Heterospheroid formation improves therapeutic efficacy of mesenchymal stem cells in murine colitis through immunomodulation and epithelial regeneration

Tissue repairing capacity and immunomodulatory effects of mesenchymal stem cells (MSCs) have been extensively utilized for treating various inflammatory disorders; however, inconsistent efficacy and therapeutic outcomes due to low survival rate after transplantation often restrain their clinical potential. To overcome these limitations, 3-dimensional culture (3D-culture) was established to augment stemness and paracrine functions of MSCs, although hypoxic stress at the core often leads to unexpected cell death. Thus, we designed a novel strategy to improve the microenvironment of MSCs by creating heterospheroids (HS) consisting of MSCs and quercetin (QUR)-loaded microspheres (MSC_HS), to achieve local drug delivery to the cells. Notably, MSC_HS exhibited resistance for senescence-associated phenotype and oxidative stress-induced apoptosis compared to 3D-cultured MSCs (MSC_3D), as well as to 2D-cultured cells (MSC_2D) in vitro. In a murine model of colitis, MSC_3D and MSC_HS exhibited enhanced anti-inflammatory impact than MSC_2Dvia attenuating neutrophil infiltration and regulating helper T cell (Th) polarization into Th1 and Th17 cells. Interestingly, MSC_HS provided better therapeutic outcomes compared to MSC_3D, partially due to their enhanced survival capacity in vivo. Moreover, we found that MSC-derived paracrine factor, prostaglandin E₂ (PGE₂), can directly drive the epithelial regeneration process by inducing specialized tissue-repairing cell generation using the intestinal organoid culture. Importantly, MSC_3D and MSC_HS displayed an outstanding regeneration-inducing potency compared to MSC_2D owing to their superior PGE₂ secretion. Taken together, we suggest a convergent strategy of MSC_HS formation with reactive oxygen species (ROS) scavenger, QUR, which can maximize the inflammation-attenuating and tissue-repairing capacity of MSCs, as well as the engraftment efficiency after transplantation.