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多万人受到影响 在美国和世界范围内正变得越来越普遍。抗肿瘤坏死因子、抗黏附分子等生物制品的应用 药物治疗显著改善了CD患者的生活质量，而生物制品往往无效 随着时间的推移，会有显著的副作用。利用免疫抑制能力的基于细胞的新疗法 成人间充质干细胞(MSC)治疗CD等免疫介导性疾病目前正在进行临床试验。一个 累积的大量数据，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 抗肿瘤坏死因子治疗无效的肛周瘘患者。然而，系统性MSC治疗腔性CD的疗效较低 疗效，这可能是由于无数的原因，其中许多没有得到充分的调查。所有的小鼠研究 在需要操作才能发展的小鼠模型中展示了MSC疗法的好处 这些药物主要用于治疗大肠炎症。尽管如此，人类近三分之二的CD 患者有小肠受累。因此，迫切需要研究MSC在相关疾病中的治疗方法。 和临床前CD小鼠模型。在本研究中，我们建议对SAMP-1/YitFc(SAMP)小鼠进行研究 人骨髓间充质干细胞(HMSC)的治疗。SAMP菌株是一种独特的模型，可以自发产生CD样细胞 小肠(SI)炎症与人类疾病有着惊人的相似之处。我的强势初选 有数据表明，SAMP小鼠建立的SI炎症治疗一次剂量的骨髓来源 人骨髓间充质干细胞(HMSC)炎症反应明显减轻，粘膜愈合良好。因此，我们的假设 HMSC治疗SAMP小鼠SI炎症的机制与其转录调控有关 宿主组织进入抗炎和组织再生途径。我将在两个具体目标上检验这一假设。 在具体目标1中，我们将使用SAMP小鼠，我们新的SAMP医学复发模型，新技术 包括3D立体显微镜(用于粘膜愈合)以检验hMSC可以治疗、预防SI的假设 炎症反应，确定hMSC给药的最佳途径。此外，使用了一种新型的hMSC优化 对于免疫抑制，我们将确定是否可以提高MSC治疗的疗效。在目标2中， 使用通过三重报告、最先进的成像技术、激光捕获 宿主细胞和间充质干细胞的显微解剖和单细胞核糖核酸测序我们将探讨其机制(S) 骨髓间充质干细胞治疗后SAMP愈合。我们的临床前实验设计，测试1)治疗 确诊疾病，2)维持缓解，3)MSC给药的最佳途径，4)使用 增强免疫抑制的hMSC 5)hMSC对粘膜愈合的作用，以及6)比较生物学方法 理解愈合的机制(S)与人类IBD具有很高的相关性。由此产生的数据 格兰特将导致一种更机械化的方法来进行治疗CD患者的人体临床试验，并可能确定 促进组织再生和愈合的新型分子，将形成机械R01的基础 未来。因此，我们已经生成了一个提案，该提案建立在我们的初步发现的基础上，通过生成新的 不仅与CD有关，而且可能与其他免疫介导性疾病相关的科学思想 骨髓间充质干细胞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.