The goal of the project is to establish a xenobiotic-free culture system that can efficiently expand human limbal stem cells (LSCs) for transplantation. We have met all of the milestones and have accomplished the following:
1) We can grow LSCs on 3T3 feeder cells (the current gold standard culture method) in our laboratory as efficiently as those described in the literature.
2) We have established two cell carrier systems for transplantation.
3) We have established four 3T3 feeder-free culture methods, including a feeder-free system to expand human LSCs as efficiently as the 3T3 feeder cells do. Three types of human feeder cells— limbal fibroblasts, bone marrow-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells—support the growth of LSCs. All of the 3T3 feeder-free cultures contain more than 3% of p63bright cells.
4) We have shown that two Wnt small molecule activators can increase the expansion efficiency of LSCs by more than 125%.
5) A nude mouse model of LSC deficiency (LSCD) has been established to test the in vivo function of cultured human LSCs.
In addition to reaching these milestones, we have derived a novel method in which feeder cells are completely separated from LSCs during culture. This novel method eliminates contamination by the feeder cells but maintains close contact between them. We have found that trypsin has detrimental effects on LSCs during isolation. Limbal epithelial cells in clusters/sheets and limbal tissue explant culture are superior to single-cell culture for the expansion of LSCs. We will continue to investigate which of the four 3T3 feeder-free systems is the most efficient in expanding the LSC population, and we will continue to refine the LSCD animal model. Before the end of the award period, we will be able to select the most efficient and consistent xenobiotic-free method of cell culture and start regulatory tests that are necessary for submission of an investigational new drug (IND) application to the FDA so that we can begin the first clinical trial to treat patients with unilateral LSCD in California.