3D spheroid culture synchronizes heterogeneous mesenchymal stem cells into an immunomodulatory phenotype with enhanced suppressive effect on inflammation

Mesenchymal stem cells (MSC) are heterogeneous in morphology and transcriptome, resulting in varying therapeutic outcomes. In this study, we found that 3D spheroid culture of heterogeneous MSC, which have undergone conventional 2D monolayer culture for 5-6 passages, synchronized the cells into a uniform cell population with dramatically reduced cell size, and considerably increased levels of immunosuppressive genes and growth factors. Sc-RNA-Seq analysis of the cells revealed that 3D MSC consisted of 2 major cell subpopulations and both expressed high levels of immunosuppressive factors, compared to 6 subpopulations in 2D MSC. In addition, 3D MSC showed a greater suppressive effect on T cells. Moreover, intravenous infusion of a large dose of 3D MSC prior to Imiquimod (IMQ) treatment significantly improved psoriatic lesion. Thus, our results indicate that 3D spheroid culture reprograms heterogeneous mesenchymal stem cells into a uniform immunosuppressive phenotype and promises a novel therapeutic potential for inflammatory diseases. Overall design: The mesenchymal stem cells were isolated from human placenta and cultured in vitro to passage 5. The mesenchymal stem cells produced by two-dimensional adhesion culture and three-dimensional spherical culture were digested with trypsin and analyzed by single cell sequencing.