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Figure 1. Schematic for the STEMdiff™ Microglia Culture System Protocol

Microglial precursors can be generated in 24 days from hPSC-derived hematopoietic progenitor cells. For the generation of hematopoietic progenitor cells, see documentation for STEMdiff™ Hematopoietic Kit (Catalog #05310). For the maturation of microglial precursors to functional microglia, see the PIS.

Figure 2. Microglia Generated Using the STEMdiff™ Microglia Culture System Exhibit Robust Expansion, Mature Phenotypic Markers, and Homeostatic Morphology

(A) Microglia generated using the STEMdiff™ Microglia Culture System undergo a four-fold expansion, on average, across four cell lines. The fold expansion was calculated by taking the total cell count at Day 24 and dividing it by the number of seeded cells at Day 0. The bars show the mean ± standard deviation. Technical replicates were averaged, n = 1 - 4 technical replicates, 1 - 9 experimental setups.

(B) Microglia generated with STEMdiff™ Microglia Culture System have CD45+ CD11b+ co-expression and P2RY12+ expression as measured by flow cytometry on Day 24. The bars show the mean ± standard deviation.Technical replicates (n = 1 - 4) were averaged, and each dot in the graph represents an experimental replicate.

(C) Normal microglial morphology, characterized by small cell bodies and ramified processes, is observed in cells generated using the STEMdiff™ Microglia Culture System. Images at Days 12 and 24 were captured prior to replate and harvest. Scale bar = 100 µm.

Figure 3. Microglia Generated with STEMdiff™ Microglia Culture System Express Disease-Relevant Genes Similar to Those from Published Differentiation and Maturation Protocols

Bulk RNA-seq datasets were extracted from 8 different publications that generated hPSC- (iMGL) and primary- (MGL) derived microglia and their transcriptional profiles compared to data from microglia generated with STEMdiff™ Microglia Culture System. The heat map displays absolute expression levels for select genes associated with Alzheimer’s disease, Parkinson’s disease, and viral encephalitis. Significant differences in gene expression between microglia generated with STEMdiff™ Microglia Culture System and any of the other 3 groups were identified by differential gene expression analysis. *= p<0.05 (DEseq2, adjusted). hPSC = human pluripotent stem cell.

Figure 4. Microglia Generated with STEMdiff™ Microglia Culture System Release Cytokines in Response to Inflammatory Signals

Microglia were generated using the STEMdiff™ Microglia Culture System and stimulated with 100 ng/mL LPS for 24 hours. The release of pro-inflammatory (TNFα, IL-6, IFN-γ, IL-1β, GM-CSF, IL-12p70, IL-2, IL-8) and anti-inflammatory (IL-10) cytokines were measured by MSD. The microglia release cytokines in response to LPS treatment, as expected. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. LPS = lipopolysaccharide; MSD = Meso Scale Discovery.

Figure 5. STEMdiff™ Microglia Culture System Generates Functional Microglia Capable of Phagocytosis at Day 34

Microglia taking up pH-sensitive bioindicator particles at a concentration of 5 μg/mL were measured over a 72-hour time period with 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. Scale bar = 400 μm.

Figure 6. PSC-Derived Microglia Incorporate into Brain Organoids After 10 Days and Display an Activated Morphology upon Injury.

(A) Representative microglia and brain organoid co-cultures after 10 days, stained with IBA1 for microglia (green) and MAP2 for neurons (magenta). The microglia integrate among the neurons and display an unactivated morphology with extended processes (arrow).

(B) The microglia display an activated amoeboid morphology upon injury as shown by IBA1 staining.