Extracellular matrix mimicking polycaprolactone-chitosan nanofibers promote stemness maintenance of mesenchymal stem cells via spheroid formation

Abstract The development of clinical applications has led to a perpetual increase in the demand for mesenchymal stem cells (MSCs). However, the ex vivo expansion of MSCs while maintaining their stemness and differentiation potential remains an immense challenge. MSCs require high cell density for their intercellular communication and specific physico-chemical cues from the surrounding environment for spheroid formation in order to maintain their stemness. Inadequacy of the traditional in vitro cell culture method (tissue culture plastic surface) to fulfill any of these special requirements is responsible for inducing the loss of stem cell properties of the MSCs over time. In this study, we propose that glucosaminoglycan (GAG) mimicking ultrafine nanofibers could support the spheroid culture for in vitro human MSC expansion. The geometrical and biochemical properties of nanofibers provide biomimicking cues to MSCs, as well as enhance cell-cell interactions and stimulate spheroid formation in MSCs, which subsequently result in increased cell proliferation, enhanced expression of stem cell markers and maintenance of their multilineage differentiation potential. Furthermore, close monitoring of the behavior of MSCs on nanofibers serves as the key to understand their mode of action in niche formation. Interestingly, GAG mimicking substrate stimulated MSCs for long-distance intercellular communication via ‘tunneling tubes’, their subsequent migration and niche formation. These kinds of cellular interactions over long distances have rarely been observed in MSCs to provide better insight for future studies on MSC niche. Furthermore, PCL-CHT nanofibers were observed to be as conducive to use as tissue culture polystyrene for stem cell expansion. Overall, these polymeric nanofibers provide a more relevant, convenient and more suitable substrate than the conventional monolayer culture for in vitro MSC expansion.