Cellular Interactions in Cell Sheets Enhance Mesenchymal Stromal Cell Immunomodulatory Properties

Immune-related applications of mesenchymal stromal cells (MSCs) in cell therapy seek to exploit immunomodulatory paracrine signaling pathways to reduce inflammation. A key MSC therapeutic challenge is reducing patient outcome variabilities attributed to insufficient engraftment/retention of injected heterogenous MSCs. To address this, we propose directly transplantable human single-cell-derived clonal bone marrow MSC (hcBMSC) sheets. Cell sheet technology is a scaffold-free tissue engineering strategy enabling scalable production of highly engraftable cell constructs retaining endogenous cell-cell and cell-matrix interactions, important to cell function. cBMSCs, as unique MSC subset populations, facilitate rational selection of therapeutically relevant MSC clones from donors. Here, we combine human cBMSCs with cell sheet technology, demonstrating cell sheet fabrication as a method to significantly upregulate expression of immunomodulatory molecules interleukin (IL)-10, indoleamine 2,3-dioxygenase (IDO-1), and prostaglandin E synthase 2 (PTGES2) across GMP-grade hcBMSC lines and whole human bone marrow-derived MSCs compared to respective conventional cell suspensions. When treated with carbenoxolone, a gap junction inhibitor, cell sheets downregulate IL-10 and IDO-1 expression, implicating functional roles for intercellular sheet interactions. Beyond producing directly transferable multicellular hcBMSC constructs, cell sheet technology amplifies hcBMSC expression of immunomodulatory factors important to therapeutic action. In addition, this work demonstrates the importance of cell-cell interactions as a tissue engineering design criterion to enhance consistent MSC functions. Impact statementMesenchymal stem/stromal cell (MSC) production of anti-inflammatory factors has motivated significant clinical interest in their therapeutic utility for immune-related diseases. Strategies that enhance the potency of culture-expanded MSCs and improve MSC engraftment hold promise to advance MSC therapy. Cell sheet technology is a tissue engineering approach enabling direct cell transplantation and localized tissue engraftment. We report the first mechanistic insights into functional roles of gap junctions within MSC sheets and demonstrate that cell sheet fabrication stimulates MSC expression of immunomodulatory factors compared to conventional MSC suspensions. These findings motivate further studies of MSC sheets in diverse immune-related applications and therapies.