Limbal Stem Cell Transplantation

1. Presenter : Dr. Om Patel
Moderator : Dr. Radhika Torgal
LIMBAL STEM CELL & AMNIOTIC
MEMBRANE TRANSPLANTATION

2. 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

3. 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

4. LIMBAL STEM CELLS

5. • 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

6. PROLIFERATION OF LIMBAL STEM CELLS
TAC
STEM CELL
PMC
TDC
STEM CELL RESERVOIR
CORNEAL EPITHELIUM

7. 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

8. • Iatrogenic:
Secondary to multiple surgeries Sectorial limbal stem cell
deficiency Secondary to
Mitomycin C treatment
TYPES

9. • 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

10. • DIFFUSE (TOTAL)
• Functional loss of the entire LESC population
• Conjunctivalization of the entire cornea

11. CLINICAL MANIFESTATIONS
• SYMPTOMS:
• Tearing
• Blepharospasm
• Photophobia
• Decreased vision
• Recurrent episodes of pain (epithelial
breakdown)
• Chronic inflammation with redness

12. SIGNS
CONJUCTIVALIZATION
INFLAMMATION
NEOVASCULARIZATION

13. • 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

14. • 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

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

16. 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

17. 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

18. 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

19.  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

20. 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

21. 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

22. • 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

23. • 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)

24. SURGICAL OPTIONS

25. 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

26. 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

27. 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

29. • 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)

30. 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

31. • 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

32. • (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

33. • 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

34. “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

35. • 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

36. 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

37. PLACENTAL ANATOMY
Amnion lines the inner cavity of the placenta. 37

38. 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

39. 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

40. 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

41. 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

42. INDICATIONS OF AMT IN OCULAR
SURGERY
•
Conjunctival surface
reconstruction
Pterygium surgery
Chemical burns
Cicatrizing conjunctivitis
Ocular surface squamous
neoplasia (OSSN)
Leaking blebs
Filtering surgery
Fornix formation
Corneal surface
reconstruction
Non-healing stromal ulcers
LSCD
Bullous keratopathy

43. 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

44. AMNIOTIC MEMBRANE DETACHED FROM
THE HUMAN PLACENTA

45. AMNION ON NITROCELLULOSE
PAPER
45
Multiple pieces of AMT from one donor

46. 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.

48. 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