3D culture reveals dual role of ICAM-1 in mediating tissue-specific human MSC spheroid formation & enhanced immunomodulation.

Abstract

Three-dimensional (3D) in vitro culture systems may better mimic in vivo physiological conditions, and are easily accessible methods to improve therapeutic effectiveness of human mesenchymal stem cells (MSCs), which with its many sources appear to harbor clinically relevant functional differences. We therefore investigated the impact and elucidated the mechanism(s) of 3D culture on the immunomodulatory capacity of two commonly used MSC sources, bone marrow (BM) and placental (P). In 3D conditions, PMSCs (PMSC 3D) form larger spheroids than BMMSCs (BMMSC 3D), with whole transcriptome profiling revealing significant enrichment of cell adhesion and immunomodulatory pathways. qPCR and functional validation demonstrated the highest expression of numerous key immunomodulatory factors and strongest capacity to inhibit T cell proliferation with PMSC 3D. Bioinformatics analyses predicted Intercellular Adhesion Molecule 1 (ICAM-1) as crucial for both PMSC 3D spheroid formation and enhanced immunomodulatory capacity, which was validated with flow cytometric analyses and further delineated with single-cell RNA sequencing data. To assess mechanistic involvement, we performed knockdown of ICAM-1 which significantly reduced PMSC 3D spheroid size as well as both in vitro and in vivo immunomodulatory capacity. These findings demonstrate that 3D culture significantly enhances the immunomodulatory potential of PMSCs, and reveal ICAM-1 as having a dual role in spheroid formation as well as modulation of immune responses. Our study also highlights the importance of understanding source-specific differences as well as the profound influence of 3D in vitro systems on MSC functions.