Augmentation of Antibacterial Activity in Mesenchymal Stromal Cells Through Systems-Level Analysis and CRISPR-mediated Activation of CD14

ABSTRACT Mesenchymal stromal cells (MSCs) have broad-ranging therapeutic capabilities, however MSC use is confounded by cell-to-cell heterogeneity, and source-to-source phenotypic inconsistencies. We utilized a systems-based approach to compare MSCs which displayed different capacity for antibacterial activity. Although MSCs from both sources satisfied traditional MSC-defining criteria, comparative transcriptomics and quantitative membrane proteomics demonstrated two unique molecular profiles. The antibacterial MSCs respond rapidly to bacterial lipopolysaccharide (LPS) and have elevated levels of the LPS co-receptor CD14. CRISPR-mediated overexpression of endogenous CD14 in non-antibacterial MSCs resulted in faster LPS response and enhanced antimicrobial activity. Single-cell transcriptome profiling of CD14-activated MSCs revealed uniform enhancement of LPS response kinetics, and a shift in the ground state of these MSCs. Our results demonstrate that systems-level analysis can reveal critical molecular targets to optimize desirable properties in MSCs, and that overexpression of CD14 in these cells can shift their state to be more responsive to future bacterial challenge.