Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

Abstract

Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSCbased therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.H uman mesenchymal stromal cells (MSCs) can potently suppress immune responses in vitro and in animal models of human disease (1, 2), but to date MSC-based therapies have produced mixed results in clinical trials for treatment of inflammatory and autoimmune diseases (3, 4). A major challenge in the development of consistently effective MSC-based immunosuppressive therapies is that MSC lines derived from different donors and manufacturing processes (i.e., cell expansion) can possess markedly dissimilar immunosuppressive function (3,5,6). Although methods exist to assess MSC immunosuppression in vitro, they are often based on only a few measured outcomes, assay culture conditions, and donor MSC samples (5, 7-9). To improve upon these methods, we developed an experimental and analytical approach to quantify MSC-mediated immune suppression using principal-component analysis (PCA) to integrate multiple measurements of T-cell activation assessed at a range of MSC densities. This approach allowed us to determine a single value for immunosuppressive capacity for MSC lines derived from two different manufacturing processes and 13 independent donors.Another major challenge associated with MSC-based immune therapies is the lack of well-defined predictive markers to identify MSC lines with therapeutically relevant biological activities or manufacturing processes that produce more effective MSCbased products. Efforts have been made to identify MSC quality attributes associated with immunosuppression (6, 7), but the majority of clinical studies (10) rely upon the surface markers described by Dominici et al. (11). Having previously shown that morphology can predict MSC mineralization capacity (12), we hypothesized that morphological features associated with immunosuppression in MSCs could be identified and used to predict their performance in our quantitative immunosuppression assay.Using our quantitative method for as...

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

“…Considering that PBMC-derived IFNγ is a key cytokine leading to MSC licensing in vitro , we examined the output of a simplified assay stimulating MSCs with recombinant IFNγ as a substitute for SEB-activated PBMCs (Galipeau and Krampera, 2015; Klinker et al, 2017). We observed that both Crohn’s disease and GvHD MSCs responded to IFNγ and upregulated the expression of IDO, CXCL9, CXCL10, CXCL11, CIITA, TRAIL, ICAM-1, HLADR, CCL5, and CCL7 in a manner closely paralleling that of the response to PBMCs (Figures 5D and 5E).…”

Commitment to Aerobic Glycolysis Sustains Immunosuppression of Human Mesenchymal Stem Cells

“…IFN‐γ at concentrations from 5 ng/ml up to 100 ng/ml has been used to induce hMSC immune polarization with most of studies using IFN‐γ at concentrations between 20 ng/ml and 50 ng/ml . In addition to immune modulation, the influence of IFN‐γ on hMSC proliferation , trilineage differentiation , migration , transcriptome profile , and secretion of angiogenic and other tropic factors has been extensively reported.…”

Single-cell high-content imaging parameters predict functional phenotype of cultured human bone marrow stromal stem cells

“…These studies actively modified cell shape by culturing the cells on a number of micropatterned surfaces, initiating differentiation or stimulating with IFNγ, which all induces morphological changes in the hBM‐MSCs. These changes could either be used to alter the differentiation outcome or predict the BM‐MSC biology . The novelty of the current study lays in the use of naive hBM‐MSC morphology.…”