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Limbal stem cell deficiency

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LIMBAL EPITHELIAL STEM CELL

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Limbal stem cell deficiency

  1. 1. Limbal stem cell deficiency:Current Management ( TAKEN FROM-Journal of Clinical Ophthalmology & Research) JAN –APRIL 2016 VOL 4 ISSUE 1 Authors:Ikeda Lal,Neha Gupta, Jyoti Purushotham,Virender S.Sangwan (by DR PAWAN JARWAL , RESIDENT DOCTOR , SSG HOSPITAL VADODARA.)
  2. 2. INTRODUCTION • Stem cells- • Undifferentiated proliferating cells • Present in all self-renewing tissues e.g blood, testis and stratified sq. epithelia. • 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. 3. RELEVANT ANATOMY • The corneal epithelium -nonkeratinised, stratified squamous epithelial cells. • Thickness ~50 μm • Limbus- • CORNEA-stratified, nonkeratinised squamous epithelium • CONJUNCTIVA-stratified, nonkeratinised columnar vascularizedepithelium with mucin-secreting goblet cells • architecture of the limbus - palisade (of Vogt) arrangement.
  4. 4. 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
  5. 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. 6. PROLIFERATION OF LIMBAL STEM CELLS TAC STEM CELL PMC TDC STESTEM CELL RESERVOIRM CORNEAL EPITHELIUM
  7. 7. 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 stratified layers of the cornea. • Z vector - shedding of squamous epithelial cells from the surface of cornea into the tear fi lm.
  8. 8. 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
  9. 9. • 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
  10. 10. • Iatrogenic: Secondary to multiple surgeries Secondary to long term glaucoma medication Sectorial limbal stem cell deficiency Secondary to Mitomycin C treatment TYPES
  11. 11. • 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
  12. 12. • DIFFUSE (TOTAL) • functional loss of the entire LESC population • conjunctivalization of the entire cornea TYPES
  13. 13. CLINICAL MANIFESTATIONS • SYMPTOMS: • Tearing • Blepharospasm • Photophobia • decreased vision • recurrent episodes of pain (epithelial breakdown) • history of chronic inflammation with redness
  14. 14. • 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
  15. 15. • 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
  16. 16. 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)
  17. 17. AVAILABLE TREATMENT OPTIONS • PARTIAL LIMBAL STEM CELL DEFICIENCY- • 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.
  18. 18. • SURGICAL OPTIONS: • 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
  19. 19. • 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
  20. 20. 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
  21. 21. 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
  22. 22. 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. • Simple limbal epithelial transplantion (SLET) SURGICAL OPTIONS
  23. 23. • CONJUNCTIVAL LIMBAL AUTOGRAFT (CLAU) • Transfer of autologous limbal tissue from the unaffected fellow eye to the stem cell deficient eye. • Imperative to exclude LSCD in the donor tissue • Optimum amount of limbal tissue. • Conservative removal to prevent iatrogenic LSCD in donor eye SURGICAL OPTIONS
  24. 24. SURGICAL OPTIONS
  25. 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. 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. 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
  28. 28. • 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 SURGICAL OPTIONS
  29. 29. Ex Vivo EXPANSION AND TRANSPLANTATION OF CULTURED LIMBAL STEM CELLS • Most exciting and promising technique currently • 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
  30. 30. • (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
  31. 31. Simple Limbal Epithelial Transplantation(SLET) • It is simplified tech of limbal transplantation,which combines the benefits of both CLAU and cultivated limbal epithelial transplant . • It is single step procedure ,doesn’t require an expensive lab set up unlike CLET and there is no danger of inducing iatrogenic LSCD in the donor eye. • A 2*2 mm autograft limbal tissue is excised from donor eye and divided in to 10-15 small pieces and placed onto the amniotic membrane over the recipient bed ,avoiding visual axis .These transplant are secured in place with fibrin glue and bandage contact lens applied .
  32. 32. Diagnosis of LSCD Treatment of adnexal and dry eye disease Unilateral Partial Repeated debridement SSCE AMT Total CLAU Cultivated limbal autograft Bilateral KLAL Lr-CLAL Cultivated limbal autograft
  33. 33. 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.
  34. 34. 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
  35. 35. • Co-cultivated limbal and conjunctival epithelium: • 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
  36. 36. THANK YOU

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