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<article article-type="research-article" dtd-version="1.3" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xml:lang="ru"><front><journal-meta><journal-id journal-id-type="publisher-id">mimmun</journal-id><journal-title-group><journal-title xml:lang="ru">Медицинская иммунология</journal-title><trans-title-group xml:lang="en"><trans-title>Medical Immunology (Russia)</trans-title></trans-title-group></journal-title-group><issn pub-type="ppub">1563-0625</issn><issn pub-type="epub">2313-741X</issn><publisher><publisher-name>SPb RAACI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.15789/1563-0625-EMO-16759</article-id><article-id custom-type="elpub" pub-id-type="custom">mimmun-3085</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="section-heading" xml:lang="ru"><subject>КРАТКИЕ СООБЩЕНИЯ</subject></subj-group><subj-group subj-group-type="section-heading" xml:lang="en"><subject>SHORT COMMUNICATIONS</subject></subj-group></article-categories><title-group><article-title>Ранние маркеры гемопоэтической дифференцировки индуцированных плюрипотентных стволовых клеток человека</article-title><trans-title-group xml:lang="en"><trans-title>Early markers of induced pluripotent stem cell hematopoietic development</trans-title></trans-title-group></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Шевелева</surname><given-names>О. Н.</given-names></name><name name-style="western" xml:lang="en"><surname>Sheveleva</surname><given-names>O. N.</given-names></name></name-alternatives><bio xml:lang="ru"><p>К.б.н., старший научный сотрудник.</p><p>Москва</p></bio><bio xml:lang="en"><p>PhD (Biology), Senior Research Associate, Koltzov Institute of Developmental Biology, Russian Academy of Sciences.</p><p>Moscow</p></bio><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Ненашева</surname><given-names>Т. А.</given-names></name><name name-style="western" xml:lang="en"><surname>Nenasheva</surname><given-names>T. A.</given-names></name></name-alternatives><bio xml:lang="ru"><p>К.б.н., старший научный сотрудник.</p><p>Москва</p></bio><bio xml:lang="en"><p>PhD (Biology), Senior Research Associate, Koltzov Institute of Developmental Biology, Russian Academy of Sciences.</p><p>Moscow</p></bio><xref ref-type="aff" rid="aff-1"/></contrib><contrib contrib-type="author" corresp="yes"><name-alternatives><name name-style="eastern" xml:lang="ru"><surname>Лядова</surname><given-names>И. В.</given-names></name><name name-style="western" xml:lang="en"><surname>Lyadova</surname><given-names>I. V.</given-names></name></name-alternatives><bio xml:lang="ru"><p>Лядова Ирина Владимировна – д.м.н., заведующая лабораторией ФГБУН «ИБР им. Н.К. Кольцова РАН»; заведующая кафедрой клеточной биомедицины и клеточных технологий ПИШ ФГАОУ ВО «РНИМУ им. Н.И. Пирогова» МЗ РФ.</p><p>119334, Москва, ул. Вавилова, 26</p><p>Тел.: 8 (499) 135-87-80</p></bio><bio xml:lang="en"><p>Irina V. Lyadova - PhD, MD (Medicine), Head of the Laboratory, Koltzov Institute of Developmental Biology, Russian Academy of Sciences; Head of the Department for Cell Biomedicine and Cell Technologies, Pirogov Russian National Research Medical University.</p><p>26 Vavilov St Moscow 119334</p><p>Phone: +7 (499) 135-87-80</p></bio><email xlink:type="simple">ivlyadova@mail.ru</email><xref ref-type="aff" rid="aff-2"/></contrib></contrib-group><aff-alternatives id="aff-1"><aff xml:lang="ru"><institution>ФГБУН «Институт биологии развития имени Н.К. Кольцова Российской академии наук»</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Koltzov Institute of Developmental Biology, Russian Academy of Sciences</institution><country>Russian Federation</country></aff></aff-alternatives><aff-alternatives id="aff-2"><aff xml:lang="ru"><institution>ФГБУН «Институт биологии развития имени Н.К. Кольцова Российской академии наук»; ФГАОУ ВО «Российский научно-исследовательский медицинский университет имени Н.И. Пирогова» Министерства здравоохранения РФ</institution><country>Россия</country></aff><aff xml:lang="en"><institution>Koltzov Institute of Developmental Biology, Russian Academy of Sciences; Pirogov Russian National Research Medical University</institution><country>Russian Federation</country></aff></aff-alternatives><pub-date pub-type="collection"><year>2024</year></pub-date><pub-date pub-type="epub"><day>21</day><month>09</month><year>2024</year></pub-date><volume>26</volume><issue>5</issue><fpage>883</fpage><lpage>890</lpage><permissions><copyright-statement>Copyright &amp;#x00A9; Шевелева О.Н., Ненашева Т.А., Лядова И.В., 2024</copyright-statement><copyright-year>2024</copyright-year><copyright-holder xml:lang="ru">Шевелева О.Н., Ненашева Т.А., Лядова И.В.</copyright-holder><copyright-holder xml:lang="en">Sheveleva O.N., Nenasheva T.A., Lyadova I.V.</copyright-holder><license xml:lang="ru" license-type="creative-commons-attribution" xlink:href="https://creativecommons.org/licenses/by/4.0/" xlink:type="simple"><license-p>Данная работа распространяется под лицензией Creative Commons Attribution 4.0.</license-p></license><license xml:lang="en" license-type="creative-commons-attribution" xlink:href="https://creativecommons.org/licenses/by/4.0/" xlink:type="simple"><license-p>This work is licensed under a Creative Commons Attribution 4.0 License.</license-p></license></permissions><self-uri xlink:href="https://www.mimmun.ru/mimmun/article/view/3085">https://www.mimmun.ru/mimmun/article/view/3085</self-uri><abstract><p>Генерация иммунокомпетентных клеток из индуцированных плюрипотентных стволовых клеток является ценной моделью для изучения механизмов регуляции гемопоэза и перспективным подходом к разработке новых методов иммунотерапии различных заболеваний, включая наследственные, онкологические и инфекционные. К настоящему времени показана возможность получения из индуцированных плюрипотентных стволовых клеток человека различных клеток иммунной системы, в том числе макрофагов, нейтрофилов, естественных киллеров и Т-лимфоцитов. Однако предложенные протоколы носят в основном экспериментальный характер и для дальнейшего применения требуют оптимизации, стандартизации и масштабирования. Решение этих задач, в свою очередь, требует наличия методов ранней оценки эффективности проводимой дифференцировки. В настоящей работе оценивали возможность использования проточной цитометрии для мониторинга эффективности ранних этапов гемопоэтической дифференцировки индуцированных плюрипотентных стволовых клеток. Гемопоэтическую и миелоидную дифференцировку индуцированных плюрипотентных стволовых клеток человека проводили с использованием двух протоколов, предложенных ранее для генерации макрофагов из индуцированных плюрипотентных стволовых клеток. Использованные протоколы различались по условиям проведения ранних и поздних стадий дифференцировки. Ранние этапы дифференцировки различались по способу индукции образования мезодермальных клеток и гемогенного эндотелия: дифференцировка в условиях 2D с добавлением экзогенных факторов, стимулирующих мезодермальное направление дифференцировки («фактор-зависимый» протокол) или в условиях 3D без добавления экзогенных факторов («спонтанный» протокол). На более поздних стадиях протоколы различались по набору экзогенных факторов, использующихся для индукции гемопоэтической и миелоидной спецификации (SCF, FGF2, IL-6, IL-3 и M-CSF или только IL-3 и M-CSF). В процессе дифференцировки с использованием обоих протоколов проводили анализ экспрессии маркеров мезодермы, гемогенного эндотелия и гемопоэтических клеток (CD309, CD34, CD31, CD43 и CD45). На начальных стадиях дифференцировки основным фенотипическим изменением было появление экспрессии на клетках рецептора к фактору роста эндотелия сосудов CD309, экспрессии сиалофорина CD43, а также антигена CD34. При использовании фактор-зависимого 2D-протокола эти изменения фиксировались раньше и были более выраженными, чем при использовании протокола, основанного на спонтанной дифференцировке клеток в условиях 3D. Полученные результаты позволяют заключить, что определение экспрессии CD309 и/или CD43 может быть использовано для ранней предикции успешности дифференцировки иПСК в гемопоэтическом направлении.</p></abstract><trans-abstract xml:lang="en"><p>The generation of immune cell populations from induced pluripotent stem cells (iPSCs) is a valuable model to study mechanisms that control hematopoietic development; it also is a promising approach to develop immunotherapeutic strategies to treat various diseases, including hereditary, oncological and infectious ones. To date, it has been demonstrated that iPSCs can differentiate into different immune cells, including macrophages, neutrophils, natural killer cells and T cells. However, the protocols suggested so far are experimental, and they require optimization, standardization and scaling. Solution to these tasks requires methods allowing to predict the efficacy of ongoing differentiation at early differentiation stages. Here, we evaluated whether iPSC hematopoietic/myeloid differentiation can be monitored by means of flow cytometry. Human iPSCs were differentiated into hematopoietic/myeloid cells using two protocols previously suggested for the generation of macrophages from iPSCs. The protocols differed by methods used to induce early and late stages of cell differentiation. At early differentiation stages, the protocols differed by approaches used to induce the generation of mesoderm and hemogenic endothelium, i.e., 2D differentiation in the presence of exogenous factors known to promote mesoderm and hemogenic endothelium development (“factor-dependent” protocol) or 3D differentiation in the absence of exogenous cytokines and growth factors (“spontaneous” protocol). At late differentiation stages, the protocols differed by factors added to the cultures to promote hematopoietic/myeloid specification (i.e., SCF, FGF2, IL-6, IL-3 and M-CSF or only IL-3 and M-CSF). At different stages of differentiation, the expressions of antigens known to be expressed by mesoderm, hemogenic endothelium and hematopoietic cells (i.e., CD309, CD34, CD31, CD43 and CD45) were evaluated. At early differentiation stages, the main phenotypic changes observed in cell cultures were an upregulation of the expression of CD309 (a receptor for vascular endothelial growth factor), the appearance of the expression of sialophorin CD43 and the expression of CD34 antigen. In cells cultured in 2D factor-dependent conditions, these changes appeared earlier and were more pronounced as compared with cells cultured in 3D “spontaneous” conditions. The results suggest that CD309 and/or CD43 are valuable markers for an early prediction of the effectiveness of iPSC differentiation into hematopoietic/ myeloid progeny.</p></trans-abstract><kwd-group xml:lang="ru"><kwd>дифференцировка иммунных клеток in vitro</kwd><kwd>индуцированные плюрипотентные стволовые клетки</kwd><kwd>мезодерма</kwd><kwd>гемогенный эндотелий</kwd><kwd>проточная цитометрия</kwd><kwd>CD309</kwd><kwd>CD43</kwd></kwd-group><kwd-group xml:lang="en"><kwd>immune cell differentiation in vitro</kwd><kwd>induced pluripotent stem cells</kwd><kwd>mesoderm</kwd><kwd>hemogenic endothelium</kwd><kwd>flow cytometry</kwd><kwd>CD309</kwd><kwd>CD43</kwd></kwd-group><funding-group><funding-statement xml:lang="ru">Работа поддержана Минобрнауки России № ГЗ 0088-2024-0013.</funding-statement></funding-group></article-meta></front><back><ref-list><title>References</title><ref id="cit1"><label>1</label><citation-alternatives><mixed-citation xml:lang="ru">Garcia-Alegria E., Menegatti S., Fadlullah M.Z.H., Menendez P., Lacaud G., Kouskoff V. Early human hemogenic endothelium generates primitive and definitive hematopoiesis in vitro. Stem Cell Rep., 2018, Vol. 11, no. 5, pp. 1061-1074.</mixed-citation><mixed-citation xml:lang="en">Garcia-Alegria E., Menegatti S., Fadlullah M.Z.H., Menendez P., Lacaud G., Kouskoff V. Early human hemogenic endothelium generates primitive and definitive hematopoiesis in vitro. Stem Cell Rep., 2018, Vol. 11, no. 5, pp. 1061-1074.</mixed-citation></citation-alternatives></ref><ref id="cit2"><label>2</label><citation-alternatives><mixed-citation xml:lang="ru">Goldenson B.H., Hor P., Kaufman D.S. iPSC-derived natural killer cell therapies – expansion and targeting. Front. Immunol., 2022, Vol. 13, 841107. doi: 10.3389/fimmu.2022.841107.</mixed-citation><mixed-citation xml:lang="en">Goldenson B.H., Hor P., Kaufman D.S. iPSC-derived natural killer cell therapies – expansion and targeting. Front. Immunol., 2022, Vol. 13, 841107. doi: 10.3389/fimmu.2022.841107.</mixed-citation></citation-alternatives></ref><ref id="cit3"><label>3</label><citation-alternatives><mixed-citation xml:lang="ru">Hu Y., Li Y., Yao Z., Huang F., Cai H., Liu H., Zhang X., Zhang J. Immunotherapy: review of the existing evidence and challenges in breast cancer. Cancers (Basel). 2023, Vol. 15, no. 3, 563. doi: 10.3390/cancers15030563.</mixed-citation><mixed-citation xml:lang="en">Hu Y., Li Y., Yao Z., Huang F., Cai H., Liu H., Zhang X., Zhang J. Immunotherapy: review of the existing evidence and challenges in breast cancer. Cancers (Basel). 2023, Vol. 15, no. 3, 563. doi: 10.3390/cancers15030563.</mixed-citation></citation-alternatives></ref><ref id="cit4"><label>4</label><citation-alternatives><mixed-citation xml:lang="ru">Klepikova A., Nenasheva T., Sheveleva O., Protasova E., Antonov D., Gainullina A., Chikina E., Sakovnich O., Gerasimova T., Nikitina I., Shevalie D., Lyadova I. iPSC-derived macrophages: the differentiation protocol affects cell immune characteristics and differentiation trajectories. Int. J. Mol. Sci., 2022, Vol. 23, no. 24, 16087. doi: 10.3390/ijms232416087.</mixed-citation><mixed-citation xml:lang="en">Klepikova A., Nenasheva T., Sheveleva O., Protasova E., Antonov D., Gainullina A., Chikina E., Sakovnich O., Gerasimova T., Nikitina I., Shevalie D., Lyadova I. iPSC-derived macrophages: the differentiation protocol affects cell immune characteristics and differentiation trajectories. Int. J. Mol. Sci., 2022, Vol. 23, no. 24, 16087. doi: 10.3390/ijms232416087.</mixed-citation></citation-alternatives></ref><ref id="cit5"><label>5</label><citation-alternatives><mixed-citation xml:lang="ru">Lange L., Morgan M., Schambach A. The hemogenic endothelium: a critical source for the generation of PSC-derived hematopoietic stem and progenitor cells. Cell. Mol. Life Sci., 2021,Vol. 78, no. 9, pp. 4143-4160.</mixed-citation><mixed-citation xml:lang="en">Lange L., Morgan M., Schambach A. The hemogenic endothelium: a critical source for the generation of PSC-derived hematopoietic stem and progenitor cells. Cell. Mol. Life Sci., 2021,Vol. 78, no. 9, pp. 4143-4160.</mixed-citation></citation-alternatives></ref><ref id="cit6"><label>6</label><citation-alternatives><mixed-citation xml:lang="ru">Lyadova I., Gerasimova T., Nenasheva T. Macrophages derived from human induced pluripotent stem cells: The diversity of protocols, future prospects, and outstanding questions. Front. Cell Dev. Biol., 2021, Vol. 9, 640703. doi: 10.3389/fcell.2021.640703.</mixed-citation><mixed-citation xml:lang="en">Lyadova I., Gerasimova T., Nenasheva T. Macrophages derived from human induced pluripotent stem cells: The diversity of protocols, future prospects, and outstanding questions. Front. Cell Dev. Biol., 2021, Vol. 9, 640703. doi: 10.3389/fcell.2021.640703.</mixed-citation></citation-alternatives></ref><ref id="cit7"><label>7</label><citation-alternatives><mixed-citation xml:lang="ru">Lyadova I., Vasiliev A. Macrophages derived from pluripotent stem cells: Prospective applications and research gaps. Cell Biosci., 2022, Vol. 12, 96. doi: 10.1186/s13578-022-00824-4.</mixed-citation><mixed-citation xml:lang="en">Lyadova I., Vasiliev A. Macrophages derived from pluripotent stem cells: Prospective applications and research gaps. Cell Biosci., 2022, Vol. 12, 96. doi: 10.1186/s13578-022-00824-4.</mixed-citation></citation-alternatives></ref><ref id="cit8"><label>8</label><citation-alternatives><mixed-citation xml:lang="ru">Malakhova A.A., Grigor’eva E.V., Pavlova S.V., Malankhanova T.B., Valetdinova K.R., Vyatkin Y.V., Khabarova E.A., Rzaev J.A., Zakian S.M., Medvedev S.P. Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population. Stem Cell Res., 2020, Vol. 48, 101952. doi: 10.1016/j.scr.2020.101952.</mixed-citation><mixed-citation xml:lang="en">Malakhova A.A., Grigor’eva E.V., Pavlova S.V., Malankhanova T.B., Valetdinova K.R., Vyatkin Y.V., Khabarova E.A., Rzaev J.A., Zakian S.M., Medvedev S.P. Generation of induced pluripotent stem cell lines ICGi021-A and ICGi022-A from peripheral blood mononuclear cells of two healthy individuals from Siberian population. Stem Cell Res., 2020, Vol. 48, 101952. doi: 10.1016/j.scr.2020.101952.</mixed-citation></citation-alternatives></ref><ref id="cit9"><label>9</label><citation-alternatives><mixed-citation xml:lang="ru">Miyauchi M., Ito Y., Nakahara F., Hino T., Nakamura F., Iwasaki Y., Kawagoshi T., Koya J., Yoshimi A., Arai S., Kagoya Y., Kurokawa M. Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment. Blood, 2021, Vol. 138, no. 24, p. 2555-2569.</mixed-citation><mixed-citation xml:lang="en">Miyauchi M., Ito Y., Nakahara F., Hino T., Nakamura F., Iwasaki Y., Kawagoshi T., Koya J., Yoshimi A., Arai S., Kagoya Y., Kurokawa M. Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment. Blood, 2021, Vol. 138, no. 24, p. 2555-2569.</mixed-citation></citation-alternatives></ref><ref id="cit10"><label>10</label><citation-alternatives><mixed-citation xml:lang="ru">Sheveleva O., Protasova E., Nenasheva T., Butorina N., Melnikova V., Gerasimova T., Sakovnich O., Kurinov A., Grigor’eva E., Medvedev S., Lyadova I. A model of iPSC-derived macrophages with TNFAIP3 overexpression reveals the peculiarities of TNFAIP3 protein expression and function in human macrophages. Int. J. Mol. Sci., 2023, Vol. 24, no. 16, 12868 . doi: 10.3390/ijms241612868.</mixed-citation><mixed-citation xml:lang="en">Sheveleva O., Protasova E., Nenasheva T., Butorina N., Melnikova V., Gerasimova T., Sakovnich O., Kurinov A., Grigor’eva E., Medvedev S., Lyadova I. A model of iPSC-derived macrophages with TNFAIP3 overexpression reveals the peculiarities of TNFAIP3 protein expression and function in human macrophages. Int. J. Mol. Sci., 2023, Vol. 24, no. 16, 12868 . doi: 10.3390/ijms241612868.</mixed-citation></citation-alternatives></ref><ref id="cit11"><label>11</label><citation-alternatives><mixed-citation xml:lang="ru">Takata K., Kozaki T., Lee C., Thion M.S., Otsuka M., Lim S., Utami K.H., Fidan K., Park D.S., Malleret B., Chakarov S., See P., Low D., Low G., Garcia-Miralles M., Zeng R., Zhang J., Goh C., Gul A., Hubert S., Lee B., Chen J., Low I., Shadan N-B., Lum J., Wei T-S., Mok E., Kawanishi S., Kitamura Y., Larbi A., Poidinger M., Renia L., Ng L-G., Wolf Y., Jung S., Önder T., Newell I., Huber T., Ashihara E., Garel S., Pouladi M., Ginhoux F. Induced-pluripotent-stem-cell-derived primitive macrophages provide a platform for modeling tissue-resident macrophage differentiation and function. Immunity, 2017, Vol. 47, pp. 183-198.</mixed-citation><mixed-citation xml:lang="en">Takata K., Kozaki T., Lee C., Thion M.S., Otsuka M., Lim S., Utami K.H., Fidan K., Park D.S., Malleret B., Chakarov S., See P., Low D., Low G., Garcia-Miralles M., Zeng R., Zhang J., Goh C., Gul A., Hubert S., Lee B., Chen J., Low I., Shadan N-B., Lum J., Wei T-S., Mok E., Kawanishi S., Kitamura Y., Larbi A., Poidinger M., Renia L., Ng L-G., Wolf Y., Jung S., Önder T., Newell I., Huber T., Ashihara E., Garel S., Pouladi M., Ginhoux F. Induced-pluripotent-stem-cell-derived primitive macrophages provide a platform for modeling tissue-resident macrophage differentiation and function. Immunity, 2017, Vol. 47, pp. 183-198.</mixed-citation></citation-alternatives></ref><ref id="cit12"><label>12</label><citation-alternatives><mixed-citation xml:lang="ru">Trump L.R., Nayak R.C., Singh A.K., Emberesh S., Wellendorf A.M., Lutzko C.M., Cancelas J.A. Neutrophils derived from genetically modified human induced pluripotent stem cells circulate and phagocytose bacteria in vivo. Stem Cells Transl. Med., 2019, Vol. 8, no. 6, pp. 557-567.</mixed-citation><mixed-citation xml:lang="en">Trump L.R., Nayak R.C., Singh A.K., Emberesh S., Wellendorf A.M., Lutzko C.M., Cancelas J.A. Neutrophils derived from genetically modified human induced pluripotent stem cells circulate and phagocytose bacteria in vivo. Stem Cells Transl. Med., 2019, Vol. 8, no. 6, pp. 557-567.</mixed-citation></citation-alternatives></ref><ref id="cit13"><label>13</label><citation-alternatives><mixed-citation xml:lang="ru">Wallis R.S., O’Garra A., Sher A., Wack A. Host-directed immunotherapy of viral and bacterial infections: past, present and future. Nat. Rev. Immunol., 2023, Vol. 23, no. 2, pp. 121-133.</mixed-citation><mixed-citation xml:lang="en">Wallis R.S., O’Garra A., Sher A., Wack A. Host-directed immunotherapy of viral and bacterial infections: past, present and future. Nat. Rev. Immunol., 2023, Vol. 23, no. 2, pp. 121-133.</mixed-citation></citation-alternatives></ref><ref id="cit14"><label>14</label><citation-alternatives><mixed-citation xml:lang="ru">van 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, Vol. 8, e71098. doi: 10.1371/journal.pone.0071098.</mixed-citation><mixed-citation xml:lang="en">van 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, Vol. 8, e71098. doi: 10.1371/journal.pone.0071098.</mixed-citation></citation-alternatives></ref></ref-list><fn-group><fn fn-type="conflict"><p>The authors declare that there are no conflicts of interest present.</p></fn></fn-group></back></article>
