•Ajay Kumar Singh•Neha Sinha•Department of Ophthalmology•King George‘s Medical University, Lucknow (INDIA)

INTRODUCTION

Stem cells-- Undifferentiated proliferating cells- Present in all self-renewing tissues- 0.5%-10% of total cell population

- Properties:- Long lived, long cell cycle time- have increased potential for error free proliferation with poor

differentiation- capability to divide in asymmetric manner

HISTORICAL BACKGROUND

1971- Davanger and Evensen found the limbus of cornea is the root of corneal epithelium proliferation and migration

1983- Schofield et al proposed “niche hypothesis”.

1986- Schermer et al found the limbus of cornea was deficient in the expression of K3.

RELEVANT ANATOMY

The corneal epithelium -nonkeratinised, stratified squamous epithelial cells. Thickness ~50 mμ

Limbus-- CORNEA-stratified, nonkeratinised squamous epithelium

- CONJUNCTIVA-stratified, nonkeratinised columnar epithelium with mucin-secreting goblet cells

architecture of the limbus - palisade (of Vogt) arrangement.

Gradual transition

SITE OF LIMBAL STEM CELL

The limbal stem cells probably reside in the basal layer of the palisades of Vogt

Limbal stem cell- boundary of cornea, conjunctiva and sclera

RELEVANT PHYSIOLOGY

The corneal epithelium undergoes a constant process of cell renewal and regeneration

regenerates approximately every 7 days

proliferative reserve in the form of multipotent stem cells

Hypothesis- stem cells flourish only in limbal area - vascularity

The prevailing view of LESC proliferation –

one division of each LESC generates a daughter TAC that migrates centrally across the cornea while the original stem cell remains within its niche in the basal epithelium of the limbus.

TACs (Transient amplifying cells)- divide rapidly in basal cell layer

PMCs (Post mitotic cells)- wing cell layer

TDCs (Terminally differentiated cells)- squamous layer

The result of this migration and differentiation is that the corneal epithelium is renewed every 7–10 days in this manner

PROLIFERATION OF LIMBAL STEM CELLS

TAC

STEM CELL

PMC

TDC

STEM CELL NICHE

CORNEAL EPITHELIUM

Thoft and Friend - “XYZ hypothesis”

only partially explains corneal epithelial renewal over the lifetime of the cornea.

The basal corneal epithelial cells are incapable of continuous replication and enter senescence after several cell divisions

The XYZ Hypothesis:

X vector - combination of proliferation and centripetal migration of the basal epithelial cells.

Xs - asymmetric division of limbal epithelial stem cells- one daughter cell that remains in the limbus as a stem cell - a second daughter cell which migrates out of the limbus into the

basal corneal epithelium - transient amplifying cell (X TA ).

Th e X TA vector arises through a combination of proliferation and centripetal migration of the basal TACs.

Y vector- As the basal epithelial cells divide they give rise to suprabasal cells that form the stratifi ed layers of the cornea.

Z vector - shedding of squamous epithelial cells from the surface of cornea into the tear fi lm.

Corneal epithelial maintenance X + Y = Z,i.e. if corneal epithelium is to be maintained, cell loss must be balanced by cell replacement

Some stem cells migrate superficially within the limbal area. These limbal stem cells serve as a proliferative barrier between corneal and

conjunctival epithelia

The LESCs - finite body of cells that cannot be replaced naturally.

A state of corneal limbal stem cell deficiency (LSCD) occurs if the number of LESCs is depleted below a critical threshold by trauma or disease.

Conditions that significantly damage the limbal stem cells can result in-- invasion of conjunctival epithelium on to the corneal surface (conjunctivalisation).

Conjunctivalisation results in-- thickened, irregular, unstable epithelium, often with secondary neovascularisation

and inflammatory cell infiltration.

SIGNIFICANCE OF LIMBAL STEM CELL THEORY

To recognize the pathogenesis of many ocular surface diseases

To find the reason of failure of rebuliding ocular surface operations and corneal transplantations

TYPES OF LIMBAL STEM CELL DEFICIENCY

According to Etiology:- Primary- Secondary

PRIMARY-- related to an insufficient stromal microenvironment to support stem cell function- presumed site of pathophysiology -limbal stromal niche- Eg.-

- CONGENITAL: aniridia, dominantly inherited keratitis, Ectodermal dysplasia

- ACQUIRED: neurotrophic (neural and ischaemic) keratopathy and chronic limbitis

SECONDARY-(more common)-- ACQUIRED:

- related to external factors that destroy limbal stem cells - presumed site of pathophysiology – Limbal cells themselves- Eg.-

Traumatic- chemical (most common) or thermal injuries,  iatrogenic (multiple surgeries or cryotherapies)

Stevens-Johnson Syndrome ocular cicatricial pemphigoid (OCP) contact lens wear extensive microbial infection

TYPES

Iatrogenic:

Secondary to multiple surgeries Secondary to long term glaucoma medication

Sectorial limbal stem cell deficiency Secondary to Mitomycin C treatment

TYPES

According to extent of involvement:- Sectorial- Diffuse

SECTORIAL (PARTIAL)

- localized deficiency of LESCs in a region of limbus but an intact population of LESCs in other areas.

- Microscopically:- columnar keratopathy - mosaic pattern of stain with impression cytology

TYPES

DIFFUSE (TOTAL)

- functional loss of the entire LESC population- conjunctivalization of the entire cornea

TYPES

Staging System*

A. Normal B. Abnormal quiet C. Inflamed

I. <50% involvement

Iatrogenic, Contact lens, Pterygium

Old mild chemical injury Recent mild chemical injury, mild SJS

II. >50% involvement

Aniridia Old severe chemical injury

Recent severe chemical injury, severe SJS, OCP

* Ana Hidalgo-Simón, Eurotimes; April 2003

CLINICAL MANIFESTATIONS

SYMPTOMS:- Tearing- Blepharospasm- Photophobia- decreased vision- recurrent episodes of pain (epithelial breakdown)- history of chronic inflammation with redness

SIGNS:

- The presence of a conjunctival phenotype on the cornea (conjunctival overgrowth, conjunctivalization) is central to the diagnosis of LSCD

- dull and irregular reflex of the corneal epithelium which varies in thickness and transparency

- an ingrowth of thickened fibrovascular pannus, chronic keratitis, scarring and calcification.

- Persistent epithelial defects- stippled fluorescein staining- melting and perforation of the cornea can occur

Diffuse LSCD: Partial LSCD (A) Diffuse illumination (B) Slit beam illumination

Partial stem cell deficiency-

- sectoral ingrowth of conjunctival epithelium from focal areas of SC deficiency

- clear line of demarcation- often, but not always, visible between corneal and conjunctival phenotype of cells

- At the line of contact of the two phenotypes, tiny "bud like projections" of normal corneal epithelium can be seen extending into the conjunctivalised area

- fluorescein pooling on the conjunctivalised side -because of its relative thinness

DIAGNOSTIC TOOLS Diagnosis is crucial because these patients are poor candidates for

conventional corneal transplantation

Histologically (impression cytology)- goblet cells containing conjunctival epithelium on the corneal surface

- In advanced disease- especially those where keratinisation of the epithelium occurs (SJS, ocular pemphigoid, Lyell syndrome), conjunctival goblet cells may be completely absent-not detectable

Immuno histo-chemically (monoclonal antibodies)- absence of a cornea-type differentiation (such as the absence of keratin CK3,12)- Presence of conjunctival phenotype (CK19)- presence of mucin in goblet cells

AVAILABLE TREATMENT OPTIONS

CONSERVATIVE OPTIONS:

- In Acute phase:- Immunosuppresion-

Topical steroids Cyclosporine

- use of intensive non-preserved lubrication- bandage contact lenses- autologous serum eye drops.- Only the latter is supported by evidence in the literature

Conservative treatment usually provides temporary remission but the condition tends to deteriorate over time.

SURGICAL OPTIONS:

PARTIAL LIMBAL STEM CELL DEFICIENCY-

- In the acute phase following injury-- repeated debridement of migrating conjunctival epithelium (sequential sector

conjunctival epitheliectomy (SSCE)- can reduce or prevent conjunctival ingrowth.

- The use of an amniotic membrane graft has also been reported to be successful

Conjunctival transplantation has been used to treat corneal stem cell deficiency.

supported by the hypothesis that conjunctival epithelium "transdifferentiates" into cornea-like epithelium.

Others believe-- Transdifferentiation does not occur in humans.- conjunctival transplantation in LESC deficiency is inferior to limbal

transplantation.

Conjunctival transplantation is, however useful in other conditions:- to reconstruct the conjunctival surface in cases of symblepharon- to treat primary and recurrent pterygia.

SURGICAL OPTIONS

TOTAL LIMBAL STEM CELL DEFICIENCY-

to restore a corneal phenotype- ocular surface reconstruction (OSR) is required

Indicated in-- bilateral blinding ocular surface diseases such as Stevens Johnson syndrome

(SJS), ocular cicatricial pemphigoid (OCP), and severe chemical/ thermal burns.

SURGICAL OPTIONS

Clinically, the process involves a sequential three-step approach.

I. Correct any dry eye disease and lid abnormality that is contributing to ocular surface failure

- correction of meibomian gland dysfunction Corneal exposure Trichiasis entropion

- Punctal occlusion- Repair of symblepharon- frequent application of preservative-free artificial tears or autologous serum

SURGICAL OPTIONS

II. Remove the conjunctival epithelium from the cornea and restore a normal stromal environment

- Debridement of abnormal conjunctival epithelium and subepithelial fi brous tissue - mechanically -combined tissue peeled off the cornea.- peritomy and resection of the conjunctival epithelium for up to 4 mm from the

limbus, with application of mitomycin C over the exposed sclera to reduce recurrence of scarring and subepithelial fibroblastic proliferation.

SURGICAL OPTIONS

III. Transplant corneal LESCs to reestablish an intact and transparent epithelium

- conjunctival limbal autograft (CLAU)- living-related conjunctival limbal allograft (Lr-CLAL)- Keratolimbal allograft (KLAL)- ex vivo expansion and transplantation of cultured LESCs.

SURGICAL OPTIONS

CONJUNCTIVAL LIMBAL AUTOGRAFT (CLAU)

- First reported by Kenyon and Tseng in 1989

- transfer of autologous limbal tissue from the unaffected fellow eye to the stem cell deficient eye

SURGICAL OPTIONS

A significant percentage of the limbus is transferred for an autograft

partial stem cell deficiency in a previously normal eye

The use of an amniotic membrane graft to cover the defect in the donor eye may promote proliferation of the remaining LESCs and reduce this risk.

SURGICAL OPTIONS

Surgical procedure for performing a conjunctival-limbal autograft :

The eye on the left hand side of the image is normal.

The eye on the right has total limbal epithelial stem cell deficiency.

SURGICAL OPTIONS

LIVING-RELATED CONJUNCTIVAL LIMBAL ALLOGRAFT TRANSPLANT (Lr-CLAL)

- In bilateral total LSCD- only potential source of LESCs- allogeneic limbus

- living-related conjuctival- limbal allograft transplantation (Lr-CLAL)- conjunctival-limbal graft is taken from a living related donor and transplanted to the recipient’s stem cell deficient eye

SURGICAL OPTIONS

- The surgical technique - identical to CLAU. - Amniotic membrane can be used similarly-

- To eliminate the concern of removing LESCs from the healthy donor eye - To augment the effect of CLAU in the recipient eye

- Risk - rejection of a Lr- CLAL- systemic immunosuppression required

SURGICAL OPTIONS

KERATOLIMBAL ALLOGRAFT TRANSPLANT (KLAL)

- using tissue from cadaveric donors- may restore the corneal phenotype in

- patients with bilateral LSCD - less commonly, in patients with unilateral LSCD who do not wish to jeopardize

the healthy eye with any surgery

- Because the tissue transplanted in KLAL is allogeneic,- systemic immunosuppression is required (like Lr-CLAL)

SURGICAL OPTIONS

most important limiting factor- Allograft rejection (despite systemic immunosuppression)

Signs of allograft rejection- - telangiectasia and engorged limbal blood vessels- epithelial rejection lines and epithelial breakdown- severe limbal inflammation

Amniotic membrane transplantation (as a corneal inlay)-- suppress inflammation- restore the damaged limbal stromal environment

SURGICAL OPTIONS

Ex Vivo EXPANSION AND TRANSPLANTATION OF CULTURED LIMBAL STEM CELLS

- Based on the pioneering work in skin of Rheinwald and Green

- First described in humans by Pellegrini et al. in 1997

SURGICAL OPTIONS

Schematic drawing of ex vivo expansion of limbal SC

(a) A limbal biopsy measuring 2 × 2 mm is performed on the donor eye

(b) This biopsy is then placed on amniotic membrane, allowed to adhere and then submerged in a culture medium

(c) Limbal epithelial cells migrate out of the biopsy onto the amnion, and after 2–3 weeks the epithelial outgrowth measures 2–3 cm in diameter

(d) After the fibrovascular pannus is removed from the recipient eye the explant is placed on the cornea

(e) sutured to the sclera

The protocols used to cultivate cells for transplantation vary widely.

- “explant culture system” – - a small limbal biopsy is placed directly onto an amniotic membrane - limbal epithelial cells migrate out of the biopsy - proliferate to form an epithelial sheet

- The amniotic membrane substrate is then purported to act as a surrogate stem cell niche environment

SURGICAL OPTIONS

- “suspension culture system” –

- limbal epithelial cells are first released from the limbal biopsy (enzyme treatment )- a suspension of individual cells is seeded

either onto amniotic membrane or onto a layer of growth-arrested 3T3 feeder cells.

- A carrier substrate such as fibrin may also be used to transfer the cells to the eye

SURGICAL OPTIONS

Theoretical advantages over CLAU and Lr-CLAL:

- substantially smaller size of the limbal biopsy is required (although more than one biopsy may be required to obtain a successful explant or cell culture).

- Minimizes the risk of precipitating stem cell failure in the donor eye and provides the option for a second biopsy if necessary

SURGICAL OPTIONS

Another advantage –

- potentially reduced risk of allograft rejection- due to the absence of antigen-presenting macrophages and Langerhan’s cells in

ex vivo cultured LEC grafts.

SURGICAL OPTIONS

ISSUES REQUIRING FURTHER INVESTIGATION:

- The exact proportion of SCs present in ex vivo cultured LEC sheets is unclear and needs to be determined.

- The behaviour of LESCs following transplantation also needs to be elucidated

SURGICAL OPTIONS

despite the diff erent methodologies employed, success rate and outcomes of ex vivo expansion and transplantation of limbal epithelium are remarkably similar.

As long as viable LESCs are transferred, the method that is used to achieve this is relatively unimportant.

The inability to identify transplanted cells on the cornea of patients more than 9 months after treatment may indicate that long-term survival of transplanted cells is not essential, and that other mechanisms are responsible for the improvement of the epithelial phenotype.

SURGICAL OPTIONS

AMNIOTIC MEMBRANE TRANSPLANTATION

first used by Kim and Tseng in1995- for corneal surface reconstruction in a rabbit model of limbal stem cell

deficiency

have also been used:- as an alternative to conjunctival flaps in treating persistent and refractory corneal

epithelial defects and ulceration- to create a limbal barrier in pterygium surgery

- for conjunctival surface reconstruction following- excision of tumours, scars and symblepharon

The amniotic membrane is a thick basement membrane and avascular stromal matrix.

As only the substrate without cells is employed, there is no adverse reaction of rejection

Action Mechanisms:

• Prolong life span and maintain clonogenicity of epithelial progenitor cells

• Promote non-goblet cell epithelial differentiation

• Promote goblet cell differentiation when combined with conjunctival fibroblasts

• Exclude inflammatory cells with anti-protease activities

• Suppress TGF- signaling system and myofibroblast differentiation of normal βfibroblasts

AMT

Observed Clinical Effects:

• Facilitate epithelialization

• Maintain normal epithelial phenotype

• Reduce inflammation

• Reduce vascularization

• Reduce scarring

AMT

LIMITATIONS:

- amniotic membrane transplantation is a substrate transplantation-- cannot be used to treat ocular surface disorders that are characterized with a

total loss of limbal epithelial stem cells or conjunctival epithelial stem cells.

- Because amniotic membrane transplantation still relies on the host tissue to supply epithelial and mesenchymal cells-- Can not be used in-

severe aqueous tear deficiency diffuse keratinization Exposure keratopathy in severe neurotrophic state Near total/toal stromal ischemia

AMT

Significance of Limbal Stem Cell Transplantation

Effective method for limbal stem cell deficiency to recover the integrity of ocular surface and rebuild limbal barrier

Successful limbal transplantation can achieve:- rapid surface healing- stable ocular surface without recurrent erosions or persistent epithelial defects- regression of corneal vascularization- restoration of a smooth and optically improved ocular surface

resulting in:- improved visual acuity- probably, increased success for subsequent keratoplasty.

RECENT ADVANCES

ALTERNATIVE SOURCES OF AUTOLOGOUS STEM CELLS

Oral mucosa:- Potential advantages-

- the cells are autologous- no risk of immune mediated rejection- immuosuppression is not required.

- oral mucosa -lower stage of differentiation than epidermal keratinocytes- they divide rapidly - can be maintained in culture for prolonged periods without keratinization.

- Theoretical disadvantage -- In treatment of autoimmune diseases (such as OCP) is that the oral and ocular

mucosa may both secrete a common basement membrane target antigen

Co-cultivated limbal and conjunctival epithelum:- Central area of HAM (human amniotic membrane) has limbal epithelial cells- Peripheral area- conjunctival epithelial cells- Advantage- total ocular surface reconstruction simultaneously

Other autologous sources:- Hemopoetic stem cells- Advantage- better long term survival without need of immunosuppression