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Title:Understanding cornea stem cells during homeostasis and wound healing in Xenopus
Author(s):Adil, Mohd Tayyab
Director of Research:Henry, Jonathan J
Doctoral Committee Chair(s):Henry, Jonathan J
Doctoral Committee Member(s):Brieher, William M; Smith-Bolton, Rachel; Yang, Jing
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Xenopus
Cornea
Cornea epithelial stem cells
Stem cell deficiency
Cornea homeostasis
Cornea wound healing
Disease model
Psoralen-UVA
Lineage tracing.
Abstract:Corneal development, homeostasis, and wound healing are supported by specialized stem cells, that include the cornea epithelial stem cells (CESCs). Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here, we focus on cornea epithelial stem cell deficiency (commonly called limbal stem cell deficiency, LSCD). Fundamental developments have been made in LSCD therapies by utilizing epithelial stem cell transplants to restore normal vision in these patients. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and maintain the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients, and questions still remain about the basic biology of CESCs, including their precise cell lineages and behaviors in the cornea. A major hurdle has been the lack of adequate vertebrate models to study CESCs. Here, we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea Stem Cell Deficiency (SCD), such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to the appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and stimulates mitosis in surviving cells within the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one-half of the cornea. These cases initially develop localized SCD characteristics and showed increased pyknosis on the treated side. The localized PUV treatment is capable of stimulating some mitosis and results in changes in the percentages of p63 expressing cells in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored, thus providing an excellent system to study healing and the contributions of CESCs. To label and lineage trace the response of these strategically spared cells in the untreated half of the cornea, we utilized a transgenic line carrying a heat-shock inducible H2B-mCherry reporter, which can be activated in a localized cluster of cells at a specific location on the cornea. We showed that in contrast to the minimal displacement observed in the labeled cells in control corneas, the cells labeled in the untreated half underwent rapid displacement into the PUV treated half. This displacement did not continue at later time points (7 day post PUV onwards). As one of the few studies utilizing lineage tracing to study SCD, this work provides insights into the corneal cell response following stem cell depletion, their displacement and their mitosis, and establishes Xenopus as an excellent model using lineage tracing for studying CESCs, stem cell deficiency, and other cornea diseases. This work will also be valuable for understanding the nature of transplanted CESCs, leading to progress in improving transplant therapies for LSCD. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.
Issue Date:2021-04-14
Type:Thesis
URI:http://hdl.handle.net/2142/110467
Rights Information:Copyright 2021 Mohd Tayyab Adil
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05


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