mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2024.001mes-66Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕORIGINAL RESEARCHIn vitro оценка иммуногенности хондроцитов, полученных из индуцированных плюрипотентных стволовых клеток с нокаутом B2MIn vitro assessment of immunogenicity in chondrocytes obtained from the B2M knockout induced pluripotent stem cellsБариноваА. А.BarinovaA. A.

Анна Александровна Баринова

ул. Малая Пироговская, д. 1а, 119435, г. Москва

Anna A. Barinova

Malaya Pirogovskaya, 1а, 119435, Moscow

barinova.anna.al@mail.ruПикинаА. С.PikinaA. S.

Москва

Moscow

ГолубинскаяП. А.GolubinskayaP. A.

Москва

Moscow

РучкоЕ. С.RuchkoE. S.

Москва

Moscow

ЕремеевА. В.EremeevA. V.

Москва

Moscow

Федеральный научно-клинический центр физико-химической медицины имени Ю. М. Лопухина Федерального медико-биологического агентстваРоссияLopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyRussian FederationИнститут биологии развития имени Н. К. КольцоваРоссияKoltzov Institute of Developmental Biology of Russian Academy of SciencesRussian FederationФедеральный научно-клинический центр физико-химической медицины имени Ю. М. Лопухина Федерального медико-биологического агентства; Институт биологии развития имени Н. К. КольцоваРоссияLopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency; Koltzov Institute of Developmental Biology of Russian Academy of SciencesRussian Federation2024231020242613242Copyright © Баринова А.А., Пикина А.С., Голубинская П.А., Ручко Е.С., Еремеев А.В., 20242024Баринова А.А., Пикина А.С., Голубинская П.А., Ручко Е.С., Еремеев А.В.Barinova A.A., Pikina A.S., Golubinskaya P.A., Ruchko E.S., Eremeev A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/66

В настоящее время клеточные технологии являются одним из инструментов по восстановлению хрящевой ткани. Создание универсального гипоиммуногенного трансплантата хрящевой ткани из дифференцированных производных индуцированных плюрипотентных стволовых клеток (ИПСК) могло бы решить проблему нехватки хрящевого клеточного продукта. Однако на сегодняшний день мало данных об иммуногенности таких тканеинженерных препаратов. Целью работы было создать хрящевой имплант из дифференцированных производных ИПСК, дефицитных по B2M, и оценить его иммуногенность. С помощью ранее разработанного протокола дифференцировали ИПСК как дикого типа, так и с нокаутом B2M в хондроцитарные производные. После проверки качества полученных линий методом полимеразной цепной реакции и иммуноцитохимическим исследованием кокультивировали полученные линии с мононуклеарными клетками периферической крови здорового донора. По окончании кокультивации методом проточной цитометрии оценивали активацию и дегрануляцию CD8+-Т-лимфоцитов по экспрессии CD69 и CD107a на поверхности клеток соответственно. Хондроцитарные производные ИПСК экспрессировали маркеры хрящевой ткани. Цитометрический анализ не выявил существенных различий между иммуногенностью хондроцитарных производных ИПСК с нокаутом и без нокаута B2M, а также клетками хрящевой ткани здорового донора. Иммуногенность хондроцитарных производных была выше, чем у гипоиммуногенных нередактированных ИПСК. Нокаутированные по B2M ИПСК демонстрировали тенденцию к большей активации CD8+-Т-лимфоцитов. Таким образом, нокаут B2M в хондроцитарных производных ИПСК не оказал существенного влияния на иммуногенность ткани. Необходимо дополнительное редактирование генов, кодирующих MHC II и CD47, для получения менее иммуногенного продукта.

Today, the cell-based technologies are one of the instruments used for the cartilage tissue repair. Creation of a universal hypoimmunogenic cartilage tissue graft from the differentiated derivatives of induced pluripotent stem cells (iPSCs) might solve the problem of the lack of the cartilage cell product. However, currently there is little data on immunogenicity of such tissue-engineered preparations. The study was aimed to create a cartilage implant from the differentiated derivatives of the B2M-deficient iPSCs and assess its immunogenicity. The previously developed protocol was used to ensure differentiation of both wild-type and B2M knockout iPSCs into chondrocyte-like cells. After quality control of the resulting cell lines by conducting polymerase chain reaction and immunocytochemical assessment, the resulting cell lines were co-cultured with the peripheral blood mononuclear cells of a healthy donor. When co-cultivation was over, activation and degranulation of CD8+ T cells was assessed by flow cytometry analysis based on the CD69 and CD107a expression on the cell surface, respectively. The iPSC-derived chondrocytes expressed the cartilage tissue markers. Flow cytometry analysis revealed no substantial differences in immunogenicity between the derivatives of wild-type and B2M knockout iPSCs, as well as from the cartilage tissue cells of a healthy donor. Immunogenicity of chondrocyte-like cells was higher than that of hypoimmunogenic non-edited iPSCs. The B2M knockout iPSCs demonstrated a trend towards greater activation of CD8+ T cells. Thus, the B2M knockout in the iPSC-derived chondrocytes had no significant effect on the tissue immunogenicity. It is necessary to further edit the genes encoding MHC II and CD47 to obtain a less immunogenic product.

ИПСКрегенеративная медицинахондрогенезхондроцитыиммуногенностьiPSCsregenerative medicinechondrogenesischondrocytesimmunogenicityполучение хондроцитарных производных из ИПСК выполнено в рамках государственного задания №122032300191-2 «Органоид-22». Иммуноцитохимический и ПЦР-анализы экспрессии хондрогенных маркеров в хондроцитарных проиводных ИПСК, а также оценка иммуногенности этих хондроцитарных производных проводились в рамках проекта РНФ #22-15-00250 «Сравнение хондрогенного потенциала хрящевой ткани, полученной с помощью первичных культур хондроцитов и дифференцированных производных индуцированных плюрипотентных стволовых клеток».chondrocyte-like cells were obtained from iPSCs within the framework of the State Assignment #122032300191-2 “Organoid-22”. 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The authors declare that there are no conflicts of interest present.