Regeneration of Functional Human Corneal Epithelial Progenitor Cells

Over 3.2 million people worldwide are bilateral blind from corneal diseases. Limbal stem cell deficiency (LSCD) has been recognized as a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders.
A healthy, transparent ocular surface is made up of non-keratinized, stratified squamous epithelium that is highly differentiated. The corneal epithelium is constantly renewed and maintained by the corneal epithelial stem cells, or limbal stem cells (LSCs) that are presumed to reside at the limbus, the junction between the cornea and conjunctiva. When the LSCs are deficient and unable to repopulate the corneal surface, the cornea surface will become opaque. Corneal transplant can’t survive and is contraindicated in LSCD.
LSC transplantation, in the form of keratolimbal allograft to restore a transparent corneal surface, has been the main therapy in the United States. The 5-year survival of these allografts is about 30%, largely due to immune rejection. Transplantation of autologous limbal epithelial stem cells that have been expanded on tissue culture has successfully restored vision and revolutionized the patient specific stem-cell based therapy as recently reported by an Italian LSC transplant team. They have achieved a 68% success rate during a mean follow up time of 3 years. The expansion process requires mouse 3T3 feeder cells to grow a sufficient amount of stem cells for transplantation. To reduce cross-contamination from animal products, LSCs that are expanded in a xenobiotic-free culture system has been established; however, the 3-year survival rate of these cells after transplantation is 50% and only 30% survive at 5 year, suggestive of inefficient expansion without the mouse feeders. Therefore, new cell engineering methods that can efficiently expand and regenerate autologous LSCs in a xenobiotic-free system are dearly needed to achieve acceptable clinical outcome and offer stem-cell based therapy to patients with this devastating blinding diseases in the United States.
The first goal of this proposed translational research is to establish a xenobiotic-free culture system by replacing the mouse feeder cells with a human feeder system to expand sufficient amount of LSCs for transplantation. This will allow immediate initiation of clinical trial. We will then further optimized the expansion efficiency by modulating the Wnt and Notch signaling pathways based on our findings that Wnt and Notch signaling regulate the proliferation and differentiation of corneal epithelial cells. In parallel, transdifferentiation of human skin epithelial stem cells to corneal epithelial cells will be induced using a similar approach. The ability and safety of these regenerated human corneal epithelial stem cells to reconstruct the ocular surface will be tested in a LSCD animal model. The results of this proposed study will pave the way for preclinical development of this novel cell engineering technique.