The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source of maintaining corneal epithelial homeostasis. Clinically, loss of limbal SCs or dysfunction of the limbal niche leads to corneal blindness due to limbal SC deficiency (LSCD). During the last funding period, our studies of three surrogate niches, i.e., a serum-free, matrix- free, and fibroblast-free niche, amniotic membrane (AM) niche, and limbal explant niche, have identified several major pitfalls in six protocols currently practiced in treating human patients with LSCD. To circumvent these pitfalls, we have discovered a clever method of isolating the entire limbal epithelial SCs together with their native niche cells (NCs) using collagenase alone. This new isolation method allows us to develop two in vitro model systems for gaining mechanistic understandings of how limbal epithelial SCs are regulated by their native NCs. The first model system breaks SC-NC contact but facilitates their reunion in vitro to generate sphere growth. Using this model system, we will examine whether SC quiescence starts with the SC- NC reunion via binding between stromal derived factor 1 SDF-1 ) expressed by NCs and its receptor (CXCR4) expressed by SCs. Moreover, we will prove that such SC-NC reunion is facilitated by bone morphogenetic protein (BMP) signaling that upregulates expression of SDF- 1 in NCs, and subsequently promotes SC renewal, while further SC renewal can be promoted by Wnt/ -catenin signaling and the FGF family signaling, but suppressed by TGF- signaling (Aim 1). We will also determine whether maintenance of the normal NC phenotype with a small round shape and expression of embryonic SC markers depends on not only biochemical but also physical properties of the basement membrane. By manipulating the basement membrane matrix component and integrity, we expect to isolate NCs and unravel a new therapeutic paradigm for treating LSCD by restoring the limbal niche health through maintenance of the normal NC phenotype (Aim 2). The introduction of exogenous mouse or human labeled cells at the time of SC-NC reunion also allows us to trace the lineage during sphere growth to understand how a normal corneal fate decision is controlled by NCs (Aim 3). Under the limbal niche influence, these studies will resolve a recent controversy whether SCs might reside in the mouse central cornea. Furthermore, they will help resolve whether conjunctival transdifferentiation can take place at the single cell level, and whether oral mucosal and epidermal epithelial progenitors have any potential to take a corneal fate. Taking advantage of an expanding repertoire of transgenic mice, we may turn this model system into a powerful assay to ascertain how human relevance can be drawn from gain or loss of a given gene function during the corneal fate decision. Finally, the aforementioned new knowledge can be applied to the second model system, which maintains SC-NC close contact during isolation and expansion by culturing collagenase-isolated limbal clusters directly on epithelially-denuded AM (Aim 4). This study will lead us achieve the ultimate goal of developing a new tissue engineering strategy to maximize ex vivo expansion of SCs by successful recapitulation of the regulatory mechanism executed by the in vivo "native" niche. Such an approach may one day help realize the considerable promise held by adult SCs in treating a number of diseases in the body.

Public Health Relevance

The ultimate goal of our proposed research is to develop a novel and effective tissue engineering strategy to maximize ex vivo expansion of adult somatic lineage-committed epithelial stem cells. Our progress made in the last funding period has led us to discover several major pitfalls in the conventional protocols used for generating a surgical graft containing limbal epithelial stem cells for treating corneal blindness caused by limbal stem cell deficiency. Furthermore, we have discovered a novel method of isolating these stem cells together with their native niche cells from the human limbus, and developed two in vitro model systems to explore the mechanistic understanding of how quiescence, self-renewal, and fate decision of limbal epithelial stem cells are controlled by the limbal niche. We believe that these studies will make a significant advance in the field of regenerative medicine by generating important knowledge that will unravel new therapeutic paradigms through restoration of limbal niche health. Furthermore, successful ex vivo expansion of limbal and other epithelial stem cells can be achieved by recapitulating the regulatory mechanism of the in vivo native niche. Such novel approaches may one day help realize the considerable promise held by adult SCs in treating a number of diseases in the body.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01EY006819-28
Application #
8716757
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Tissuetech, Inc.
Department
Type
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33173
Chen, Szu-Yu; Mahabole, Megha; Horesh, Elan et al. (2014) Isolation and characterization of mesenchymal progenitor cells from human orbital adipose tissue. Invest Ophthalmol Vis Sci 55:4842-52
Han, Bo; Chen, Szu-Yu; Zhu, Ying-Ting et al. (2014) Integration of BMP/Wnt signaling to control clonal growth of limbal epithelial progenitor cells by niche cells. Stem Cell Res 12:562-73
Zhang, Suzhen; Zhu, Ying-Ting; Chen, Szu-Yu et al. (2014) Constitutive expression of pentraxin 3 (PTX3) protein by human amniotic membrane cells leads to formation of the heavy chain (HC)-hyaluronan (HA)-PTX3 complex. J Biol Chem 289:13531-42
Kawakita, Tetsuya; Espana, Edgar M; Higa, Kazunari et al. (2013) Activation of Smad-mediated TGF-ýý signaling triggers epithelial-mesenchymal transitions in murine cloned corneal progenitor cells. J Cell Physiol 228:225-34
He, Hua; Zhang, Suzhen; Tighe, Sean et al. (2013) Immobilized heavy chain-hyaluronic acid polarizes lipopolysaccharide-activated macrophages toward M2 phenotype. J Biol Chem 288:25792-803
Xie, Hua-Tao; Chen, Szu-Yu; Li, Gui-Gang et al. (2012) Isolation and expansion of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 53:279-86
Li, Gui-Gang; Chen, Szu-Yu; Xie, Hua-Tao et al. (2012) Angiogenesis potential of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 53:3357-67
Chen, Szu-Yu; Hayashida, Yasutaka; Chen, Mei-Yun et al. (2011) A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells. Tissue Eng Part C Methods 17:537-48
Tan, Ek Kia; He, Hua; Tseng, Scheffer C G (2011) Epidermal differentiation and loss of clonal growth potential of human limbal basal epithelial progenitor cells during intrastromal invasion. Invest Ophthalmol Vis Sci 52:4534-45
Xie, Hua-Tao; Chen, Szu-Yu; Li, Gui-Gang et al. (2011) Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation. Stem Cells 29:1874-85

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