Mature Microglia Research Articles

The Generation and Functional Characterization of Human Microglia-Like Cells Derived from iPS and Embryonic Stem Cells.

The following protocol describes the generation of microglia cells from human-induced pluripotent stem cells (hiPSCs) using commercially available kits by StemCell Technologies. This protocol consists of three major steps: (1) Differentiation of hematopoietic precursor cells, (2) Microglia differentiation, and (3) Microglia maturation. Assays are described to characterize hematopoietic precursor cells and mature microglia.

A Step-by-step Protocol for Obtaining Mature Microglia from Mice.

In mice, microglial precursors in the yolk sac migrate to the brain parenchyma through the head neuroepithelial layer between embryonic days 8.5 (E8.5)-E16.5 and acquire their unique identity with a ramified form. Based on the microglial developmental process, we dissected the neuroepithelial layer (NEL) of E13.5 mice, which is composed of microglial progenitor and neuroepithelial cells. The NEL was bankable and expandable. In addition, microglial precursors were matured according to NEL culture duration. The matured microglia (MG; CD11b-positive cells) were easily isolated from the cultured NEL using a magnetic-activated cell sorting system and named NEL-MG. In conclusion, we obtained higher yields of adult-like microglia (mature microglia: NEL-MG) compared to previous in vitro surrogates such as neonatal microglia and microglial cell lines. Graphical abstract.

Regulation of intracellular innate immune amyloid beta (aβ) clearance

AbstractBackgroundInnate immune Aβ clearance may be involved in Alzheimer’s disease (AD) etiology and treatment response following anti‐amyloid immunotherapy. Regulation of intracellular Aβ clearance is not measured in AD risk‐ or diagnostic assessments, and pathways and mechanisms are insufficiently mapped. We present assays for Aβ catalysis in myeloid cells (THP‐1 cells and human iPSC microglia).MethodTHP‐1 cells were differentiated toward macrophages, and treated with various inhibitors including IDE inhibitor (6bK, Tocris),BACE1 inhibitor (LY2886721, Tocris), Nep inhibitor (Phosphoramidon, Tocris), Bafilomycin A1(Sigma)(1h) and Rapamycin (Sigma) (18h) before adding 100 ng/ml Aβ40 (2h), followed by 4h chase. For AD patient derived microglia, reprogrammed iPSCs were differentiated through a myeloid progenitor stage. Mature microglia were characterized by qPCR, Immunofluorescence and phagocytosis assay. Two immunoassays were developed on the Simoa platform using proprietary sheep monoclonal antibodies (Pre Diagnostics, Norway). Both assays employ an Aβ c‐terminal antibody for capture (AB.4D7). Assay 20‐x employs a specific Aβ20’ cut point antibody (NH2‐20.2B8) and assay x‐40 a linear mid‐domain antibody (AB.2A9) binding full length Aβ as detector.Result1) Innate immune cells (THP1, microglia) catabolize Aβ 1‐40 producing 20‐x fragments. 2) Inhibition of Nep and BACE1 reduces the production of 20‐x fragment of Aβ in THP1 cells. 3) Autophagy inhibitor BafA1 increases both 1‐40 and 20‐x fragments indicating the role of the endo‐lysosomal system in the degradation of full length and 20‐x Aβ. 4) Autophagy inducer, Rapamycin reduces both 1‐40 and 20‐x fragments indicating the role of mTOR mediated autophagy and the endo‐lysosomal system in the degradation of full length and 20‐x Aβ.Conclusion1) We demonstrate a novel assay for Aβ degradation by Innate immune cells including THP‐1 and iPSC derived microglia. 2) Inhibition of Nep and BACE1 reduces 20‐x peptide generation indicating this as a potential marker for Aβ. 3) Modulation of autophagy affects 20‐x peptide generation indicating the potential of targeting endo‐lysosomal and autophagic pathway against AD.

Biodegradable polymeric nanoparticles administered in the cerebrospinal fluid: Brain biodistribution, preferential internalization in microglia and implications for cell-selective drug release

Cell-selective drug release in the central nervous system (CNS) holds great promise for the treatment of many CNS disorders but it is still challenging. We previously demonstrated that polymeric nanoparticles (NPs) injected intra-parenchyma in the CNS can be internalized specifically in microglia/macrophages surrounding the injection site. Here, we explored NPs administration in the cerebrospinal fluid (CSF) to achieve a wider spreading and increased cell targeting throughout the CNS; we generated new NPs variants and studied the effect of modifying size and surface charge on NPs biodistribution and cellular uptake. Intra-cerebroventricular administration resulted in prevalent localization of the NPs in proximity to stem-cell niches, such as around the lateral ventricles, the subventricular zone and the rostral migratory stream. NPs internalization occurred preferentially in brain myeloid cells/microglia. We demonstrated that brain biodistribution and extent of internalization in microglia are influenced by NPs dimensions and can be improved by applying a transient disruption of the blood-brain barrier with mannitol, leading to NPs internalization in up to 25% of brain myeloid/microglia cells. A fraction of the targeted cells was positive for markers of proliferation or stained positive for stemness/progenitor-cell markers such as Nestin, c-kit, or NG2. Interestingly, through these newly formulated NPs we obtained controlled and selective release of drugs otherwise difficult to formulate (such as busulfan and etoposide) to the target cells, preventing unwanted side effects and the toxicity obtained by direct brain delivery of the not encapsulated drugs. Overall, these data provide proof of concept of the applicability of these novel NP-based drug formulations for achieving internalization not only in mature microglia but also possibly in more immature myeloid cells in the brain and pave the way for brain-restricted microglia-targeted drug delivery regimens.

AN AMYLOID-BETA-DERIVED PEPTIDE TRANSFORMS RAT GLIAL PROGENITOR CELLS INTO MICROGLIA

Septapeptides (“septas”), previously identified as meningitis-specific antigens, defined by a rubella virus monoclonal antibody, were found in human Monocyte Chemoattractant Protein (hMCP-1) and on the surface of meningitis-causing bacteria, viruses and spirochetes.1-3 Some bacterial septas were tested for Ca+2 mobilization through receptor-associated heterotrimeric G-protein binding on THP-1 cells, progenitor cells of circulating peripheral macrophages. Certain of the free septas acted on their own as mild agonists of Ca+2 mobilization. Their signal transduction activity may be mediated through a single (or a single class) of receptor, but the data do not link this with the MCP-1 receptor on THP-1 cells. These data support the notion that (1) infectious organisms may conserve and employ these sequences in order to facilitate their transport through the BBB to infect the CNS and (2) the hypothesis that the septas represent muteins of the MCP-1 active site for monocyte stem cell activation to macrophages. Other MCP muteins have since been identified in the AD-associated agents, amyloid beta and prions, as well as in viruses like HIV, known to establish chronic infections in the CNS. Rat glial progenitor cells in tissue culture 4-6 were used to test for hMCP-1 activity in septas derived from amyloid beta. Nanomolar concentrations of the amyloid beta septa 13HHQKLVF19 were found to transform more than 60% of rat glial progenitor cells into mature microglia in tissue culture7.8. These data support the hypothesis that an amyloid beta-derived septa may serve to activate stem cells to increase the number of microglia available to combat chronic infection in the CNS. 1VanAlstyne & Sharma. April 23, 1996. US Patent No. 5,510,264. 2VanAlstyne & Sharma. Sept. 17, 1996. US Patent No. 5,556,757. 3Van Alstyne & Sharma. Dec. 6, 2011. US Patent No. 8,071,102. 4Singh and Van Alstyne. 1978.Brain Res. 155:418-421. 5Pope and Van Alstyne. 1981.Virology 113(2):776-780. 6Van Alstyne and Paty. 1983.Virology 124:173-180 7Van Alstyne, Jan. 4, 2018. US Patent Application 62/613,621. 8Van Alstyne. June 20, 2018 Poster(3035) presentation at Keystone Symposia Conference “Advances in Neurodegenerative Disease Research and Therapy”. Session Z3.

Microglial dynamics during brain development.

Microglial dynamics during brain development.

Microglial-specific transcriptome changes following chronic alcohol consumption

Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer's disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.

Activation of neonatal microglia can be influenced by other neural cells.

During development, microglial progenitor cells migrate into the brain from the periphery, a process critical to the maturation of the developing brain. Although they perform functions similar to mature, adult microglia, immature microglia are distinct from mature microglia. Activation of immature microglia, via an early-life immune challenge, can lead to persistent changes in microglial function, resulting in long-term neuronal and cognitive dysfunction. Early-life immune activation is associated with multiple neurodevelopmental disorders, including autism, ADHD, schizophrenia, and cerebral palsy - disorders with known or suspected immune etiologies, and strong sex biases for males. Activation of immature microglia requires further examination to determine its potential role in these neurodevelopmental disorders. More work is also necessary to better understand the relationship between developing microglia and other developing neural cells during this critical period of development. Thus, we treated freshly isolated, sex-specific microglia from the rat hippocampus with lipopolysaccharide (LPS) on P4, in either the presence or absence of other neural cells. Mixed and microglial-specific cultures were analyzed for inflammatory gene expression to determine whether immature microglia exhibited a sex-specific response to immune activation, and if the presence of all other neural cells influenced that response. We found that the microglial response to an LPS-induced immune activation differed depending on the presence of other neural cells in the culture. We found very few sex differences in the cytokine response, except that the microglial expression of IL-6 following immune activation was more robust in male microglia that were in the presence of other neural cells than female microglia in the same condition.

Expression of angiotensin II and its receptors in activated microglia in experimentally induced cerebral ischemia in the adult rats

Expression of angiotensin II (Ang II) and its receptors (AT1/AT2) is undetected in the mature microglia in normal brain. We report here that the immunoexpression of Ang II and AT1/AT2 was altered in activated microglia notably at 1 week in rats subjected to middle cerebral artery occlusion (MCAO). Immunolabeled activated microglia were widely distributed in the infarcted cerebral tissue after MCAO. By enzyme immunoassay, Ang II protein expression levels of the ischemic tissues were decreased drastically at 12 h after ischemia, then rose rapidly at 3 days and 1 week after MCAO when compared with the control. On the other hand, AT1 and AT2 receptor mRNA and protein levels were up-regulated after MCAO, peaking at 12 h, but declined thereafter. Expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein levels was concomitantly increased. Edaravone significantly suppressed Ang II and AT1/AT2 receptor expression as well as that of TNF-α and IL-1β suggesting that microglia-derived Ang II can act through an autocrine manner via its receptor that may be linked partly to the production of proinflammatory cytokines. We conclude that neuroinflammation in MCAO may be attenuated by Edaravone which acts through suppression of expression of Ang II and its receptors and proinflammatory cytokines in activated microglia.

Development of polymeric and cyclodextrin nanoparticles for camptothecin delivery

Cell-selective drug release in the central nervous system (CNS) holds great promise for the treatment of many CNS disorders but it is still challenging. We previously demonstrated that polymeric nanoparticles (NPs) injected intra-parenchyma in the CNS can be internalized specifically in microglia/macrophages surrounding the injection site. Here, we explored NPs administration in the cerebrospinal fluid (CSF) to achieve a wider spreading and increased cell targeting throughout the CNS; we generated new NPs variants and studied the effect of modifying size and surface charge on NPs biodistribution and cellular uptake. Intra-cerebroventricular administration resulted in prevalent localization of the NPs in proximity to stem-cell niches, such as around the lateral ventricles, the subventricular zone and the rostral migratory stream. NPs internalization occurred preferentially in brain myeloid cells/microglia. We demonstrated that brain biodistribution and extent of internalization in microglia are influenced by NPs dimensions and can be improved by applying a transient disruption of the blood-brain barrier with mannitol, leading to NPs internalization in up to 25% of brain myeloid/microglia cells. A fraction of the targeted cells was positive for markers of proliferation or stained positive for stemness/progenitor-cell markers such as Nestin, c-kit, or NG2. Interestingly, through these newly formulated NPs we obtained controlled and selective release of drugs otherwise difficult to formulate (such as busulfan and etoposide) to the target cells, preventing unwanted side effects and the toxicity obtained by direct brain delivery of the not encapsulated drugs. Overall, these data provide proof of concept of the applicability of these novel NP-based drug formulations for achieving internalization not only in mature microglia but also possibly in more immature myeloid cells in the brain and pave the way for brain-restricted microglia-targeted drug delivery regimens.

Expression of purine metabolism-related enzymes by microglial cells in the developing rat brain.

The nucleoside triphosphatase (NTPase), nucleoside diphosphatase (NDPase), 5'-nucleotidase (5'-Nase), and purine nucleoside phosphorylase (PNPase) activity has been examined in the cerebral cortex, subcortical white matter, and hippocampus from embryonic day (E)16 to postnatal day (P)18. Microglia display all four purine-related enzymatic activities, but the expression of these enzymatic activities differed depending on the distinct microglial typologies observed during brain development. We have identified three main morphologic typologies during the process of microglial differentiation: ameboid microglia (parenchymatic precursors), primitive ramified microglia (intermediate forms), and resting microglia (differentiated cells). Ameboid microglia, which were encountered from E16 to P12, displayed the four enzymatic activities. However, some ameboid microglial cells lacked 5'-Nase activity in gray matter, and some were PNPase-negative in both gray and white matter. Primitive ramified microglia were already observed in the embryonic period but mostly distributed during the first 2 postnatal weeks. These cells expressed NTPase, NDPase, 5'-Nase, and PNPase. Similar to ameboid microglia, we found primitive ramified microglia lacking the 5'-Nase and PNPase activities. Resting microglia, which were mostly distinguishable from the third postnatal week, expressed NTPase and NDPase, but they lacked or displayed very low levels of 5'-Nase activity, and only a subpopulation of resting microglia was PNPase-positive. Apart from cells of the microglial lineage, GFAP-positive astrocytes and radial glia cells were also labeled by the PNPase histochemistry. As shown by our results, the differentiation process from cell precursors into mature microglia is accompanied by changes in the expression of purine-related enzymes. We suggest that the enzymatic profile and levels of the different purine-related enzymes may depend not only on the differentiation stage but also on the nature of the cells. The use of purine-related histoenzymatic techniques as a microglial markers and the possible involvement of microglia in the control of extracellular purine levels during development are also discussed.

Microglia development in the quail cerebellum

We used the QH1 antibody to study changes in the morphological features and distribution of microglial cells throughout development in the quail cerebellum. Few microglial precursors were present in the cerebellar anlage before the ninth incubation day (E9), whereas many precursors apparently entered the cerebellum from the meninges in the basal region of the cerebellar peduncles between E9 and E16. From this point of entry into the nervous parenchyma, they spread through the cerebellar white matter, forming a 'stream' of labeled cells that could be seen until hatching (E16). The number of microglial cells in the cerebellar cortex increased during the last days of embryonic life and first posthatching week, whereas microglial density within the white matter decreased after hatching. As a consequence, the differences in microglial cell density observed in the cerebellar cortex and the white matter during embryonic life diminished after hatching, and microglia showed a nearly homogeneous pattern of distribution in adult cerebella. Ameboid and poorly ramified microglial cells were found in developing stages, whereas only mature microglia appeared in adult cerebella. Our observations suggest that microglial precursors enter the cerebellar anlage mainly by traversing the pial surface at the basal region of the peduncles, then migrate along the white matter, and finally move radially to the different cortical layers. Differentiation occurs after the microglial cells have reached their final position. In other brain regions the development of microglia follows similar stages, suggesting that these steps are general rules of microglial development in the central nervous system.

Microglia development in the quail cerebellum

We used the QH1 antibody to study changes in the morphological features and distribution of microglial cells throughout development in the quail cerebellum. Few microglial precursors were present in the cerebellar anlage before the ninth incubation day (E9), whereas many precursors apparently entered the cerebellum from the meninges in the basal region of the cerebellar peduncles between E9 and E16. From this point of entry into the nervous parenchyma, they spread through the cerebellar white matter, forming a ‘stream’ of labeled cells that could be seen until hatching (E16). The number of microglial cells in the cerebellar cortex increased during the last days of embryonic life and first posthatching week, whereas microglial density within the white matter decreased after hatching. As a consequence, the differences in microglial cell density observed in the cerebellar cortex and the white matter during embryonic life diminished after hatching, and microglia showed a nearly homogeneous pattern of distribution in adult cerebella. Ameboid and poorly ramified microglial cells were found in developing stages, whereas only mature microglia appeared in adult cerebella. Our observations suggest that microglial precursors enter the cerebellar anlage mainly by traversing the pial surface at the basal region of the peduncles, then migrate along the white matter, and finally move radially to the different cortical layers. Differentiation occurs after the microglial cells have reached their final position. In other brain regions the development of microglia follows similar stages, suggesting that these steps are general rules of microglial development in the central nervous system. J. Comp. Neurol. 389:390–401, 1997. © 1997 Wiley-Liss, Inc.

2-04-14 Pneumococcal cell wall components induce an outward potassium current only in mature microglia

2-04-14 Pneumococcal cell wall components induce an outward potassium current only in mature microglia

Patterns of proliferation in cerebral lymphoreticular tumours.

A study of eighteen cases of primary lymphoreticular tumours in the brain is described. In four of these there were extraneural lesions and in one macroglobulinaema. The use of whole brain sections embedded in celloidin, and of metallic impregnation methods, revealed certain constant patterns of proliferation. The tumours were diffuse and multicentric; the leptomeninges and perivascular spaces especially in the subependymal regions were frequently involved. Mature microglia were active both in infiltrated and in apparently tumour-free regions.