Alexander Richardson1, Mijeong Park1, Stephanie L Watson2,3, Denis Wakefield1, Nick Di Girolamo1. 1. School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia. 2. Save Sight Institute, University of Sydney, Sydney, New South Wales, Australia. 3. Sydney Eye Hospital, Sydney, New South Wales, Australia.
Abstract
Purpose: Therapies for limbal stem cell deficiency (LSCD) include stem cell (SC) grafts that regenerate the damaged ocular surface. However, the fate of transplanted cells is ill-defined. We addressed this limitation using primary corneal epithelial cells from K14-Confetti mice. Methods: Cultures of primary corneo-limbal epithelia were generated from K14-Confetti (n = 6) and wild-type (WT) (n = 3) mice. Cell phenotype and function was ascertained by immunofluorescence, flow cytometry, quantitative PCR and colony formation. K14-Confetti cells were nurtured on fibrin and transferred onto WT mice with experimentally induced LSCD (n = 16) to determine the site of implantation, longevity, and phenotype. Results: Transgenic and WT cells derived from explanted corneal tissue displayed no phenotypic or functional differences. K14-Confetti corneo-limbal epithelia that engrafted in recipient LSCD WT mice formed 107 ± 36 fluorescent clones at 2 weeks postprocedure, which decreased to 70.0 ± 5.5 by 6 weeks (P = 0.15). Furthermore, cells commonly implanted in the periphery (P < 0.05) and some generated clones that migrated centripetally. However, a normal corneal epithelial phenotype was not restored. We speculate this is due to insufficient SCs being seeded within grafts, and shows evidence of both cell loss from the implants and transdifferentiation into K8+-conjunctival and K10+-cutaneous epithelia after transplantation. Conclusions: This study successfully tracked the fate of transplanted corneo-limbal epithelia in a mouse model of LSCD by intravital microscopy. Our data shed new light into how donor cells behave, the positions they take, how long they survive, and potential mechanisms of loss from the ocular surface. This information is important for improving future animal models, to render them clinically relevant.
Purpose: Therapies for limbal stem cell deficiency (LSCD) include stem cell (SC) grafts that regenerate the damaged ocular surface. However, the fate of transplanted cells is ill-defined. We addressed this limitation using primary corneal epithelial cells from K14-Confetti mice. Methods: Cultures of primary corneo-limbal epithelia were generated from K14-Confetti (n = 6) and wild-type (WT) (n = 3) mice. Cell phenotype and function was ascertained by immunofluorescence, flow cytometry, quantitative PCR and colony formation. K14-Confetti cells were nurtured on fibrin and transferred onto WT mice with experimentally induced LSCD (n = 16) to determine the site of implantation, longevity, and phenotype. Results: Transgenic and WT cells derived from explanted corneal tissue displayed no phenotypic or functional differences. K14-Confetti corneo-limbal epithelia that engrafted in recipient LSCD WT mice formed 107 ± 36 fluorescent clones at 2 weeks postprocedure, which decreased to 70.0 ± 5.5 by 6 weeks (P = 0.15). Furthermore, cells commonly implanted in the periphery (P < 0.05) and some generated clones that migrated centripetally. However, a normal corneal epithelial phenotype was not restored. We speculate this is due to insufficient SCs being seeded within grafts, and shows evidence of both cell loss from the implants and transdifferentiation into K8+-conjunctival and K10+-cutaneous epithelia after transplantation. Conclusions: This study successfully tracked the fate of transplanted corneo-limbal epithelia in a mouse model of LSCD by intravital microscopy. Our data shed new light into how donor cells behave, the positions they take, how long they survive, and potential mechanisms of loss from the ocular surface. This information is important for improving future animal models, to render them clinically relevant.
Authors: Mijeong Park; Alexander Richardson; Elvis Pandzic; Erwin P Lobo; J Guy Lyons; Nick Di Girolamo Journal: Proc Natl Acad Sci U S A Date: 2019-12-16 Impact factor: 11.205