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 used expressed the astrocytic markers vimentin, S100β, ALDH1L1 and GFAP, and human microglia expressed the microglial markers Iba1 and P2Y12.
“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]
The Results section on engraftment (Fig. 5; page unavailable) reports that the derived human microglia express Iba1 and P2RY12. The paper does not report astrocytes expressing vimentin, S100β, ALDH1L1 and GFAP, or establish that the astrocytes and microglia in the citing study came from the same hiPSC line. The citing study points to its own supplementary figures for the marker observations. (LLM, conf=0.86)
Experimental — accuracy not yet measuredE3 and E3S/S iPSCs were differentiated into microglia-like cells (iMGLs), confirmed by the expression of microglia markers IBA1 and TMEM119.
“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 <i>APOE3</i> - <i>R136S</i> mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
TREM2 deficiency leads to impaired microglial response to β-amyloid plaques, accumulation of lipid droplets, and increased β-amyloid load in the brain.
“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]
The Results section on IDE1-treated microglia reports TREM2 expression, and the functional-validation Results report uptake of beta-amyloid fibrils by cells made using two differentiation methods (page numbers unavailable). Neither passage addresses TREM2 deficiency, responses to plaques, lipid droplets, or brain amyloid load. Searches for the claimed deficiency and those outcomes found no passage that does. (LLM, conf=0.76)
Experimental — accuracy not yet measuredAD patients exhibit myelin loss, and higher myelin expression is associated with tau pathology protection in AD.
“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]
The Background and Discussion discuss microglia in Alzheimer’s disease, while the Results examine stem-cell-derived microglia. Searches for myelin loss, myelin expression, and tau pathology found no passage addressing either part of the claim; no page numbers are available in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredHigher levels of myelin are associated with protection against tau pathology in AD.
“higher levels of myelin are associated with protection against tau pathology in AD43,44”
From <i>APOE3</i> - <i>R136S</i> mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
The Background and Discussion address microglia and Alzheimer’s disease, while the Results describe microglial differentiation and function. Searches for myelin, tau, and protection against tau pathology found no passage in our copy addressing the claimed association. (LLM, conf=0.77)
Experimental — accuracy not yet measured
AD patients exhibit increased myelin loss.
“AD patients exhibit increased myelin loss”
From <i>APOE3</i> - <i>R136S</i> mutation confers resilience against tau pathology via cGAS-STING-IFN inhibition (2024) · cites this paper as [43]
The Background and Discussion discuss microglia in Alzheimer’s disease, and the Results examine phagocytosis of beta-amyloid by stem-cell-derived microglia. None reports myelin loss in AD patients; searches for myelin loss and demyelination found no supporting passage in our copy. No page numbers were provided. (LLM, conf=0.76)
Experimental — accuracy not yet measuredHighly efficient techniques exist to derive microglia-like cells from human induced pluripotent stem cells (iPSCs).
“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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“iPSCs were passaged every week using ReLeSR dissociation reagent (StemCell Technologies) and used for hematopoietic stem cell differentiation with STEMdiff Hematopoietic kit (STEMCELL Technologies #05310) followed by differentiation to induced microglial-like cells (iMGLs) using a previously published protocol by (McQ…”
Large-Scale Production of Human iPSC-Derived Macrophages for Drug Screening
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- 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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