Corneal blindness affects millions of individuals world-wide and usually can only be treated by transplantation of donated tissue. In many regions of the world donor tissue is not available;in the US, the supply of corneas for transplant is threatened by increasing numbers of eyes with refractive surgeries. The long-term goal of this project is to address corneal blindness by developing therapies based on stem cells. This project has already identified and characterized adult human corneal stromal stem cells (CSSC), which can be expanded in culture while retaining the ability to adopt a keratocyte phenotype and to elaborate abundant stroma-like connective tissue. When CSSC were cultured on a scaffolding of aligned paralel nanofibers of poly(ester urethane)urea (PEUU), a biodegradable polymer, they secreted connective tissue with aligned collagen similar to that of normal corneal stroma. When CSSC were injected into stromas of lumican knockout mice, collagen in the stroma became more organized and corneal transparency was increased. Importantly, human CSSC exhibited an immune privilege in mice, remaining viable for months without immune rejection. The new proposal will test specific hypotheses emanating directly from these exciting findings.
Aim 1 documents the process of tissue regeneration by CSSC by testing the predictions that (a) CSSC first remove existing tissue by temporally regulated expression of a specific group of matrix metalloproteinases;(b) after this removal process, CSSC rebuild stromal tissue by deposition of new connective tissue;and (c) this regeneration process can remodel scar tissue similar to that causing most human corneal blindness.
Aim 2 combines CSSC with PEUU scaffolding to produce a novel bioengineered tissue and tests the predictions that (a) CSSC monolayers on PEUU nanofiber scaffolding can integrate and function as transparent stromal tissue in mouse corneal transplants;(b) CSSC can be incorporated into multilayered PEUU scaffolding with the thickness and strength of a corneal stroma;and (c) the multilayered constructs can functionally replace stroma after transplantation into rabbit corneas using deep anterior lamellar keratoplasty. The scientific impact of this study will be a demonstration of effectiveness of two novel stem-cell based therapeutic approaches for corneal scarring, indicating a readiness for translation of each to clinical trials.

Public Health Relevance

Corneal blindness is a health problem world-wide. For many individuals with this condition there is no option for restoration of vision. This project will use adult human stem cells to develop therapy for corneal scarring, a major source of blindness. These stem cells can be produced in large numbers, are safe, and do cause tissue rejection. Successful development of these therapies may open new opportunity to restore vision to large numbers of people.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY016415-08
Application #
8450197
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
2005-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2013
Total Cost
$422,920
Indirect Cost
$143,765
Name
University of Pittsburgh
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Karamichos, Dimitrios; Funderburgh, Martha L; Hutcheon, Audrey E K et al. (2014) A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells. PLoS One 9:e86260
Wu, Jian; Rnjak-Kovacina, Jelena; Du, Yiqin et al. (2014) Corneal stromal bioequivalents secreted on patterned silk substrates. Biomaterials 35:3744-55
Wu, Jian; Du, Yiqin; Mann, Mary M et al. (2014) Corneal stromal stem cells versus corneal fibroblasts in generating structurally appropriate corneal stromal tissue. Exp Eye Res 120:71-81
Wu, Jian; Du, Yiqin; Mann, Mary M et al. (2013) Bioengineering organized, multilamellar human corneal stromal tissue by growth factor supplementation on highly aligned synthetic substrates. Tissue Eng Part A 19:2063-75
Chan, Audrey A; Hertsenberg, Andrew J; Funderburgh, Martha L et al. (2013) Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype. PLoS One 8:e56831
Du, Yiqin; Yun, Hongmin; Yang, Enzhi et al. (2013) Stem cells from trabecular meshwork home to TM tissue in vivo. Invest Ophthalmol Vis Sci 54:1450-9
Roh, Danny S; Du, Yiqin; Gabriele, Michelle L et al. (2013) Age-related dystrophic changes in corneal endothelium from DNA repair-deficient mice. Aging Cell 12:1122-31
Fujita, Minoru; Mehra, Ruhina; Lee, Seung Eun et al. (2013) Comparison of proliferative capacity of genetically-engineered pig and human corneal endothelial cells. Ophthalmic Res 49:127-38
Boote, Craig; Du, Yiqin; Morgan, Sian et al. (2012) Quantitative assessment of ultrastructure and light scatter in mouse corneal debridement wounds. Invest Ophthalmol Vis Sci 53:2786-95
Wu, Jian; Du, Yiqin; Watkins, Simon C et al. (2012) The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells. Biomaterials 33:1343-52

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