Modulation of therapeutic cells exosome content by autophagy

项目摘要 我们目前正在研究人骨髓间充质干细胞(MSCs)在组织修复中的应用 骨髓间充质干细胞植入受损器官。最近的发现表明，骨髓间充质干细胞的许多治疗益处可以 归因于各种生物分子的分泌，这些生物分子可以通过外切体、小膜分泌 内吞的小泡。大多数细胞类型都会分泌含有蛋白质、DNA、信使核糖核酸和 MicroRNA，所有这些都被认为在细胞间的交流中发挥作用。虽然之前的研究 我们目前的研究主要集中在外切体中发现的蛋白质的特性上。 外体RNA。越来越多的证据表明，外切体的形成和释放受 自噬途径，一种通过识别回收蛋白质和细胞器的自稳质量控制途径， 隔离和溶酶体降解。刺激自噬途径的条件可以抑制外体 释放，但同时自噬的药物抑制剂促进外切体的释放。为 在我们的研究中，我们建议使用一种称为“骨髓分离的成人多系诱导”的MSCs亚型(迈阿密)。 细胞由于其易于从骨髓中分离、分化能力、免疫调节和组织 修复能力，以及分泌各种趋化因子/生长因子的能力。 我们假设自噬调节了迈阿密细胞外切体的释放，调节了它们的RNA。 含量及其免疫调节能力。 为了刺激迈阿密细胞，我们将同时将它们暴露在炎症反应刺激剂干扰素下。 使用自噬的药物刺激剂或抑制剂。随后，我们将分离和鉴定 用纳米显微镜、电子显微镜和免疫印迹技术鉴定迈阿密细胞来源的外切体 比较外切体大小分布、外切体产量和标记(CD9、CD63和CD81)(目标1)。到时候我们会的 通过鉴定和验证Long来确定自噬的调节如何调节外体RNA含量 迈阿密细胞来源的外切体中的短(200个核苷酸以上)和短(200个核苷酸以下)RNA(AIM 2)。最后，我们将评估迈阿密细胞在调节自噬时的免疫调节特性。 迈阿密细胞-T细胞共培养及随后的流式细胞仪分析以评估T细胞增殖标志物 和细胞因子分泌(目标3)。这一机制研究的结果将加深我们对 自噬在调节外体中的RNA含量方面发挥作用，它也将揭示靶向自噬是否可以 被用来操纵RNA含量，并随后进行免疫调节。归根结底，这种知识是 预计将促进组织再生细胞疗法的进一步发展。 1

Project Summary We are currently investigating the use of human mesenchymal stromal cells (MSCs) for tissue repair by injecting MSCs into the damaged organ. Recent discoveries indicate that many of the therapeutic benefits of MSCs can be attributed to secretion of various biomolecules which can be secreted via exosomes, small membrane vesicles of endocytic origin. Most cell types secrete exosomes which contain proteins, DNA, mRNA, and microRNA, all of which which are thought to play a role in cell-cell communication. While previous research efforts largely focused on the characterization of proteins found in exosomes, our current research focuses on exosomal RNAs. Increasing evidence suggests that exosome formation and release are regulated by the autophagy pathway, a homeostatic quality control pathway that recycles proteins and organelles via recognition, sequestration, and lysosomal degradation. Conditions that stimulate autophagy pathway can inhibit exosome release, but at the same time pharmacological inhibitors of autophagy enhance the release of exosomes. For our study, we propose utilizing a subtype of MSCs called “marrow-isolated adult multilineage inducible” (MIAMI) cells due to their ease of isolation from bone marrow, differentiation capacity, their immunomodulatory and tissue repair capacities, and ability to secrete various chemokines/growth factors. We hypothesize that autophagy mediates release of exosomes from MIAMI cells, regulates their RNA content and their immunomodulatory capacity. To stimulate MIAMI cells, we will expose them to inflammatory response stimulator, IFN, while simultaneously applying pharmacologic stimulators or inhibitors of autophagy. Subsequently, we will isolate and characterize MIAMI cell-derived exosomes by using NanoSight, electron microscopy and immunoblotting to characterize and compare exosomes size distributions, exosome yield and markers (CD9, CD63 and CD81) (Aim 1). We will then determine how modulation of autophagy regulates exosomal RNA content by identifying and validating long (more than 200 nucleotides) and short (less than 200 nucleotides) RNAs in MIAMI cells-derived exosomes (Aim 2). Lastly, we will evaluate immunoregulatory properties of MIAMI cells upon modulation of autophagy using MIAMI cell-T cell co-cultures and subsequent flow cytometry analysis to assess T cell markers of proliferation and cytokine secretion (Aim 3). Results of this mechanistic study will increase our understanding of the role that autophagy plays in regulating RNA content in exosomes and it will also reveal whether targeting autophagy could be used to manipulate RNA content and subsequently immunomodulation. Ultimately, such knowledge is anticipated to foster further development of cell therapies for tissue regeneration. 1