Generation of pure monocultures of human microglia-like cells from induced pluripotent stem cells
Poulomi Banerjee1, Evdokia Paza1, Emma M Perkins1
1Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK; UK Dementia Research Institute at University of Edinburgh, Edinburgh, UK.
Abstract:
Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y12+ 95% TMEM119+ ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease.
Insights
A new protocol generates pure, high-quality human microglia-like cells from induced pluripotent stem cells. These cells mimic primary human microglia, aiding research into central nervous system diseases.
Area of Science:
- Neuroscience
- Stem Cell Biology
- Immunology
Background:
- Microglia are crucial for central nervous system (CNS) development, homeostasis, and disease response.
- Generating pure human microglia in vitro from induced pluripotent stem cells (hiPSCs) is challenging.
- Existing methods for in vitro human microglia generation raise concerns about purity and quality.
Purpose of the Study:
- To develop an improved protocol for generating pure human microglia-like cells (hiPSC-MG) from hiPSCs.
- To characterize the phenotype and function of hiPSC-MG generated by the new protocol.
- To assess the suitability of hiPSC-MG for transplantation studies.
Main Methods:
- hiPSCs were differentiated into microglia-like cells without co-culture with neural cells.
- Whole-transcriptome analysis was performed to identify microglia signature genes.
- hiPSC-MG were stimulated with lipopolysaccharide to assess functional responses.
- Phagocytic activity and in vivo/ex vivo engraftment were evaluated.
Main Results:
- The protocol yielded 100% P2Y12+ and 95% TMEM119+ ramified hiPSC-MG.
- hiPSC-MG expressed key human microglia signature genes (SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB, SIGLEC11).
- Stimulation induced expected functional changes, and cells showed phagocytic activity and engraftment capacity.
Conclusions:
- A novel, efficient protocol for generating high-purity human microglia-like cells from hiPSCs has been established.
- These hiPSC-MG accurately model human microglial characteristics and functions.
- This advancement will significantly aid research into human microglial roles in health and disease.
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