Presenter : Dr. Om PatelModerator : Dr. Radhika Torgal

LIMBAL STEM CELL & AMNIOTIC MEMBRANE TRANSPLANTATION

STEM CELLS• Small, quiescent subpopulation of specialized

undifferentiated, self-renewing cells, which are capable of indefinite proliferation to large number of differentiated progeny, responsible for the cellular replacement and regeneration in all the self-renewing tissues

• Maintains a steady-state population of healthy cells within tissues during the lifespan of the organism

• Poorly differentiated with a slow cell cycle, long life span and high capacity for error-free self renewal

LIMBAL STEM CELLS• Resides in Palisades Of Vogt

• Responsible for corneal epithelial renewal and regeneration

• Acts as a barrier, preventing conjunctival epithelium from growing onto the cornea

LIMBAL STEM CELLS

• 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

LESC Proliferation

PROLIFERATION OF LIMBAL STEM CELLS

TAC

STEM CELL

PMC

TDC

STEM CELL RESERVOIR

CORNEAL EPITHELIUM

LIMBAL STEM CELLS DEFICIENCY

Primary LSCD

• Absence of identifiable external factors

• Inappropriate microenvironment to support the limbal stem cells

• Aniridia, Multiple endocrinal deficiency

• Destruction of limbal stem cells by external factors

• Chemical /thermal injuries, Stevens-Johnson syndrome, Ocular Pemphigoid, Multiple ocular surgeries, Contact lens wear

PRIMARY SECONDARY

• Iatrogenic:

Secondary to multiple surgeries 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

• DIFFUSE (TOTAL)

• Functional loss of the entire LESC population• Conjunctivalization of the entire cornea

CLINICAL MANIFESTATIONS

• SYMPTOMS:• Tearing• Blepharospasm• Photophobia• Decreased vision• Recurrent episodes of pain (epithelial

breakdown)• Chronic inflammation with redness

SIGNS

CONJUCTIVALIZATION

INFLAMMATION

NEOVASCULARIZATION

• SIGNS:• Conjuctivalization is central to the diagnosis of LSCD

• 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

• Fibrovascular pannus, chronic keratitis, scarring and calcification

• SIGNS:• Persistent epithelial defects- stippled fluorescein

staining

• Fluorescein pooling on the conjunctivalised side because of its relative thinness

• Melting and perforation of the cornea can occur

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

DIAGNOSTIC TOOLS• 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), conjunctival goblet cells may be completely absent

• 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

Diagnosis of LSCD

Treatment of adnexal and dry eye disease

Unilateral

Partial

Repeated debridement

AMT

CLAU

Total

CLAU

Cultivated limbal autograft

Bilateral

KLAL

Lr-CLAL

Cultivated limbal autograft

TREATMENT OPTIONS• CONSERVATIVE OPTIONS:

• In Acute phase:• Immunosuppresion-

• Topical steroids• Cyclosporine

• Use of intensive non-preserved lubrication• Bandage contact lenses• Autologous serum eye drops

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

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

1. 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

SURGICAL OPTIONS

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

• Debridement of abnormal conjunctival epithelium and subepithelial fibrous tissue

• Mechanically - combined tissue peeled off the cornea

• Peritomy and resection of the conjunctival epithelium for up to 4 mm from the limbus

SURGICAL OPTIONS

III. Transplant corneal LESCs to re-establish 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

• Unilateral partial or total LSCD

SURGICAL OPTIONS

• Imperative to exclude LSCD in the donor tissue

• Optimum amount of limbal tissue

• Conservative removal to prevent iatrogenic LSCD in donor eye

SURGICAL OPTIONS

CONJUNCTIVAL LIMBAL AUTOGRAFT (CLAU)

SURGICAL OPTIONS

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

• In bilateral total LSCD - only potential source of LESCs - allogenic limbus

• 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 ( KLAL)

• Cadaveric tissue, the stem cell carrier may be either conjunctiva or cornea

• Donor Tissue Selection : • Youngest possible donor with an upper limit of 50 years

• Surgery should be performed within 72 hours

• Systemic Immuno-suppression required

SURGICAL TECHNIQUE• Corneo scleral rim (4-5mm wide) of cadaveric eye is taken &

central 7.5mm of corneal rim is removed

• Corneo-scleral rim is cut into 2 equal halves

• For 4 crescents, we require both eyes of the cadaver

• By lamellar dissection , posterior half of each lenticule is removed (post sclera + stroma + Dm + endothelium)

• Cover 360 degrees of recipient with donor tissue

• Suture the edges – patch eye with shield

• 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• Punctate epithelial keratopathy• Severe limbal inflammation• Elevated Perilimbal area

• Amniotic membrane transplantation (as a corneal inlay)-• Suppress inflammation• Restore the damaged limbal stromal environment

SURGICAL OPTIONS

Keratolimbal allograft ( KLAL)

Ex Vivo EXPANSION AND TRANSPLANTATION OF CULTURED LIMBAL STEM CELLS

• Most exciting and promising technique currently

• Based on the pioneering work of Rheinwald and Green

SURGICAL OPTIONS

• Advantages

• 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

• Less chances of rejection as only epithelial cells are transplanted

SURGICAL OPTIONS

• (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) Suture

Technique

• Protocols used to cultivate cells for transplantation

• “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)

• Suspension of individ1ual cells is seeded• On amniotic membrane• or onto a layer of growth-arrested 3T3 feeder cells

• Medium - Dulbecco’s minimum essential medium (DMEM) and Ham’s F12 medium

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

SURGICAL OPTIONS

• POINTS OF CONCERN:

• The exact proportion of SCs required in ex vivo cultured LEC sheets is unclear and needs to be determined

• Multiple transplantation may be required

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

• 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 in 1995• For corneal surface reconstruction in a rabbit model of

limbal stem cell deficiency

• Also been used:• 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

PLACENTAL ANATOMY

Amnion lines the inner cavity of the placenta. 37

AMNIOTIC MEMBRANE• The innermost layer of the placenta• Amnion is a 0.2 mm to 0.5 mm five-layered

membrane• Lacks nerves, lymphatics and blood vessels• Composed of three basic layers:

• Epithelial monolayer • Thick basement membrane • Avascular, hypocellular stromal matrix

HISTOLOGY• Epithelium : single layer of cuboidal cells with large

number of microvilli

• Basement membrane • Histochemically it closely resembles conjunctiva

• Fibroblast layer : thickest layer of the AM -- made up of a loose fibroblast network

PROPERTIES OF AMNIOTIC MEMBRANE

I. Promote epithelial cell migration, adhesion and differentiation

II. Contains anti-angiogenic proteins, which inhibit neovascularization by inhibiting vascular endothelial cell growth

III. Promote non-goblet cell differentiation of the conjunctival epithelium

IV. Do not express HLA & does not cause any rejection reaction

V. Produces basic fibroblast, hepatocyte and transforming growth factor

VI. Stromal matrix is rich in fetal hyaluronic acid -- suppresses TGF B signaling & proliferation of fibroblast

VII. Suppresses expression of inflammatory cytokines : IL-1a, IL -2, IL-8, interferon γ, tumor necrosis factor-β & PDGF

VIII. The AM attracts and sequesters inflammatory cells infiltrating the ocular surface

IX. Basement membrane of AM has Type IV collagen and laminin : cell adhesion

PROPERTIES OF AMNIOTIC MEMBRANE

INDICATIONS OF AMT IN OCULAR SURGERY

•

Conjunctival surface reconstructionPterygium surgeryChemical burnsCicatrizing conjunctivitisOcular surface squamous neoplasia (OSSN)Leaking blebsFiltering surgeryFornix formation

Corneal surface reconstruction

Non-healing stromal ulcers

LSCD

Bullous keratopathy

AMNIOTIC MEMBRANE GRAFT (AMG)

• Amniotic membrane is obtained from prospective donors undergoing Caesarean section

• Strict asepsis

• Screening for communicable diseases HIV, hepatitis,syphilis and human T cell leukemia virus

AMNIOTIC MEMBRANE DETACHED FROM THE HUMAN PLACENTA

AMNION ON NITROCELLULOSE PAPER

45Multiple pieces of AMT from one donor

SUTURE LESS AMG Prokera 11 : AM attached to a soft contact lens-sized

conformer( PMMA/ polycarbonate) – 15mm diameter for easy

insertion

11.Kheirkhah A ,Johnson DA et al. Temporary sutureless amniotic membrane patch for acute alkaline burns. Arch Ophthalmol. 2008 Aug;126(8):1059-66.

12. Casas V, Raju VK et al. Sutureless amniotic membrane transplantation for partial limbal stem cell deficiency . Am J Ophthalmol. 2008 May;145(5):787-94.

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

• 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

Thank You…