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Cited by 593Neuroinflammation and Neurodegeneration MechanismsPluripotent Stem Cells ResearchNeurogenesis and neuroplasticity mechanisms

A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response

Open access
Haenseler, Walther, Sansom, Stephen N., Buchrieser, Julian, Newey, Sarah E., Moore, Craig S., Nicholls, Francesca J., Chintawar, Satyan, Schnell, Christian +6 more·2017·Stem Cell Reports
DOI 10.1016/j.stemcr.2017.05.017PMC PMC5470330
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Abstract

Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper establishes a highly efficient human iPSC-derived microglia–neuron co-culture model that recapitulates embryonic microglial ontogeny and yields cells with a fetal microglia-like transcriptome, dynamic ramified morphology, phagocytic capacity, and inflammatory responses. Co-culture microglia downregulate pathogen-response pathways and show a more anti-inflammatory/pro-remodeling cytokine profile than monocultures.

Key findings

  • iPSC-derived macrophage precursors were generated at a cumulative yield 10- to 43-fold higher than input iPSCs, and co-culture with iPSC cortical neurons produced ramified microglia-like cells that persisted for at least 42 days while neurons maintained spontaneous electrical activity.
  • Co-culture microglia expressed key microglia markers and neurodegenerative disease-relevant genes, clustered with human cortex myeloid cells and fetal microglia, and downregulated type I interferon, TLR1/TLR2, and antigen-presentation pathways while upregulating differentiation, chemotaxis/migration, cell-cell adhesion, and metal ion response pathways.
  • Co-culture microglia were motile, moving an accumulated distance of 251 ± 21 μm over 5 hr versus 119 ± 6 μm for pMGL and 53 ± 11 μm for pMac; upon LPS stimulation they clustered and adopted ameboid morphology, with blinded activated-morphology scores increasing from 4.1 ± 0.6 at 0 hr to 7.6 ± 1.1 at 10 hr and 8.1 ± 0.8 at 20 hr.
  • In cytokine assays, co-culture microglia showed dampened chemokine/cytokine secretion versus pMGL but significantly higher activated levels of SerpinE1, VEGF-A, and anti-inflammatory IL-10, indicating co-culture-specific anti-inflammatory and pro-remodeling responses.

Methods

  • iPSCs from four donors were differentiated into embryonic-like, MYB-independent macrophage precursors using defined-size embryoid bodies with BMP4, VEGF, and SCF, followed by M-CSF and IL-3; precursors were harvested weekly from supernatant.
  • Precursors were co-cultured with iPSC-derived cortical neurons in Advanced DMEM/F12 + N2 with 100 ng/mL IL-34 and optional 10 ng/mL GM-CSF; assays were mostly performed after 2 weeks of co-culture.
  • Characterization included Illumina HT12v4 transcriptome analysis against fetal microglia, pMGL, pMac, pMacpre, and blood monocytes; qRT-PCR; flow cytometry; live imaging; phagocytosis assays; and cytokine profiling by Proteome Profiler (102 cytokines) and Luminex multiplex array (22 cytokines).

Limitations

  • The paper does not state a formal limitations section; evident limitations from the text include that co-pMG isolated with CD11b beads showed a weak neural cell signature, and TMEM119/P2RY12 antibody staining in co-culture had background staining.
  • HLA-DR was undetectable in all PSC-derived myeloid lineages, reflecting unprimed culture conditions, and qRT-PCR comparison with adult human microglia used n = 1 cultured and n = 1 directly isolated/processed sample with technical triplicates.
  • Most assays were conducted after 2 weeks of co-culture because neuronal progenitors continued to proliferate and increase culture density, while co-pMG proliferation was very low.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.stemcr.2017.05.017

Cites

47 of 47 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

12 of 51 cited sources checked · 8 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    All the cell types in the current study cluster with human cortex myeloid cells and not with cortical oligodendrocytes, astrocytes, neurons, or endothelial cells.

    “Comparison with a previously published expression dataset for cells derived from human brain tissue (Zhang et al., 2016) showed that all the cell types in the current study cluster with human cortex myeloid cells and not with cortical oligodendrocytes, astrocytes, neurons, or endothelial cells (Figure 2B).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Zhang et al., 2016 source
  • Findingown data

    Genes previously identified to be associated specifically with microglia but not with blood monocytes showed similar expression in co-pMG and fetalMG.

    “As a set, genes previously identified to be associated specifically with microglia but not with blood monocytes (Bennett et al., 2016, Butovsky et al., 2014, Melief et al., 2012) (Figure 2C) showed similar expression in co-pMG and fetalMG.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Bennett et al., 2016 source
    • cites Butovsky et al., 2014 source
    • cites Melief et al., 2012 source
  • Premisecitation

    Microglia rapidly lose their unique identity when removed from the brain environment and cultured in monoculture in vitro.

    “Moreover, microglia rapidly lose their unique identity when removed from the brain environment and cultured in monoculture in vitro (Butovsky et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Butovsky et al., 2014 source

      In Results (‘Microglia cell lines do not express a microglia signature’; page not provided), the paper reports that primary cultured newborn microglia and microglial cell lines did not express the adult microglial signature. It does not establish that microglia rapidly lose that identity upon removal from the brain: newborn microglia were not shown to have first acquired the adult signature, and another Results section reports that adult microglia cultured with MCSF and TGF-β1 closely resembled freshly isolated cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premiseown data

    Our protocol yield is 10-43 per starting iPSC, versus 0.5-4 for Muffat et al. (2016) and Pandya et al. (2017).

    “It is robust and efficient: time frame 1 month, versus 2 months for Muffat et al. (2016); yield 10–43 per starting iPSC for our protocol, versus 0.5–4 for Muffat et al. (2016) and Pandya et al. (2017); manipulation consisting of once-weekly feeding of differentiation”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Muffat et al., 2016 source
    • cites Pandya et al., 2017 source
  • Premisecitation

    Microglia derive from primitive, yolk sac macrophages that migrate into the developing brain.

    “Our own work (Buchrieser et al., 2017) and that of others (Hoeffel et al., 2015) indicates that this protocol produces MYB-independent myeloid cells, recapitulating an embryonic ontogeny, and lineage-tracking studies in mouse have demonstrated that microglia derive from primitive, yolk sac macrophages that migrate into the developing brain (Ginhoux et al., 2010, Ginhoux et al., 2013).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Ginhoux et al., 2010 source
    • cites Ginhoux et al., 2013 source
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    The six key microglia-specific genes identified by Butovsky et al. (2014), MERTK, GPR34, PROS1, C1QA, GAS6, and P2RY12, were all strongly expressed in co-pMG, whereas bloodMono poorly expressed most of these markers.

    “Most notably, the six key microglia-specific genes identified by Butovsky et al. (2014), MERTK, GPR34, PROS1, C1QA, GAS6, and P2RY12, were all strongly expressed in co-pMG, whereas bloodMono poorly expressed most of these markers (highlighted in magenta, Figure 2C)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Butovsky et al., 2014 source

  • May combine two claims.

    Premisecitation

    PSC macrophages derive from MYB-independent, RUNX1- and PU.1-dependent precursors, characteristic of yolk sac-derived macrophages.

    “We have recently directly demonstrated that these derive from MYB-independent, RUNX1- and PU.1-dependent precursors, characteristic of yolk sac-derived macrophages (Buchrieser et al., 2017, Vanhee et al., 2015).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Buchrieser et al., 2017 source

      The Results section shows that iPSC-derived monocytes/macrophages develop without MYB but require RUNX1 and SPI1 (PU.1). The Discussion, however, says their derivation from these progenitors is suggested rather than directly established, and notes that other primitive macrophage progenitors cannot be excluded without clonal analysis. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Vanhee et al., 2015 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    In humans, yolk sac-derived macrophages appear at E17, enter the brain from E31 onward, and mature together with neurons to fully functional ramified microglia.

    “Yolk sac-derived macrophages appear at E17 (Tavian and Peault, 2005), enter the brain from E31 onward (Rezaie et al., 2005, Monier et al., 2007), and mature together with neurons to fully functional ramified microglia (Figure 1A).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Tavian and Peault, 2005 source

      In “The yolk sac” (page not provided), the paper reports that macrophages have been observed in the human yolk sac. It does not date their appearance to embryonic day 17; it describes blood-island precursors at about day 16 and its own observations beginning at day 19. The brain-entry and microglial-maturation clauses are not attributed to this source in the printed sentence. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Rezaie et al., 2005 source
    • cites Monier et al., 2007 source

  • May combine two claims.

    Premisecitation

    Schwartz et al. (2015) successfully seeded iPSC macrophages into 3D iPSC-neuronal structures for toxicity testing, but did not characterize the resulting cells extensively.

    “Schwartz et al. (2015) recently successfully seeded iPSC macrophages into 3D iPSC-neuronal structures for toxicity testing, but did not characterize the resulting cells extensively.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Schwartz et al., 2015 source

      The Results (page not available) report adding microglia/macrophage precursors to 3D neural constructs for neurotoxicity testing. However, the cells came from the H1 human embryonic stem-cell line, not iPSCs. The Results also describe FACS, phagocytosis, gene-expression, and morphology assessments, so the claim that the cells were not characterized extensively is not established by this copy. (LLM, conf=0.89)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    co-pMG roughly tile within the neuronal culture, making direct contacts with the neurons.

    “Co-pMG roughly “tile” within the neuronal culture, making direct contacts with the neurons (Figure 5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Co-pMG moved constantly, most of them roughly maintaining their territories.

    “Co-pMG moved constantly, most of them roughly maintaining their territories.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    pMacpre, pMac, and pMGL also expressed high levels of most of these genes, along with many of those identified by Melief et al. (2012).

    “Notably, pMacpre, pMac, and pMGL also expressed high levels of most of these genes, along with many of those identified by Melief et al. (2012).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Melief et al., 2012 source

  • May combine two claims.

    Findingown data

    CD11c was well expressed in monoculture cells, but weakly expressed in co-pMG.

    “CD11c (integrin alpha X, part of the inactivated-C3b receptor 4, CR4), was well expressed in monoculture cells, but weakly expressed in co-pMG.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Other key AD-related genes expressed in fetalMG and co-pMG (and bloodMono) included APP, PICALM, and CD33; PD-related genes included PARK15, PINK1, SNCA, and DJ-1; and MND-related genes included C9orf72, TDP43, and SOD1.

    “Other key AD-related genes expressed in fetalMG and co-pMG (and bloodMono) included APP, PICALM, and CD33; PD-related genes included PARK15, PINK1, SNCA, and DJ-1; and MND-related genes included C9orf72, TDP43, and SOD1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Blood monocyte-derived macrophages also upregulated several of these genes, but mostly did not reach the same expression level.

    “These expression profiles were confirmed by qRT-PCR (Figure 2D), which also showed that co-pMG had comparable levels of expression of these microglial genes with cultured adult human microglia and with directly processed adult human microglia, and that while blood monocyte-derived macrophages also upregulated several of these genes, they mostly did not reach the same expression level.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Quantification of movement of co-pMG, pMGL, and pMac over 5 hr showed co-pMG moved a significantly greater accumulated distance (251 ± 21 μm; mean ± SEM; n = 6) than pMGL (119 ± 6 μm; n = 5) and pMac (53 ± 11 μm; n = 5), which hardly moved at all.

    “Quantification of movement of co-pMG, pMGL, and pMac over 5 hr showed co-pMG moved a significantly greater accumulated distance (251 ± 21 μm; mean ± SEM; n = 6) than pMGL (119 ± 6 μm; n = 5) and pMac (53 ± 11 μm; n = 5), which hardly moved at all (Figures 5B–5F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    APOE was among the most strongly differentially expressed genes, being very low in bloodMono and high in all other populations.

    “APOE, variants of which are major risk factors for AD, was among the most strongly differentially expressed genes, being very low in bloodMono and high in all other populations (Figure S3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A targeted principal components analysis of these samples revealed a weak neural cell signature in the co-cultured pMG isolated with CD11b beads, but otherwise demonstrated the close similarity of these cells to fetal microglia.

    “A targeted principal components analysis of these samples revealed a weak neural cell signature in the co-cultured pMG isolated with CD11b beads, but otherwise demonstrated the close similarity of these cells to fetal microglia (Figures S4A and S4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MERTK was expressed highly on all PSC-derived macrophage and microglia conditions, while CD33 was detected albeit at low levels.

    “The microglia-associated protein MERTK was expressed highly on all PSC-derived macrophage and microglia conditions, while the AD-associated protein CD33 was detected albeit at low levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Upon activation co-culture microglia become more ameboid and release multiple microglia-relevant cytokines.

    “Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Co-culture could be extended for at least 42 days, during which time neurons maintained spontaneous electrical activity at least as well as neurons cultured alone.

    “Co-culture could be extended for at least 42 days, during which time neurons maintained spontaneous electrical activity at least as well as neurons cultured alone (Figure S1E), and calcium flux was observable upon addition of potassium ions (Movie S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An unconjugated polyclonal antibody to TMEM119 gave modest staining of pMGL but background staining was evident in co-culture cells, and an unconjugated antibody to the purinergic receptor P2YR12 showed strong staining of pMGL although background staining was similarly evident in co-culture.

    “An unconjugated polyclonal antibody to TMEM119 gave modest staining of pMGL but background staining was evident in co-culture cells, and an unconjugated antibody to the purinergic receptor P2YR12 showed strong staining of pMGL although background staining was similarly evident in co-culture (Figure S5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    FERMT2, TREM2, APOE, and UCHL1 were expressed in fetalMG and co-pMG but not in bloodMono (although TREM2 was upregulated in bloodMono-derived macrophages).

    “FERMT2, TREM2, APOE, and UCHL1 were expressed in fetalMG and co-pMG but not in bloodMono (although TREM2 was upregulated in bloodMono-derived macrophages, Figure 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The pMacpre yield is consistent with previously reported yields for this protocol, similar to Abud et al. 2017 (typically 40-fold), and 10-fold higher than two other recently published hiPS-microglia protocols.

    “The cumulative yield of pMacpre for the lines used in this study was 10- to 43-fold higher than the number of input iPSCs, consistent with yields previously reported for this protocol (van Wilgenburg et al., 2013), similar to a recently published hiPS-microglia protocol (typically 40-fold) (Abud et al., 2017), and 10-fold higher than two other recently published hiPS-microglia protocols (0.5- to 4”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites van Wilgenburg et al., 2013 source

      Not checked in the free run.

    • cites Abud et al., 2017 source

      Not checked in the free run.

    • cites Muffat et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    pH-sensitive fluorescent zymosan particles added to co-cultures became visible inside co-pMG within 1 hr, comparable with pMac.

    “pH-sensitive fluorescent zymosan particles added to co-cultures became visible inside co-pMG, within 1 hr, comparable with pMac (Movie S4), indicating the competent development of mature phagosomes in co-pMG.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    There was broad correspondence across Proteome Profiler and Luminex platforms.

    “There was broad correspondence across Proteome Profiler and Luminex platforms.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Co-pMG can be maintained in co-culture, retaining neuronal maturity and functionality for many weeks.

    “Co-pMG can be maintained in co-culture, retaining neuronal maturity and functionality for many weeks.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-34 is an alternative CSF1R ligand supporting microglia survival and differentiation.

    “In the brain, interleukin-34 (IL-34) is an alternative CSF1R ligand supporting microglia survival and differentiation (Greter et al., 2012), and microglia adopt an increasingly ramified morphology and continued maturation far beyond birth.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Greter et al., 2012 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In mice, two waves of embryonic macrophages are produced in the yolk sac blood islands at embryonic day 7.5 and E8.25, and the first wave migrate into the developing brain and differentiate to microglia.

    “In mice, two waves of embryonic macrophages are produced in the yolk sac blood islands at embryonic day 7.5 (E7.5) and E8.25, and the first wave migrate into the developing brain and differentiate to microglia (Ginhoux et al., 2010, Gomez Perdiguero et al., 2015, Hoeffel et al., 2015, Palis et al., 1999).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Ginhoux et al., 2010 source

      Not checked in the free run.

    • cites Gomez Perdiguero et al., 2015 source

      Not checked in the free run.

    • cites Hoeffel et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    Taken together, these results show the transcriptomic similarity of co-pMG with primary microglia, with expression of key microglial markers and genes in relevant homeostatic pathways, and downregulation of antimicrobial pathways in co-pMG.

    “Taken together, these results show the transcriptomic similarity of co-pMG with primary microglia, with expression of key microglial markers and genes in relevant homeostatic pathways, and downregulation of antimicrobial pathways in co-pMG.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Human microglial ontogeny processes are analogous to those in mice.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Yolk sac-derived macrophages are Myb independent but dependent on PU.1 and Irf8.

    “These yolk sac-derived macrophages are Myb independent but dependent on PU.1 and Irf8 (Kierdorf et al., 2013, Schulz et al., 2012).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kierdorf et al., 2013 source
    • cites Schulz et al., 2012 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Microglia in the developing brain proliferate locally at a low rate and are not normally replaced by other monocytes and macrophages from outside the brain, unlike most other tissue-resident macrophages.

    “Microglia in the developing brain proliferate locally at a low rate and are not normally replaced by other monocytes and macrophages from outside the brain, in contrast to most other tissue-resident macrophages (which also initially originate from yolk sac-derived macrophages, but are partially or fully replaced by fetal liver- or blood monocyte-derived macrophages [Bain et al., 2014, Calderon et”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Bain et al., 2014 source

      Not checked in the free run.

    • cites Calderon et al., 2015 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Epelman et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    pMac are competent at phagocytosing particles, progressive acidification of the maturing phagosome being detectable using particles coupled to pH-sensitive fluorophores.

    “We have previously demonstrated that pMac are competent at phagocytosing particles, progressive acidification of the maturing phagosome being detectable using particles coupled to pH-sensitive fluorophores (Kapellos et al., 2016).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kapellos et al., 2016 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationown data

    Genes that have been previously identified as being specific to microglia versus blood monocytes are not necessarily exclusive to microglia, but a subset of them are more likely correlates of primitive macrophages, since they are also highly expressed in pMac.

    “However, these results also highlight a previously unappreciated detail, which is that genes that have been previously identified as being specific to microglia versus blood monocytes are not necessarily exclusive to microglia, but a subset of them are more likely correlates of primitive macrophages, since they are also highly expressed in pMac.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Myb independence distinguishes yolk sac-derived macrophages from adult, definitive, blood monocyte-derived macrophages.

    “Myb independence, therefore, distinguishes yolk sac-derived macrophages from adult, definitive, blood monocyte-derived macrophages.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Co-culture conditions were compatible for co-pMG and had no detrimental effect on the maturity and functionality of the neurons.

    “Together, these observations indicate that co-culture conditions were compatible for co-pMG and had no detrimental effect on the maturity and functionality of the neurons.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Human microglial ontogeny processes are analogous to those in mice.

  • May combine two claims.

    Interpretationown data

    co-pMG and their primitive precursors express expected microglia and myeloid-associated proteins, although PSC macrophages and microglia have not been exposed to the cytokine milieu of the body and thus have low basal levels of proteins such as MHC antigens.

    “These results show that co-pMG and their primitive precursors express expected microglia and myeloid-associated proteins, although PSC macrophages and microglia have not been exposed to the cytokine milieu of the body and thus have low basal levels of proteins such as MHC antigens.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    These features were a direct result of physical contact with neurons, as cells monocultured on tissue-culture plastic did not display such dynamic microglial characteristics.

    “These features were a direct result of physical contact with neurons, as cells monocultured on tissue-culture plastic did not display such dynamic microglial characteristics.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    co-pMG display the morphology and dynamic behavior expected of microglia, continually sensing and responding to their neuronal environment.

    “Together, these results show that co-pMG display the morphology and dynamic behavior expected of microglia, continually sensing and responding to their neuronal environment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Figure 7 used mean ± SD, three genetic backgrounds.

    “Mean ± SD, three genetic backgrounds.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    The Beutner/Almeida method directs EBs through a neuronal differentiation pathway and thus does not replicate yolk sac myelopoiesis.

    “However, this method directs the EBs through a neuronal differentiation pathway and thus does not replicate yolk sac myelopoiesis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Abud et al. (2017) uses low oxygen concentrations/several replatings/five different cytokine cocktails/FAC sorting progenitors.

    “versus sequential trituration/replating steps for Muffat et al. (2016), fluorescence-activated cell (FAC) sorting/plating on astrocytes/second FAC sorting for Pandya et al. (2017), and low oxygen concentrations/several replatings/five different cytokine cocktails/FAC sorting progenitors for Abud et al. (2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Abud et al., 2017 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The protocol produces MYB-independent myeloid cells, recapitulating an embryonic ontogeny.

    “Our own work (Buchrieser et al., 2017) and that of others (Hoeffel et al., 2015) indicates that this protocol produces MYB-independent myeloid cells, recapitulating an embryonic ontogeny, and lineage-tracking studies in mouse have demonstrated that microglia derive from primitive, yolk sac macrophages that migrate into the developing brain (Ginhoux et al., 2010, Ginhoux et al., 2013).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Buchrieser et al., 2017 source

      Not checked in the free run.

    • cites Hoeffel et al., 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Figure 6 quantitative analysis used two videos each of three cultures, i.e., six replicates per condition.

    “(C–E) Quantitative analysis (two videos each of three cultures, i.e., six replicates per condition).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pandya et al. (2017) uses FAC sorting/plating on astrocytes/second FAC sorting.

    “versus sequential trituration/replating steps for Muffat et al. (2016), fluorescence-activated cell (FAC) sorting/plating on astrocytes/second FAC sorting for Pandya et al. (2017), and low oxygen concentrations/several replatings/five different cytokine cocktails/FAC sorting progenitors for Abud et al. (2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pandya et al., 2017 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Beutner et al. (2013) described a method for deriving microglia from mouse embryonic stem cells.

    “Beutner et al. (2013) described a method for deriving microglia from mouse embryonic stem cells, and, using the same methodology, Almeida et al. (2012) derived microglia from human iPSCs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Beutner et al., 2013 source

  • May combine two claims.

    Interpretationreasoning

    Some functions and disease-relevant genes can be studied in monoculture conditions, but others involving crosstalk between microglia and neurons will be better studied using the co-culture model.

    “Some of these functions and disease-relevant genes can be studied in the monoculture conditions detailed here, but others, involving crosstalk between microglia and neurons, such as paired receptor engagement, paracrine signaling, damage responses, synaptic surveillance, and pruning, will be better studied using the co-culture model we have described.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The protocol is now widely used by others.

    “In 2008, we derived human PSC macrophages using a very simple methodology without OP9 feeders (Karlsson et al., 2008), which we subsequently adapted to a fully defined, robust serum-free protocol (van Wilgenburg et al., 2013), now widely used by others (Aflaki et al., 2014, Alasoo et al., 2015, Gupta et al., 2016).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Aflaki et al., 2014 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Alasoo et al., 2015 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Gupta et al., 2016 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pandya et al. (2017) characterized their iPSC microglia in monoculture following isolation from astrocytes.

    “Muffat et al. (2016) characterized their iPSC microglia in monoculture, briefly assessing co-culture, and Pandya et al. (2017) characterized their iPSC microglia in monoculture following isolation from astrocytes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pandya et al., 2017 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The authors subsequently adapted the protocol to a fully defined, robust serum-free protocol.

    “In 2008, we derived human PSC macrophages using a very simple methodology without OP9 feeders (Karlsson et al., 2008), which we subsequently adapted to a fully defined, robust serum-free protocol (van Wilgenburg et al., 2013), now widely used by others (Aflaki et al., 2014, Alasoo et al., 2015, Gupta et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites van Wilgenburg et al., 2013 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Morphology scoring was performed blind by three independent assessors.

    “Micrographs were scored blind by three independent assessors for number of microglia with activated morphology.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All human material was obtained with informed consent and with approval of relevant institutions.

    “All human material (iPSCs, adult blood, fetal and adult microglia) was obtained with informed consent and with the approval of the relevant institutions (see Supplemental Information for full details).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Muffat et al. (2016) uses sequential trituration/replating steps.

    “versus sequential trituration/replating steps for Muffat et al. (2016), fluorescence-activated cell (FAC) sorting/plating on astrocytes/second FAC sorting for Pandya et al. (2017), and low oxygen concentrations/several replatings/five different cytokine cocktails/FAC sorting progenitors for Abud et al. (2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Muffat et al., 2016 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In 2008, the authors derived human PSC macrophages using a simple methodology without OP9 feeders.

    “In 2008, we derived human PSC macrophages using a very simple methodology without OP9 feeders (Karlsson et al., 2008), which we subsequently adapted to a fully defined, robust serum-free protocol (van Wilgenburg et al., 2013), now widely used by others (Aflaki et al., 2014, Alasoo et al., 2015, Gupta et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Karlsson et al., 2008 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Almeida et al. (2012) derived microglia from human iPSCs using the same methodology.

    “Beutner et al. (2013) described a method for deriving microglia from mouse embryonic stem cells, and, using the same methodology, Almeida et al. (2012) derived microglia from human iPSCs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Almeida et al., 2012 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Interpretationnone given

    The system is amenable to scaling for the development of drug-screening assays to identify compounds that can improve microglial homeostatic clearance functions and dampen chronically activated microglia.

    “The system is also amenable to scaling for the development of drug-screening assays to identify compounds that can improve microglial homeostatic clearance functions and dampen chronically activated microglia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Muffat et al. (2016) characterized their iPSC microglia in monoculture, briefly assessing co-culture.

    “Muffat et al. (2016) characterized their iPSC microglia in monoculture, briefly assessing co-culture, and Pandya et al. (2017) characterized their iPSC microglia in monoculture following isolation from astrocytes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Muffat et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    The authors extensively characterized iPSC microglia in co-culture with iPSC cortical neurons, where cells have highly ramified, dynamic characteristics, and compared directly to intermediate and parallel monoculture stages of differentiation.

    “We have extensively characterized iPSC microglia in co-culture with iPSC cortical neurons, where cells have highly ramified, dynamic characteristics, and compared directly to the intermediate and parallel monoculture stages of differentiation.”
    Quote in the paperSubstantial claim
53 of 53

  • The authors state this as an assumption.

    Human microglial ontogeny processes are analogous to those in mice.

    “In humans there are few opportunities to investigate the ontogeny of microglia, but it is assumed that the processes are analogous to those in mice.”

  • The paper relies on this without support.

    Fresh primary human microglia are very limited in availability and cannot be propagated.

    “Study of microglia has been largely restricted to non-human models (mostly mouse), since availability of fresh primary human microglia is very limited and they cannot be propagated.”

  • The paper relies on this without support.

    Primary human microglia can only be obtained from fresh brain material.

    “Since primary human microglia can only be obtained from fresh brain material, previous efforts have been made by others to develop methods for deriving microglia in vitro.”

  • The paper relies on this without support.

    The blood monocyte pathway poorly recapitulates microglia ontogenesis.

    “but it is now known that this pathway poorly recapitulates microglia ontogenesis and that blood is not a limitless source of cells, unlike PSCs.”

  • The paper relies on this without support.

    Blood is not a limitless source of cells, unlike PSCs.

    “but it is now known that this pathway poorly recapitulates microglia ontogenesis and that blood is not a limitless source of cells, unlike PSCs.”

  • The paper relies on this without support.

    B27 contains cortisone, SOD, and catalase.

    “Our microglia medium avoids B27, which contains cortisone, SOD, and catalase (likely to compromise microglia function).”

  • The paper relies on this without support.

    B27 compromises microglia function.

    “Our microglia medium avoids B27, which contains cortisone, SOD, and catalase (likely to compromise microglia function).”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Astrocyte and microglia iPSC models partially recapitulate the transcriptomes of their in vivo counterparts and may capture authentic neurodegenerative disease phenotypes.

    “Astrocyte and microglia iPSC models partially recapitulate the transcriptomes of their in vivo counterparts and may capture authentic neurodegenerative disease phenotypes [345,406,480,481,482,483,484].”

    From Revisiting the Pathogenesis of X-Linked Adrenoleukodystrophy (2025) · cites this paper as [484]

    The Results report transcriptomic similarity between iPSC-derived co-culture microglia and primary microglia; the Discussion says their profile resembles fetal microglia and identifies disease-relevant genes. The paper presents the model as useful for studying neurodegenerative disease genes, but it does not establish that it captures disease phenotypes or assess iPSC-derived astrocyte models. Page numbers were not supplied in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • These cells closely resemble human foetal microglia with regards to transcriptional signature and express high levels of microglial genes.

    “These cells closely resemble human foetal microglia with regards to transcriptional signature and express high levels of microglial genes29,30.”

    From PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages (2021) · cites this paper as [29]

  • The differentiated hiPSC-derived microglia exhibit functional and transcriptional profiles consistent with human microglia.

    “We used CRISPR editing to generate PLCγ2HET and PLCγ2HOM hiPSC cells, and differentiated them into microglia that exhibit functional and transcriptional profiles consistent with human microglia [21].”

    From Heterozygous expression of the Alzheimer’s disease-protective PLCγ2 P522R variant enhances Aβ clearance while preserving synapses (2022) · cites this paper as [21]

  • Genomic integrity of iPSC lines was verified by Illumina Omniexpress 24 v1.2 SNP microarray analysis.

    “An Illumina Omniexpress 24 v1.2 SNP microarray analysis was performed to verify genomic integrity, as previously described in Haenseler et al.29.”

    From PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages (2021) · cites this paper as [29]

    The Results section’s Figure 2 caption describes Illumina HT12v4 transcriptome analysis, not an OmniExpress SNP microarray used to verify genomic integrity. Searches for “OmniExpress,” “SNP microarray analysis,” and genomic-integrity testing found no passage in our copy describing the claimed procedure; page numbers were unavailable. (LLM, conf=0.74)

    Experimental — accuracy not yet measured

  • Protocols have been developed to produce human iPSC-derived microglial-like cells (iMG).

    “Recently, protocols for induce human iPSC-derived microglial-like cells (iMG) have been developed [67-71].”

    From Transcriptional and Epigenetic Regulation of Microglia in Health and Disease (2019) · cites this paper as [70]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Replicate counts disagree for the motility comparison

    Minor

    The motility results report five replicates for pMGL and pMac, but the Figure 5 caption states that there were six replicates per condition.

1 of 1

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Authors
Haenseler, W., Sansom, S. N., Buchrieser, J., Newey, S. E., Moore, C. S., Nicholls, F. J., Chintawar, S., Schnell, C., Antel, J. P., Allen, N. D., Cader, M. Z., Wade-Martins, R., James, W. S., & Cowley, S. A.
Affiliations
  • University of Oxford (GB)
  • Memorial University of Newfoundland (CA)
  • Warneford Hospital (GB)
  • Cardiff University (GB)
  • Montreal Neurological Institute and Hospital (CA)
Corresponding author
Cowley, Sally A.
ORCID
Venue
Stem Cell Reports
Publisher
Elsevier BV
Published
2017-06
Volume
8
Issue
6
Pages
1727-1742
ISSN
2213-6711 (ISSN-L)
DOI
10.1016/j.stemcr.2017.05.017
PMC
PMC5470330
OpenAlex
W2623352127
Cited by
593
References
47
Citations by year
2026: 54 · 2025: 70 · 2024: 98 · 2023: 62 · 2022: 87 · 2021: 74 · 2020: 59 · 2019: 41 · 2018: 39 · 2017: 7
FWCI
19.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Stem Cell Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ORCA Online Research @Cardiff (Cardiff University) (acceptedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by)
  • Zurich Open Repository and Archive (University of Zurich)
Funders
  • Wellcome Trust (WTISSF121302)
  • Medical Research Council (MC_EX_MR/N50192X/1, MC_PC_16034, MR/N013255/1)
  • National Institute for Health and Care Research
  • Seventh Framework Programme (FP7/2007e2013)
  • Innovative Medicines Initiative (115439)
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (148607)
  • Alzheimer’s Research UK
  • Oxford Martin School, University of Oxford (LC0910-004)
  • Royal Society
  • Parkinson's UK (2581970, SC037554)
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, iPSC-derived macrophages, microglial differentiation, neurodegenerative diseases, inflammation, cytokine expression, phagocytosis, anti-inflammatory cytokines, cortical neurons
MeSH terms
Cells, Cultured, Humans, Macrophages, Models, Biological, Neurons, Phagocytosis, Down-Regulation, Cytokines, Microglia, Coculture Techniques, Neurodegenerative Diseases, Proto-Oncogene Proteins c-myb, Pluripotent Stem Cells, fms-Like Tyrosine Kinase 3, Transcriptome

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

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Statistics and arithmetic are recomputed, not judged.

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