The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities.

Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but maintaining the stemness of ASCs during in vitro culture is still a challenging issue. In this study, human ASCs spontaneously formed three-dimensional spheroids on chitosan films. Most ASCs within the spheroid were viable, and the cells produced more extracellular molecules, like laminin and fibronectin. Comparing to monolayer culture, ASC spheroids also exhibited enhanced cell survival in serum deprivation condition. Although cell proliferation was inhibited in spheroids, ASCs readily migrated out and proliferated upon transferring spheroids to another adherent growth surface. Moreover, spheroid-derived ASCs exhibited higher expansion efficiency and colony-forming activity. Importantly, we demonstrated that spheroid formation of human ASCs on chitosan films induced significant upregulation of pluripotency marker genes (Sox-2, Oct-4 and Nanog). By culturing the ASC spheroids in proper induction media, we found that ASC differentiation capabilities were significantly enhanced after spheroid formation, including increased transdifferentiation efficiency into neuron and hepatocyte-like cells. In a nude mice model, we further showed a significantly higher cellular retention ratio of ASC spheroids after intramuscular injection of spheroids and dissociated ASCs. These results suggested that ASCs cultured as spheroids on chitosan films can increase their therapeutic potentials.