FITNESS AND IMMUNOREGULATORY FUNCTIONS OF HUMAN MESENCHYMAL STROMAL CELLS FROM IMMUNOLOGICALLY PRIVILEGED VERSUS IMMUNOLOGICALLY ACTIVE TISSUES

Abstract

Mesenchymal Stem/Stromal Cells (MSCs) are non-hematopoietic, immunomodulatory cells with therapeutic potential. MSCs have been extensively researched; however, many mysteries hinder their progression to clinical application. This project investigated the immunoregulatory characteristics of MSCs from immunologically active tissue, bone marrow, and immunologically privileged tissue, cornea. An assay matrix consisting of flow cytometry, metabolic assays, secretory analysis, immunosuppressive assays, and qPCR was implemented to conduct this research. Aim one characterizes the immunosuppressive functionality of Human-Cornea derived Mesenchymal Stromal Cells (cMSCs). cMSCs demonstrate the capability to suppress the proliferation of T-cells in stimulated peripheral blood mononuclear cells (PBMCs) when cocultured or separated by a transwell membrane, which allows for the exchange of soluble factors. Further mechanistic analysis shows that activated PBMCs induce expression of the immunomodulatory enzymes of the tryptophan catabolizing pathway, specifically, indoleamine 2,3-dioxygenase (IDO). Knocking down of IDO abolishes cMSCs’ immunosuppression on the proliferation of T-cells. These results suggest that immunosuppression via cMSCs occurs in a paracrine manner using IDO as the principal mechanism. Therapeutic utilization of MSCs in the treatment of inflammatory bowel disease (IBD) patients is currently being tested and has been approved in Europe. As such, aim two elucidates the impact of confounding factors on the potency mechanisms of Human Bone Marrow-derived MSCs (BM-MSCs) in the large intestine. The secretory analysis determined that BM-MSCs secrete copious amounts of the angiogenic factor Vascular Endothelial Growth Factor-A (VEGF-A). Dose-dependent inhibition of VEGF-A is observed when BM-MSCs are cocultured with Human Large-intestinal Microvascular Endothelial Cells (HLMVECs). BM-MSCs’ immunosuppressive functionality is not compromised regardless of the presence of HLMVECs. Further analysis confirmed the expression of endothelial Nitric Oxide Synthase (eNOS) in HLMVECs. Blocking of eNOS by a pharmacological inhibitor restores BM-MSCs’ VEGF-A secretion and does not modulate MSCs’ immunosuppressive capabilities. These findings suggest wound healing and immunosuppression are two separable MSC potency mechanisms. Overall, this research has illustrated that regardless of anatomical tissue site, MSCs display non-overlapping immunosuppressive and wound-healing mechanisms.