Towards a consensus potency assay for mesenchymal stromal cells: a matrix analysis of cell source, donor variability and inflammatory stimuli to refine surrogate markers of immunomodulation

Abstract

Background & Aim Reliable metrics to assess the therapeutic potential of mesenchymal stromal cell (MSC) batches are needed for successful translation of MSC-based products. Here, the veracity of proposed biomarkers was evaluated by matrix analysis of 3 donor populations each of umbilical cord-derived (UC) and bone marrow-derived (BM) MSCs primed by 3 different proinflammatory cytokines. Methods, Results & Conclusion High quality UC MSCs (Tissue Regeneration Therapeutics Inc., CAN) and BM MSCs (RoosterBio Inc. (RBI), USA) were cultured in xeno-free media (RBI). MSCs were primed by 24 hour exposure to TNF-α (50 ng/ml), IFN-γ (50 ng/ml) or IL-1β (80 pg/ml) in protein-free media (RBI) (n=3 replicates). Cells from replicate flasks were pooled for RNA extraction and transcriptome analysis by Affymetrix GeneChip™ U133A 2.0 microarray. Secreted proteins were analyzed by multiplex ELISA (BioPlex 27-plex Human Inflammation Panel II). The data were interrogated against the ISCT's proposed panel of 47 reference genes and proteins that report MSC priming to IFN-γ. The only gene markers of activation shared across MSC types and stimulants were ICAM1 (p 0.05), which increased >2-fold in primed versus resting cells. Eighteen genes responded significantly (p Eight secreted biomarkers proposed by the ISCT panel were represented in the multiplex data; none were induced by IFN-γ. TNF-α elicited significantly more IL-8 (CXCL8) and IL-1ra (IL1RN) from both MSC types, and specifically increased RANTES (CCL5) and IL-6 output from BM MSCs and MCP-1 (CCL2) from UC MSCs. In BM MSCs only, priming with TNF-α or IL-1β elevated IP-10 (CXCL10) levels, as well as IL-8 and IL-1ra. These outputs correlated with the array data, except for IL-1ra and IP-10 whose mRNA levels were unchanged. These data suggest that only a subset of proposed biomarkers may have broad utility for developing standardized potency assays for a range of MSC products. Ongoing work seeks to identify additional surrogate markers with applicability across MSC types, variable donors and inflammatory stimuli.