This document discusses limbal stem cells, which reside in the basal layer of the palisades of Vogt in the limbus and are responsible for renewing the corneal epithelium every 7 days. It outlines the historical background and relevant anatomy and physiology of limbal stem cell renewal. It also describes limbal stem cell deficiency, including types, clinical manifestations, diagnostic tools, and available treatment options such as conservative treatments and surgical procedures like limbal stem cell transplantation.

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

2. 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

3. 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.

4. RELEVANT ANATOMY
 The corneal epithelium -nonkeratinised, stratified squamous epithelial cells.
 Thickness ~50 μm
 Limbus-
- CORNEA-stratified, nonkeratinised squamous epithelium
Gradual transition
- CONJUNCTIVA-stratified, nonkeratinised columnar epithelium with mucin-
secreting goblet cells
 architecture of the limbus - palisade (of Vogt) arrangement.

5. 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

6. 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

7. 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

8. PROLIFERATION OF LIMBAL STEM CELLS
CORNEAL EPITHELIUM
STEM CELL
TAC
PMC
STEM CELL NICHE
TDC

9.  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

10. 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. Corneal epithelial maintenance
X + Y = Z,
 Z vector - shedding of squamous epithelial cells from the surface of i.e. if corneal epithelium is to be
cornea into the tear fi lm. maintained, cell loss must be
balanced by cell replacement

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

12.  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.

13.  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.

14. 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

15. 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

16. TYPES
 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

17. TYPES
 Iatrogenic:
Secondary to multiple surgeries Secondary to long term Sectorial limbal stem cell
glaucoma medication deficiency Secondary to
Mitomycin C treatment

18. 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

19. TYPES
 DIFFUSE (TOTAL)
- functional loss of the entire LESC population
- conjunctivalization of the entire cornea

20. Staging System*
A. Normal B. Abnormal quiet C. Inflamed
I. <50% Iatrogenic, Contact lens, Old mild chemical injury Recent mild chemical
involvement Pterygium injury, mild SJS
II. >50% Aniridia Old severe chemical Recent severe
involvement injury chemical injury,
severe SJS, OCP
* Ana Hidalgo-Simón, Eurotimes; April 2003

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

22.  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

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

24.  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

25. 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

26. 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.

27.  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

28. SURGICAL OPTIONS
 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.

29. 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.

30. 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

31. 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.

32. 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.

33. 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

34. 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.

35. 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.

36. 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

37. 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

38. 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)

40. 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

41. 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

42.  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

43. SURGICAL OPTIONS
 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

44. 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

45. 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

46. 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.

47. 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

48. 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.

49. 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

50. AMT
 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-β signaling system and myofibroblast differentiation of normal
fibroblasts

51. AMT
 Observed Clinical Effects:
• Facilitate epithelialization
• Maintain normal epithelial phenotype
• Reduce inflammation
• Reduce vascularization
• Reduce scarring

52. 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

54. 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.

55. 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

56.  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