Abstract
Purpose:
Corneal opacity, a major source of world blindness, is frequently treated by keratoplasty using allogenic tissue. Although generally successful, these allografts often succumb to immune rejection. Stem cells from autologous tissue offer an avenue for corneal therapy that could avoid this risk. Dental pulp is the center-most soft tissue of the tooth, containing a population of highly potent mesenchymal stem cells. Similar to the cornea, the dental pulp is derived from the cranial neural crest. This tissue can be obtained in a non-operating room setting and the cells expand rapidly in vitro. Here we investigated the potential of dental pulp stem cells (DPSC) to generate corneal stromal cells and tissues.
Methods:
DPSC were isolated from dental pulp of human adult 3rd molars by collagenase and dispase digestion. Keratocyte differentiation was induced by culture as cell pellets in a growth factor-supplemented serum-free keratocyte differentiation medium (KDM, PMID: 17962455). Gene expression was documented by qRT-PCR. Matrix organization was induced by culture on substrates of aligned polycaprolactone nanofibers in KDM. Protein expression was detected by immunohistochemistry and western blotting. Response of DPSC to a stromal microenvironment was assessed after injection of these cells into mouse corneal stroma for 2-4 weeks.
Results:
DPSCs expressed pluripotency and neural crest progenitor genes. Inducing keratocyte differentiation, upregulated expression of keratocyte genes (ALDH3A1, AQP1, CHST6, KERA, PTGDS and B3GNT7) and secretion of keratocyte-specific keratan sulfate proteoglycans including keratocan. DPSC cultured on aligned nanofibers generated a stroma-like tissue of uniform aligned fibrils of Type I collagen. Orientation of the collagen fibrils was rotated in adjacent layers similar to that of lamellae of corneal stroma. Injection of human DPC into mouse corneal stromas did not elicit corneal haze as detected by OCT, and the in vivo corneal stromal microenvironment induced keratocyte differentiation from DPC as detected by the expression of human keratocyte-specific markers.
Conclusions:
Dental pulp stem cells exhibit the potential to differentiate into keratocytes and generate stroma-like tissue. These cells can serve as a platform for development of novel autologous treatments for corneal blindness using engineered stromal tissue or stem cell-based therapy.
Keywords: 484 cornea: stroma and keratocytes •
721 stem cells