Transcriptome and Secretome Based Assay Matrix to Define Mesenchymal Stromal Cell Immune Potency

Assays that inform on MSC's immune potency need to be defined in the cell therapy arena in an effort to meet the requirement of such by Regulatory authorities as part of advanced clinical trials. MSCs possess a plurality of putative regenerative and immunomodulatory properties and hence an assay matrix approach would likely best capture the summation effector pathways involved in immunomodulation. Here we have developed two assay systems that can serve as complementary surrogate measures of potency: (1) Quantitative RNA based array for genes specific to MSC's immunomodulatory and homing properties; and, (2) MSC Secretome analysis of cytokines and morphogens. Both assay systems are applied to resting and cytokine activated MSC lots and differential expression of surrogate markers serve as the quantitative metric of potency. Considering the well described responsiveness of MSCs to IFNγ and TNFα, we systematically analyzed MSCs activated with IFNγ, TNFα, IFNγ + TNFα and performed nanoscale qRT-PCR Fluidigm® arrays which allows us to interrogate expression levels of 48 transcripts selected on the basis their published association with MSC immune function, homing and regenerative properties. Volcano plot analysis identified a unique signature of genes that are transcriptionally upregulated by IFNγ, TNFα and IFNγ + TNFα. Indoleamine 2,3 Dioxygenase (IDO), CXCL9, CXCL10, CXCL11 and CIITA are upregulated by IFNγ while TNFα upregulates CXCL12, TSG6, A20, IL8, ULBP3 and CCL5. IFNγ + TNFα significantly (Negative Log P value 3) enhances the expression such as IDO, ICAM1, PDL1, CXCL9, CXCL10, CXCL11, HLAG5, and COX2 . For MSC Secretome Analysis, we have cultured MSCs in the presence and absence of aCD3aCD28 activated Peripheral Blood Mononuclear Cells (PBMCs) and analysed their secretome using Luminex® 30plex cytokine analysis. We have found that MSCs cultured with activated PBMCs significantly upregulate the cytokines MCP-1, VEGF, GCSF and CXCL10. In addition, MSCs activation correlates with decrease the production of cytokines such as IFNγ, TNFα, IL2R, IL1Ra, IL17 and IL13 by activated PBMCs. Combination of concise transcriptome and secretome assays as part of a matrix assay can be utilized to robustly define surrogate measures of potency when performing paired analysis of resting and IFNγ activated MSC lots. We propose that this approach obviates the need of a universal cell ruler and may serve as a stand-alone potency release strategy.