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• W2794760100 abstract "•Microglia can be efficiently differentiated from human iPSCs•iPSC microglia resemble human primary microglia transcriptionally and functionally•Disease-causing mutations in TREM2 affect receptor processing and expression•TREM2 mutant microglia differentiate, respond to pathogenic stimuli, and phagocytose The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2), which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. Microglia are brain-resident immune cells that perform key functions during nervous system development and homeostasis. After colonization and maturation in the developing CNS (Ginhoux et al., 2010Ginhoux F. Greter M. Leboeuf M. Nandi S. See P. Gokhan S. Mehler M.F. Conway S.J. Ng L.G. Stanley E.R. et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3174) Google Scholar, Ransohoff and Cardona, 2010Ransohoff R.M. Cardona A.E. The myeloid cells of the central nervous system parenchyma.Nature. 2010; 468: 253-262Crossref PubMed Scopus (611) Google Scholar), microglia shape synaptic connections between neurons through synapse pruning and provision of trophic support (Parkhurst et al., 2013Parkhurst C.N. Yang G. Ninan I. Savas J.N. Yates 3rd, J.R. Lafaille J.J. Hempstead B.L. Littman D.R. Gan W.B. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.Cell. 2013; 155: 1596-1609Abstract Full Text Full Text PDF PubMed Scopus (1523) Google Scholar, Schafer et al., 2012Schafer D.P. Lehrman E.K. Kautzman A.G. Koyama R. Mardinly A.R. Yamasaki R. Ransohoff R.M. Greenberg M.E. Barres B.A. Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.Neuron. 2012; 74: 691-705Abstract Full Text Full Text PDF PubMed Scopus (2244) Google Scholar). As dynamic surveyors of the brain, microglia respond to damage- and pathogen-associated signals to maintain homeostasis (Koizumi et al., 2007Koizumi S. Shigemoto-Mogami Y. Nasu-Tada K. Shinozaki Y. Ohsawa K. Tsuda M. Joshi B.V. Jacobson K.A. Kohsaka S. Inoue K. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis.Nature. 2007; 446: 1091-1095Crossref PubMed Scopus (591) Google Scholar, Nimmerjahn et al., 2005Nimmerjahn A. Kirchhoff F. Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (3938) Google Scholar). In addition to developmental and homeostatic functions, it is now well established that microglia and associated neuroinflammation play roles in the progression of a number of neurodegenerative conditions (Perry et al., 2010Perry V.H. Nicoll J.A. Holmes C. Microglia in neurodegenerative disease.Nat. Rev. Neurol. 2010; 6: 193-201Crossref PubMed Scopus (1190) Google Scholar, Ransohoff, 2016Ransohoff R.M. How neuroinflammation contributes to neurodegeneration.Science. 2016; 353: 777-783Crossref PubMed Scopus (1067) Google Scholar), with variants or mutations in microglia-expressed genes linked directly to disease in some cases (Malik et al., 2015Malik M. Parikh I. Vasquez J.B. Smith C. Tai L. Bu G. LaDu M.J. Fardo D.W. Rebeck G.W. Estus S. Genetics ignite focus on microglial inflammation in Alzheimer's disease.Mol. Neurodegener. 2015; 10: 52Crossref PubMed Scopus (99) Google Scholar, Paloneva et al., 2002Paloneva J. Manninen T. Christman G. Hovanes K. Mandelin J. Adolfsson R. Bianchin M. Bird T. Miranda R. Salmaggi A. et al.Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype.Am. J. Hum. Genet. 2002; 71: 656-662Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, Sims et al., 2017Sims R. van der Lee S.J. Naj A.C. Bellenguez C. Badarinarayan N. Jakobsdottir J. Kunkle B.W. Boland A. Raybould R. Bis J.C. et al.Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease.Nat. Genet. 2017; 49: 1373-1384Crossref PubMed Scopus (516) Google Scholar). While microglial function and dysfunction are involved in many neurodegenerative conditions, their precise contributions to disease pathogenesis and progression are not well understood. Avenues for the study of microglial biology in disease have primarily been limited to animal models and immortalized cell lines, both of which carry limitations in their ability to approximate primary human microglia. As more is understood about the developmental origin and unique identity of microglia, recent studies have attempted to circumvent this issue by deriving microglia from human induced pluripotent stem cells (iPSCs) in order to study human and cell-type-specific biology and disease (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, Douvaras et al., 2017Douvaras P. Sun B. Wang M. Kruglikov I. Lallos G. Zimmer M. Terrenoire C. Zhang B. Gandy S. Schadt E. et al.Directed differentiation of human pluripotent stem cells to microglia.Stem Cell Reports. 2017; 8: 1516-1524Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, Haenseler et al., 2017Haenseler 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. et al.A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response.Stem Cell Reports. 2017; 8: 1727-1742Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Muffat et al., 2016Muffat J. Li Y. Yuan B. Mitalipova M. Omer A. Corcoran S. Bakiasi G. Tsai L.H. Aubourg P. Ransohoff R.M. et al.Efficient derivation of microglia-like cells from human pluripotent stem cells.Nat. Med. 2016; 22: 1358-1367Crossref PubMed Scopus (393) Google Scholar, Pandya et al., 2017Pandya H. Shen M.J. Ichikawa D.M. Sedlock A.B. Choi Y. Johnson K.R. Kim G. Brown M.A. Elkahloun A.G. Maric D. et al.Differentiation of human and murine induced pluripotent stem cells to microglia-like cells.Nat. Neurosci. 2017; 20: 753-759Crossref PubMed Scopus (237) Google Scholar, Takata et al., 2017Takata K. Kozaki T. Lee C.Z.W. Thion M.S. Otsuka M. Lim S. Utami K.H. Fidan K. Park D.S. Malleret B. et al.Induced-pluripotent-stem-cell-derived primitive macrophages provide a platform for modeling tissue-resident macrophage differentiation and function.Immunity. 2017; 47: 183-198.e6Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Here we describe and characterize a robust method for the derivation of microglia from human stem cells, which we then used to investigate the expressional and functional consequences of mutations in the microglia-expressed triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 is a transmembrane receptor expressed on cells of myeloid lineage, including osteoclasts and tissue-specific macrophages such as microglia (Colonna and Wang, 2016Colonna M. Wang Y. TREM2 variants: new keys to decipher Alzheimer disease pathogenesis.Nat. Rev. Neurosci. 2016; 17: 201-207Crossref PubMed Scopus (229) Google Scholar). Homozygous mutations in TREM2 or its intracellular signaling partner DAP12 are causal for Nasu-Hakola disease (NHD), which is associated with bone cysts and an early-onset dementia (Paloneva et al., 2000Paloneva J. Kestila M. Wu J. Salminen A. Bohling T. Ruotsalainen V. Hakola P. Bakker A.B. Phillips J.H. Pekkarinen P. et al.Loss-of-function mutations in TYROBP (DAP12) result in a presenile dementia with bone cysts.Nat. Genet. 2000; 25: 357-361Crossref PubMed Scopus (359) Google Scholar, Paloneva et al., 2002Paloneva J. Manninen T. Christman G. Hovanes K. Mandelin J. Adolfsson R. Bianchin M. Bird T. Miranda R. Salmaggi A. et al.Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype.Am. J. Hum. Genet. 2002; 71: 656-662Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar), while a frontotemporal dementia (FTD)-like syndrome without bone dysfunction has also been described in patients carrying certain TREM2 mutations (Chouery et al., 2008Chouery E. Delague V. Bergougnoux A. Koussa S. Serre J.L. Megarbane A. Mutations in TREM2 lead to pure early-onset dementia without bone cysts.Hum. Mutat. 2008; 29: E194-E204Crossref PubMed Scopus (96) Google Scholar, Giraldo et al., 2013Giraldo M. Lopera F. Siniard A.L. Corneveaux J.J. Schrauwen I. Carvajal J. Munoz C. Ramirez-Restrepo M. Gaiteri C. Myers A.J. et al.Variants in triggering receptor expressed on myeloid cells 2 are associated with both behavioral variant frontotemporal lobar degeneration and Alzheimer's disease.Neurobiol. Aging. 2013; 34: 2077.e11-2077.e18Crossref PubMed Scopus (113) Google Scholar, Guerreiro et al., 2013bGuerreiro R.J. Lohmann E. Bras J.M. Gibbs J.R. Rohrer J.D. Gurunlian N. Dursun B. Bilgic B. Hanagasi H. Gurvit H. et al.Using exome sequencing to reveal mutations in TREM2 presenting as a frontotemporal dementia-like syndrome without bone involvement.JAMA Neurol. 2013; 70: 78-84Crossref PubMed Scopus (255) Google Scholar). The recent discovery that heterozygous coding variants in TREM2 confer an increased risk of Alzheimer's disease (AD) (Guerreiro et al., 2013aGuerreiro R. Wojtas A. Bras J. Carrasquillo M. Rogaeva E. Majounie E. Cruchaga C. Sassi C. Kauwe J.S. Younkin S. et al.TREM2 variants in Alzheimer's disease.N. Engl. J. Med. 2013; 368: 117-127Crossref PubMed Scopus (1885) Google Scholar, Jin et al., 2014Jin S.C. Benitez B.A. Karch C.M. Cooper B. Skorupa T. Carrell D. Norton J.B. Hsu S. Harari O. Cai Y. et al.Coding variants in TREM2 increase risk for Alzheimer's disease.Hum. Mol. Genet. 2014; 23: 5838-5846Crossref PubMed Scopus (201) Google Scholar, Jonsson et al., 2013Jonsson T. Stefansson H. Steinberg S. Jonsdottir I. Jonsson P.V. Snaedal J. Bjornsson S. Huttenlocher J. Levey A.I. Lah J.J. et al.Variant of TREM2 associated with the risk of Alzheimer's disease.N. Engl. J. Med. 2013; 368: 107-116Crossref PubMed Scopus (1639) Google Scholar) has reignited interest in understanding the role of this receptor in microglial function. While the endogenous ligand has not been confirmed, in vitro studies have demonstrated binding of TREM2 to lipoprotein, apolipoprotein, and pathogen- and damage-associated ligands (Atagi et al., 2015Atagi Y. Liu C.C. Painter M.M. Chen X.F. Verbeeck C. Zheng H. Li X. Rademakers R. Kang S.S. Xu H. et al.Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2).J. Biol. Chem. 2015; 290: 26043-26050Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, Bailey et al., 2015Bailey C.C. 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Mazaheri F. et al.TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.Sci. Transl. Med. 2014; 6: 243ra286Crossref Scopus (473) Google Scholar, Kober et al., 2016Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.Elife. 2016; 5https://doi.org/10.7554/eLife.20391Crossref PubMed Scopus (110) Google Scholar, Park et al., 2015Park J.S. Ji I.J. An H.J. Kang M.J. Kang S.W. Kim D.H. Yoon S.Y. Disease-associated mutations of TREM2 alter the processing of N-linked oligosaccharides in the Golgi apparatus.Traffic. 2015; 16: 510-518Crossref PubMed Scopus (45) Google Scholar), while AD-associated variants are thought to reduce the affinity of TREM2 for its ligands (Kober et al., 2016Kober D.L. Alexander-Brett J.M. Karch C.M. Cruchaga C. Colonna M. Holtzman M.J. Brett T.J. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms.Elife. 2016; 5https://doi.org/10.7554/eLife.20391Crossref PubMed Scopus (110) Google Scholar, Yeh et al., 2016Yeh F.L. Wang Y. Tom I. Gonzalez L.C. Sheng M. TREM2 binds to apolipoproteins, including APOE and CLU/APOJ, and thereby facilitates uptake of amyloid-beta by microglia.Neuron. 2016; 91: 328-340Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Extensive studies have ascribed a number of functions to TREM2, including regulation of phagocytosis (Hsieh et al., 2009Hsieh C.L. Koike M. Spusta S.C. Niemi E.C. Yenari M. Nakamura M.C. Seaman W.E. A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia.J. Neurochem. 2009; 109: 1144-1156Crossref PubMed Scopus (298) Google Scholar, Kleinberger et al., 2014Kleinberger G. Yamanishi Y. Suarez-Calvet M. Czirr E. Lohmann E. Cuyvers E. Struyfs H. Pettkus N. Wenninger-Weinzierl A. Mazaheri F. et al.TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.Sci. Transl. Med. 2014; 6: 243ra286Crossref Scopus (473) Google Scholar, Takahashi et al., 2005Takahashi K. Rochford C.D. Neumann H. Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2.J. Exp. Med. 2005; 201: 647-657Crossref PubMed Scopus (763) Google Scholar), cytokine release (Hamerman et al., 2006Hamerman J.A. Jarjoura J.R. Humphrey M.B. Nakamura M.C. Seaman W.E. Lanier L.L. Cutting edge: inhibition of TLR and FcR responses in macrophages by triggering receptor expressed on myeloid cells (TREM)-2 and DAP12.J. Immunol. 2006; 177: 2051-2055Crossref PubMed Scopus (319) Google Scholar, Turnbull et al., 2006Turnbull I.R. Gilfillan S. Cella M. Aoshi T. Miller M. Piccio L. Hernandez M. Colonna M. Cutting edge: TREM-2 attenuates macrophage activation.J. 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Holtzman D.M. Altered microglial response to Abeta plaques in APPPS1-21 mice heterozygous for TREM2.Mol. Neurodegener. 2014; 9: 20Crossref PubMed Scopus (205) Google Scholar, Yuan et al., 2016Yuan P. Condello C. Keene C.D. Wang Y. Bird T.D. Paul S.M. Luo W. Colonna M. Baddeley D. Grutzendler J. TREM2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy.Neuron. 2016; 92: 252-264Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), the precise role of TREM2 in microglial biology and the consequences of its dysregulation in neurodegenerative disease pathogenesis remain to be determined. Therefore, we used our method for generating human microglia to study the expression, cellular localization, and function of TREM2 in microglia differentiated from iPSCs derived from individuals carrying TREM2 mutations causal for FTD-like syndrome and NHD. Microglia differ from other adult tissue-resident macrophages in two key ways; firstly, their yolk-sac-derived progenitors arise early in development from a program of primitive hematopoiesis rather than the later definitive hematopoiesis that replaces many tissue-resident macrophages in the developed adult (Ginhoux et al., 2010Ginhoux F. Greter M. Leboeuf M. Nandi S. See P. Gokhan S. Mehler M.F. Conway S.J. Ng L.G. Stanley E.R. et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3174) Google Scholar, Ginhoux et al., 2013Ginhoux F. Lim S. Hoeffel G. Low D. Huber T. Origin and differentiation of microglia.Front. Cell. Neurosci. 2013; 7: 45Crossref PubMed Scopus (524) Google Scholar, Kierdorf et al., 2013Kierdorf K. Erny D. Goldmann T. Sander V. Schulz C. Perdiguero E.G. Wieghofer P. Heinrich A. Riemke P. 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The PMP generation phase can continue indefinitely and is particularly efficient: in the longest ongoing differentiation in this study, one million PSCs produced between 23 and 52 million PMPs in seven PSC lines over 80 days, similar to previously reported PMP yields using the same method (Haenseler et al., 2017Haenseler 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. et al.A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response.Stem Cell Reports. 2017; 8: 1727-1742Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, van Wilgenburg et al., 2013van Wilgenburg B. Browne C. Vowles J. Cowley S.A. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.PLoS One. 2013; 8: e71098Crossref PubMed Scopus (166) Google Scholar) and microglia yields using a recently described alternative method (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Using complete RPMI1640 containing a combination of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34) (Ohgidani et al., 2014Ohgidani M. Kato T.A. Setoyama D. Sagata N. Hashimoto R. Shigenobu K. Yoshida T. Hayakawa K. Shimokawa N. Miura D. et al.Direct induction of ramified microglia-like cells from human monocytes: dynamic microglial dysfunction in Nasu-Hakola disease.Sci. Rep. 2014; 4: 4957Crossref PubMed Scopus (84) Google Scholar), we differentiated PMPs over 6–10 days to produce monocultures that morphologically resemble microglia (Figure 1A). Analysis of the proportion of these cells expressing canonical macrophage/microglia markers indicates that this protocol has a high level of efficiency across genetic backgrounds, producing cells 95.6% ± 3.6% positive for Iba1 (mean ± SD, n = 6), 95.0% ± 3.6% positive for CD45 (mean ± SD, n = 6), and 99.5% ± 0.4% positive for TREM2 (mean ± SD, n = 5) (Figure 1B). To investigate the transcriptional identity of our stem cell-derived microglia in the context of the wider myeloid family, we used RNA sequencing (RNA-seq) to compare the transcriptome of these microglia with a number of published datasets: primary ex vivo CNS CD45+ microglia/macrophages (Zhang et al., 2016Zhang Y. Sloan S.A. Clarke L.E. Caneda C. Plaza C.A. Blumenthal P.D. Vogel H. Steinberg G.K. Edwards M.S. Li G. et al.Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.Neuron. 2016; 89: 37-53Abstract Full Text Full Text PDF PubMed Scopus (1120) Google Scholar) and microglia (Gosselin et al., 2017Gosselin D. Skola D. Coufal N.G. Holtman I.R. Schlachetzki J.C.M. Sajti E. Jaeger B.N. O'Connor C. Fitzpatrick C. Pasillas M.P. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; 356https://doi.org/10.1126/science.aal3222Crossref PubMed Scopus (588) Google Scholar), primary microglia and fetal microglia cultured in vitro for 7–10 days (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, Gosselin et al., 2017Gosselin D. Skola D. Coufal N.G. Holtman I.R. Schlachetzki J.C.M. Sajti E. Jaeger B.N. O'Connor C. Fitzpatrick C. Pasillas M.P. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; 356https://doi.org/10.1126/science.aal3222Crossref PubMed Scopus (588) Google Scholar), myeloid cells of alternate lineages (monocyte-derived macrophages (Zhang et al., 2015Zhang H. Xue C. Shah R. Bermingham K. Hinkle C.C. Li W. Rodrigues A. Tabita-Martinez J. Millar J.S. Cuchel M. et al.Functional analysis and transcriptomic profiling of iPSC-derived macrophages and their application in modeling Mendelian disease.Circ. Res. 2015; 117: 17-28Crossref PubMed Scopus (79) Google Scholar), CD14+/CD16− monocytes (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) and dendritic cells (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar)), and iPSC-microglia derived using an alternative method (Abud et al., 2017Abud E.M. Ramirez R.N. Martinez E.S. Healy L.M. Nguyen C.H.H. Newman S.A. Yeromin A.V. Scarfone V.M. Marsh S.E. Fimbres C. et al.iPSC-derived human microglia-like cells to study neurological diseases.Neuron. 2017; 94: 278-293.e9Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar) (Figure 2). The recently published dataset from Gosselin et al., 2017Gosselin D. Skola D. Coufal N.G. Holtman I.R. Schlachetzki J.C.M. Sajti E. Jaeger B.N. O'Connor C. Fitzpatrick C. Pasillas M.P. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; 356https://doi.org/10.1126/science.aal3222Crossref PubMed Scopus (588) Google Scholar is a particularly useful addition to our understanding of microglial identity, as it compares the transcriptomes of freshly isolated ex vivo microglia with the same cells cultured in vitro for 7–10 days, allowing assessment of how culture environment alters the transcriptome. PMPs and microglia derived from three independent differentiations from two genetic backgrounds were harvested for RNA-seq analysis, in order to assess reproducibility of the differentiation process both within and between genetic backgrounds. At the whole-transcriptome level, PMPs and microglia generated by the method reported here most closely resemble cultured primary microglia (Figure 2A). Due to a lack of unique surface markers, it has historically been difficult to distinguish microglia from other macrophages and cells of myeloid lineage. It is only recently that a distinct transcriptomic profile of microglia has emerged (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar, Butovsky et al., 2014Butovsky O. Jedrychowski M.P. Moore C.S. Cialic R. Lanser A.J. Gabriely G. Koeglsperger T. Dake B. Wu P.M. Doykan C.E. et al.Identification of a unique TGF-beta-dependent molecular and functional signature in microglia.Nat. Neurosci. 2014; 17: 131-143Crossref PubMed S" @default.
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