cryopreserved Human iPSC-Derived Microglia generated from SCTi003-A iPSCs were thawed and cultured using STEMdiff? Microglia Maturation Kit for 1 day. Phagocytosis was assessed by adding pH-sensitive bioindicator particles (25 ?g/mL), and particle uptake was tracked over 24 hours using live-cell imaging. As the particles are phagocytosed, the particles turn red and are concentrated within the cells. Over time, the microglia display an activated ameboid morphology. iPSC = induced pluripotent stem cell.

Figure 5. Human iPSC-Derived Microglia Can Be Tri-Cultured with Neurons and Astrocytes to Model Cell-Cell Interactions In Vitro

Representative immunocytochemistry images display Human iPSC-Derived Microglia tri-cultured with Human iPSC-Derived Astrocytes and Human iPSC-Derived Forebrain Neurons for 7 or 20 days using BrainPhys? hPSC Neuron Kit supplemented with STEMdiff? Microglia Supplement 2 (Component #100-0023 of STEMdiff? Microglia Differentiation Kit). Cells were stained for MAP2 (cyan, neuronal dendrite marker), IBA1 (pink, microglia marker), GFAP (green, astrocyte marker), and DAPI (grey, nuclei). The presence of MAP2-positive neurons, GFAP-positive astrocytes, and IBA1-positive microglia confirms the successful establishment of tri-culture conditions. iPSC = induced pluripotent stem cell.