Towards a consensus potency assay for mesenchymal stromal cells: identification of activation markers reliable across media formulations, donor and tissue source

Abstract

Background & Aim Reliable metrics to assess therapeutic potential of mesenchymal stromal cell (MSC) batches are needed for successful translation of MSC-based products. We previously reported that 1 of 47 ISCT-proposed surrogate markers of immunomodulation, ICAM-1, reports activation in 3 donor populations each of human umbilical cord (UC) and bone marrow (BM) MSCs primed by TNF-α, IL-1β or IFN-γ. Seventeen other transcript markers are reliable in at least 1 context. Here we expanded the test matrix to include protein-free (PF), xeno-free (XF) and FBS supplemented (FS) media, and combined TFN-α+IFN-γ. Methods, Results & Conclusion UC-MSCs (Tissue Regeneration Therapeutics Inc., CAN) and BM-MSCs (RoosterBio Inc. (RBI), USA) were cultured in XF or FS media (RBI). MSCs were primed for 24 hours with TNF-α (50ng/ml), IFN-γ (50ng/ml), IL-1β (80pg/ml) or TNF-α+IFN-γ (10ng/ml each) in PF, XF or FS media (n=3). RT-qPCR was performed for the 18 curated ISCT genes and 3 potential new markers identified in the first study. Data was normalized to mean expression of ACTB, GAPDH, RPS18 and YWHAZ reference genes (ΔCq), and relative RNA abundance of primed versus resting MSCs assessed using 2−ΔΔCq. Statistically significant differences were determined by false discovery rate-adjusted p The refined panel of surrogate markers was responsive in all 3 media formulations within the test matrices, although relative levels of 9 biomarkers were significantly affected by media type in at least 1 context. Notably, ICAM-1 and 1 candidate gene remained reliable in all conditions. Stimulation with TFN-α+IFN-γ can synergistically enhance MSC immune-modulatory potency. Here, only CCL5, CXCL11, IL-6 and TNFSF10 reflected this phenomenon with a synergistic increase in transcript levels in all media types, although IL-6 increased more in BM-MSCs. Intriguingly, CXCL8 and HLA-DRB1 specifically decreased in FS media, while CIITA and HLA-DRA decreased in both supplemented formulations but not in PF media. CCL2 was the only reduced transcript in PF media, although it synergistically increased in FS media. The 3 putative activation biomarkers also synergistically increased in XF and FS media treated with TFN-α+IFN-γ. Taken together, we have identified ICAM-1 and a new gene marker as suitable candidates for further development of standardized MSC performance assays. Ongoing work seeks to correlate levels of surrogate markers to clinical outcomes and define thresholds that functionally qualify MSC batches.