Derived Human Microglia Manage Without Functional TREM2

Microglia. [Courtesy of National Institutes of Health.]

In the March 29 Stem Cell Reports, researchers led by Frederick Livesey at the University of Cambridge in England described a new protocol for generating microglia-like cells from human stem cells. Compared with a handful of other recently published microglia recipes, this one yields maximal numbers of cells using a relatively simple process. The microglia expressed genes on par with primary microglia cultured directly from the brain, buddied up with neurons in coculture, and responded to inflammatory stimuli. In microglia generated from people harboring pathogenic mutations in TREM2, the immune receptor’s trafficking was profoundly altered, yet the microglia gobbled up bacteria and pumped out cytokines just fine. However, some researchers wondered if these cells were tested in a physiologically meaningful paradigm.

The pivotal role of microglia in neurodegenerative disease makes the immune cells a key subject of study, but efforts to understand them outside of their physiological environment have been fraught with challenges. For one, the highly responsive cells are shifty by nature—removing them from the brain evokes almost immediate changes in gene expression (see Jun 2017 news). “You only have to look at them funny and they’ll get aggressive,” Livesey joked. The cells are also sparse in the mouse brain, and mouse microglia can only approximate the characteristics of their human counterparts. An emerging strategy to sidestep these issues is to make human microglia from scratch. In recent years, researchers have developed a handful of protocols that generate microglia from human stem cells, including the induced pluripotent variety (iPSCs) (see Jul 2016 conference news). Reasoning that the environment of the brain is essential for microglial development, some protocols utilize complex coculture systems to coax precursors into microglia (Haenseler et al., 2017; Takata et al., 2017; Pandya et al., 2016). Others rely entirely on cocktails of specific growth factors to steer the cells toward microgliahood (Muffat et al., 2016; Abud et al., 2017; Douvaras et al., 2017).

First author Philip Brownjohn and colleagues went with the cocktail approach, first generating primitive macrophage precursors (PMPs) from iPSCs. These cells approximate the macrophage precursors that arise from erythro-myeloid precursors in the yolk sac during fetal development and that give rise to brain-resident microglia. Other researchers previously developed protocols to establish them in culture (Karlsson et al., 2008; van Wilgenburg et al., 2013). Brownjohn found that iPSCs expanded by 20- to 50-fold into PMPs over 80 days. Then, by adding in other factors, including granulocyte colony-stimulating factor (GM-CSF) and IL-34, to replicate the environment of the developing brain, the researchers steered the PMPs into microglia-like cells within roughly a week. Ninety-five percent of the cells expressed typical microglial markers, including Iba-1 and CD45, while almost 100 percent of them expressed TREM2.

Making Microglia. Pluripotent stem cells grown in clumps (embryoid bodies) differentiated into primitive macrophage precursors and, finally, microglia. [Courtesy of Brownjohn et al., 2018, Stem Cell Reports.]

Brownjohn compared gene expression profiles to see how the derived cells measured up to primary microglia and to those developed via alternate methods (Zhang et al., 2016; Gosselin et al., 2017; Abud et al., 2017). Broadly, the transcriptome of the new microglia aligned well with those of other induced microglia, and of primary microglia that had been maintained in culture for a week. The transcriptome diverged from those reported for other types of myeloid cells such as monocytes and dendritic cells. While it also differed substantially from the transcriptome of freshly extracted microglia, it matched where it counts most: in expression of microglial signature genes (Hickman et al., 2013; Butovsky et al., 2014; Bennett et al., 2016). Livesey told Alzforum that while it would be preferable to generate microglia exactly like those in the brain, it may not be possible to escape what he called the “culture artifact.” He believes the PMP-derived microglia are a useful model to study processes in the brain.

In keeping with the status of such professional phagocytes, the induced microglia efficiently engulfed bacterial particles. And, when doused with lipopolysaccharide, the cells ramped up expression of inflammatory cytokines, including IL-1β, TNF-α, and IL-6. To see if the cells would interact appropriately with neurons, the researchers added them to three-dimensional cortical organoids. The microglia migrated deeply into the organoids and set up shop, extending probing processes to monitor their new environs.

Make Yourself at Home.
Microglia in culture (left) migrate deep into three-dimensional cortical organoids (right), spread out evenly, and take on a ramified shape. [Courtesy of Brownjohn et al., 2018, Stem Cell Reports.]

In a first stab at using these microglia to investigate processes related to neurodegeneration, the researchers next turned their sights on TREM2. They generated microglia from four iPSC sources: a patient with frontotemporal dementia who carried two copies of the T66M mutation in TREM2; two unaffected family members who carried only one copy of the mutant gene; and one person who carried two copies of the W50C mutation that causes Nasu-Hakola disease (NHD). All of the iPSC lines appeared to differentiate into microglia-like cells as well as those carrying two normal copies of TREM2. Using antibodies specific for different regions of TREM2, the researchers found that neither the W50C nor T66M variant trafficked normally to the cell surface, where the extracellular N-terminal domain is often shed following cleavage via ADAM10 protease. This cleavage leaves behind a C-terminal stub. Lysates from microglia bearing homozygous T66M or W50C mutations were devoid of this C terminal fragment. Overall, the findings indicated that the mutations disrupted TREM2 trafficking and processing, in agreement with studies reporting reduced maturation of the mutant protein in a mouse microglial cell line and in human embryonic kidney cells (Kleinberger et al., 2014).

How would this stunted maturation affect microglial responses? To address this, the researchers first provoked the T66M and W50C TREM2 microglia with lipopolysaccharide (LPS). They found no deficits in the inflammatory responses as compared with induced microglia carrying wild-type TREM2. Normal and mutant microglia also engulfed fluorescently labeled E. coli with equal gusto. A previous study reported that TREM2 facilitates the uptake of acetylated lipoproteins, along with associated Aβ, and that pathogenic TREM2 mutations or TREM2 deficiency impaired this process (Yeh et al., 2016). However, the mutant cells had no problem engulfing these lipoproteins. The results are seemingly at odds with previous studies reporting that the mutations affect phagocytic and other microglial functions (see May 2017 news).

Brownjohn and Livesey told Alzforum that the findings point to compensatory mechanisms—myriad other phagocytic receptors, for example—that could make up for TREM2’s loss of function in the mutant microglia.

In a joint comment to Alzforum, Christian Haass of the German Center for Neurodegenerative Diseases and Dominik Paquet of University Hospital, both in Munich, agreed that compensatory mechanisms were likely afoot, pointing out that they, too, had observed similar compensatory effects when assessing antibody-mediated phagocytosis of Aβ plaques in TREM2-deficient cells (Xiang et al., 2016).

Damian Crowther at AstraZeneca in Cambridge, England, offered a cautionary note. He referred to LPS as the “nuclear option,” pointing out that it maxes out pro-inflammatory responses and even downregulates TREM2 expression. This could mask differences between TREM2 mutants and wild-type cells, he said. “Of greater interest in neurodegenerative disease might be the interaction of the TREM2 pathway with weaker TLR4 agonists, such as amyloid-containing debris (reviewed in Molteni et al., 2016),” he wrote.

Likewise, Oleg Butovsky of Brigham and Women’s Hospital in Boston said he would like to see how the transcriptomes of the microglial cells change in response to disease-relevant stimuli, such as material from dying neurons. He previously reported that resting microglia express a homeostatic gene-expression signature, which is rapidly lost upon stimulation of TREM2 by neuronal detritus (see Feb 2015 conference news; Sep 2017 news). Whether the TREM2 mutant microglia developed by Brownjohn and colleagues are deficient in changing gears will be a crucial next question to address with this beautiful cellular model, he said.

Brownjohn and Livesey told Alzforum that these experiments are already in the works, assessing how the cells migrate and respond to multiple threats in coculture with neurons.

How do Brownjohn’s microglia measure up to cells developed by other protocols? The jury is still out on that, according to Haass and Paquet. “At the current—still pioneering— state of the field, independent studies confirming transferability of protocols, validity of cellular fates/transcriptomics, as well as applicability for disease research are crucial. The recent paper by the Livesey lab therefore is a welcome addition,” they wrote.

Featured Paper

Brownjohn PW, Smith J, Solanki R, Lohmann E, Houlden H, Hardy J, Dietmann S, Livesey FJ. Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia. Stem Cell Reports, March 29, 2018. PubMed.


Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response. Stem Cell Reports. 2017 Jun 6;8(6):1727-1742. PubMed.

Takata K, Kozaki T, Lee CZ, Thion MS, Otsuka M, Lim S, Utami KH, Fidan K, Park DS, Malleret B, Chakarov S, See P, Low D, Low G, Garcia-Miralles M, Zeng R, Zhang J, Goh CC, Gul A, Hubert S, Lee B, Chen J, Low I, Shadan NB, Lum J, Wei TS, Mok E, Kawanishi S, Kitamura Y, Larbi A, Poidinger M, Renia L, Ng LG, Wolf Y, Jung S, Önder T, Newell E, Huber T, Ashihara E, Garel S, Pouladi MA, Ginhoux F. Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function. Immunity. 2017 Jul 18;47(1):183-198.e6.  PubMed.

Pandya H, Shen MJ, Ichikawa DM, Sedlock AB, Choi Y, Johnson KR, Kim G, Brown MA, Elkahloun AG, Maric D, Sweeney CL, Gossa S, Malech HL, McGavern DB, Park JK. Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nat Neurosci. 2017 May;20(5):753-759. PubMed.

Muffat J, Li Y, Yuan B, Mitalipova M, Omer A, Corcoran S, Bakiasi G, Tsai LH, Aubourg P, Ransohoff RM, Jaenisch R. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nat Med. 2016 Nov;22(11):1358-1367. Epub 2016 Sep 26 PubMed.

Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017 Apr 19;94(2):278-293.e9. PubMed.

Karlsson KR, Cowley S, Martinez FO, Shaw M, Minger SL, James W. Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3. Exp Hematol. 2008 Sep;36(9):1167-75. Epub 2008 Jun 11  PubMed.

van Wilgenburg B, Browne C, Vowles J, Cowley SA. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions. PLoS One. 2013;8(8):e71098. Epub 2013 Aug 12 PubMed.

Douvaras P, Sun B, Wang M, Kruglikov I, Lallos G, Zimmer M, Terrenoire C, Zhang B, Gandy S, Schadt E, Freytes DO, Noggle S, Fossati V. Directed Differentiation of Human Pluripotent Stem Cells to Microglia. Stem Cell Reports. 2017 Jun 6;8(6):1516-1524. PubMed.

Zhang Y, Sloan SA, Clarke LE, Caneda C, Plaza CA, Blumenthal PD, Vogel H, Steinberg GK, Edwards MS, Li G, Duncan JA 3rd, Cheshier SH, Shuer LM, Chang EF, Grant GA, Gephart MG, Barres BA. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse. Neuron. 2016 Jan 6;89(1):37-53. Epub 2015 Dec 10 PubMed.

Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JC, Sajti E, Jaeger BN, O’Connor C, Fitzpatrick C, Pasillas MP, Pena M, Adair A, Gonda DD, Levy ML, Ransohoff RM, Gage FH, Glass CK. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017 Jun 23;356(6344) Epub 2017 May 25  PubMed.

Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang LC, Means TK, El Khoury J. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. 2013 Dec;16(12):1896-905. Epub 2013 Oct 27 PubMed.

Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014 Jan;17(1):131-43. Epub 2013 Dec 8 PubMed.

Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A, Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG, Barres BA. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A. 2016 Mar 22;113(12):E1738-46. Epub 2016 Feb 16 PubMed.

Kleinberger G, Yamanishi Y, Suárez-Calvet M, Czirr E, Lohmann E, Cuyvers E, Struyfs H, Pettkus N, Wenninger-Weinzierl A, Mazaheri F, Tahirovic S, Lleó A, Alcolea D, Fortea J, Willem M, Lammich S, Molinuevo JL, Sánchez-Valle R, Antonell A, Ramirez A, Heneka MT, Sleegers K, van der Zee J, Martin JJ, Engelborghs S, Demirtas-Tatlidede A, Zetterberg H, Van Broeckhoven C, Gurvit H, Wyss-Coray T, Hardy J, Colonna M, Haass C. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci Transl Med. 2014 Jul 2;6(243):243ra86. PubMed.

Yeh FL, Wang Y, Tom I, Gonzalez LC, Sheng M. TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia. Neuron. 2016 Jul 20;91(2):328-40. PubMed.

Molteni M, Gemma S, Rossetti C. The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation. Mediators Inflamm. 2016;2016:6978936. Epub 2016 May 18 PubMed.

Further Reading

Salter MW, Stevens B. Microglia emerge as central players in brain disease. Nat Med. 2017 Sep 8;23(9):1018-1027. PubMed.

Serio, A, Patani R. The Cellular Conspiracy of Amyotrophic Lateral Sclerosis. Stem Cells. 2018 Mar;36(3):293-303. PubMed.

Myszczynska M, Ferraiuolo L. New In Vitro Models to Study Amyotrophic Lateral Sclerosis. Brain Pathol. 2016 Mar;26(2):258-65. PubMed.

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disease-ad disease-als inflammation iPSCs microglia neuroinflammation organoids phagocytosis topic-newmethods topic-preclinical topic-researchmodels
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