Transplantation of autologous corneal epithelial stem/progenitor cells (CESCs) expanded in tissue culture has successfully restored vision and revolutionized the treatment for limbal stem cell deficiency (LSCD) which is a major cause, either primary or secondary, of significant visual loss and blindness in many common corneal disorders. A higher expansion efficiency of the stem/progenitor cell population in culture corresponds to a better long-term graft survival. The most efficient expansion method requires mouse 3T3 feeder cells which are grown using calf serum and are the source of mouse RNA that contaminates the expanded cells. This cross-contamination by animal products poses considerable potential health hazards and therefore makes this culture method unlikely to be approved by the US Food and Drug Administration to be used in humans. New cell engineering methods that achieve the same or better efficiency of expanding CESCs under xenobiotic-free conditions are needed to achieve acceptable clinical outcomes. The long-term goal of my laboratory is to elucidate the regulatory factors that govern CESC self-renewal and differentiation, and to develop patient- specific stem cell-based therapies for LSCD. The objective of this particular application is to identify optimal cell engineering systems that can specifically and efficiently expand the stem/progenitor cell population of human corneal epithelial cells for transplantation. The central hypothesis is that appropriate human feeder cells can replace mouse 3T3 cells to provide a proper microenvironment to support the growth of CESCs, and upon receiving additional appropriate external signals the expansion of CESCs could be further optimized in culture. The hypothesis has been formulated based on the data produced in my laboratory. To achieve the objective of this translational research application, two specific aims are proposed: 1) Establish a xenobiotic-free culturing system using a human feeder layer that can efficiently expand CESCs;and 2) Identify an optimal expansion condition of CESCs by modulating the Wnt and/or Notch signaling pathway using small molecules or bioengineered human feeder cells.) Under the first aim, five human feeder candidates will be tested for their ability to grow CESCs. The functional aspect of these bioengineered CESCs will be tested in a well-established mouse model of LSCD. Under the second aim, proliferation of CESCs will be further optimized using human feeder cells that are engineered to over express limbal-specific Wnt molecules and Notch ligands, and small molecules of Wnt activators and Notch inhibitors. The approach is innovative, because it utilizes a novel method to bioengineer CESCs without permanent genetic alternation of the target cells. This approach has three major advantages: reversible, specific and translational. This will eliminate any potential permanent side effects or toxicity. The proposed translational research is significant, because the results from any of the two aims can be readily adapted for clinical development.

Public Health Relevance

The goal of this proposed research is to establish an ex vivo expansion protocol for limbal stem cells, or corneal epithelial stem/progenitor cells (CESCs), in xenobiotic-free conditions for transplantation into humans and a novel bioengineering approach to increase the efficiency of expansion of functional human CESCs. Thus, the proposed research will enable the initiation of a safe and effective patient specific stem cell-based therap for limbal stem cell deficiency.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY021797-02
Application #
8530236
Study Section
Special Emphasis Panel (BVS)
Program Officer
Mckie, George Ann
Project Start
2012-09-01
Project End
2017-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$365,750
Indirect Cost
$128,250
Name
University of California Los Angeles
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Pellegrini, Graziella; Lambiase, Alessandro; Macaluso, Claudio et al. (2016) From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU. Regen Med 11:407-20
González, Sheyla; Mei, Hua; Nakatsu, Martin N et al. (2016) A 3D culture system enhances the ability of human bone marrow stromal cells to support the growth of limbal stem/progenitor cells. Stem Cell Res 16:358-64
Chan, Eric; Le, Qihua; Codriansky, Andres et al. (2016) Existence of Normal Limbal Epithelium in Eyes With Clinical Signs of Total Limbal Stem Cell Deficiency. Cornea 35:1483-1487
Chuephanich, Pichaya; Supiyaphun, Chantaka; Aravena, Carolina et al. (2016) Characterization of the Corneal Subbasal Nerve Plexus in Limbal Stem Cell Deficiency. Cornea :
Restuccia, Agnese; Yang, Feikun; Chen, Changyan et al. (2016) Mps1 is SUMO-modified during the cell cycle. Oncotarget 7:3158-70
Le, Qihua; Yang, Yujing; Deng, Sophie X et al. (2016) Correlation between the existence of the palisades of Vogt and limbal epithelial thickness in limbal stem cell deficiency. Clin Exp Ophthalmol :
Wang, Ling; González, Sheyla; Dai, Wei et al. (2016) Effect of Hypoxia-regulated Polo-like Kinase 3 (Plk3) on Human Limbal Stem Cell Differentiation. J Biol Chem 291:16519-29
Hong, Jiaxu; Yu, Zhiqiang; Cui, Xinhan et al. (2015) Meibomian Gland Alteration in Patients with Primary Chronic Dacryocystitis: An In vivo Confocal Microscopy Study. Curr Eye Res 40:772-9
Chan, Eric H; Chen, Luxia; Yu, Fei et al. (2015) Epithelial Thinning in Limbal Stem Cell Deficiency. Am J Ophthalmol 160:669-77.e4
Chan, Eric H; Chen, Luxia; Rao, Jian Yu et al. (2015) Limbal Basal Cell Density Decreases in Limbal Stem Cell Deficiency. Am J Ophthalmol 160:678-84.e4

Showing the most recent 10 out of 22 publications