Spheroid cultures promote the stemness of corneal stromal cells

•Rabbit and mouse CSCs could form suspension spheroids in methylcellulose medium.•Rabbit CSCs could form adherent spheroids by proteins of Oct4, Klf4, Sox2 and VPA.•CSC suspension and adherent spheroids were positive for vimentin, CD34 and nestin.•The gene expression of Nanog was positive for CSC adherent spheroids by proteins.•CSC suspension and adherent spheroids possess mesenchymal and stem cell phenotypes.

Several culture methods generated spheroids of rabbit and mouse corneal stromal cells (CSCs) in vitro. In this study, rabbit CSC spheroids were positively expressed the mesenchymal and stem cell phenotypes, which contained immunopositive for vimentin (a mesenchymal cell marker) and CD34 (a stem cell marker), as well as mRNA expression of nestin (a neural stem cell marker) and Nanog (a stem cell marker), in suspension or adherent cultures that were induced by methylcellulose, a rotary cell culture system (RCCS) or reprogramming proteins and VPA. Mouse CSCs showed poor growth and hardly formed spheroids after treatment with methylcellulose or reprogramming proteins and VPA. Our work has laid a promising foundation to elucidate CSCs and the further use of CSC spheroids for reprogramming, bioprinting and tissue engineering