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.