Institute for Cardiovascular Science and Department of Cardiovascular Surgery, First Affiliated Hospital of Soochow University, Suzhou, 215123, Jiangsu, People's Republic of China.
Department of Cardiac Surgery, Fuwai Hospital, Beijing, 100037, People's Republic of China.
Cell Mol Life Sci. 2020 Mar;77(5):937-952. doi: 10.1007/s00018-019-03220-3. Epub 2019 Jul 16.
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 β-microglobulin (B2M) in UMSC to enhance stem cell engraftment and survival after transplantation.
We developed an innovative strategy using CRISPR/Cas9 to generate UMSC with B2M deletion (B2MUMSC). AlloUMSC injection induced CD8 T cell-mediated immune rejection in immune competent rats, whereas no CD8 T cell-mediated killing against B2MUMSC 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 B2MUMSC 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 B2MUMSCs. 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 B2MUMSCs are mediated by the exosome/miR-24/Bim pathway.
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.
同种异体人脐带间充质干细胞（alloUMSC）是干细胞治疗的方便细胞来源。然而，免疫排斥是同种异体 UMSC 在心梗后用于心脏修复的临床应用的主要障碍。免疫排斥是由于人白细胞抗原（HLA）I 类分子的存在所致，该分子在心肌梗死后增加。本研究旨在敲除 UMSC 中的 HLA 轻链β-微球蛋白（B2M），以增强移植后干细胞的植入和存活。
我们开发了一种使用 CRISPR/Cas9 的创新策略，生成了 B2M 缺失的 UMSC（B2MUMSC）。alloUMSC 注射在免疫功能正常的大鼠中诱导 CD8 T 细胞介导的免疫排斥，而即使在用 IFN-γ处理时，也未观察到针对 B2MUMSC 的 CD8 T 细胞杀伤。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，并且源自 B2MUMSC 的外泌体比源自 UMSC 的外泌体更有效，表明通过调节外泌体的印迹可以增强外泌体的有益作用。机制上，miRNA 测序确定 miR-24 为 B2MUMSC 来源的外泌体的主要成分。生物信息学分析确定 Bim 为 miR-24 的潜在靶标。细胞水平的功能丧失研究和外泌体的功能获得方法表明，B2MUMSC 的有益作用是通过外泌体/miR-24/Bim 途径介导的。
我们的研究结果表明，通过 B2M 敲除调节外泌体的印迹是防止同种异体 UMSC 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。
