Funding opportunities

Parthenogenetic Stem Cell-Derived Human Cornea Models for In-Vitro Toxicity Testing

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01878
Funds requested: 
$1 860 606
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
In the United States, over 65,000 people suffer from work-related eye injuries and illnesses each year. Another 125,000 Americans each year suffer eye injuries from household products. Worldwide, over 10 million people suffer from loss of sight because the cornea, the eye's transparent outer shell, becomes cloudy or opaque through injury or disease. Because eye disease and eye injury affect so many people, there is a need for human corneal tissue that can be transplanted into patients or used for safety testing. Currently, there is not enough corneal tissue of high enough quality to meet these needs. Some patients receive transplanted corneas from human cadavers, but the supply of transplant-quality corneas is inadequate. Some safety testing is done using cornea-like tissues derived from cells taken from adult donors, but these tissue models do not adequately mimic the microscopic structure of the human cornea. Safety testing is also done on animals, but this practice is controversial and these animal models are also a poor match for the human cornea. Our main goal is to use human stem cells to provide cornea-like tissue that can be used as a tool to improve the safety testing of chemicals and consumer products, and as a model system for researchers to study eye injuries. Ultimately, this cornea model may also be developed into a source of new, healthy corneal tissue to replace damaged corneas in human patients. Our corneal tissue model, which we call a corneal orb, is a liquid-filled globe with an outer shell of tissue similar to the cornea. We derive these corneal orbs from human parthenogenetic stem cells (hpSC). These hpSC are derived from unfertilized eggs, so that no human embryo is destroyed in producing them. They therefore avoid the ethical dilemmas surrounding research on human embryonic stem cells. Our research has shown that these hpSC can develop naturally into corneal orbs that have the same types of cells, arranged in approximately the same structures, as those found in the human cornea. The corneal orbs also have optical qualities similar to those of the human cornea. However, to supply corneal tissue for transplantation or safety testing, we need to control the process of transforming hpSC into corneal tissue, so that we can produce larger numbers of corneal orbs with more consistent size and properties. This program is designed to make that next crucial step. As described in the full proposal, our main strategy for achieving this goal is to identify the right mix of certain proteins, called growth factors, which must be present in the culture medium to guide the development of corneal tissue. We also propose to manipulate the physical environment of the developing cell cultures in ways that may improve the strength and size of the corneal orbs. By these methods, we hope to meet the need for corneal tissue for transplantation and safety testing.
Statement of Benefit to California: 
Large economic and health benefits will accrue to California through the completion of a living stem-cell-based model of the human eye (the cornea) as a research tool to study eye injury. It will provide a safer environment for California consumers and workers through more accurate eye safety testing of chemical and consumer products. It will provide a laboratory model for researchers to study healing of the eye for clinical research. World-wide demand for such a product will provide manufacturing and employment opportunities for Californians and added income to California. Finally, this model will give tangible validation to the public of the benefits that proposition 71 is bringing to California. There is a world-wide demand for a product that models the human eye for safety testing of chemicals and consumer products. Damage to the cornea is estimated to affect over 10 million people worldwide and household products are involved in 125,000 eye injuries in the USA each year. In June of 2007 The European Union started its “REACH” regulation (Registration, Evaluation and Authorization of Chemicals) requiring approximately 3.9 million test animals be used to assess the safety of chemicals (18% for eye irritation) at a cost of over 1.5 billion EURO (data published by the European Commission, Institute for Health and Consumer Protection, Nov., 2004). Current models use living animals and are regarded by scientists as being flawed and by the public as cruel. Even with these flaws, alternative tests are not effective substitutes for them. In the beginning of 2008, top officials from the U.S. National Institutes of Health (NIH) and Environmental Protection Agency (EPA) announced a five-year deal promising to share technology, information and other resources that will improve the toxicity testing of chemical compounds using current technologies rather than lab animals. This effort is designed to expand the use of human cells for testing and represents the "birth of a new approach to a crucial problem in public health." Laboratory models of living human cornea will provide a valuable tool for California researchers to study corneal damage and will bypass the current and projected shortages of acceptable human adult corneal tissue for research due to the increased use of laser vision corrective surgery, the increased longevity of the general population, and the increase in incidence of transmissible diseases. Intellectual property is likely to be generated through this work, resulting in increased valuation for California research organizations involved. This will generate further growth and investment and result in increased employment for Californians and added tax revenue.
Review Summary: 
The outer layer of the human eye, or cornea, is subject to injury and disease. This project seeks to improve the generation of a cornea model from human parthenogenetic stem cells (hpSC), i.e. pluripotent stem cells derived from unfertilized eggs. The primary use of this tissue is envisioned as an in vitro model for toxicity testing, and in the long term it is hoped this tissue may be suitable for corneal transplantations. The applicant provides preliminary data showing three-dimensional (3D) tissues with histological features similar to those of human cornea. These structures, termed corneal orbs, spontaneously arise at relatively low efficiencies over extended periods in undefined culture conditions. In Aim 1, the applicants seek to improve this efficiency by testing whether conditioned medium from mature orb cultures can improve orb yield. The investigators will then perform proteomic analysis on the conditioned medium to identify factors that direct corneal orb development, and test the role of these factors by manipulating their concentrations during differentiation. The goal of Aim 2 is to identify physical conditions that lead to more consistent production of orbs with optimal size and properties. Reviewers endorsed in vitro tissue modeling as one of the most promising short-term applications of human pluripotent stem cells to science and medicine. They agreed that the focus on corneal tissue is significant, given the lack of sufficient primary human corneal tissue for such studies and the inability of animal models to accurately predict human tissue behavior. In addition, in vitro human models alleviate the ethical concerns associated with animal testing for safety. Despite these assets, reviewers raised serious doubts as to whether corneal orbs would be suitable for high throughput toxicology studies, as it takes a long time to generate the orbs in culture, and the applicant did not propose to determine how they compare to existing, commercially available, much more rapidly produced corneal constructs. Reviewers suggested that rather than advancing translational goals, the proposed project has value in creating a research tool for answering fundamental questions regarding the development of a cornea-like tissue, but that is not the purpose of this application. Reviewers expressed concerns regarding the feasibility of this effort. Although success rests on the premise that mature or maturing corneal tissues release factors that stimulate differentiation of hpSCs to cornea, there is little evidence to suggest that this is the case. Furthermore, as several cell types are important for corneal tissue development and the process requires several months, it seems unlikely that conditioned media collected at a few time points would direct appropriate tissue development and morphogenesis. Preventing unwanted differentiation may be necessary to achieve the desired result, and the absence of certain factors may be important, a concept that would not be addressed by the proposed proteomic analysis. The notion that certain physical culture parameters may improve epithelial stratification is better substantiated. Reviewers questioned the conclusions drawn from the preliminary studies, citing a lack of compelling evidence that hpSC-derived orbs are corneal equivalents. For most of the histological analysis, no normal human tissue is shown for comparison, and some of the histological details provided do not conform well to those of authentic human cornea. Validation of the model needs to be substantiated by rigorous analysis of cornea-specific markers and by functional assessment. Reviewers suggested that a prior or simultaneous focus on model quality would improve the likelihood of generating a representative model system, but without that validation, optimization of orb production is not justified. The principal investigator has expertise in generating corneal tissue models from hpSCs and has a strong background in molecular and cellular biology. Other personnel provide expertise in hpSC culture and differentiation and in proteomics, and a collaborator, a well-established ophthalmologist, will provide expertise in validating corneal structures. This team is capable of executing the proposed project and is supported by a suitable infrastructure at the applicant institution. Reviewers judged the travel budget to be excessive. Overall, reviewers expressed substantial enthusiasm for the development and improvement of corneal tissues from human pluripotent stem cells, but this enthusiasm was diminished by the concern that the experiments described in this proposal will not identify factors that improve efficiency or quality of these tissues. Without further validation, reviewers judged the proposed culture optimization to be premature and did not recommend this application for funding.
Conflicts: 

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