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Development and validation of a simplified method to generate human microglia from pluripotent stem cells
Open accessAbstract
BACKGROUND: Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS: hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFβ-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION: We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.
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“Human astrocytes, expressing the astrocytic markers vimentin, S100β, ALDH1L1 and GFAP (Supplementary Figure 3 A), and human microglia, expressing the microglial markers Iba1 and P2Y12 (Supplementary Figure 3 B) were derived from the same hiPSC line [27].”
From Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates (2021) · cites this paper as [27]
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“E3 and E3S/S iPSCs were differentiated into microglia-like cells (iMGLs)43 (Fig. 6A), confirmed by the expression of microglia markers, IBA1 and TMEM119 (Fig. 6B).”
From APOE3 - R136S mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
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“HiPSCs were differentiated to hiPSC‐derived microglial cells (iMGL) according to the protocol previously published by McQuade et al., with minor adjustments.22,23”
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“TREM2 deficiency leads to impaired microglial response to β-amyloid plaques, accumulation of the lipid droplets, and increased β-amyloid load in the brain [11, 41].”
From Protective PLCG2 variants associate with a delayed onset of Alzheimer’s disease among heterozygous APOE ε4 carriers (2026) · cites this paper as [41]
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“AD patients exhibit myelin loss, and higher myelin expression is associated with tau pathology protection in AD43,44.”
From The R136S mutation in the APOE3 gene confers resilience against tau pathology via inhibition of the cGAS-STING-IFN pathway (2025) · cites this paper as [43]
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“higher levels of myelin are associated with protection against tau pathology in AD43,44”
From APOE3 - R136S mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
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“AD patients exhibit increased myelin loss”
From APOE3 - R136S mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
Experimental — accuracy not yet measuredIn the citing paper
“This has resulted in highly efficient techniques to derive microglia-like cells from human induced pluripotent stem cells (iPSCs) [278, 279].”
From Genetic architecture of Alzheimer's disease (2020) · cites this paper as [278]
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Additional file 4 of An integrated toolkit for human microglia functional genomics
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Additional file 1 of An integrated toolkit for human microglia functional genomics
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Heuer, S. E., Ahsan, F. M., Terzioğlu, G., Misra, A., Duong, D., Katkar, C., Giera, M. A., van der Kant, R., Seyfried, N. T., & Young-Pearse, T. L. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.10.16.759681
Harej, A., Rački, V., Belančić, A., Križ, J., Vitezić, D., Mršić‐Pelčić, J., Acharya, G., Varshney, M., & Pilipović, K. (2026) · Stem Cells International · doi:10.1155/sci/1130043
Endogenous versus exogeneous expression of Tau in iPSC-derived immune microglia
Ananthanarayana, V., Narayanappa, T. S., & Chinnathambi, S. (2026) · Advances in pharmacology · doi:10.1016/bs.apha.2026.08.007
Metadata
- Authors
- McQuade, A., Coburn, M., Tu, C. H., Hasselmann, J., Davtyan, H., & Blurton-Jones, M.
- Affiliations
- University of California, Irvine (US)
- Corresponding author
- Blurton-Jones, Mathew
- ORCID
- Venue
- Molecular Neurodegeneration
- Published
- 2018-12
- Volume
- 13
- Issue
- 1
- Pages
- 67-67
- ISSN
- 1750-1326 (ISSN-L)
- PMC
- PMC6303871
- OpenAlex
- W2905656859
- Cited by
- 449
- References
- 33
- Citations by year
- 2026: 64 · 2025: 98 · 2024: 73 · 2023: 53 · 2022: 54 · 2021: 43 · 2020: 40 · 2019: 23
- FWCI
- 11.92
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Neurodegeneration (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- National Institutes of Health (AG048099, AG056303, AG016573)
- California Institute for Regenerative Medicine (RT3-07893)
- National Institute of Neurological Disorders and Stroke (NS082174)
- Topics
- Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Keywords
- microglia differentiation, induced pluripotent stem cells, iPSC-derived microglia, hematopoietic progenitor cells, macrophage colony-stimulating factor, IL-34, TGF-β1, TGF-beta signaling, microglial transcriptome, neurodegeneration
- MeSH terms
- Animals, Brain, Cell Differentiation, Humans, Cytokines, Microglia, Pluripotent Stem Cells, Induced Pluripotent Stem Cells
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