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Sudan Puri, Mingxia Sun, Kazadi Nadine Mutoji, Tarsis Ferreira Gesteira, Vivien Jane Coulson-Thomas; Deciphering limbal stem cell microenvironment for optimizing limbal stem cell culture for treating limbal stem cell deficiency. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4712.
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© ARVO (1962-2015); The Authors (2016-present)
A major hurdle in limbal stem cell (LSC) transplantation for the treatment of LSC deficiency (LSCD) is ensuring the LSCs remain stem cell-like during ex vivo expansion. Therefore, this study aims to understand the role of hyaluronan (HA) in the LSC microenvironment, how the structural properties of the hyaluronan synthases (HASs) dictate the molecular weight and composition of HA and to reproduce the substrate in vitro, thereby generating optimal ex vivo culture conditions for LSCs.
For in silico experiments, we modeled HASs using cellulose synthases by iterative threading assembly refinement using Rosetta 3.10 and performed molecular dynamics simulations using the molecular simulation package Amber. For in vitro experiments, limbal rims from freshly enucleated murine eyes were dissected and underwent enzymatic digestion to obtain single cells that were cultured ex vivo on pre-coated plates. The ability of different substrates, such as poly-L-lysine (PLL), collagen IV and HA, to maintain the LSC phenotype were compared by flow cytometry, quantitative PCR and immunofluorescence for putative LSC markers. For in vivo experiments, the regeneration potential of LSCs was tested by culturing them on fibrin gels that were used as carriers for LSC transplantation onto mice with experimentally induced LSCD.
Our data indicate the length of the growing HA chain is influenced by amino acids at both glucuronyl transferase domain and translocation pore of the HASs. The LSCs cultured on HA coated dishes had significantly higher expression level and more cells positive for putative LSC markers. The transplantation of LSCs expanded on HA effectively repopulated the corneal epithelium of the LSCD mouse model and maintained the healthy cornea for longer period after treatment, compared to other substrates.
These results are consistent with our hypothesis that HA has an important role in the limbal stem cell microenvironment to maintain LSC phenotype and function. HA is required in the optimal substrate for ex vivo LSC expansion that could be used to treat patients with visual impairment due to LSCD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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