Abstract
Purpose :
To develop an effective approach for the physical reprogramming of human corneal stromal cells (hCSCs, also called keratocytes) derived from SMILE lenticules into stem-like cells without using transcription factors. Then, further, prove that these cells can be good seed cells for tissue engineering corneal construction and cell therapy.
Methods :
Primary hCSCs were isolated from SMILE-derived lenticules and cultured in a low-serum RIFA medium with soluble human corneal stromal extract (hCSE). A novel platform with 62 V-bottom micro-cavities and essential 8 (E8) medium was used to generate adherent 3D keratocyte spheroids in the polydimethylsiloxane (PDMS) microwell. The reprogramming results were compared between 3D spheroids with 2D cultures via qPCR and Immunofluorescence (IF) staining.
Results :
The hCSCs were successfully isolated from SMILE-derived lenticules, exhibiting dendritic or stellate shapes（1-A）. IF analysis showed that these cells strongly expressed keratocyte markers (ALDH3A1 and Lumican) and were weakly stained with (myo)fibroblast markers (fibronectin and α-SMA) after subculturing in RIFA + hCSE（1-B）. We successfully generated efficient and homogeneous 3D spheroids in the PDMS microwell（1-C, D）. The average diameter of 3D spheroids is 433.40 ± 68.66 µm. qPCR analysis showed that the expression of stem cell marker genes (SOX2, PAX6, OCT4, NANOG, NGFR, and NESTIN) was significantly greater in 3D spheroids than those in 2D cells at different times after spherical cultivation, especially at 1 week and 4 weeks（1-E）. Some keratocyte genes (Lumincan and Keratocan) showed an increase at 1 week while decreasing at 4 weeks of 3D spheroids. Furthermore, IF staining revealed that KLF4, NANOG, SOX2, and PAX6 were positively expressed in adherent 3D spheroids but negatively in 2D cells（1-F）.
Conclusions :
Our results indicated that the physiological cells and culture conditions enhance reprogramming. Therefore, the adherent 3D spheroid culture of keratocytes using the PDMS microwell platform is a promising and safe strategy to promote reprogramming and enhance stemness, suggesting its potential application for developing implants in tissue engineering and regenerative medicine.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.