Cell and Tissue BiologyCell and Tissue BiologyЦитология0041-37713034-6061The Russian Academy of Sciences66964610.31857/S0041377123030082VDTYOWArticlesСтатьиMicroglia Cell Line SIM-A9 Features – New DataХАРАКТЕРИСТИКА КЛЕТОК МИКРОГЛИИ ЛИНИИ SIM-A9 – НОВЫЕ ДАННЫЕShaposhnikovaD. A.ШапошниковаД. А.dasha-shap13@mail.ruMoskalevaE. Yu.МоскалеваЕ. Ю.dasha-shap13@mail.ruSemochkinaYu. P.СёмочкинаЮ. П.dasha-shap13@mail.ruVysotskayaO. V.ВысоцкаяО. В.dasha-shap13@mail.ruKomovaO. V.КомоваО. В.dasha-shap13@mail.ruNasonovaE. A.НасоноваЕ. А.dasha-shap13@mail.ruKoshlanI. V.КошланьИ. В.dasha-shap13@mail.ruNational Research Center “Kurchatov Institute”НИЦ “Курчатовский институт”Joint Institute for Nuclear ResearchОбъединенный институт ядерных исследований0105202365325927227022025Copyright ©; 2023, Д.А. Шапошникова, Е.Ю. Москалева, Ю.П. Сёмочкина, О.В. Высоцкая, О.В. Комова, Е.А. Насонова, И.В. КошланьCopyright ©; 2023, Д.А. Шапошникова, Е.Ю. Москалева, Ю.П. Сёмочкина, О.В. Высоцкая, О.В. Комова, Е.А. Насонова, И.В. Кошлань2023Д.А. Шапошникова, Е.Ю. Москалева, Ю.П. Сёмочкина, О.В. Высоцкая, О.В. Комова, Е.А. Насонова, И.В. КошланьД.А. Шапошникова, Е.Ю. Москалева, Ю.П. Сёмочкина, О.В. Высоцкая, О.В. Комова, Е.А. Насонова, И.В. Кошланьhttps://journals.eco-vector.com/0041-3771/article/view/669646

SIM-A9 is a line of spontaneously immortalized mouse microglia cells obtained from newborn C57BL/6 mice’s cerebrum. The aim of this work is to characterize SIM-A9 line by the ratio of cells with the resting and activated microglia phenotype, to analyze the expression of stem/progenitor cell markers CD133 and nestin, growth factors receptors CSF-1R and EGFR, and the karyotype of this line. The light microscopy, immunocytochemistry, flow cytometry and RT/PCR were used to analyze the morphology, phenotype, and gene expression levels of pro-inflammatory cytokines, and the mFISH method was used to analyze the karyotype. It was shown for the first time that SIM-A9 cells express a high level of TSPO protein, CD68, CD11b and CD45 markers on the surface membrane of cells, which corresponds to the phenotype of activated microglia. Despite this, the cells of this line respond with additional activation to LPS stimulation, which leads to an increase in the pro-inflammatory cytokine genes IL-1β, TNFα, IL-6 expression and a high level of active oxygen and nitrogen metabolites formation. It was shown that SIM-A9 cells express stem and progenitor cells markers, CD133+ and nestin, which allows us to consider the cells of this line as early poorly differentiated progenitor cells, despite their phenotype corresponding to activated microglia. It was also found that SIM-A9 cells express receptors of two growth factors CSF-1 and EGF, CSF-1R and EGFR, which indicates the possibility of SIM-A9 cells proliferation stimulation by two alternative mechanisms under the action of the corresponding factors. SIM-A9 cells have a hypotetraploid karyotype with a large number of structural and quantitative chromosome anomalies.

SIM-A9 – линия спонтанно иммортализованных клеток микроглии мыши, полученных из головного мозга новорожденных мышат линии С57BL/6. Цель настоящей работы – характеристика микроглии мыши линии SIM-A9 по соотношению клеток с фенотипом покоящейся и активированной микроглии в культуре, анализ экспрессии маркеров стволовых (прогениторных) клеток CD133 и нестина, рецепторов факторов роста CSF-1R и EGFR, а также анализ кариотипа этой линии. Использованы методы световой микроскопии, иммуноцитохимии в сочетании с проточной цитометрией и ОТ-ПЦР для анализа морфологии, фенотипа и уровня экспрессии генов провоспалительных цитокинов и метод mFISH для анализа кариотипа. Впервые показано, что клетки линии SIM-А9 экспрессируют высокий уровень белка TSPO, маркеров CD68, CD11b и CD45high на поверхностной мембране клеток, что соответствует фенотипу активированной микроглии. Несмотря на это, клетки линии отвечают дополнительной активацией в ответ на стимуляцию ЛПС, которая приводит к повышению экспрессии генов провоспалительных цитокинов IL-1β, TNFα, IL-6 и образованию высокого уровня активных метаболитов кислорода и азота. Показано, что клетки линии SIM-A9 экспрессируют маркеры стволовых и прогениторных клеток CD133+ и нестин, что позволяет рассматривать их как ранние низко дифференцированные прогениторные клетки, несмотря на их фенотип, соответствующий активированной микроглии. Обнаружено также, что клетки линии SIM-A9 экспрессируют рецепторы двух факторов роста CSF-1 и EGF – CSF-1R и EGFR, что свидетельствует о возможности стимуляции пролиферации клеток SIM-A9 по двум альтернативным механизмам под действием соответствующих факторов. У клеток SIM-A9 установлен гипотетраплоидный кариотип с большим числом структурных и количественных аномалий хромосом.

microgliacell line SIM-A9karyotypestem cellsCSF-1 receptorEGF receptorgene expression, cytokinesмикроглиялиния SIM-A9кариотипстволовые клеткирецептор CSF-1рецептор EGFэкспрессия генов, цитокиныМоскалева Е.Ю., Родина А.В., Семочкина Ю.П., Высоцкая О.В. 2022. Анализ маркеров окислительного повреждения нейронов и нейровоспаления в отдаленный период после γ-облучения головы мышей в разных дозах Радиационная биология. Радиоэкология. Т. 62 № 2. С. 171. (Moskaleva E.Yu., Rodina A.V., Semochkina Ju.P., Vysotskaya O.V. 2022. Analysis of neurons damage and level of neuroinflammation late after γ-irradiation of mice head at different doses. Radiacionnaja biologija. Radioekologija. V. 62 № 2. Р. 171.) https://doi.org/10.31857/S0869803122020059Новиков В.Е., Левченкова О.С., Пожилова Е.В. 2014. Роль активных форм кислорода в физиологии и патологии клетки и их фармакологическая регуляция. Обзоры по клинической фармакологии и лекарственной терапии. Т. 12. № 4. С. 13. (Novikov V.E., Levchenkova O.S., Pozhilova Y.V. 2014. Role of reactive oxygen species in cell physiology and pathology and their pharmacological regulation. Rev. Clin. Pharmacol. Drug Ther. Vol. 12. № 4. P. 13.) https://doi.org/10.17816/RCF12413-21Askew K., Li K., Olmos-Alonso A., Garcia-Moreno F., Liang Y., Richardson P., Tipton T., Chapman M.A., Riecken K., Beccari S., Sierra A., Molnár Z., Cragg M.S., Garaschuk O., Perry V.H., Gomez-Nicola D. 2017. Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain. Cell Rep. V. 18. P. 391. https://doi.org/10.1016/j.celrep.2016.12.041Bachiller S., Jiménez-Ferrer I., Paulus A., Yang Y., Swanberg M., Deierborg T., Boza-Serrano A. 2018. Microglia in neurological diseases: a road map to brain-disease dependent-inflammatory response. Front. Cell Neurosci. V. 12. P. 488. https://doi.org/10.3389/fncel.2018.00488Becher B., Antel J.P. 1996. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia. V. 18. P. 1. https://doi.org/10.1002/(SICI)1098-1136(199609)18:1<1::AID-GLIA1>3.0.CO;2-6Bennett 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., Weissman I.L., Chang E.F., Li G., Grant G.A., Hayden Gephart M.G., Barres B.A. 2016. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA. V. 113. P. 1738. https://doi.org/10.1073/pnas.1525528113Bernal A., Arranz L. 2018. Nestin-expressing progenitor cells: function, identity and therapeutic implications. Cell. Mol. Life Sci. V. 75. P. 2177. https://doi.org/10.1007/s00018-018-2794-zBlasi E., Barluzzi R., Bocchini V., Mazzolla R., Bistoni F. 1990. Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J. Neuroimmunol. V. 27. P. 229. https://doi.org/10.1016/0165-5728(90)90073-VBohnert S., Seiffert A., Trella S., Bohnert M., Distel L., Ondruschka B., Monoranu C.M. 2020. TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination. Int. J. Legal Med. V. 134. P. 2167. https://doi.org/10.1007/s00414-020-02384-zBonsack F., Sukumari-Ramesh S. 2018. TSPO: an evolutionarily conserved protein with elusive functions. Int. J. Mol. Sci. V. 19. P. 1694. https://doi.org/10.3390/ijms19061694Cornforth M.N. 2001. Analyzing radiation-induced complex chromosome rearrangements by combinatorial painting. Radiat. Res. V. 155. P. 643. https://doi.org/10.1007/978-1-4939-9432-8_15Coniglio S.J., Eugenin E., Dobrenis K., Stanley E.R., West B.L., Symons M.H., Segall J.E. 2012. Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling. Mol. Med. V. 18. P. 519. https://doi.org/10.2119/molmed.2011.00217Coskun V., Wu H., Blanchi B., Tsao S., Kim K., Zhao J., Biancotti J.C., Hutnick L., Krueger R.C. Jr., Fan G., de Vellis J., Sun Y.E. 2008. CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain. Proc. Natl. Acad. Sci. USA. V. 105. P. 1026. https://doi.org/10.1073/pnas.0710000105Douglas M.R., Morrison K.C., Jacques S.J., Leadbeater W.E., Gonzalez A.M., Berry M., Logan A., Ahmed Z. 2009. Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth. Brain. V. 132. P. 3102. https://doi.org/10.1093/brain/awp240Elmore M.R., Najafi A.R., Koike M.A., Dagher N.N., Spangenberg E.E., Rice R.A., Kitazawa M., Matusow B., Nguyen H., West B.L., Green K.N. 2014. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron. V. 82. P. 380. https://doi.org/10.1016/j.neuron.2014.02.040Eyo U.B., Dailey M.E. Microglia: key elements in neural development, plasticity, and pathology. 2013. J. Neuroimmune Pharmacol. V. 8. P. 494. https://doi.org/10.1007/s11481-013-9434-zFischer O.M., Hart S., Ullrich A. 2006. Dissecting the epidermal growth factor receptor signal transactivation pathway. Methods Mol. Biol. V. 327. P. 85. https://doi.org/10.1385/1-59745-012-X:85Green K.N., Crapser J.D., Hohsfield L.A. 2020. To kill a microglia: A case for CSF1R inhibitors. Trends Immunol. V. 41. P. 771. https://doi.org/10.1016/j.it.2020.07.001Hagan N., Kane J.L., Grover D., Woodworth L., Madore C., Saleh J., Sancho J., Liu J., Li Y., Proto J., Zelic M., Mahan A., Kothe M., Scholte A.A., Fitzgerald M. 2020. CSF1R signaling is a regulator of pathogenesis in progressive MS. Cell Death Dis. V. 11. P. 904. https://doi.org/10.1038/s41419-020-03084-7Han J., Chitu V., Stanley E.R., Wszolek Z.K., Karrenbauer V.D., Harris R.A. 2022. Inhibition of colony stimulating factor-1 receptor (CSF-1R) as a potential therapeutic strategy for neurodegenerative diseases: opportunities and challenges. Cell. Mol. Life Sci. V. 79. P. 219. https://doi.org/10.1007/s00018-022-04225-1Huang Y., Xu Z., Xiong S. 2018. Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nat. Neurosci. V. 21. P. 530. https://doi.org/10.1038/s41593-018-0090-8Jenkins S.J., Ruckerl D., Thomas G.D., Hewitson J.P., Duncan S., Brombacher F., Maizels R.M., Hume D.A., Allen J.E. 2013. IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. J. Exper. Med. V. 210. P. 2477. https://doi.org/10.1084/jem.20121999Jones S., Rappoport J.Z. 2014. Interdependent epidermal growth factor receptor signalling and trafficking. Int. J. Biochem. Cell. Biol. V. 51. P. 23. https://doi.org/10.1016/j.biocel.2014.03.014Jurga A.M., Paleczna M., Kuter K.Z. 2020. Overview of general and discriminating markers of differential microglia phenotypes. Front. Cell Neurosci. V. 14. P. 198. https://doi.org/10.3389/fncel.2020.00198Kalm M., Andreasson U., Björk-Eriksson T., Zetterberg H., Pekny M., Blennow K., Pekna M., Blomgren K. 2016. C3 deficiency ameliorates the negative effects of irradiation of the young brain on hippocampal development and learning. Oncotarget. V. 7. P. 19382. https://doi.org/10.18632/oncotarget.8400Lei F., Cui N., Zhou C., Chodosh J., Vavvas D.G., Paschalis E.I. 2020. CSF1R inhibition by a small-molecule inhibitor is not microglia specific; affecting hematopoiesis and the function of macrophages. Proc. Natl. Acad. Sci. USA. V. 22. P. 23336. https://doi.org/10.1073/pnas.1922788117Lively S., Schlichter L.C. 2018. Microglia responses to pro-inflammatory stimuli (LPS, IFNγ + TNFα) and reprogramming by resolving cytokines (IL-4, IL-10). Front. Cell Neurosci. V. 12. P. 215. https://doi.org/10.3389/fncel.2018.00215Liu G.J., Middleton R.J., Hatty C.R., Kam W.W., Chan R., Pham T., Harrison-Brown M., Dodson E., Veale K., Banati R.B. 2014. The 18 kDa translocator protein, microglia and neuroinflammation. Brain Pathol. V. 24. P. 631. https://doi.org/10.1111/bpa.12196Mansour H.M., Fawzy H.M., El-Khatib A.S., Khattab M.M. 2022. Repurposed anti-cancer epidermal growth factor receptor inhibitors: mechanisms of neuroprotective effects in Alzheimer’s disease. Neural Regen. Res. V. 17. P. 1913. https://doi.org/10.4103/1673-5374.332132Michalczyk K., Ziman M. 2005. Nestin structure and predicted function in cellular cytoskeletal organisation. Histol. Histopathol. V. 20. P. 665. https://doi.org/10.14670/HH-20.665Muñoz-Garcia J., Cochonneau D., Télétchéa S., Moranton E., Lanoe D., Brion R., Lézot F., Heymann M.F., Heymann D. 2021. The twin cytokines interleukin-34 and CSF-1: masterful conductors of macrophage homeostasis. Theranostics. V. 11. P. 1568. https://doi.org/10.7150/thno.50683Nagamoto-Combs K., Kulas J., Combs C.K. 2014. A novel cell line from spontaneously immortalized murine microglia. J. Neurosci. Methods. V. 15. P. 187. https://doi.org/10.1016/j.jneumeth.2014.05.021Onyango I.G., Jauregui G.V., Čarná M., Bennett J.P. Jr., Stokin G.B. 2021. Neuroinflammation in Alzheimer’s Disease. Biomedicines. V. 9. P. 524. https://doi.org/10.3390/biomedicines9050524Pannell M., Economopoulos V., Wilson T.C., Kersemans V., Isenegger P.G., Larkin J.R., Smart S., Gilchrist S., Gouverneur V., Sibson N.R. 2020. Imaging of translocator protein upregulation is selective for pro-inflammatory polarized astrocytes and microglia. Glia. V. 68. P. 280. https://doi.org/10.1002/glia.23716Prater K.E., Aloi M.S., Pathan J.L., Winston C.N., Chernoff R.A., Davidson S., Sadgrove M., McDonough A., Zierath D., Su W., Weinstein J.R., Garden G.A. 2021. A Subpopulation of microglia generated in the adult mouse brain originates from Prominin-1-expressing progenitors. J. Neurosci. V. 41. P. 7942. https://doi.org/10.1523/JNEUROSCI.1893-20.2021Qu W.S., Liu J.L., Li C.Y., Li X., Xie M.J., Wang W., Tian D.S. 2015. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia. Neurochem. Int. V. 90. P. 85. https://doi.org/10.1016/j.neuint.2015.07.007Qu W.S., Tian D.S., Guo Z.B., Fang J., Zhang Q., Yu Z.Y., Xie M.J., Zhang H.Q., Lü J.G., Wang W. 2012. Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury. J. Neuroinflammation. V. 9. P. 178. https://doi.org/10.1186/1742-2094-9-178Ramprasad M.P., Terpstra V., Kondratenko N., Quehenberger O., Steinberg D. 1996. Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein. Proc. Natl. Acad. Sci. USA. V. 93. P. 14833. https://doi.org/10.1073/pnas.93.25.14833Sasaki Y., Ohsawa K., Kanazawa H., Kohsaka S., Imai Y. 2001. Iba1 is an actin-cross-linking protein in macrophages/microglia. Biochem. Biophys. Res. Commun. V. 286. P. 292. https://doi.org/10.1006/bbrc.2001.5388Stanley E.R., Chitu V. 2014. CSF-1 receptor signaling in myeloid cells. Cold Spring Harb. Perspect Biol. V. 6. P. a021857. https://doi.org/10.1101/cshperspect.a021857Stansley B., Post J., Hensley K. 2012. A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease. J. Neuroinflammation. V. 9. P. 115. https://doi.org/10.1186/1742-2094-9-115Takamori Y., Mori T., Wakabayashi T., Nagasaka Y., Matsuzaki T., Yamada H. 2009. Nestin-positive microglia in adult rat cerebral cortex. Brain Res. V. 1270. P. 10. https://doi.org/10.1016/j.brainres.2009.03.014Waller R., Baxter L., Fillingham D.J., Coelho S., Pozo J.M., Mozumder M., Frangi A.F., Ince P.G., Simpson J.E., Highley J.R. 2019. Iba-1-/CD68+ microglia are a prominent feature of age-associated deep subcortical white matter lesions. PLoS One. V. 25. P. e0210888. https://doi.org/10.1371/journal.pone.0210888Wohl S.G., Schmeer C.W., Friese T., Witte O.W., Isenmann S. 2011. In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo. PLoS One. V. 6. P. e22408. https://doi.org/10.1371/journal.pone.0022408Wong A.M., Patel N.V., Patel N.K., Wei M., Morgan T.E., de Beer M.C., de Villiers W.J., Finch C.E. 2005. Macrosialin increases during normal brain aging are attenuated by caloric restriction. Neurosci. Lett. V. 390. P. 76. https://doi.org/10.1016/j.neulet.2005.07.058Yan P., Wu X., Liu X., Cai Y., Shao C., Zhu G. 2019. A Causal relationship in spinal cord injury rat model between microglia activation and EGFR/MAPK detected by overexpression of microRNA-325-3p. J. Mol. Neurosci. V. 68. P. 181. https://doi.org/10.1007/s12031-019-01297-wYang Y., Sun Y., Hu R., Yan J., Wang Z., Li W., Jiang H. 2021. Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway. PLoS One. V. 16 P. e0256870. https://doi.org/10.1371/journal.pone.0256870