Novel immunosuppressive mechanism of human bone marrow mesenchymal stem cells in experimental Crohn's disease

摘要 克罗恩病（CD）是一种慢性肠道炎症性疾病，在美国有70多万人感染。 美国，并在世界范围内变得越来越普遍。抗肿瘤坏死因子、抗粘附分子等生物制剂的应用 药物治疗显著改善了CD患者的生活质量，但生物制剂往往失去疗效 并有显著的副作用。利用免疫抑制能力的新细胞疗法 成人间充质干细胞（MSC）的免疫介导的疾病，如CD目前在临床试验。一 累积的数据，包括我们自己的工作表明，局部注射MSC是一种有前途的治疗方法， 抗TNF难治性肛周瘘患者。然而，全身MSC治疗管腔CD具有较低的 有效性，这可能是由于无数的原因与许多不充分的调查。所有的鼠类研究 进行的研究证实了MSC治疗在需要操作以发育的小鼠模型中的益处。 炎症，并专注于治疗大肠炎症。然而，近三分之二的人类CD 患者有小肠受累。因此，迫切需要研究MSC治疗与疾病相关的疾病， 和CD的临床前鼠模型。在这项研究中，我们建议研究SAMP-1/YitFc（SAMP）小鼠， SAMP菌株是一种独特的模型，其自发地发展CD样细胞， 小肠（SI）炎症，其与人类疾病具有令人印象深刻的相似性。我强大的初步 数据显示，用一剂骨髓来源的 人MSC（hMSC）具有显著较低炎症并具有粘膜愈合。因此，我们的假设 hMSC是通过涉及调节 宿主组织的抗炎和组织再生途径。我将在两个具体目标中检验这个假设。 在具体目标1中，我们将使用SAMP小鼠，我们的新SAMP医学复发模型，新技术 包括3D立体显微镜检查（用于粘膜愈合），以检验hMSC可以治疗、预防SI 炎症和确定hMSC施用的最佳途径。此外，使用新的hMSC优化， 对于免疫抑制，我们将确定我们是否可以增强MSC疗法的治疗效果。在目标2中， 使用三重报告基因转导的人和小鼠MSC，最先进的成像技术，激光捕获， 通过显微切割、宿主细胞和MSC的单细胞RNA测序，我们将研究 MSC治疗后SAMP愈合。我们的临床前实验设计，测试1）治疗 确定的疾病，2）缓解的维持，3）MSC施用的最佳途径，4）使用 增强免疫抑制性hMSC 5）hMSC对粘膜愈合的影响，以及6）比较生物学方法 理解愈合机制对于人类IBD具有高度相关性。由此产生的数据 一笔赠款将为治疗CD患者的人体临床试验提供一种更机械的方法， 促进组织再生和愈合的新分子，将成为R 01机制的基础， 未来因此，我们提出了一项建议，该建议以我们的初步调查结果为基础， 不仅与CD相关，而且可能与其他免疫介导疾病相关的科学思想， MSC目前处于临床前或早期临床试验阶段。

Abstract Crohn's disease (CD) is chronic intestinal inflammatory disease that affects more than 700,000 individuals in the US and is becoming more common worldwide. The use of biologics such as anti-TNF and anti-adhesion molecule medications has significantly improved the quality of life of CD patients, however biologics tend to lose efficacy with time and have significant side effects. New cell based therapies that utilize the immunosuppressive capacity of adult mesenchymal stem cells (MSC) for immune mediated diseases like CD are currently in clinical trials. A cumulative body of data, including our own work shows that locally injected MSC are a promising therapy for patients with perianal fistulae refractory to anti-TNF's. However, systemic MSC therapy for luminal CD has lower efficacy, which may be due to myriad reasons with many inadequately investigated. All the murine studies performed demonstrate the benefit of MSC therapy in mouse models that require manipulation to develop inflammation and are focused on treating large intestinal inflammation. Though, nearly two thirds of human CD patients have small intestinal involvement. Thus, there is a critical need to study MSC therapy in disease relevant and preclinical murine models of CD. In this study, we propose to study the SAMP-1/YitFc (SAMP) mouse for treatment with human MSC (hMSC).The SAMP strain is a unique model that spontaneously develops CD-like small intestinal (SI) inflammation which has impressive similarities to the human disease. My strong preliminary data has shown that SAMP mice with established SI inflammation treated with one dose of bone marrow derived human MSC (hMSC) had significantly lower inflammation and had mucosal healing. Therefore, our hypothesis is that hMSC treat SI inflammation in SAMP mice by a mechanism involving the modulation of transcriptome of host tissue to an anti-inflammatory and tissue regeneration pathway. I will test this hypothesis in 2 specific aims. In specific aim 1, we will use the SAMP mice, our new SAMP medical recurrence model, novel techniques including 3D stereomicroscopy (for mucosal healing) to test the subhypothesis that hMSC can treat, prevent SI inflammation and determine the optimal route of hMSC administration. In addition, using a novel hMSC optimized for immunosuppression, we will determine if we can enhance the treatment efficacy of MSC therapy. In aim 2, using human and mouse MSCs transduced with triple reporter, state-of-the-art imaging techniques, laser capture microdissection, single cell RNA sequencing of host cells and MSCs we will investigate the mechanism(s) of healing in SAMP after treatment with MSC. Our preclinical experimental design that tests 1) treatment of established disease, 2) maintenance of remission, 3) the optimal route of MSC administration, 4) use of enhanced immunosuppressive hMSC 5) hMSC effect on mucosal healing, and 6) comparative biology approach to understand the mechanism (s) of healing has high relevance for human IBD. The data generated from this grant will lead to a more mechanistic approach to human clinical trials for treating CD patients and may identify novel molecules that promote tissue regeneration and healing that will form the basis for mechanistic R01s in the future. Thus, we have generated a proposal that builds upon our preliminary findings by generating new scientific ideas that have relevance not only to CD, but potentially to other immune mediated diseases where MSCs are currently in preclinical or early-stage clinical trials.