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

Abstract

There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery.

An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

“…MEMα containing physiological concentration of glucose was used as the base of the 2.9 microglia differentiation medium, instead of DMEM/F12 in the 2.0 medium. The growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) at low concentration has been previously shown to be beneficial in increasing iMGL cell yield (26) and was therefore incorporated as a mitogenic factor, in addition to IL-34, M-CSF and TGF-ý1. While C-X3-C motif chemokine ligand 1 (CX3CL1) and cluster of differentiation 200 (CD200) had been used in the 2.0 protocol as modulators of microglia function, CD200 was omitted in the 2.9 protocol as the expression of its receptor is almost absent in human microglia (15, 27).…”

“…While C-X3-C motif chemokine ligand 1 (CX3CL1) and cluster of differentiation 200 (CD200) had been used in the 2.0 protocol as modulators of microglia function, CD200 was omitted in the 2.9 protocol as the expression of its receptor is almost absent in human microglia (15, 27). Finally, monothioglycerol and N2 supplements were omitted, as they have been shown to provide no benefit in microglia identity acquisition (26).…”

“…While C-X3-C motif chemokine ligand 1 (CX3CL1) and cluster of differentiation 200 (CD200) had been used in the 2.0 protocol as modulators of microglia function, CD200 was omitted in the 2.9 protocol as the expression of its receptor is almost absent in human microglia (15, 27). Finally, monothioglycerol and N2 supplements were omitted, as they have been shown to provide no benefit in microglia identity acquisition (26). From the same starting number of iHPCs, the new 2.9 protocol resulted on average in ~3 times higher number of viable iMGL compared to the 2.0 protocol (Figure 1B), with some variability in yield improvement observed between different iPSC lines (between 2 to 8fold increase; Additional file 1: Figure S2A).…”

Proximity proteomics reveals UCH-L1 as an NLRP3 interactor that modulates IL-1β production in human macrophages and microglia

“…After 4-5 weeks of differentiation, microglial precursors shed into the media are harvested weekly by collection of supernatant and pelleting at 400 x g for 5 minutes. Microglial precursors are then resuspended in the final microglia maturation media ITMG (ADMEM/F12 (LifeTech, 12634-010), 1x GlutaMAX (Lifetech, 25050-061), 100 ng/mL IL-34 (PeproTech, 200-34-500), 50 ng/mL TGFβ-1 (PeproTech, 100-21C-250), 25 ng/mL M-CSF (Gibco, PHC0033), 10 ng/mL GM-CSF (Gibco, PHC2013), as previously described (Washer et al, 2022), and differentiated to hiPSC-microglia for 14 days, with 50% media changes every 3 to 4 days. All differentiation steps are carried out at 37°C, 5% CO2.…”

Innovations advancing our understanding of microglia in Alzheimer's disease: From in vitro to in vivo models

“…Primary human microglia transplanted into 3D organoids showed an increase in homoeostatic signature by scRNA‐seq, suggesting that organoids are able to provide an environment for microglia that is more representative of the brain than can be achieved in 2D cultures (Popova et al, 2021). The hPSC‐derived microglia (hPSC‐MG) are subjected to different culture conditions, such as different media conditions, presence of serum and growth factors, which will affect microglial identity and function, transcriptomic profile and phagocytic ability (Washer et al, 2022; as reviewed by Hedegaard et al, 2020).…”