ABSTRACT

An immune reaction is a protector of our body but a target to be overcome for all non-self-derived medicine. Extracellular Vesicles (EVs), noted as a primary alternative to cell therapy products that exhibit immune rejection due to mismatching-major histocompatibility complex (MHC), were discovered to have excellent curative effects through the delivery of various biologically active substances. Although EVs are sure to incur immune reaction by immunogenicity due to alloantigens from their parental cells, their immune rejection is rarely known. Hence, to develop cell lines and EVs as medicines with no immune rejection, we noted the immune tolerance where the foetus, as semi-allograft, is perfectly protected from the maternal immune system. We designed the ex-vivo culture systems to simulate in-vivo environmental factors inducing extravillous trophoblast (EVT)-specific Human Leukocyte Antigen-G (HLA-G) expression and secretion of HLA-G-bearing EVs at the mother-foetus interface. Using them, we confirmed that immune-tolerized stem cells (itSCs) continuously expressing and secreting HLA-G like EVTs during pregnancy can be induced. Also, EVs secreted from itSCs are verified as immune-tolerized EVs (itSC-EVs) containing HLA-G and not causing immune rejection through various analytical methods. These findings can provide a new perspective on the local and extensive immune tolerance environment where HLA-G is expressed and secreted by pregnancy-related hormones and different biological conditions. Furthermore, they show the new way to develop itSCs-EVs-based therapeutics that are free from time, space, and donor limitation causing immune rejection.

Abbreviations

CFSE: carboxyfluorescein succinimidyl ester; DC: dendritic cells; ELISA: enzyme-linked immunosorbent assay; EV: extracellular vesicles; EVT: extravillous trophoblast; FSH: follicle stimulating hormone; HA: hyaluronic acid; hCG: human chorionic gonadotropin; HLA-G: human leukocyte antigen G; iPSC: induced pluripotent stem cells; itSC-EVs: immune-tolerized extracellular vesicles from itSCs; itTBC-EVs: immune-tolerized extracellular vesicles from itTBCs; itSCs: immune tolerized stem cells; itTBCs: immune-tolerized trophoblast cells; LH: luteinizing hormone; MHC: major histocompatibility complex; MSC: mesenchymal stem cells; NK: natural killer cells; NTA: nanoparticle tracking analysis; PBMC: peripheral blood mononuclear cells; PHA: phytohemagglutinin; SP-IRIS: single particle interferometric reflectance imaging sensing; STB: syncytiotrophoblast

中文翻译：

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妊娠期免疫耐受细胞系和细胞外囊泡诱导环境促进半移植物免疫耐受中HLA-G持续表达和分泌的研究。

摘要

免疫反应是我们身体的保护者，但也是所有非自源性药物都需要克服的目标。细胞外囊泡（EVs）被认为是细胞治疗产品的主要替代品，由于主要组织相容性错配复合物（MHC）而表现出免疫排斥，被发现通过递送各种生物活性物质具有出色的疗效。尽管 EV 肯定会由于来自其亲代细胞的同种异体抗原的免疫原性而引起免疫反应，但它们的免疫排斥反应鲜为人知。因此，为了将细胞系和 EV 开发为没有免疫排斥的药物，我们注意到了免疫耐受性，其中胎儿作为半同种异体移植物可以完全免受母体免疫系统的影响。我们设计了离体培养系统来模拟体内环境因素，诱导绒毛外滋养层 (EVT) 特异性人类白细胞抗原-G (HLA-G) 表达和母胎界面处带有 HLA-G 的 EV 的分泌。使用它们，我们证实可以诱导免疫耐受干细胞 (itSC) 在怀孕期间持续表达和分泌 HLA-G 样 EVT。此外，通过各种分析方法，从 itSC 分泌的 EV 被证实为含有 HLA-G 且不会引起免疫排斥的免疫耐受 EV (itSC-EV)。这些发现可以为了解妊娠相关激素和不同生物学条件下 HLA-G 表达和分泌的局部和广泛的免疫耐受环境提供新的视角。此外，

缩写

CFSE：羧基荧光素琥珀酰亚胺酯；DC：树突状细胞；ELISA：酶联免疫吸附试验；EV：细胞外囊泡；EVT：绒毛外滋养层；FSH：促卵泡激素；HA：透明质酸；hCG：人绒毛膜促性腺激素；HLA-G：人白细胞抗原G；iPSC：诱导多能干细胞；itSC-EVs：来自 itSCs 的免疫耐受的细胞外囊泡；itTBC-EVs：来自 itTBCs 的免疫耐受细胞外囊泡；itSCs：免疫耐受干细胞；itTBCs：免疫耐受的滋养层细胞；LH：促黄体激素；MHC：主要组织相容性复合体；MSC：间充质干细胞；NK：自然杀伤细胞；NTA：纳米粒子跟踪分析；PBMC：外周血单个核细胞；PHA：植物血凝素；SP-IRIS：单粒子干涉反射成像传感；STB：合体滋养细胞