CoChat
Sign Up
Cited by 651Pluripotent Stem Cells ResearchNeuroinflammation and Neurodegeneration MechanismsNeurogenesis and neuroplasticity mechanisms

Efficient derivation of microglia-like cells from human pluripotent stem cells

Open access
Muffat, Julien, Li, Yun, Yuan, Bingbing, Mitalipova, Maisam, Omer, Attya, Corcoran, Sean, Bakiasi, Grisilda, Tsai, Li-Huei +3 more·2016·Nature Medicine
DOI 10.1038/nm.4189PMC PMC5101156
Open this paper in CoChat — free accountOpen PDF

Abstract

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell–derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper establishes a defined, serum-free protocol to derive microglia-like cells (pMGLs) from human ES and iPS cells, including disease-specific lines, and shows they resemble primary human fetal microglia phenotypically and transcriptionally. pMGLs are phagocytic, respond to inflammatory stimuli, integrate into 3D neuroglial cultures, and can model injury responses and isogenic disease phenotypes such as MECP2 mutant size differences.

Key findings

  • pMGLs were generated from 20 different human ES and iPS cell lines representing healthy and disease genotypes, with successful derivation from all lines; after 8 weeks, example yields from 2×10^6 pluripotent stem cells included 1×10^6 (hES-wibr1), 1×10^6 (iPS-wt1), 3×10^6 (iPS-wt4), 2×10^6 (iPS-ALD4), and 8×10^6 (iPS-ALD1) microglia-like cells.
  • pMGLs expressed microglial markers including TMEM119, P2RY12, IBA1, CD11b, CD45, and PU.1; transcriptomic clustering placed pMGLs with primary fetal and adult human microglia, separate from other macrophages, and they expressed high levels of microglial genes such as APOE, CD33, TREM2, HEXB, GPR34, and P2RY12/13.
  • Functionally, pMGLs were highly phagocytic and motile; early pMGLs showed EdU incorporation in 60% of nuclei, falling to less than 5% after 2 months in culture. IFN-γ/LPS stimulation increased release of CXCL10, MIP1a, IL-6, and TNFα more than 2-fold, with corresponding increases in IL-6 and TNFα mRNA.
  • pMGLs derived from isogenic MECP2 mutant hES cells were significantly smaller than wild-type controls; in 3D organotypic cultures, GFP-labeled pMGLs integrated, tiled the volume, extended highly branched ramifications, and responded to focal laser injury by extending processes and migrating toward damage within minutes.

Methods

  • Differentiation used human ES/iPS cells grown on murine embryonic fibroblasts, collagenase-passaged into uniform clusters, and seeded in ultra-low-adherence plates in NGD serum-free medium containing 10 ng/mL CSF1 and 10 ng/mL IL-34 (MGdM). After one week, yolk sac-like embryoid bodies (YS-EBs) formed; semi-adherent cells were harvested onto Primaria plates and maintained in MGM (NGD + 100 ng/mL IL-34 + 5 ng/mL CSF1).
  • Characterization included immunofluorescence, flow cytometry, phagocytosis assays, EdU click-it proliferation assays, cytokine antibody arrays, qPCR, and RNA-seq (SMART-Seq v4, hg19 mapping, TopHat, FeatureCounts, DESeq, PCA, hierarchical clustering). 3D organotypic cultures were made by transducing pMGLs with GFP lentivirus and re-aggregating them with pre-differentiated neural cultures, followed by live imaging and two-photon laser injury.
  • The study states no statistical tests were used to pre-determine sample size; experimenters were not blinded to genotypes or culture conditions except for transcriptomic analysis where line genotype was not explicit. Two-tailed Student’s t-tests were used where appropriate, with p<0.05 considered significant.

Limitations

  • Stated: primary human microglia are poorly characterized, not easily accessible, and current isolation methods likely alter their defining characteristics; the protocol yields “microglia-like” cells (pMGLs) rather than bona fide primary microglia, and the genetic background of the pluripotent donor affects kinetics and yield.
  • Stated: no statistical tests were used to pre-determine sample size, and experimenters were not blinded to genotypes or culture conditions except for transcriptomic analysis.
  • Evident: the transcriptomic comparison relies on primary fetal human microglia maintained in the same culture conditions, and the MECP2 mutant size difference is reported as a finding worthy of further investigation rather than a functional mechanism; long-term in vivo behavior and disease relevance remain to be established.

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

Integrity signals

Registry

Crossref · 10.1038/nm.4189

Cites

50 of 50 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    pMGLs were generated from multiple disease-specific cell lines.

    “We generated these cells from multiple disease-specific cell lines”
    Quote in the paperNot a substantial claim
  • Findingown data

    pMGLs derived from MeCP2 mutant hES cells are smaller than their isogenic controls.

    “find that pMGLs derived from MeCP2 mutant hES cells are smaller than their isogenic controls.”
    • Also stated in: § Phenotypic and functional characterization of pMGLs · plain
      “We derived pMGLs from isogenic male wild-type and MECP2 mutant cells, and observed that mutant cells were significantly smaller than the wild-type cells (Fig. 3c).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Human fetal microglia adopted a more ramified morphology in the presence of high concentrations of IL-34.

    “with hFMGs adopting a more ramified morphology in the presence of high concentrations of IL-34 (Supplementary Fig 1a, b).”
    Quote in the paperNot a substantial claim
  • Findingown data

    The majority of cells physically selected by the growth conditions were positive for all markers.

    “with the majority of cells physically selected by the growth conditions positive for all markers.”
    Quote in the paperSubstantial claim
  • Findingcitation

    Neural differentiation can be initiated in NGD by adding dorsomorphin.

    “Neural differentiation can be initiated in NGD by addition of a small molecule inhibitor of TGFβ signaling (dorsomorphin) 15.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Zhou, J. et al. High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors. Stem Cells 28, 1741–1750 (2010). source

      Not checked in the free run.

  • May combine two claims. 36
  • Classified as probably not a substantial claim. 20
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Mature pMGLs stained positive for markers of murine and human microglia, with TMEM119 and P2RY12 displaying broad membrane and punctate vesicular expression.

    “Mature pMGLs stained positive for markers of murine and human microglia 10,11,29,30, with both TMEM119 and P2RY12 displaying a broad membrane and punctate vesicular expression pattern”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [10] Bennett, M.L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA 113, E1738–E1746 (2016). source

      Not checked in the free run.

    • cites [11] Hickman, S.E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 (2013). source

      Not checked in the free run.

    • cites [29] Butovsky, O. et al. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131–143 (2014). source

      Not checked in the free run.

    • cites [30] Haynes, S.E. et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat. Neurosci. 9, 1512–1519 (2006). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Immature pMGLs initially delaminating from YS-EBs are round yet already highly phagocytic, as observed by uptake of fluorescent latex beads and appearance of filopodia and membrane ruffles.

    “The immature pMGLs initially delaminating from YS-EBs are round yet already highly phagocytic, as observed by the uptake of fluorescent latex beads and the appearance of filopodia and membrane ruffles”
    Quote in the paperSubstantial claim

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

    Findingnone given

    Neural progenitors can be expanded by adding FGF2 to the NGD base.

    “Neural progenitors can be expanded by addition of FGF2 to the NGD base”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both human fetal microglia and mouse neonatal microglia died when no CSF1R agonist was present.

    “Both hFMGs and mNMGs died in the absence of any CSF1R agonist”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The cells can be sorted by FACS and are homogeneously positive for IBA1, CD45 and CD11b.

    “These cells can be further sorted by FACS and are homogeneously positive for IBA1, CD45 and CD11b (Fig 2d, e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The cells are highly motile and will tax to and encapsulate foreign bodies such as fibers present in the well.

    “The cells are highly motile and will readily tax to and encapsulate foreign bodies such as fibers present in the well (Fig. 2f).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Harvested semi-adherent cells plated on Primaria appear vacuolated, round and motile, and stain positive for PU.1, CD11b and IBA1.

    “When harvested and plated separately on Primaria, semi-adherent cells appear vacuolated, round and motile, and stain positive for PU.1, as well as CD11b and IBA1 (AIF1) ((Fig. 2b,c, Supplementary Movie 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Clusters of cells delaminated towards the lumen of cysts and directly into the outer medium or onto the plastic culture surface.

    “clusters of cells were seen to delaminate towards the lumen of the cysts (Fig. 1e) as well as directly into the outer medium or on the plastic culture surface.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    When plated early on poly-D-lysine, neuralized embryoid bodies flattened into rosette-forming neuroepithelium, while cystic embryoid bodies flattened into cell lawns resembling endothelial cells.

    “When plated at these early stages on poly-D-lysine, the neuralized EBs flattened into typical rosette-forming neuroepithelium, while the small cystic EBs flattened into cell lawns reminiscent of endothelial cells 18 (Supplementary Fig. 1b).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Palis, J., McGrath, K.E. & Kingsley, P.D. Initiation of hematopoiesis and vasculogenesis in murine yolk sac explants. Blood 86, 156–163 (1995). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    GFP-labeled pMGLs integrated into 3D cultures, tiled the volume, and projected highly branched ramifications.

    “GFP-labeled pMGLs, which had adopted rounded to first-order ramified morphologies when cultured on plastic, integrated into the three-dimensional cultures, tiled the volume (Fig. 6c), and projected highly branched ramifications (Fig. 6d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    pMGLs migrate to and encapsulate a site of localized injury.

    “Furthermore, they respond to localized injury by migrating and encapsulating the damaged area.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    pMGLs kept in mono-culture express high levels of SELPLG, TXNIP, CST3 and OLFML3, suggesting they adopt a relatively more mature profile than primary fetal cells.

    “pMGLs kept in mono-culture express high levels of SELPLG, TXNIP, CST3 and OLFML3, suggesting they adopt a relatively more mature profile than primary fetal cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Exposure to undifferentiated neural progenitor conditioned medium maintains the rounded phenotype of immature microglia and promotes their proliferation.

    “We observed that exposure to undifferentiated neural progenitor conditioned medium maintains the rounded phenotype of immature microglia and promotes their proliferation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    pMGLs form a cluster with both fetal and mature primary microglia, while iPS-derived neural cells cluster with primary neurons and glia, close to fetal astrocytes.

    “We find that pMGLs form a cluster with both fetal and mature primary microglia, while iPS-derived neural cells cluster with primary neurons and glia, close to fetal astrocytes (Figure 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    pMGLs bypass a colony-forming monocytic stage, are initially amoeboid and capable of proliferation, extensive migration and phagocytosis.

    “In particular, they bypass a colony-forming monocytic stage, are initially amoeboid and capable of proliferation, extensive migration and phagocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In cultures, SELPLG, CST3 and TXNIP are already expressed at high levels in both FMGs and pMGLs, while OLFML3 and P2RY13 are more than 4-fold higher in pMGLs compared to FMGs.

    “In our cultures, we find that SELPLG, CST3 and TXNIP are already expressed at high levels in both FMGs and pMGLs, while OLFML3 and P2RY13 are more than 4-fold higher in pMGLs compared to FMGs.”
    Quote in the paperSubstantial claim

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

    Findingown data

    As pMGLs populate the substrate, they isolate from their nearest neighbors, an in vitro behavior that may correspond to the ability of microglia to tile the CNS in vivo.

    “As pMGLs populate the substrate, they clearly isolate from their nearest neighbors, an in vitro behavior which may correspond to the ability to tile the CNS by their in vivo counterparts.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    These genes are expressed at low levels in cultured macrophage cell lines and previously described mouse ES-derived microglia.

    “Notably, these genes are expressed at low levels in cultured macrophage cell lines as well as in previously described mouse ES-derived microglia 33.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Beutner, C., Roy, K., Linnartz, B., Napoli, I. & Neumann, H. Generation of microglial cells from mouse embryonic stem cells. Nat. Protoc. 5, 1481–1494 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Neural progenitors progressively differentiated into neurons and macro-glia and would be devoid of microglia without exogenous pMGLs.

    “The neural progenitors progressively differentiated into neurons and macro-glia (astrocytes, oligodendrocytes), and would be devoid of any microglia without the exogenous addition of pMGLs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    pMGLs display high expression of PU.1 and IRF8, but very low levels of MYB.

    “We also note that pMGLs display high expression of both PU.1 and IRF8, but very low levels of MYB (Suppl. Fig 3a)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The pluripotent stem cell-derived microglia-like cells faithfully recapitulate the expected ontogeny and characteristics of in vivo microglia and resemble primary fetal human and mouse microglia.

    “These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts and resemble primary fetal human and mouse microglia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Primary human microglia are poorly characterized, differ greatly from mature mouse microglia, are not easily accessible, and current isolation methods likely alter their defining characteristics.

    “primary human microglia remain poorly characterized as they differ greatly from mature mouse microglia, are not easily accessible and current methods employed for isolation likely alter their defining characteristics6.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Smith, A.M., Gibbons, H.M., Lill, C., Faull, R.L. & Dragunow, M. Isolation and culture of adult human microglia within mixed glial cultures for functional experimentation and high-content analysis. Methods Mol. Biol. 1041, 41–51 (2013). source

      Not checked in the free run.

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

    Premisecitation

    Microglia leave the yolk sac as erythromyeloid precursors or primitive macrophages before peripheral hematopoiesis begins.

    “They emigrate as erythromyeloid precursors or primitive macrophages from the yolk sac, before further hematopoiesis takes place in the periphery.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Robust protocols exist for differentiating human ES or iPS cells into neurons, astrocytes and oligodendrocytes.

    “Robust protocols have been developed to induce human ES or iPS cells to differentiate into the different neuroectodermal cell types of the CNS including neurons, astrocytes and oligodendrocytes 7,8.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [7] Hu, B.Y., Du, Z.W., Li, X.J., Ayala, M. & Zhang, S.C. Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452 (2009). source

      Not checked in the free run.

    • cites [8] Hu, B.Y. & Zhang, S.C. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat. Protoc. 4, 1295–1304 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The differentiation protocol is efficient and robust, though the genetic background of the pluripotent donor affects the kinetics and yield of microglia-like cells.

    “We conclude that the differentiation protocol is efficient and robust, though the genetic background of the pluripotent donor affects the kinetics and yield of the microglia-like cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Monocultures of pMGLs can serve as a substrate to study functional and morphological changes in microglia derived from isogenic parental cells, or to screen for modulators of inflammation.

    “These results indicate that monocultures of pMGLs can be a substrate to study the functional and morphological changes in microglia, derived from isogenic parental cells, or to screen for modulators of inflammation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The modular differentiation system allows microglia to be studied under highly defined conditions as they mature in response to developmental cues and provides a framework for studying long-term interactions in a tissue-like environment.

    “This modular differentiation system allows the study of microglia in highly defined conditions, as they mature in response to developmentally relevant cues, and provides a framework to study the long-term interactions of microglia residing in a tissue-like environment.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Mouse genetic lineage tracing indicates that microglia colonize the developing neural tube shortly before the blood-brain barrier closes.

    “recent genetic lineage tracing in the mouse determined that microglia take up residence in the developing neural tube shortly before closure of the blood-brain barrier 4.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010). source

      Not checked in the free run.

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

    Premisecitation

    These markers have been extensively used to label or define microglia in various systems.

    “These markers have been extensively used to label or define microglia in various systems 24.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Ito, D. et al. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res. Mol. Brain Res. 57, 1–9 (1998). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Myeloid cells can be generated from mouse and human ES cells using protocols that recapitulate classical hematopoiesis followed by monocyte expansion.

    “Protocols recapitulating classical hematopoiesis, followed by expansion of monocytes have shown that myeloid cells can be generated from mouse and human ES cells 9.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9] Grigoriadis, A.E. et al. Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells. Blood 115, 2769–2776 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Microglial identity combines a unique mesodermal ontogeny from primitive myelopoiesis with lifelong residence in an immune-privileged neuro-glial environment.

    “microglial identity is a combination of unique mesodermal ontogeny (primitive myelopoiesis) and lifelong residence in an immune-privileged neuro-glial environment.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    PU.1 is necessary for microglial differentiation and maintenance and can induce macrophages from fibroblasts.

    “which is necessary for microglial differentiation and maintenance 21,22 and can induce macrophages from fibroblasts 23.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [21] McKercher, S.R. et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15, 5647–5658 (1996). source

      Not checked in the free run.

    • cites [22] Smith, A.M. et al. The transcription factor PU.1 is critical for viability and function of human brain microglia. Glia 61, 929–942 (2013). source

      Not checked in the free run.

    • cites [23] Feng, R. et al. PU.1 and C/EBPα/β convert fibroblasts into macrophage-like cells. Proc. Natl. Acad. Sci. USA 105, 6057–6062 (2008). source

      Not checked in the free run.

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

    Premisecitation

    Microglia are the resident macrophages of the central nervous system.

    “Microglia are the resident macrophages of the central nervous system (CNS) 1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Tremblay, M.E. et al. The role of microglia in the healthy brain. J. Neurosci. 31, 16064–16069 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Inflammation mediated by microglia can harm the brain.

    “Microglia mediated inflammation can have deleterious consequences for the brain and much evidence indicates that microglial activation contributes to neurodegenerative disease such as AD, PD and ALD 40,41.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Aubourg, P. Cerebral adrenoleukodystrophy: a demyelinating disease that leaves the door wide open. Brain 138, 3133–3136 (2015). source

      Not checked in the free run.

    • cites [41] Ransohoff, R.M. & El Khoury, J. Microglia in health and disease. Cold Spring Harb. Perspect. Biol. 8, a020560 (2015). source

      Not checked in the free run.

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

    Premisecitation

    In ALD, phagocytes from autologous bone marrow transplants likely act beneficially by replacing mutant microglia, thereby halting terminal neuro-inflammation and demyelination.

    “In ALD, phagocytes derived from autologous bone marrow transplants likely elicit their beneficial effects by replacing mutant microglia, as they stall terminal neuro-inflammation and demyelination 42.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009). source

      Not checked in the free run.

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

    Study design and conductnone given

    All parental cell lines were maintained beyond 50 passages, verified for stable Oct3/4, Nanog, and TRA1-60 expression, and routinely tested for mycoplasma.

    “All parental lines were maintained for over 50 passages, verified for stable expression of Oct3/4, Nanog and TRA1-60, and routinely tested for mycoplasma negativity.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The sample size was not pre-determined using statistical tests.

    “no statistical tests were used to pre-determine sample size.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All experiments with human donor cells and animals complied with established IRB protocols at the Whitehead Institute.

    “all experiments involving cells from human donors and animals were performed in compliance with established IRB protocols at the Whitehead institute.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    pMGLs can integrate into organotypic neural cultures and mature into what is currently defined as resting yet dynamically motile microglia.

    “These results suggest that pMGLs can integrate into organotypic neural cultures, and mature into what is currently defined as resting yet dynamically motile microglia.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 3D organotypic culture and focal laser injury model reproduce relevant in vivo microglial behavior.

  • May combine two claims.

    Interpretationown data

    The difference between NPCs and pMGLs along PC1 may reflect the main myeloid versus neural identity and accounts for about 77% of sample variance.

    “The difference between NPCs and pMGLs along PC1 may reflect the main myeloid vs neural identity, accounting for ~77% of sample variance”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationown data

    The absence of canonical myeloid ontology terms among differentially expressed genes supports the use of pMGLs as a surrogate for human microglia.

    “supporting their use as surrogate for human microglia.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Microglial activation contributes to neurodegenerative diseases such as AD, PD, and ALD.

    “Microglia mediated inflammation can have deleterious consequences for the brain and much evidence indicates that microglial activation contributes to neurodegenerative disease such as AD, PD and ALD 40,41.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Aubourg, P. Cerebral adrenoleukodystrophy: a demyelinating disease that leaves the door wide open. Brain 138, 3133–3136 (2015). source

      Not checked in the free run.

    • cites [41] Ransohoff, R.M. & El Khoury, J. Microglia in health and disease. Cold Spring Harb. Perspect. Biol. 8, a020560 (2015). source

      Not checked in the free run.

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

    Study design and conductnone given

    Experimenters were not blinded to genotype or culture conditions, except in transcriptomic analysis where line genotype was not explicit.

    “Experimenters were not blinded to the genotypes or culture conditions, except for transcriptomic where line genotype was not explicit for analysis.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    In mouse, several microglia-specific genes are upregulated in brain-resident microglia through early postnatal development.

    “In the mouse, several microglia-specific genes appear to be upregulated in brain-resident microglia through early postnatal development 10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bennett, M.L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA 113, E1738–E1746 (2016). source

      Not checked in the free run.

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

    Premisecitation

    Microglia carry out key functions in brain development, including circuit refinement, neurogenesis, and neuronal growth.

    “It is well established that microglia perform key functions in the development of the brain such as in circuit refinement, neurogenesis, and neuronal growth 39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Wu, Y., Dissing-Olesen, L., MacVicar, B.A. & Stevens, B. Microglia: dynamic mediators of synapse development and plasticity. Trends Immunol. 36, 605–613 (2015). source

      Not checked in the free run.

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

    Interpretationcitation

    This is consistent with the current understanding that microglia derive from non-monocytic primitive myeloid precursors, unlike adult bone marrow-derived macrophages but similarly to some other tissue-resident macrophages.

    “This is consistent with our current understanding of microglial development, which supports the notion that microglia derive from non-monocytic primitive myeloid cells acting as precursors, unlike adult bone marrow-derived macrophages, but similarly to some other tissue-resident macrophages 37.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    pMGLs are highly phagocytic and, based on phenotypic and gene expression profiles, resemble primary fetal and adult human microglia.

    “We demonstrated that pMGLs are highly phagocytic and, based on their phenotypic and gene expression profiles, resemble primary fetal and adult human microglia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Iron salts were omitted and iron-loaded transferrin used instead to prevent dangerous redox cycling via Fenton chemistry.

    “Iron salts were excluded, instead using iron-loaded transferrin in Neurobasal, in order to avoid dangerous redox cycling through Fenton chemistry.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Biotin and lipid-loaded albumin were added to support de novo synthesis of lipid bilayers such as axons and myelin sheaths.

    “Biotin and lipid-loaded albumin (Albumax) were added to support de novo synthesis lipid bilayers (e.g. axons, myelin sheaths).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Ionic concentrations were set to match human cerebrospinal fluid osmolality, providing extracellular sodium levels required for proper electrophysiological function.

    “Ionic concentrations were chosen to match human cerebrospinal fluid osmolality 45, thus providing extracellular sodium concentrations necessary for proper electrophysiological function.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Harrington, M.G. et al. Cerebrospinal fluid sodium rhythms. Cerebrospinal Fluid Res. 7, 3 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The NGD medium component concentrations were adjusted to match human cerebrospinal fluid, supplying metabolic substrates needed by individual cell types grown without others.

    “We adjusted the component concentrations to match those of human cerebrospinal fluid, providing metabolic substrates necessary for individual cell types growing in the absence of others.”
    Quote in the paperSubstantial claim
51 of 51

  • The paper relies on this without support.

    Neuroglial derivatives from neural progenitors are poorly adherent on Primaria and are therefore selected against by this substrate.

    “In contrast, neuroglial derivatives from neural progenitors are poorly adherent, and thus selected against on this substrate (not shown).”

  • The paper relies on this without support.

    Human fetal microglia maintained in the same culture conditions retain characteristics of bona fide primary microglia and are a valid reference for comparison.

    “Isolated human fetal microglia and pMGLs were grown in the same maintenance medium for a week (MGM).”

  • The paper relies on this without support.

    The 3D organotypic culture and focal laser injury model reproduce relevant in vivo microglial behavior.

    “We took advantage of our 3D culture system to model such localized damage and observe microglial behavior in real-time.”
3 of 3

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?

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Muffat, J., Li, Y., Yuan, B., Mitalipova, M., Omer, A., Corcoran, S., Bakiasi, G., Tsai, L.-H., Aubourg, P., Ransohoff, R. M., & Jaenisch, R.
Affiliations
  • Whitehead Institute for Biomedical Research (US)
  • Université Paris-Sud (FR)
  • Bryn Mawr College (US)
  • Massachusetts Institute of Technology (US)
  • Biogen (United States) (US)
Corresponding author
Jaenisch, Rudolf
ORCID
Venue
Nature Medicine
Publisher
Springer Science and Business Media LLC
Published
2016-09-26
Volume
22
Issue
11
Pages
1358-1367
ISSN
  • 1078-8956 (ISSN-L)
  • 1546-170X
DOI
10.1038/nm.4189
PMC
PMC5101156
OpenAlex
W2525538087
Semantic Scholar
7b38bfb134631f7ab7fe2f4f2b3bf5fb0475b9a5
Cited by
651
References
50
Citations by year
2026: 45 · 2025: 58 · 2024: 70 · 2023: 48 · 2022: 76 · 2021: 86 · 2020: 74 · 2019: 59 · 2018: 80 · 2017: 44 · 2016: 8 · 2015: 1
FWCI
33.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
  • Archivio Istituzionale della Ricerca - Università Vita-Salute San Raffaele (submittedVersion)
  • PMC
Funders
  • Howard Hughes Medical Institute
  • Simons Foundation (SFARI 204106)
  • International Rett Syndrome Foundation
  • Amgen
  • Biogen
  • National Alliance for Research on Schizophrenia and Depression
  • Simons Foundation Autism Research Initiative
  • Emerald Foundation
  • Association Européenne contre les Leucodystrophies
  • National Institutes of Health (2r01ca084198-10, 1rf1ag048029-01, 1rf1ag042978-01, HD 045022)
  • Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
  • Bhabha Atomic Research Centre
  • National Institute on Aging (RF1-AG048029, RF1-AG042978)
  • National Institute of Mental Health (R01MH104610)
  • National Cancer Institute (R01-CA084198, R37CA084198)
  • National Institute of Neurological Disorders and Stroke (R01NS088538)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD045022, R37HD045022)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
Keywords
microglia-like cells, human pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, microglia differentiation, serum-free culture, Rett syndrome, neurodegenerative diseases
MeSH terms
Human Embryonic Stem Cells, Cell Differentiation, Humans, Organ Culture Techniques, Rett Syndrome, Microglia, Induced Pluripotent Stem Cells, In Vitro Techniques

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 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review