Potency analysis of Mesenchymal Stromal Cells using a phospho-STAT matrix loop analytical approach

Abstract

Potency assays for Mesenchymal Stromal Cells (MSCs) need to be defined in advanced clinical trials. Here, we have developed an assay matrix approach that captures the STAT phosphorylation of MSCs upon stimulation with their combined secretome that arose with the interaction of activated peripheral blood mononuclear cells (PBMCs). Secretome of heat inactivated (HI) MSCs cocultured with and without activated PBMCs were used as an internal reference. We have compared the short-term phosphorylation status of STAT1, STAT3, STAT4, STAT5 and STAT6 on MSCs derived from human bone marrow, adipose tissue and umbilical cord using phosflow technology. Secretome of live MSCs cocultured with activated PBMCs down regulate STAT1 and STAT3 phosphorylation on MSCs while the secretome of HI-MSCs or PBMCs do not. Thus, investigation of the combined secretome of MSC and PBMC interaction on MSCs determine the potency of MSCs as the generator and sensor of the secretome. Bone marrow, adipose and umbilical cord MSCs are comparable in modulating STAT1 and STAT3 responses. Measurements of STAT1 and STAT3 phosphorylation on MSCs as responder cells correlate and predict allogeneic T cell suppression. Our comparative phospho matrix approach between live and reference HI MSCs defines the potency of MSCs as both stimulators and responders as part of a robust platform for predictive potency analysis.

Graphical Abstract:

We have demonstrated a potency testing approach using MSCs as the stimulator and sensor of the secretome upon interaction with Peripheral Blood Mononuclear Cells. Heat inactivated MSCs serve as the reference control for live MSCs in this loop analytical approach.