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Management Protocols:
Limbal Stem Cell Deficiency:
Management Protocol
Alok Sati
MS
Alok Sati MS, DNB, 2Virender S. Sangwan MS, 3Vijay K. Sharma MS 2Dhanyasree Nair B.Sc,
1
Sandeep Shankar MS, DNB, 1Ashok Jha MS, DNB, 1Deepak Kalra MS
1
1. Command Hospital (EC), Alipore Road, Kolkata
2. Cornea and Anterior Segment Services, L.V. Prasad Marg, Banjara Hills, Hyderabad
3. Dr. Rajendra Prasad Centre for Ophthalmic Sciences,
All India Institute of Medical Sciences, New Delhi
M
ost experts agree that the limbal stem cells reside
in a complex microenviornment at the limbus
and are evident by both the clinical observation and the
basic scientific research1,2. Clinically, it is evident by the
observation that the corneal epithelial wound heals from
the peripheral aspect of the cornea3. Moreover, the basic
scientific research such as DNA labelling studies of mouse
cornea, have identified a special group of cells in the basal
layer of the corneal epithelium that invariably remains
quiescent and get activated whenever there is a requirement
to replenish the damaged corneal epithelial cells4. Any
insult to this complex microenvironment (niche), whether
congenital or acquired, leads to limbal stem cell deficiency
(LSCD).
surface is conjunctivalized. This thick corneal pannus is
often associated with inflammation and vascularization.
Diagnosis of LSCD is mainly clinical. However, it can be
confirmed by either impression cytology or by In vivo laser
scanning confocal microscopy (IVCM)5.
Etiology of LSCD
Partial limbal stem cell deficiency
Congenital: Aniridia, congenital erythrokerato-dermia,
keratitis associated with multiple endocrine deficiencies
and epidermal dysplasia. It is the stromal microenvironment
that is insufficient in these congenital causes.
Asymptomatic patients can be kept under observation.
However, symptomatic patients can be treated by
sequential sector conjunctival epitheliectomy (SSCE)6,
amniotic membrane transplantation (hAM)7 or ipsilateral
limbal translocation8. In SSCE, the conjunctival epithelium
from the cornea is repeatedly scraped so as to allow the
normal corneal epithelial cells to grow over the denuded
surface. In ipsilateral limbal translocation, a healthy portion
of limbus is transferred to the burned area of the same eye,
without intervening in the contralateral eye.
Acquired: Ocular surface burns, prolonged contact lens
wear, multiple surgeries involving the limbal region,
ocular surface inflammatory disorders like Stevens Johnson
syndrome, ocular cicatrical pemphigoid, chronic vernal
keratoconjunctivitis, microbial infections involving limbus,
radiotherapy and toxicity by topical medications like
mitomycin C and 5-Fluorouracil. These acquired causes
not only affect the niche but directly destroy the stem cells.
Clinical features and Diagnosis of LSCD
In partial limbal stem cell deficiency, where a sector of cornea
is conjunctivalized, patients often remain asymptomatic.
In total limbal stem cell deficiency, the entire corneal
Management protocol
Ocular surface optimization
This initial step involves lubrication of the ocular surface,
suppressing the ocular surface inflammation and correction
of lid structural abnormalities. Oflate, scleral contact lens is
gaining popularity which help in providing ocular comfort
and in resolution of kertaopathy.
Total limbal stem cell deficiency
The current treatment strategies include conjunctival limbal
autograft (CLAU), cultivated limbal epithelial transplantation
(CLET) and simple limbal epithelial transplantation (SLET)
for unilateral cases. Keratolimbal allograft (KLAL), allogenic
CLET, keratoprosthesis and cultivated oral mucosal
transplantation (COMET) are meant for bilateral cases.
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Limbal Stem Cell Deficiency
Figure 1: Preoperative photograph showing total LSCD (1A). Postoperative photographs
at day 10 (1B) and at six month (1C) after CLET.
Conjunctival limbal autograft (CLAU)
In this technique, the limbal graft tissues (2 o’clock hours
each) are harvested along with conjunctival carrier from
the superior and inferior limbal zones at 12 and 6 o’clock
positions respectively. The donor tissue is then sutured on
to the recipient bed with interrupted 10-0 monofilament
nylon sutures at the corneal and scleral margin. However,
this procedure is associated with a potential risk of
iatrogenic limbal stem cell deficiency in the donor eye9.
Cultivated limbal epithelial transplantation (CLET)
(Figure 1)
The main concern with CLAU is the development of donor
site LSCD. To overcome this, Pellegrini et al10, proposed a
method in which a small limbal biopsy is harvested from
the donor site followed by its ex vivo expansion in culture
and then transplanted onto the limbal stem cell deficient
eye. Two types of culture techniques exist i.e. ‘suspension’
and ‘explant’ culture techniques. In suspension technique,
the limbal cells are separated from the limbal niche by
enzyme digestion and are supported by the feeder cells
(Mouse NIH3T3-J2 fibroblasts). In this technique, there is
a theoretical chance of transmission of prion disease and
viruses to the recipients. In explants technique, the limbal
biopsy specimen is transported to laboratory in modified
human corneal epithelium medium (HCE) and shredded
into multiple pieces on to the hAM and cultured in HCE
medium with 10% autologous serum at 37ºC, 5% CO2
and 95% air. The growth of limbal stem cells is monitored
daily under an inverted phase contrast microscope and
the medium is changed on alternate days. A confluent
monolayer is typically formed in 10 to 14 days. This
cultured epithelial sheet is then transplanted onto the
corneal surface after removal of pannus and made adhered
to it with the help of fibrin glue. The periphery of the
cultured sheet is tucked underneath the conjunctival edge.
40 l DOS Times - Vol. 20, No. 9 March, 2015
Most of the studies have stated a favorable outcome
following CLET11-13. However, the factors affecting the
outcomes have been sparingly described. These factors
could be clinical or histological. Etiology of LSCD and age
has been slated as the clinical factors in the literature14. Of
late, a retrospective study by Sati and associates, has found
that the presence of either calcific deposits or hyperplasia
in the excised corneal pannus indicate poor prognosis15.
The strengths of this procedure, besides the clinical success
are twofold. Firstly, it does not induce LSCD in donor
eyes. Secondly, a repeat CLET can be easily performed.
The limitation lies in establishing the expensive culture
laboratory facilities.
Simple limbal epithelial transplantation (SLET)
(Figure 2)
This is the latest technique for unilateral LSCD and is being
introduced by Sangwan and associates. Its advantages over
CLET is that it avoids the need of expensive laboratory
facilities. In this technique, a 2x2 mm limbal graft is
harvested from the unaffected eye and is then divided
into multiple small pieces. Amniotic membrane is made
to adhere to the recipient cornea with the help of fibrin
glue after removal of pannus. The limbal graft pieces (10 to
12 in number) are then placed on the amniotic membrane
in a double concentric ring pattern without involving the
visual axis and secured by fibrin glue. A Bandage contact
lens (BCL) is then placed over the grafted material. In the
pioneered published series from L V Prasad Eye Institute,
Hyderabad, it is observed that this technique was quite
effective in six patients who underwent this procedure for
unilateral LSCD for ocular surface burns16.
Keratolimbal allograft (KLAL)
This procedure involves the transplantation of limbal tissue
with a corneal carrier from the cadaver to the stem cell
Management Protocols
(a)
(b)
(c)
Figure 2: Preoperative photograph showing total LSCD (2A). Postoperative photographs at day 10
(2B) and at six month (2C) after SLET.
Figure 3: Preoperative photograph showing total LSCD (3A) including symblepharon.
Postoperative photographs (3B) after Keratoprosthesis.
deficient eye. The success of this procedure relies on, in part,
the administration of systemic immunosuppression, though
the prolonged administration of immunosuppression has
its own limitations. Moreover, the graft survival following
KLAL has been reported to be inferior to autologous
transplantation17.
Living Related Conjunctival Limbal Allograft
(lr-CLAL)
Unlike CLAU, this procedure is meant for bilateral cases and
involves transplantation of limbal tissue with conjunctival
carrier from the live related donor to the stem cell deficient
eye. Unlike CLAU, this technique requires prolonged
immunosuppression to reduce the risk of rejection.
Moreover, unlike KLAL, a limited amount of limbus can be
harvested resulting in transplantation of fewer stem cells.
Allogenic cultivated limbal epithelial
transplantation (CLET)
Considering the low graft survivability of KLAL and a low
proliferative potential of cadaveric donor tissue both in vivo
and ex vivo, a live related allogenic CLET has gained wider
acceptance for patients with bilateral LSCD. Though the
success rate is quite variable, the issues that are bothersome
include the requirement of expensive laboratory facilities
and the need of long term immunosuppression with its
adverse effects.
Cultivated oral mucosal transplantation (COMET)
The concept of using cultivated oral mucosal cells was
pioneered by Nakamura and associates18.
The technique begins with the collection of oral mucosal
biopsy specimens, each measuring 2 mm2, from the patient
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Limbal Stem Cell Deficiency
and is divided into multiple small explants after removing
the submucosal connective tissue. These explants are then
immersed thrice in phosphate buffered saline solution
followed by treatment with trypsin-EDTA 0.05% solution.
The separated oral mucosal cells are then spread onto
the denuded amniotic membrane, present at the bottom
of culture inserts thereby forming the culture sheets. This
culture sheet is then made adhered to the cornea with
the help of fibrin glue. Though this technique has found
to be quite effective, the biggest challenge is its long term
effectiveness that has been found to be poorer due to
persistent epithelial defect (PED) and a tendency to develop
a varying degree of vascularisation with time.
Boston keratoprosthesis (BKPro) (Figure 3)
Boston type I keratoprosthesis (Massachusetts Eye and Ear
Infirmary, Boston, MA) is an emerging option for patients
with bilateral LSCD with wet ocular surface. The other
types of prosthesis i.e. osteoodontokeratoprosthesis and
Boston type 2 keratoprosthesis are reserved for patients
with bilateral LSCD with minimal or no tear function.
Emerging therapies
3.
Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J
Ophthalmol 1994;78:401-408.
4. Cotsarelis G, Cheng SZ, Dong G et al. Existence of slow-cycling
limbal epithelial basal cells that can be preferentially stimulated to
proliferate: Implications on epithelial stem cells. Cell 1989;57:201–
209.
5. Sejpal K, Bakhtiari P, Deng SX. Presentation, diagnosis and
management of limbal stem cell deficiency. Middle East Afr J
Ophthalmol 2013;20:5-10.
6. Dua HS, Saini JS, Azuara-Blanco A, Gupta P. Limbal stem cell
deficiency: Concept, aetiology, clinical presentation, diagnosis and
management. Indian J Ophthalmol 2000;48:83-92.
7. Anderson DF, Ellies P, Pires RT, Tseng SC. Amniotic membrane
transplantation for partial limbal stem cell deficiency. Br J
Ophthalmol 2001;85:567-75.
8. Nishiwaki-Dantas MC, Dantas PE, Reggi JR. Ipsilateral limbal
translocation for treatment of partial limbal deficiency secondary to
ocular alkali burn. Br J Ophthalmol 2001;85:1031-3.
9.
Biber JM, Holland EJ, Neff KD. Management of stem cell disease.
International Ophthalmology Clinics 2010;50(3):25-34.
10. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R,
De Luca M. Long-term restoration of damaged corneal surfaces
with autologous cultivated corneal epithelium. Lancet. 1997 Apr
5;349(9057):990-3.
Since each of the above mentioned ocular surface
reconstructive strategy has limitation, so a search is on
for a more idealistic treatment strategy. In this regard,
both the clinicians and the scientists are working on the
tissue engineering approaches along with stem cell based
regenerative therapies and the ocular surface regenerative
techniques using embryonic, induced pluripotent and
mesenchymal stem cells. Such strategies are expected to
enhance the outcomes of limbal stem cell transplantation
in the future.
11. Zakari N, Possemiers T, Dhubhghaill SN, et al. Results of a phase I/
II clinical trial: standardized, non-xenogenic, cultivated limbal stem
cell transplantation. J Transl Med. 2014;3:12:58.
Conclusion
14. Sejpal K, Bakhtiari P, Deng SX. Presentation, diagnosis and
management of limbal stem cell deficiency. Middle East Afr J
Ophthalmol 2013;20:5-10.
Amongst the above mentioned techniques, the most
favourable strategy for both the unilateral or bilateral cases,
is a matter of debate. So far, literature is scant on comparative
evaluation between the different strategies. Moreover,
the actual mechanism by which limbal transplantation
works is also debated. It is unclear whether the treatment
revives the surviving stem cells by improving the complex
microenvironment or replenishes the stem cell reserve.
References
1.
Akpek EK, Ilhan-Sarac O. Cicatrizing conjunctivitis. In: Foster CS,
Azar DT, Dholmn CH. The Cornea; Scientific Foundations and
Clinical Practice. 4th ed., Philadephia: Lippincott Williams and
Wilkins; 2005:483-488.
2.
Davanger M, Evensen A. Role of the pericorneal papillary structure
in renewal of corneal epithelium. Nature. 1971;229:560-561.
42 l DOS Times - Vol. 20, No. 9 March, 2015
12. Sharma S, Tandon R, Mohanty S, Kashyap S, Vanathi M. Phenotypic
evaluation of severely damaged ocular surface after reconstruction
by cultured limbal epithelial cell transplantation. Ophthalmic Res.
2013;50:59-64.
13. Dobrowolski D, Wylegala E, Orzechowska-Wylegala B, Wowra B,
Wróblewska-Czajka E. Application of autologous cultivated corneal
epithelium for corneal limbal stem cell insufficiency--short-term
results. Klin Oczna. 2011;113(10-12):346-51.
15. Sati A, Basu S, Sangwan VS, Vemuganti GK. Correlation between
the histological features of corneal surface pannus following ocular
surface burns and the final outcome of cultivated limbal epithelial
transplantation. Br J Ophthalmol 2014;0:1-5.
16. Sangwan VS, Basu S, MacNeil S, Balasubramanian D. Simple limbal
epithelial transplantation (SLET): a novel surgical technique for the
treatment of unilateral limbal stem cell deficiency. Br J Ophthalmol
2012;96 (7):931–934
17. Miri A, Al-Deiri B, Dua HS. Long-term outcomes of autolimbal and
allolimbal transplants. Ophthalmology 2010;117:1207-13.
18. Nakamura T, Inatomi T, Sotozono C, et al. Transplantation of
cultivated autologous oral mucosal epithelial cells in patients with
severe ocular surface disorders. Br J Ophthalmol 2004; 88:1280–4.