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.
This proposal is to develop a stem-cell based transplantation therapy for treating a blinding corneal disorder, limbal stem cell deficiency (LSCD). Corneal diseases are the second leading cause of treatable blindness in the world and over 3.2 million people worldwide are bilateral blind from corneal diseases. LSCD has been recognized as a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders, such as chemical/thermal burn, keratopathy related to contact lens wear, and severe infection and inflammation. Due to visual impairment, LSCD patients lose the ability to drive, read, and watch TV. In addition, they would experience recurrent corneal erosion that causes severe pain and sensitivity to light. Frequent break down of the corneal surface increases the risk of infection that requires frequent medical interventions. All of these can also exert psychological impact to the patients and their family members. Therefore, LSCD imposes significant social and economical impact on our society.
California is the most populated state in the US. There are more than 36 million people in the State of California and the population will increase to 46 million in 2030. Accordingly, the number of residents with limbal stem cell deficiency is likely disproportionately elevated due to the environmental risk factors. Thus, this disease affects a large population of patients in the state of California. A new treatment to restore vision would represent an important benefit to the people of California.
Further, the project would train new stem-cell researchers and advance innovative technology in stem cell therapy. This technology has application to other stem-cell related diseases. When this project enters the clinical phase, it will bring together new physicians and scientists and attract funding by the federal government. In addition, it will undoubtedly attract biotechnology investment in California. The stem-cell based transplantation to treat a stem-cell related disease like limbal stem cell deficiency is well-aligned with the broad mission of CIRM and the objectives of the Early Translational Research Award program.
Over last year, our research team has made significant progress in achieving our research goals and reaching the appropriate milestones. We first have demonstrated that we are able to grow the human limbal stem cells under the standard method using mouse 3T3 cells as feeder at the same efficiency level as the leading group in the world. This is the first milestone that we have reached.
We then proceeded with the initial testing of all the proposed human feeder candidates for their ability to support the growth of human limbal stem cells. We found that the current standard culture method on 3T3 cells did not work for human feeder cells. We then investigated four new culture methods to maximize the growth of limbal epithelial cells on human feeder candidates. We also have included a new human feeder cell candidate, adipose-derived mesenchymal stem cells. We are very excited to find that two of the human feeder cell types could support the growth of limbal epithelial cells with a significantly higher efficiency than the 3T3 cells using our two new culture methods. We are in the process of further refining the culture methods and characterize the expanded limbal epithelial cells. We believe that we are able to establish a xenobiotic free culture system to efficiently expand limbal stem cells for transplantation.
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.
During the last six months, my laboratory has successfully established a xenobiotic-free and feeder-free culture method to efficiently expand human corneal epithelial stem cells in culture by removing the feeder cells and replacing the fetal bovine serum with human serum. The amount of stem cells produced using this method is comparable to the standard culture method using mouse 3T3 cells as feeder cells and fetal bovine serum. We also have tested the feasibility of using fibrin gel and amniotic membrane as cell carrier and found that amniotic membrane is superior to fibrin as cell carrier. We are establishing an animal model to test the disease modifying effects of these cultivated stem cells. Lastly, we can increase the limbal stem cell expansion efficiency by modulating the Wnt and Notch signaling pathway.
- Invest Ophthalmol Vis Sci (2014) Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells. (PubMed: 25277234)
- Stem Cells (2013) Frizzled 7 maintains the undifferentiated state of human limbal stem/progenitor cells. (PubMed: 24170316)
- Tissue Eng Part C Methods (2013) A Three-Dimensional Culture Method to Expand Limbal Stem/Progenitor Cells. (PubMed: 24047104)
- Exp Eye Res (2013) Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture. (PubMed: 24016868)
- Methods Mol Biol (2013) Enrichment of human corneal epithelial stem/progenitor cells by magnetic bead sorting using SSEA4 as a negative marker. (PubMed: 23690006)
- PLoS One (2013) Preferential biological processes in the human limbus by differential gene profiling. (PubMed: 23630617)