Abstract: Mesenchymal stromal cells (MSCs) offer enormous therapeutic potential for immune-mediated disorders through their profound immunosuppressive effects. However, their potency varies, which may be associated with specific phenotypic and molecular features. This study characterizes resting and IFNg primed clinical-grade MSCs from adipose (ADSC) and decidua (DSC) and integrates the predictive phenotypic, genetic, and epigenetic markers linked to better potency. The immunosuppressive potential of MSCs was evaluated by co-culturing them with PBMCs (n=4) at various concentrations. Key metrics included activation marker expression, proliferation indices, and Tregs analyzed by FACS, alongside cytokine levels measured by Luminex. These data were integrated using PCA and PLSDA analysis, with the area under the curve (AUC) of PC1 and LV1 calculated across MSC concentrations to represent PBMC functional activity and hence, of the MSC immunosuppressive potential. The phenotypic and molecular features of MSCs predictive for AUC were determined using integrative analysis of morphology markers (n=84), cytokines (n=46), gene expression markers (n=25), miRNA markers (n=800) and surfaceome (n=361). Scores were determined by statistical analysis via mixOmics R package on experimental set (n= 6: 3 ADSCs & 3 DSCs). Our results showed lower AUC for ADSCs compared to DSCs and for primed versus resting MSCs, indicating greater potency of ADSCs and primed MSCs. We identified five highly correlated scores distinguishing between ADSC & DSC; these included the morphology score composed of cytoplasmic eccentricity and zernike_8_0 & 1_1and nuclear zernike_5_5 & 4_2; surface markers scores - EphA2, CD274, CD49c, MSC and CD99; RNA score- IFIT1, CD83, PDL1and VCAM1; miRNA score-miR99b.5p, miR23b.3p, let7i.5p, miR148a.3p and miR29c.3p; cytokines score- GROb, PDGF-AB/BB, PDGF-AA, EGF and IP-1. RNA and cytokines scores were validated with high accuracy in the validation set. We additionally determined scores distinguishing between high and low performing ADSCs in the experimental set, which are being tested in the validation set (n=32). Through an extensive characterization of clinical grade products, we identified scores that can guide selection and manufacturing of MSC products with enhanced immune suppressive potency.
Funding Source: Qatar National Research Fund (QNRF)
