Journal: Scientific Reports

Article Title: Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSC to microglia

doi: 10.1038/s41598-022-23477-2

Figure Lengend Snippet: A flowchart of the differentiation process from iPSC to Microglia as described by Haenseler et al. ( A ) iPSC are cultured in OxE8 medium for 7 days before differentiation into embryoid bodies in Aggrewell plates for 6 days in embryoid body medium (BMP4/VEGF/SCF). After 6 days embryoid bodies are transferred to T175 flasks and cultured in myeloid differentiation media of IL-3 and M-CSF. At approximately d40 these factories produce microglial precursor cells which are harvested and plated in microglial media and differentiated for 14 days. This final step is the target for optimisation in this manuscript. iPSC induced pluripotent stem cell, EB embryoid body, SCF stem cell factor, BMP4 bone morphogenetic protein 4, VEGF vascular endothelial growth factor, IL-3 Interleukin 3, M-CSF macrophage colony stimulation factor, d day. Created with BioRender.com. ( B ) A flowchart describing the systematic identification of factors required for iPSC-microglial differentiation. After the first triage of 15 media combinations, the morphology analysis removed three conditions due to poor differentiation. Following qPCR analysis four factors could be removed from further medium development. The second set of differentiations focused on IL-34, TGF-β1, M-CSF, and GM-CSF as these were shown to have an effect in the first round of differentiations. Several conditions were removed due to poor microglial identity following scRNA-seq and morphology analysis. In the final differentiation, we identified that our new medium (ITMG) produced iPSC-microglia with a microglial-like transcriptome, improved survival, and performed similarly to our previous medium in functional assays. Created with BioRender.com. ( C ) Conditions and media tested and their reason for exclusion. ( D ) Representative images of d14 microglia cultured in our previous media (IGBN) and the media presented in this manuscript (ITMG). Scale bar 100 µm.

Article Snippet: Rinse solution was then removed and the wells washed with 1 mL PBS before the addition of 1 mL of 2 × concentrated EB medium (1 × EB medium: OxE8 medium supplemented with 50 ng/mL BMP4 (PeproTech no PHC9534), 50 ng/mL VEGF (PeproTech no PHC9394), and 20 ng/mL SCF (Miltenyi Biotec no 130-096-695)) supplemented with 10 μM Y-27632 ROCK inhibitor. iPSC were cultured to 70–80% confluency before washing with 1 mL PBS then incubation in TrypLE Express (Gibco, no 12604013) for 3–5 min at 37 °C, 5% CO 2 .

Techniques: Cell Culture, Produced, Functional Assay