Stemness Transcription Factor Nanog is Expressed During Spheroid Culture of Corneal Stromal Cells

Abstract

Purpose: Corneal stromal cells transform to precursor cells in spheroid culture. We determined whether stemness genes (Nanog, Oct4, Sox2, Klf4, and Myc) are expressed during this transformation.

Methods: Stromal cells were isolated from murine corneas by sequential collagenase digestion and adherent culture to generate a population of mixed stromal cell phenotypes. After trypsinization, spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were analyzed for expression of stemness genes. Spheroids in culture were induced to differentiate to keratocytes, fibroblasts, and myofibroblasts using the appropriate growth medium.

Results: Sphere formation occurred in ultra-low attachment plates but not in adherent culture of stromal cells. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids, but not Sox2, Klf4, and Myc. On immunofluorescence staining analysis, Oct4 was localized in the cytoplasm. Forced differentiation of spheroids reverted them to keratocytes, fibroblasts, and myofibroblasts. Keratocytes were more efficiently induced from spheroids and appeared more phenotype specific with higher expression of Keratocan and ALDH3A1, and a lower expression of α-SMA compared to keratocytes induced from adherent cells.

Conclusions: Through spheroid culture, adherent corneal stromal cells transform to a less differentiated precursor form, possibly due to upregulation of stemness genes. This finding suggests that terminally differentiated stromal cells possess inherent plasticity that can be activated in certain conditions.

Keywords: 484 cornea: stroma and keratocytes