Early Translational II
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.
Statement of Benefit to California:
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.
This application is a Development Candidate Feasibility (DCF) proposal aimed at regeneration of corneal epithelial stem/progenitor cells from autologous ex vivo expansion or epidermal epithelial stem cells to achieve patient specific restoration of a transparent ocular surface. The target population is patients with corneal epithelial stem cell deficiency, known as limbal stem cell deficiency (LSCD). The applicants propose to optimize culture growth and expand human limbal stem cells (LSCs) under xenobiotic-free conditions, modulate the Notch and Wnt pathways to regenerate human corneal LSCs, and regenerate human corneal epithelial stem cells via transdifferentiation of epidermal epithelial stem cells. The target product is an LSC therapy, and if successful, this proposal will establish an ex vivo expansion protocol with human feeder cells for LSCs transplantation in the United States. Although LSC expansion and transplantation procedures have been developed in Europe, they utilize xenobiotic reagents that impede their acceptance in the US. Reviewers acknowledged that the proposed project could address a medical need but suggested that the project’s impact would be only incremental and, although aimed broadly at corneal disease, would likely only be appropriate for a limited number of corneal disease patients. Additionally, reviewers questioned the applicant’s assertion that an alternative cell source was needed to treat bilateral disease, since a recent study described treatment of a patient with bilateral disease using autologous cells grown from a spared limbal region. Reviewers raised a number of concerns about the feasibility of the proposed research. Although specific aims appear straightforward, clear endpoints and go/no go criteria for many activities were not adequately defined. For example, The selection criteria for establishing human feeder cell lines are poorly defined, and there was no discussion of alternatives if appropriate lines were not identified. Reviewers felt that the proposal lacked compelling preliminary data in support of the use of Wnt and Notch modulation to regulate LSC differentiation. Additionally, the absence of published or preliminary data supporting the transdifferentiation of epidermal epithelial stem cells into corneal epithelial cells raised critical doubts about the feasibility of the third specific aim. Although reviewers found this last goal intriguing, they felt is was more appropriate for a program focused on fundamental cellular mechanisms. Reviewer judged the PI as generally well-trained but lacking experience in coordinating collaborative research teams. Additionally, he/she has only a modest publication record. The research team has some experience culturing LSCs, but lacks expertise in stem cell biology. There were concerns also as to whether the PI’s percent effort was large enough for a project of this scope. The budget appeared appropriate for the studies described in the application. The research environment was viewed as excellent, and the institutional support is strong. In summary, this application describes a DCF proposal to derive and expand LSCs for application in autologous corneal regeneration. The reviewers found significant deficiencies in the preliminary data and stem cell expertise, and expressed serious concerns about the project’s feasibility. Consequently, the application was not recommended for funding.