Publication Date

2019-01-11

Availability

Embargoed

Embargo Period

2021-01-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2018-11-09

First Committee Member

Feng Gong

Second Committee Member

Thomas Harris

Third Committee Member

Ming Jiang

Fourth Committee Member

Wen Shen

Abstract

On the ocular surface, corneal epithelial stem cells (SC) reside in the limbus bordered between the cornea and the conjunctiva. To explore how limbal epithelial progenitor/stem cells (LEPC) control quiescence, self-renewal, and corneal fate decision by their native niche, our laboratory was the first to develop the in vitro isolation and expansion methods of limbal niche cells (LNC) and established a SC-LNC reunion model on 3D Matrigel that mimics the in vivo where there is close contact between LNC and LEPC. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. We showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulting in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche. For two decades, transplantation of cryopreserved amniotic membrane (AM) has been shown to exert anti-inflammatory, anti-scarring, and anti-angiogenic actions to promote epithelial wound healing and nerve regeneration in ocular surface reconstruction. Our lab has previously successfully purified the active component from AM that is responsible for the aforementioned therapeutic actions and characterized “HC-HA/PTX3” as a unique matrix formed by hyaluronan covalently linked to heavy chain 1 of inter-alpha-inhibitor and non-covalently linked to pentraxin. To understand how LNC regulates LEPC under the context of HC-HA/PTX3, we created a similar in vitro reunion assay in immobilized HC-HA/PTX3 in this study. The resultant spheres exhibited similar suppression of the corneal fate decision but upregulation of quiescence markers, including nuclear translocation of Bmi-1, and negligible clonal growth of LEPC. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (PCP) Wnt signaling as well as bone morphogenetic protein (BMP) signaling in both LEPC and LNC. The activation of BMP signaling in LNC was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPC when reunioned with mLNC of conditionally deleted Bmpr1a; Acvr1DCKO mice. Furthermore, ablation of BMP signaling in LEPC led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPC. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNC, which leads to BMP signaling in LEPC to achieve quiescence, helping explain how AM transplantation is clinically useful as a matrix for ex vivo expansion of LEPC and to treat corneal blindness caused by limbal SC deficiency.

Keywords

Limbal stem cells; Niche Regulation; Pax6; Neurogensis; HC-HA/PTX3

Available for download on Sunday, January 10, 2021

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