A novel approach for treating diabetes using pulsed focused ultrasound and intra-arterial delivery of mesenchymal stem cell based therapies directly into the pancreas

项目摘要 1型糖尿病（T1 D）的治疗包括终身每日注射胰岛素，以确保严格的代谢控制。 然而，最近的研究表明，糖尿病患者在其体内仍有残余的功能性β细胞。 胰腺和因此可以恢复和/或再生β细胞数量和功能的治疗干预 会对这些病人产生深远的影响。保存和再生β细胞的一种有希望的方法是 将间充质干细胞（MSC）疗法直接输送到胰腺。MSC可以释放 免疫调节、血管生成、抗炎、抗凋亡和抗纤维化因子进入其周围 调节免疫系统以及刺激受损组织再生的微环境; 这些旁分泌因子以可溶形式或在细胞外囊泡（EV）内释放。研究 显示亲本MSC（即细胞疗法）和MSC衍生的EV（即无细胞疗法）两者都可以 改善胰岛的存活和功能。不幸的是，基于MSC的治疗的临床转化 糖尿病的治疗一直是次优的，主要是由于大多数MSC和EV被困在 肺和网状内皮系统，分别在常规静脉内（IV）注射。因此，我们认为， 在本提案中，我们将：（i）研究一种使用脉冲聚焦超声（pFUS）的新方法， 轻轻摇动胰腺，释放出能够吸引和保留MSC治疗的化学物质 通过动脉内（IA）注射直接递送到腺体中，以及（ii）确定哪种来源和 一种基于MSC的治疗最适合于再生和保护糖尿病胰腺。在目标1中，我们 研究pFUS对胰腺的生物学效应，pFUS是一种临床可用的技术， 单个胰岛和不同来源的MSC。在初步研究中，我们发现声波可以 不仅刺激胰岛和MSC，而且它们还可以诱导化学引诱物（即， 细胞因子，营养因子和细胞粘附分子）;后者将有助于促进胰腺中的细胞因子， 基于MSC的治疗对糖尿病胰腺内挣扎的胰岛的归巢、渗透和保留。在 目的2（亲本MSC）和3（MSC衍生的EV），我们将评估MSC衍生的基于MSC的疗法的效果。 从不同来源（即骨髓、脂肪组织和脐带）对再生糖尿病 当它们通过IA注射直接给予胰腺时，在用“预充”胰腺之前和之后， pFUS。为了实现这些目标，我们开发了一种技术，通过 它的动脉血液供应，旨在模拟介入放射科医生可以使用 血管内技术。此外，我们将使用一种名为ExoTIC的新型装置来分离MSC衍生的EV， 纯度和产率都很高。我们希望共同确定最佳参数（即基于MSC的 治疗、递送途径和pFUS参数），其可以在临床上转化用于T1 D患者的治疗。

PROJECT SUMMARY Treatment for type 1 diabetes (T1D) involves life-long daily injections of insulin to ensure tight metabolic control. However, recent studies have shown that diabetic patients still have residual functional β cells within their pancreas and hence therapeutic interventions that could recover and/or regenerate β cell quantity and function would have a profound impact on these patients. A promising approach to preserve and regenerate β cells is to deliver mesenchymal stem cell (MSC)-based therapies directly to the pancreas. MSCs can release immunomodulatory, angiogenic, anti-inflammatory, anti-apoptotic and anti-fibrotic factors into their surrounding microenvironment to modulate the immune system as well as stimulate the regeneration of damaged tissues; these paracrine factors are released either in a soluble form or within extracellular vesicles (EVs). Studies have shown that both parent MSCs (i.e. a cellular therapy) and MSC-derived EVs (i.e. a cell free therapy) can improve the survival and function of islets. Unfortunately, the clinical translation of MSC-based therapies for the treatment of diabetes has been sub-optimal, predominantly due to majority of MSCs and EVs getting trapped in the lung and reticuloendothelial system, respectively, following conventional intravenous (IV) injection. Hence, in the present proposal, we will: (i) investigate a novel approach using pulsed focused ultrasound (pFUS) to gently shake and “prime” the pancreas to release chemicals which can attract and retain MSC-based therapies that are delivered directly into the gland by intra-arterial (IA) injection and (ii) determine which source and type of MSC-based therapy is best suited to regenerate and protect the diabetic pancreas. In Aim 1, we will investigate the biological effects of pFUS, which is a clinically available technology, on the pancreatic gland, individual pancreatic islets and different sources of MSCs. In pilot studies, we have found that soundwaves can not only stimulate pancreatic islets and MSCs, but they can also induce the expression of chemoattractants (i.e. cytokines, trophic factors, and cell adhesion molecules) in the pancreas; the latter will help to facilitate the homing, permeation and retention of MSC-based therapies to struggling islets within the diabetic pancreas. In Aims 2 (parent MSCs) and 3 (MSC-derived EVs), we will evaluate the effect of MSC-based therapies derived from different sources (i.e. bone marrow, adipose tissue and umbilical cord) on regenerating the diabetic pancreas when they are given directly into the gland via IA injection, before and after “priming” the pancreas with pFUS. To achieve these aims, we developed a technique to deliver therapeutics directly into the pancreas, via its arterial blood supply, which is designed to simulate what Interventional Radiologists can do using endovascular techniques. In addition, we will use a novel device called ExoTIC to isolate MSC-derived EVs with high purity and yield for our studies. Together, we hope to determine the optimal parameters (i.e. MSC-based therapy, delivery route and pFUS parameters) that can be clinically translated for the treatment of T1D patients.