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      Development and validation of a simplified method to generate human microglia from pluripotent stem cells

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          Abstract

          Background

          Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia.

          Results

          iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43 + hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFβ-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered.

          Conclusion

          We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.

          Electronic supplementary material

          The online version of this article (10.1186/s13024-018-0297-x) contains supplementary material, which is available to authorized users.

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          Most cited references13

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          Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.

          An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.
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            TGF-β signaling through SMAD2/3 induces the quiescent microglial phenotype within the CNS environment.

            Microglia are myeloid-derived cells that colonize the central nervous system (CNS) at early stages of development and constitute up to 20% of the glial populations throughout life. While extensive progress has been recently made in identifying the cellular origin of microglia, the mechanism whereby the cells acquire the unique ramified and quiescent phenotype within the CNS milieu remains unknown. Here, we show that upon co-culturing of either CD117(+) /Lin(-) hematopoietic progenitors or CD11c(+) bone marrow derived cells with organotypic hippocampal slices or primary glia, the cells acquire a ramified morphology concomitant with reduced levels of CD86, MHCII, and CD11c and up-regulation of the microglial cell-surface proteins CX(3) CR1 and Iba-1. We further demonstrate that the transforming growth factor beta (TGF-β) signaling pathway via SMAD2/3 phosphorylation is essential for both primary microglia and myeloid-derived cells in order to acquire their quiescent phenotype. Our study suggests that the abundant expression of TGF-β within the CNS during development and various inflammatory processes plays a key role in promoting the quiescent phenotype of microglia and may thus serve as a target for therapeutic strategies aimed at modulating the function of microglia in neurodegenerative diseases such as Alzheimer's and prion. Copyright © 2012 Wiley Periodicals, Inc.
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              The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia

              Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two progressive, fatal neurodegenerative syndromes with considerable clinical, genetic and pathological overlap. Clinical symptoms of FTD can be seen in ALS patients and vice versa. Recent genetic discoveries conclusively link the two diseases, and several common molecular players have been identified (TDP-43, FUS, C9ORF72). The definitive etiologies of ALS and FTD are currently unknown and both disorders lack a cure. Glia, specifically astrocytes and microglia are heavily implicated in the onset and progression of neurodegeneration witnessed in ALS and FTD. In this review, we summarize the current understanding of the role of microglia and astrocytes involved in ALS and FTD, highlighting their recent implications in neuroinflammation, alterations in waste clearance involving phagocytosis and the newly described glymphatic system, and vascular abnormalities. Elucidating the precise mechanisms of how astrocytes and microglia are involved in ALS and FTD will be crucial in characterizing these two disorders and may represent more effective interventions for disease progression and treatment options in the future.
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                Author and article information

                Contributors
                amcquade@uci.edu
                macoburn@uci.edu
                htu@uci.edu
                jhasselm@uci.edu
                hdavtyan@uci.edu
                949-824-5243 , mblurton@uci.edu
                Journal
                Mol Neurodegener
                Mol Neurodegener
                Molecular Neurodegeneration
                BioMed Central (London )
                1750-1326
                22 December 2018
                22 December 2018
                2018
                : 13
                : 67
                Affiliations
                [1 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Department of Neurobiology & Behavior, , University of California, ; 3014 Gross Hall, 845 Health Science Rd, Irvine, CA 92697-4545 USA
                [2 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Sue and Bill Gross Stem Cell Research Center, , University of California, ; 3014 Gross Hall, 845 Health Science Rd, Irvine, CA 92697-4545 USA
                [3 ]ISNI 0000 0001 0668 7243, GRID grid.266093.8, Institute for Memory Impairments and Neurological Disorders, , University of California, ; 3014 Gross Hall, 845 Health Science Rd, Irvine, CA 92697-4545 USA
                Article
                297
                10.1186/s13024-018-0297-x
                6303871
                30577865
                41bfe2e3-d65f-469d-be56-96101364334b
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 3 August 2018
                : 28 November 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: AG048099
                Award ID: AG056303
                Award ID: AG016573
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000900, California Institute for Regenerative Medicine;
                Award ID: RT3-07893
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;
                Award ID: NS082174
                Award Recipient :
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2018

                Neurosciences
                microglia,neurodegeneration,gwas,stem cells,ipscs,hematopoietic precursor cells,phagocytosis,tgfb,ide1,differentiation

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