Elisabeth CA MacdonaldMaria Elena Gregory, David Lockington, Fiona Roberts,

Kanna Ramaesh

Authors have no financial interest

BackgroundThe keratocyte is the most abundant cell in the

corneal stromaResponsible for synthesizing the stromal ECM In health the keratocytes remain quiescent

Following injury keratocytes become activated They may assume a repair fibroblast

phenotypeOr regeneration may occur by keratocyte

proliferation and migration to replace lost cells

BackgroundThe keratocyte appears to exist in a dynamic stateThe 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

Purpose and MethodsTo 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

Time interval 1st graft to

regraft

Reason for 1st graft

Reason for

regraft

Host corneal cells identified in the

centre of the explanted button

Epithelial cells

Stromal keratocyte

s

1 9 mths HSV keratitis

Graft rejection

2 2 yrs Keratoconus

Graft rejection

3 7 yrs HSV keratitis

Endothelial failure

4 8 yrs Aphakic bullous

keratopathy

Endothelial failure

x(specimen highly

inflamed impairing analysis)

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)

e

dm

s

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)

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

s

e

s

ImplicationsStromal 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 emergeResearch regarding the location and niche is evolving

Does the progenitor cell for the keratocyte exist in the peripheral cornea or limbus ?

HypothesisThe 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?

Clinical ImplicationsCorneal transplantationMigration of residual abnormal cells

from host rim to the graft may lead to: Recurrence of the genetic stromal dystrophiesRecurrence 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

ConclusionCorneal stromal keratocytes are capable of

centripetal movement in vivoThis adds to the emerging evidence regarding the

existence of keratocyte progenitor cellsOur data suggests that the peripheral cornea or

limbus is a likely locationDefining 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