Abstract

Background and aims

Allogeneic human umbilical mesenchymal stem cells (alloUMSC) are convenient cell source for stem cell-based therapy. However, immune rejection is a major obstacle for clinical application of alloUMSC for cardiac repair after myocardial infarction (MI). The immune rejection is due to the presence of human leukocyte antigen (HLA) class I molecule which is increased during MI. The aim of this study was to knockout HLA light chain β₂-microglobulin (B2M) in UMSC to enhance stem cell engraftment and survival after transplantation.

Methods and results

We developed an innovative strategy using CRISPR/Cas9 to generate UMSC with B2M deletion (B2M^–UMSC). AlloUMSC injection induced CD8⁺ T cell-mediated immune rejection in immune competent rats, whereas no CD8⁺ T cell-mediated killing against B2M^–UMSC was observed even when the cells were treated with IFN-γ. Moreover, we demonstrate that UMSC-derived exosomes can inhibit cardiac fibrosis and restore cardiac function, and exosomes derived from B2M^–UMSC are more efficient than those derived from UMSC, indicating that the beneficial effect of exosomes can be enhanced by modulating exosome’s imprinting. Mechanistically, microRNA sequencing identifies miR-24 as a major component of the exosomes from B2M^–UMSCs. Bioinformatics analysis identifies Bim as a putative target of miR-24. Loss-of-function studies at the cellular level and gain-of-function approaches in exosomes show that the beneficial effects of B2M^–UMSCs are mediated by the exosome/miR-24/Bim pathway.

Conclusion

Our findings demonstrate that modulation of exosome’s imprinting via B2M knockout is an efficient strategy to prevent the immune rejection of alloUMSCs. This study paved the way to the development of new strategies for tissue repair and regeneration without the need for HLA matching.

中文翻译：

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敲除 β2 微球蛋白可通过调节外泌体印记和抑制干细胞诱导的免疫排斥来增强心脏修复

 抽象的

 背景和目标

同种异体人脐带间充质干细胞（alloUMSC）是干细胞治疗的便捷细胞来源。然而，免疫排斥是alloUMSC临床应用用于心肌梗死（MI）后心脏修复的主要障碍。免疫排斥是由于人白细胞抗原 (HLA) I 类分子的存在而导致的，该分子在 MI 期间会增加。本研究的目的是敲除 UMSC 中的 HLA 轻链 β _{2 -微}球蛋白 (B2M)，以增强干细胞的植入和移植后的存活率。

 方法和结果

我们开发了一种创新策略，使用 CRISPR/Cas9 生成具有 B2M 删除的 UMSC（B2M ^– UMSC）。 AlloUMSC 注射在免疫能力强的大鼠中诱导 CD8 ⁺ T 细胞介导的免疫排斥，而即使用 IFN-γ 处理细胞，也没有观察到 CD8 ⁺ T 细胞介导的针对 B2M ^– UMSC 的杀伤。此外，我们证明UMSC衍生的外泌体可以抑制心脏纤维化并恢复心脏功能，并且B2M ^- UMSC衍生的外泌体比UMSC衍生的外泌体更有效，这表明可以通过调节外泌体的印迹来增强外泌体的有益作用。从机制上讲，microRNA 测序将 miR-24 识别为 B2M ^– UMSC 外泌体的主要成分。生物信息学分析将Bim确定为 miR-24 的推定靶标。细胞水平的功能丧失研究和外泌体的功能获得方法表明，B2M ^– UMSC 的有益作用是由外泌体/miR-24/Bim 途径介导的。

 结论

我们的研究结果表明，通过 B2M 敲除调节外泌体的印记是防止 alloUMSC 免疫排斥的有效策略。这项研究为开发无需 HLA 匹配的组织修复和再生新策略铺平了道路。