Elisabeth CA Macdonald Maria Elena Gregory, David Lockington, Fiona Roberts, Kanna Ramaesh

1. Observation of the in vivo movement of human host keratocytes into donor tissue following corneal graft; A novel technique Elisabeth CA Macdonald Maria Elena Gregory, David Lockington, Fiona Roberts, Kanna Ramaesh Authors have no financial interest

2. Background • The keratocyte is the most abundant cell in the corneal stroma • Responsible for synthesizing the stromal ECM • In health the keratocytes remain quiescent • Following injury keratocytes become activated • They may assume a repair fibroblast phenotype • Or regeneration may occur by keratocyte proliferation and migration to replace lost cells

3. Background • The keratocyte appears to exist in a dynamic state • The natural history of the keratocyte is not clear • However considerable cell loss and renewal is recognised to occur in certain situations • The process of renewal may depend on a sub-population of progenitor or stem cells • Demonstration of keratocytes capable of movement within the corneal stroma would add support to this concept

4. Purpose and Methods • To investigated the in vivo movement of corneal stromal and epithelial cells using CISH technique • Four explanted human sex-mismatched corneal buttons were studied • Corneal epithelial and keratocyte cells containing the Y chromosome were identified • The sex mismatch of donor (XX) and host (XY) meant any identified Y chromosomes cells were of host origin having migrated into the donor tissue

5. Patient details and results

6. e Corneal epithelium showing Y chromosome (brown dot; black arrows) and X chromosome (blue dot; red arrows) in explanted sex-mismatched corneal button studied using CISH (CISH X/Y; Magnification x1000)

7. s dm Stromal keratocytes showing Y chromosome (brown dot; black arrows) and X chromosome (blue dot; red arrows) in explanted sex-mismatched corneal button studied using CISH (CISH X/Y; Magnification x1000)

8. Immunohistochemical analysis confirmed the identified stromal cells as keratocytes • Positive staining in the stroma for keratocytes (CD34) • Negative staining for dendritic markers (CD21, CD23 , CD35), lymphocytes (CD45) and macrophages (CD68) e e s s

9. Implications • Stromal keratocytes undergo centripetal movement in vivo • Complete replacement of the donor cells with host cells may ultimately occur Does the donor cornea serve as a framework which is repopulated with recipient cells over time ? • Evidence regarding the potential keratocyte progenitor/ precursor cells is starting to emerge • Research regarding the location and niche is evolving Does the progenitor cell for the keratocyte exist in the peripheral cornea or limbus ?

10. Hypothesis • The corneal stromal cells are not a static population • Renewal depends on a source of precursors cells • They may exist in the peripheral cornea or limbus Are epithelial and keratocyte stem cells resident in a reciprocal niche at the corneal limbus?

11. Clinical Implications Corneal transplantation • Migration of residual abnormal cells from host rim to the graft may lead to: • Recurrence of the genetic stromal dystrophies • Recurrence of HSV keratitis • Immunosuppression in ant lamellar grafts may not be necessary long term if all donor cells are ultimately replaced by host cells Conditions affecting the limbus • Epithelial and stromal stem cells may coexist in a reciprocal niche, possibly at limbus, stem cells deficiency may ultimately affect both

12. Conclusion • Corneal stromal keratocytes are capable of centripetal movement in vivo • This adds to the emerging evidence regarding the existence of keratocyte progenitor cells • Our data suggests that the peripheral cornea or limbus is a likely location • Defining the corneal cell movements and the location of the progenitor or stem cells has important clinical implications • CISH technique may allow further investigation of the corneal stromal dynamics using archival tissue