Development and validation of a potency assay matrix for optimized and consistent manufacture of clinical mesenchymal stem/stromal cells.

INTRODUCTION: Mesenchymal stem/stromal cells (MSCs) are being evaluated as cell-based therapies for inflammatory and immune-mediated disorders. However, variability in clinical efficacy and a lack of validated potency assays have impeded regulatory approval for commercialization. Here, we report on our success with developing a matrix of potency assays for evaluating the therapeutic fitness of bone marrow-derived MSCs and demonstrate that the cells consistently suppress T cell proliferation, induce regulatory T cell differentiation, and polarize monocytes into anti-inflammatory M2 macrophages. METHODS: Vertebrae were recovered from consented and screened organ donors by Organ Procurement Organizations and shipped on ice to a central processing facility for isolation of vertebral body bone marrow. MSCs were cultured in a xenogeneic-free medium and characterized based on established markers and expanded for 4 passages. Modulation of immune cells isolated from peripheral blood was evaluated using T cell suppression assays, macrophage polarization, regulatory T cell (Treg) induction and monocyte/macrophage chemoattraction assays. RESULTS: Mechanistic studies revealed that potency is mediated by MSC-secreted immunoregulatory molecules, including macrophage colony-stimulating factor (M-CSF), transforming growth factor-β1 (TGFβ1), and the chemokine CCL2, as well as by tryptophan depletion via the cytoplasmic protein indoleamine 3,4 dioxygenase-1 (IDO1). Additionally, we show that MSCs secrete high levels of extracellular vesicles which potently induce an anti-inflammatory phenotype in T cells and monocytes. These findings were employed to develop a matrix of surrogate potency assays which consistently demonstrated predicted in vitro functionality of MSCs derived from 10 donors. DISCUSSION: This potency assay platform provides a critical tool for ensuring the quality and consistency of MSC products and will facilitate clinical translation by demonstrating comparability between MSC donors as well as manufactured lots and potentially predicting therapeutic efficacy in clinical trials.