iPSC-derived human microglia-like cells to study neurological diseases

The approach successfully produced iMGLs from 10 independent iPSC lines.

“This approach was used to successfully produce iMGLs from 10 independent iPSC lines (Figure S1A–C).”

iMGLs express microglial-enriched proteins including Mertk, Itgb5, Cx3cr1, Tgfbr1, and Pros1.

“By day 38, iMGLs resemble human microglia, but not monocytes nor macrophages by cytospin/Giemsa staining (Figure 1G) and express many other microglial-enriched proteins including Mertk, Itgb5, Cx3cr1, Tgfbr1, and Pros1 protein (Figures 1H and 4S).”

iMGLs developing in vitro express PU.1, TREM2, and CD11bint/CD45low, like murine microglia in vivo.

“Like murine microglia development in vivo, iMGLs developing in vitro express PU.1, TREM2, and CD11bint/CD45low (Figure 1Aiii, D–F).”

iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo.

“Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo.”

As iMGLs mature in vitro, they become more ramified, similar to microglia in vivo.

“As iMGLs mature in vitro, they also become more ramified, similar to microglia in vivo (Figure 1Aiv, 1H).”

FACS sorting for CD43+ cells produces iHPCs with greater than 90% purity.

“FACS sorting for CD43+ cells reveal that our approach produces iHPCs with a >90% purity (Figure 1Bii).”

A representative differentiation across the entire probeset (n=383) showed high correlation between iPSC and iMGL genomes (r2 > 0.92).

“A comparison of a representative differentiation across the entire probeset (n=383, nCounter® Human Karyotype Panel) revealed a high correlation between the iPSC and iMGL genomes (r2 > 0.92, Figure S1F).”

iMGLs secrete cytokines in response to inflammatory stimuli.

“Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates.”

Day 14 early iMGLs are c-kit−/CD45+.

“Day 14 early iMGLs were c-kit−/CD45+ (Figure 1C), suggesting commitment towards a myeloid lineage.”

The iMGL transcriptome is highly similar to human primary fetal and adult microglia.

“Correlational analysis and Principal Component Analysis (PCA) revealed striking similarity of iMGLs (blue) to Fetal MG (orange) and Adult MG (green) (Figures 2A and S3A).”

A two-step fully-defined protocol generates iMGLs from iPSCs in just over five weeks.

“A two-step fully-defined protocol was developed to efficiently generate microglia-like cells (iMGLs) from iPSCs in just over five weeks (Figure 1A).”

iMGLs develop in vitro in a manner similar to microglia in vivo.

“We find that iMGLs develop in vitro similarly to microglia in vivo and whole transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia.”

CD43+ iHPCs grown with CSF-1, IL-34, and TGFβ1 express PU.1 and TREM2 by day 14.

“Next, CD43+ iHPCs were grown in serum-free differentiation medium (formulated in house) containing CSF-1, IL-34, and TGFβ1. By day 14, cells expressed the myeloid-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (Figure 1Aiii) demonstrating an early commitment toward microglial fate.”

The protocol typically yielded 30-40 million iMGLs from one million iPSCs.

“Importantly, this protocol typically yielded 30–40 million iMGLs from one million iPSCs, suggesting that our approach can be readily scaled-up for high content screening.”

Whole transcriptome analysis shows that iMGLs are highly similar to cultured adult and fetal human microglia.

“We find that iMGLs develop in vitro similarly to microglia in vivo and whole transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia.”

• Also stated in: § iMGLs resemble human fetal and adult microglia · boosted

  “Correlational analysis and Principal Component Analysis (PCA) revealed striking similarity of iMGLs (blue) to Fetal MG (orange) and Adult MG (green) (Figures 2A and S3A).”

P2ry12 and Trem2 co-expression is enriched in iMGLs compared to monocytes.

“Furthermore, purinergic receptor P2ry12 and Trem2 co-expression was enriched in iMGLs when compared to monocytes and quantification reveals our protocol yielded iMGLs of high purity (>97.2%, n=5) (Figures 1I and S2A, B).”

iMGLs robustly phagocytose CNS substrates.

“Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates.”

The resulting iHPCs resembled a commercial source from Cellular Dynamics International.

“The resulting iHPCs resembled a commercial source (Cellular Dynamics International) and represent the hematopoietic progenitor used to generate iMGLs.”

The protocol yields primitive iHPCs that are CD43+/CD235a+/CD41+ after 10 days.

“Our protocol (depicted in Figure 1Bi) yields primitive iHPCs that are CD43+/CD235a+/CD41+ after 10 days (Kennedy et al., 2007; Sturgeon et al., 2014).”

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The protocol yielded iMGLs of high purity (>97.2%, n=5).

“Furthermore, purinergic receptor P2ry12 and Trem2 co-expression was enriched in iMGLs when compared to monocytes and quantification reveals our protocol yielded iMGLs of high purity (>97.2%, n=5) (Figures 1I and S2A, B).”

Co-culturing increased expression of microglial genes CABLES1, TRIM14, MITF, MMP2, and SLC2A5.

“Additionally, we saw increased expression of microglial genes CABLES1, TRIM14, MITF, MMP2, and SLC2A5.”

In their basal state, iMGLs and microglia express many AD-GWAS-related genes including those without murine orthologs, such as CD33, MS4A4A, and CR1.

“In their investigated basal state, iMGLs and microglia express many AD-GWAS-related genes including those without murine orthologs i.e. CD33, MS4A4A, CR1.”

The Mertk inhibitor UNC569 only marginally decreased iMGL human synaptosome phagocytosis by 12.6% (p<0.05).

“In contrast, an inhibitor of Mertk (UNC569), also implicated in synaptic pruning, only marginally decreased iMGL hS phagocytosis (−12.6%, *p<0.05)(Figure 3H, I).”

Differential analysis between fetal and adult microglia identified pathways related to responses to environment such as migration and phagocytosis regulation, but not key myeloid genes in fetal microglia.

“Differential analysis between Fetal MG and Adult MG identified pathways related to responses to environment like migration and phagocytosis regulation, but not key myeloid genes in Fetal MG.”

Six genes were differentially elevated in fAβ compared to BDTOs.

“In addition, 6 genes were differentially elevated in fAβ compared to BDTOs (Table S3).”

CD200 and CX3CL1 modulated iMGL response to CNS stimuli such as fAβ.

“Importantly, we found that CD200 and CX3CL1 modulated iMGL response to CNS stimuli, such as fAβ.”

iMGLs recognize and internalize pHrodo-labeled brain-derived tau oligomers.

“iMGLs also recognize and internalize pHrodo-labled brain–derived tau oligomers (BDTOs) (Figure 4B).”

Co-culturing microglia with neurons increased expression of Siglec11 and Siglec12.

“In contrast, co-culturing microglia with neurons increased expression of Siglec11 and 12, human-specific sialic-acid binding proteins that interact with the neuronal glycocalyx, function in neuroprotection, and suppress pro-inflammatory signaling, and thus maintain a microglia homeostatic state (Linnartz-Gerlach et al., 2014; Wang and Neumann, 2010).”

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Neuronal co-culturing upregulated 156 and downregulated 244 iMGL genes.

“Differential gene expression analysis revealed that neuronal co-culturing upregulated 156 and downregulated 244 iMGL genes (Figure 5C,D).”

iMGLs express other CNS disease-related genes including APP, PSEN1/2, HTT, GRN, TARDBP, LRRK2, C9orf72, SOD1, VCP, and FUS.

“In addition to AD GWAS genes, iMGLs express other CNS disease-related genes including APP, PSEN1/2, HTT, GRN, TARDBP, LRRK2, C9orf72, SOD1, VCP, and FUS and therefore, can likely be used to study other neurological diseases such as ALS, HD, FTD, and DLB in which microglia may play a prominent role (Bachstetter et al., 2015; Crotti et al., 2014; Lui et al., 2016; O’Rourke et al., 2016)(Figure S6C).”

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iMGLs, fetal microglia, and adult microglia express canonical microglial genes such as P2RY12, GPR34, C1Q, CABLES1, BHLHE41, TREM2, ITAM PROS1, APOE, SLCO2B1, SLC7A8, PPARD, and CRYBB1.

“Importantly, iMGLs, Fetal MG, and Adult MG express canonical microglial genes such as P2RY12, GPR34, C1Q, CABLES1, BHLHE41, TREM2, ITAM PROS1, APOE, SLCO2B1, SLC7A8, PPARD, and CRYBB1 (Figure 2C and Table S1).”

iMGLs differentially release cytokines and chemokines based on cell-surface receptor stimuli, closely aligning with responses observed in acutely isolated primary microglia.

“Collectively, iMGLs differentially release cytokines/chemokines based on their cell-surface receptor stimuli, a finding that closely aligns with the responses observed in acutely isolated primary microglia (Rustenhoven et al., 2016).”

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BDTOs increased expression of 4 genes including CD2AP (4.62 ± 0.45).

“BDTOs increased expression of 4 genes including CD2AP (4.62 ± 0.45), previously implicated in tau-mediated toxicity (Shulman et al., 2014).”

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iMGL phagocytosis of pHrodo-labeled human synaptosomes was less robust than that of MD-Mφ.

“In comparison to MD-Mφ, iMGL phagocytosis of pHrodo-labeled hS was less robust (Figure 3F, G).”

Many differentially expressed genes are core microglial signature targets including P2RY12, TGFβR1, CD33, transcription factors EGR1 and ETV5, and APOE.

“Many of the differentially expressed genes are identified as core microglial signature targets including P2RY12, TGFβR1, and CD33, and transcription factors EGR1 and ETV5, and APOE (Figure S5A–C).”

Cells exposed to CD200 and CX3CL1 responded differentially to fAβ, increasing expression of genes involved in neuronal cell surface motif recognition or phagocytosis of CNS substrates, including MS4A genes, TREM2, TYROBP, CD33, and ABCA7.

“Whereas cells exposed to these two factors responded differentially to fAβ, increasing expression of genes involved in neuronal cell surface motif recognition, or phagocytosis of CNS substrates, including MS4A genes, TREM2, TYROBP, CD33, and ABCA7 (Bradshaw et al., 2013; Fu et al., 2016; Greer et al., 2016)(Figure S6B).”

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iMGLs preferentially internalized human synaptosomes compared to E. coli particles when normalized to MD-Mφ.

“However, iMGLs preferentially internalized hS when compared to E. coli particles and normalized to MD-Mφ (Figure S4D, E) supporting the notion that iMGLs and microglia are more polarized toward homeostatic functions than MD-Mφ.”

iMGLs express the microglial-enriched purinergic receptor P2ry12.

“iMGLs express the microglial-enriched purinergic receptor P2ry12, which sense extracellular nucleotides from degenerating neurons, and is critical for microglial homeostatic function (De Simone et al., 2010; Moore et al., 2015) (Figure S2A,B).”

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In response to IFNγ or IL-1β, iMGLs secrete 8 different chemokines including TNFα, CCL2, CCL4, and CXCL10.

“However, in response to IFNγ or IL-1β, iMGLs secrete 8 different chemokines including TNFα, CCL2, CCL4, and CXCL10.”

In the absence of CD200 and CX3CL1, fAβ stimulated expression of AD-GWAS genes such as CLU (APOJ).

“In the absence of CD200 and CX3CL1, fAβ stimulated the expression of AD-GWAS genes implicated in interacting with misfolded folded protein, surface receptors, or anti-apoptotic events such as CLU (APOJ) (Yeh et al., 2016).”

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Using an additive-free CD11b antibody, iMGL phagocytosis of human synaptosomes was significantly reduced by 40.0% (p<0.0001).

“Using an additive-free CD11b antibody, iMGL phagocytosis of hS was significantly reduced (−40.0%, ***p<0.0001)(Figure 3H, I).”

Basally, iMGLs secrete 10 of the examined cytokines at low but detectable levels.

“Basally, iMGLs secrete 10 of the examined cytokines at low but detectable levels (Table S2).”

iMGLs internalize fluorescently labeled fibrillar Aβ like primary microglia.

“Like primary microglia, iMGLs internalize fluorescently labeled fibrillar Aβ (Figure 4B).”

iMGLs robustly responded to LPS with induction of all measured cytokines except CCL3.

“As expected, iMGLs robustly responded to LPS with induction of all measured cytokines except for CCL3 (Table S2 for values).”

Gene ontology highlights neurodegenerative disease pathways including Alzheimer's, Parkinson's, and Huntington's diseases that are TGFβ dependent.

“Examination of gene ontology highlight neurodegenerative disease pathways including AD, Parkinson’s, and Huntington’s diseases that are TGFβ dependent (Figure S5D).”

Hierarchical clustering using 25 AD-GWAS genes demonstrates that iMGLs resemble microglia and not peripheral myeloid cells.

“Hierarchical clustering using just these 25 AD-GWAS genes also demonstrates that iMGLs resemble microglia and not peripheral myeloid cells (Figure 4A).”

Following fAβ exposure, iMGLs increased expression of 10 genes including ABCA7 (5.79 ± 0.44), CD33 (6.02 ± 0.41), TREM2 (4.86 ± 0.50), and APOE (2.52 ± 0.19).

“Following fAβ exposure, iMGLs increased expression of 10 genes (Table S3) including ABCA7 (5.79 ± 0.44), CD33 (6.02 ± 0.41), TREM2 (4.86 ± 0.50, and APOE (2.52 ± 0.19), genes implicated in Aβ clearance/degradation.”

P2ry12 is expressed in highly ramified iMGLs resembling quiescent cortical microglia.

“At higher magnification, P2ry12 is expressed in highly ramified iMGLs resembling quiescent cortical microglia; the membrane distribution accentuates the finer extended processes (Figure 7B–D)(Baron et al., 2014).”

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A homeostatic microglial signature dependent on TGFβ was found in iMGLs.

“a TGFβ-dependent homeostatic microglia signature was identified”

By day 7, iMGLs tiled and extended varying degrees of ramified processes within the 3D organoid environment.

“By day 7, iMGLs (green) also tiled and extended varying degrees of ramified processes within the 3D organoid environment (Figure 6D–F).”

iMGLs secrete multiple cytokines after stimulation with IFNγ, IL-1β, and LPS.

“iMGLs secrete a variety of cytokines in response to IFNγ, IL-1β, and LPS stimulation.”

iMGLs express functional P2ry12.

“we likewise find that iMGLs robustly express functional P2ry12”

• AssumptionRests on an assumption: Microglia sense ADP release via purinergic receptors.

Blocking CR3 via anti-CD11b in iMGLs reduces phagocytosis of human synaptosomes.

“Here, we found that blocking CR3, via anti-CD11b, in iMGLs reduces phagocytosis of human synaptosomes.”

Whole-transcriptome PCA highlights the differentiation trajectory of iPSCs toward iHPCs and then iMGLs.

“Our whole-transcriptome PCA also highlights the differentiation trajectory of iPSCs toward iHPCs, and then iMGLs.”

A commercial hematopoietic progenitor source can be differentiated into iMGLs using the authors' protocol.

“We have also identified a commercial HPC source that can be readily differentiated to iMGLs with our protocol”

Aβ fibrils raise the expression of APOE, TREM2, CD33, and APOJ in iMGLs.

“Aβ fibrils upregulated APOE, TREM2, CD33, and APOJ”

Aβ fibrils raise MS4A6A expression in iMGLs.

“We also found that MS4A6A gene expression was increased.”

CD200 and CX3CL1 are important developmental CNS signals for producing iMGLs that respond correctly to Aβ.

“CD200 and CX3CL1 were important developmental CNS cues for generating iMGLs that can respond appropriately to Aβ.”

Tmem119 and Iba1 were expressed in both hCyto+ soma and highly arborized iMGL processes.

“Tmem119 and Iba1 were also expressed in both hCyto+ soma and in highly arborized iMGL processes (Figure 7E–L).”

A number of iMGLs began to phagocytose fibrillar Aβ.

“A number of iMGLs also began to phagocytose fibrillar Aβ (Figures 7N–P and S7E–H).”

Transplanted human iMGLs co-expressing ku80 and P2ry12 were abundant within MITRG brains, suggesting long-term engraftment potential.

“Transplanted human iMGLs co-expressing both ku80 and P2ry12 were abundant within MITRG brains suggesting long-term engraftment potential (Figure 7A–D).”

Co-culture with neurons changes microglial gene expression.

“neuronal co-culture can further modulate microglial gene expression”

Contact with the neuronal glycocalyx raises Siglec expression in iMGLs.

“interactions with the neuronal glycocalyx increase Siglec expression”

iMGLs take up several neuronal substrates, including beta-amyloid, tau oligomers from brain, and human synaptosomes.

“iMGLs phagocytose a variety of neuronal substrates including beta-amyloid, brain-derived tau oligomers, and human synaptosomes”

The authors developed a reproducible method that yields iPSC-derived hematopoietic progenitors and microglia-like cells in large numbers for functional testing.

“we have developed a highly reproducible protocol to generate iPSC-derived hematopoietic progenitor cells and microglia-like cells in high yield that can be used in functional assays”

The authors report a serum-free, fully defined method for making microglial cells from iPSCs.

“Our study is one of the first to describe a fully-defined, serum-free protocol for generating microglial cells from induced pluripotent stem cells”

iMGLs migrated and extended processes toward Aβ plaques to begin walling them off.

“In many instances, iMGLs migrated and extended processes towards Aβ plaques to begin walling them off (Figure 7N–P, and Supplementary Video 1).”

The TGFβ-dependent homeostatic signature matched findings in murine studies.

“that paralleled murine studies”

iMGLs grown without neurons differentially express early microglial genes, including FFAR2.

“iMGLs not cultured with neurons differentially expressed early microglia genes including FFAR2”

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Within the brain, transplanted iMGLs with many branches varied in morphology.

“transplanted, highly ramified iMGLs were morphological heterogeneous within the brain”

iMGLs react in an appropriate way to accumulating neurotoxic Aβ.

“iMGLs can respond appropriately to the neurotoxic build-up of Aβ”

Microglia originate from yolk sac erythromyeloid progenitors generated during primitive hematopoiesis.

“Elegant lineage tracing studies show that microglia originate from yolk sac erythromyeloid progenitors (EMP) generated during primitive hematopoiesis (Ginhoux et al., 2010; Kierdorf et al., 2013; Schulz et al., 2012).”

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Murine studies identified key cytokines, cell receptors, and transcription factors required for microglia development and survival in vivo.

“Recently, murine studies identified key cytokines, cell receptors, and transcription factors required for microglia development and survival in vivo.”

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Microglia are implicated in the neuroinflammatory component of Alzheimer's disease etiology, including cytokine and chemokine secretion that worsens pathology.

“Microglia are also implicated in the neuroinflammatory component of AD etiology, including cytokine/chemokine secretion, which exacerbate disease pathology (Guillot-Sestier and Town, 2013).”

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In Alzheimer's disease, microglia cluster around beta-amyloid plaques, indicating an inability to clear beta-amyloid.

“In AD, microglia cluster around beta-amyloid plaques highlighting their inability to clear beta-amyloid (Hickman et al., 2008; Liu et al., 2010).”

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The c-kit−/CD45+ phenotype of day 14 iMGLs suggests commitment toward a myeloid lineage.

“Day 14 early iMGLs were c-kit−/CD45+ (Figure 1C), suggesting commitment towards a myeloid lineage.”

Robust differentiation of iPSCs to hematopoietic progenitors is a critical prerequisite for generating iMGLs.

“A critical prerequisite is the robust differentiation of iPSCs to hematopoietic progenitors (iHPCs).”

The approach can be readily scaled up for high-content screening.

“Importantly, this protocol typically yielded 30–40 million iMGLs from one million iPSCs, suggesting that our approach can be readily scaled-up for high content screening.”

IL-34 and TGFβ-1 are among the factors required for microglia development and survival in vivo.

“These factors include IL-34 and TGFβ-1 (Butovsky et al., 2014; Greter et al., 2012; Wang et al., 2012; Yamasaki et al., 2014).”

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Expression of PU.1 and TREM2 by day 14 demonstrates early commitment toward a microglial fate.

“By day 14, cells expressed the myeloid-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (Figure 1Aiii) demonstrating an early commitment toward microglial fate.”

Many identified AD GWAS genes function in the maintenance of microglia homeostasis.

“suggesting many identified AD GWAS genes function in the maintenance of microglia homeostasis (Figure S5E) and underscoring the utility of iMGLs to interrogate AD GWAS gene function.”

CX3CL1 and CD200 are neuronal- and endothelial-derived cues that can further educate iMGLs toward an endogenous microglia phenotype.

“CX3CL1 and CD200 are both neuronal- and endothelial-derived cues that can further educate iMGLs toward an endogenous microglia phenotype (reviewed in (Kierdorf and Prinz, 2013).”

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iMGLs can likely be used to study other neurological diseases such as ALS, HD, FTD, and DLB.

“In addition to AD GWAS genes, iMGLs express other CNS disease-related genes including APP, PSEN1/2, HTT, GRN, TARDBP, LRRK2, C9orf72, SOD1, VCP, and FUS and therefore, can likely be used to study other neurological diseases such as ALS, HD, FTD, and DLB in which microglia may play a prominent role (Bachstetter et al., 2015; Crotti et al., 2014; Lui et al., 2016; O’Rourke et al., 2016)(Figure S6C).”

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iMGLs could be utilized to identify compounds in high-throughput drug-screening assays that enhance Aβ degradation or block exosome-mediated tau release.

“Together, these findings suggest that iMGLs could be utilized to identify compounds in high-throughput drug-screening assays that enhance Aβ degradation or block exosome-mediated tau release.”

iMGLs and microglia are more polarized toward homeostatic functions than MD-Mφ.

“However, iMGLs preferentially internalized hS when compared to E. coli particles and normalized to MD-Mφ (Figure S4D, E) supporting the notion that iMGLs and microglia are more polarized toward homeostatic functions than MD-Mφ.”

Both soluble and surface CNS cues are factors in microglia maturation.

“Overall, these results implicate both soluble and surface CNS cues as factors in microglia maturation (Biber et al., 2007)(Figure 5E,F).”

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Whole transcriptome RNA-seq confirmed the importance of CX3CL1, CD200, and TGFβ for establishing microglia in vitro.

“Whole transcriptome RNA-seq analysis confirmed the importance of these factors for establishing microglia in vitro (Figures S5 and S6).”

iMGLs can be used to study how altered expression of AD-GWAS genes influences microglia phenotype in a way that cannot be recapitulated in transgenic mice.

“Thus, iMGLs can be used to study how altered expression of these genes influence microglia phenotype in a way that cannot be recapitulated in transgenic mice.”

iMGLs can be used to study how AD genetic risk factors, including novel TREM2 variants, affect microglial function.

“that can be used to study the influence of AD genetic risk factors, including recently identified novel TREM2 variants (Sirkis et al., 2016) on microglia function”

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• AssumptionRests on an assumption: Several AD-GWAS genes, including CD33 and CR1, lack functionally similar murine orthologs.

The authors' data suggest that MS4A6A may influence interactions with Aβ.

“our data suggests a potential role in modulating Aβ interactions”

iMGLs are highly similar to cultured human adult and fetal microglia by transcriptomic and functional analyses.

“Importantly, iMGLs are highly similar to cultured human adult and fetal microglia by both transcriptomic and functional analyses.”

Cytokines released by microglia can alter the inflammatory environment of the CNS.

“Microglial-mediated cytokine secretion can further influence the inflammatory milieu in the CNS”

The series of microglial functional assays strengthens how iMGLs can be used to investigate microglia genes implicated in disease and understand physiological function in vitro and in vivo.

“Moreover, our series of microglial functional assays, only possible with a high yield and pure protocol, further strengthens how iMGLs can be used to investigate microglia genes implicated in disease and understand physiological function both in vitro and in vivo.”

The Muffat et al. approach uses hematopoietic cells derived from embryoid bodies as microglial precursors.

“their approach uses hematopoietic cells derived from embryoid bodies (EB) as microglia precursors”

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Pluripotency of all iPSC lines was verified by PluriTest and a functional identification kit.

“Pluripotency of all lines was confirmed via (http://pluritest.org) and further confirmed using the Human Pluripotent Stem Cell Functional Identification Kit”

An embryoid-body-based method with adhesion selection risks contamination by other cell types that arise spontaneously from embryoid bodies, such as neuroectoderm including astrocytes.

“One challenge with the use of an EB-based method and selection by cell adhesion (Muffat et al protocol) is the potential contamination by other cell types that spontaneously arise from EBs i.e. neuroectoderm including astrocytes.”

Transplanted iMGLs closely resemble quiescent microglia.

“closely resembling quiescent microglia”

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iMGLs may be useful for finding therapeutic compounds through high-throughput measurements of microglial physiology.

“Our data highlights the potential utility of iMGLs to identify therapeutic compounds via high throughput assessment of microglia physiology.”

AD GWAS genes act in microglial homeostasis.

“highlight that AD GWAS genes function in microglia homeostasis”

iMGLs offer a model for studying microglial roles in various proteinopathies.

“thus providing a model system to investigate the role of microglia in various proteinopathies”

iMGLs are a renewable source of patient-derived microglia-like cells for studying microglial roles in neurodegenerative disease.

“these results underscore the potential of iMGLs as a renewable source of patient-derived microglial-like cells for studying the role of microglia in neurodegenerative diseases”

Migration, calcium imaging, and cytokine secretion assays together indicate that iMGLs closely resemble brain-derived microglia.

“Together, migration, calcium imaging, and cytokine secretion assays not only validate iMGLs to be highly similar to brain-derived microglia”

• AssumptionRests on an assumption: Microglia sense ADP release via purinergic receptors.

The iPSC lines were verified as sterile and karyotypically normal by G-banding.

“iPSCs were confirmed to be sterile and karyotypically normal via G-banding (WiCell.org).”

The findings provide one of the first examples of quantitative evidence showing that human microglia engulf human synaptosomes predominantly via the CR3 axis, as implicated by transgenic mouse studies.

“Our findings provide one of the first examples, to our knowledge, of quantitative evidence showing human microglia engulfing human synaptosomes predominately via the CR3 axis, as implicated by transgenic mouse studies (Hong et al., 2016a).”

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CD200 and CX3CL1 likely act on promoter or enhancer regions to allow responses to stimuli previously seen in macrophages.

“These data implicate these factors in modulating promoter/enhancer regions that enables appropriate responses to stimuli previously shown for macrophages”

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Additional factors are required to educate microglia further into brain tissue-resident macrophages.

“suggesting that other factors are needed to further educate microglia as tissue-resident macrophages of the brain”