Predictive Analysis of the Immunosuppressive Functionality of Human Bone Marrow Derived Mesenchymal Stem Cells as Cellular Therapeutics

Abstract

Human Mesenchymal Stem/Stromal Cells (MSCs) of bone marrow carry immunomodulatory and regenerative properties and are being tested as a cellular therapy for inflammatory and degenerative disorders. They are involved with the paracrine secretion of anti-inflammatory cytokines and chemokines and the promotion of anti-inflammation in tissue microenvironments by dampening inflammatory T-cells. However, the mechanism of action of MSCs on T-cells has yet to be understood. Here we aim to identify the pattern of chemokine secretion in human bone marrow MSCs and their regulation and functions on T-cell responses and immune suppression. MSCs were derived from healthy human bone marrow aspirates. MSC secretome was collected systematically under defined cell densities and subjected to multiplex secretome analysis with or without exogenous stimulation to identify inherently secreted MSC chemokines. MSC derived chemokines’ immunosuppressive role on T-cells was further determined with a PBMC and MSC coculture and siRNA chemokine transfection strategies. MSC secretome was further tested on human peripheral blood mononuclear cells (PBMCs) derived from blood and early phosphorylation of signaling molecules in T-cells were specifically analyzed utilizing PhosflowTM technology in flow cytometry. Of thirty tested chemokines nine (CXCL16, CCL2, CXCL6, CCL7, CXCL1, CCL13, CCL5 CXCL2 and CCL1) are secreted inherently by MSCs suggesting that MSC potency and immunosuppressive potential can be determined by the presence of these chemokines. In addition, MSC mediated blocking of T cell proliferation predominantly inversely correlates with chemokines. Knockdown of chemokines have demonstrated that MSC sourced inherent chemokines do not actively play a role in T cell suppression and thus are the bystander predictors of T cell suppression. The present analysis of MSC’s matrix chemokine responses can be deployed in the advanced potency determination of MSCs. As well, little difference was seen between chemokine levels from intestinal organoid secretome samples from IBD and non-IBD cultures. Seven signaling molecules [PLCγ1, PLCγ2, PKCα, JNK, P38 MAPK, Erk ½, pAkt (pS473)] were analyzed for phosphorylation events in T-cells when stimulated with MSC secretome. Our results provided evidence that MSC derived chemokines and secretome predicts T-cell suppression. These mechanistic understandings will help us to improve MSC based cellular therapy.