IC-55:Limbal stem cell deficiency and its management
Limbal stem cell deficiency is a painful and blinding disease characterised by recurrent epithelial defects, corneal vascularisation and corneal conjunctivalisation. There are many causes including hereditary (such as aniridia) and acquired causes (such as chemical eye burns and inflammatory eye diseases). There are medical and surgical measures for this debilitating disease, including cultured limbal stem cell therapy. This course will describe the conventional management, including conservative management and whole tissue limbal grafting, as well as more contemporary management such as cultured limbal stem cell transplantation. The different methods and regulatory issues involved in limbal stem cell culture will also be highlighted.
Objectives:
- Limbal stem cell deficiency and its causes
- Diagnosis of limbal stem cell deficiency
- Medical management of limbal stem cell deficiency
- Surgical management of limbal stem cell deficiency: whole tissue limbal grafting and cultured limbal stem cell transplantation
- Different methods for culturing limbal stem cells
- Regulatory requirements for cultured limbal stem cell transplantation
Schedule:
0-20 minutes Limbal stem cell deficiency, its causes and diagnostic tools
20-30 minutes Questions
30-50 minutes Management options for limbal stem cell deficiency
50-60 minutes Questions
60-75 minutes Culture methods for limbal stem cells
75-90 minutes Transplantation of cultured limbal stem cells
90-105 minutes The regulation of cultured limbal stem cell transplantation
105-120 minutes Questions
1) Limbal stem cell deficiency, its causes and diagnostic tools
Limbal stem cell deficiency
The limbus has two important functions with regards to the corneal epithelium. Firstly, the corneal epithelium is renewed by stem cells located at the limbus, the so-called limbal stem cells. Secondly, the limbus also acts as a barrier preventing the conjunctival epithelium and its blood vessels from encroaching on to the corneal surface. When the limbal stem cells become deficient or dysfunctional, the disease of limbal stem cell deficiency results. The corneal epithelium cannot be maintained and conjunctivalisation ensues. The result is a disease which causes chronic ocular surface inflammation and epithelial breakdown, and visual impairment.
Causes
Limbal stem cells become deficient or dysfunctional from a variety of causes. These can be broken down into primary or hereditary causes and secondary or acquired causes. Primary causes include aniridia and ectodermal dysplasia. Secondary causes include chemical and thermal burns, inflammatory ocular surface disease (Stevens-Johnson syndrome and ocular cicatricial pemphigoid), iatrogenic causes (extensive limbal surgery and cryotherapy, and radiotherapy) and contact lens related keratitis.
Diagnosis
The diagnosis of limbal stem cell deficiency is very much made on natural history of the disease and clinical signs of conjunctivalisation of the corneal surface. Delayed fluorescein staining of the corneal surface is a characteristic feature. Corneal surface impression cytology is a useful diagnostic tool to confirm conjunctivalisation of the corneal surface. Corneal impression cytology staining for goblet cells (which are present in conjunctival but not corneal epithelium) and for cytokeratin 19 (a marker for conjunctival epithelium) are commonly used. Imaging of the cornea and limbus, such as confocal microscopy, are also being investigated but their role in diagnosis is not yet clear.
2) Management options for limbal stem cell deficiency
Medical or conservative: Lubricants (preservative free) and bandage contact lens wear are the mainstay. Topical steroids and antibiotics (preservative free) for periods of significant ocular surface inflammation and epithelial breakdown. Autologous serum drops have a role in epithelial wound healing but the exact nature of their action is unclear. Punctal plugs for improvement of dry eye also have a role
Surgical:Prior to any surgical management of the limbal stem cell deficiency, any eyelid disease and dry eye need to be optimised. Lid margin malposition (cicatricial entropion) and trichiasis need to be treated. Punctal occlusion and lateral tarsorrhaphy may be necessary to reduce dry eye. Only once eyelid disease and dry eye have been treated effectively should one consider surgical management of limbal stem cell deficiency. The surgical options for limbal stem cell deficiency are based on the transplantation of healthy limbal epithelium. This can be in the form of whole tissue grafts or cultured cells. The source of the tissue can be autologous (in unilateral disease) or allogeneic from matched living donors or cadaveric donors.
3) Culture methods for limbal epithelium
The culture of human limbal epithelium has many variations:
Suspension versus explant
3T3 fibroblast co-culture versus amniotic membrane versus other methods
Foetal calf serum versus autologous serum
The use of airlifting
The duration of culture expansion
Each of these different culture methods will be described in detail (Osei-Bempong et al Regenerative Medicine 2009).
4) Transplantation of cultured limbal epithelium
The use of cultured limbal epithelial cell therapy for severe limbal stem cell deficiency essentially involves three steps:
Procurement of limbal epithelium from donor eye: The removal of a small amount of limbal tissue (1-2mm X 1-2mm)
Culture: Ex vivo expansion of the limbal epithelium.
Transplantation of ex vivo expanded limbal epithelium: After performing a superficial keratectomy to remove the conjunctival tissue from the corneo-limbal surface, the cultured limbal epithelial sheet is sutured to the eye.
There are some significant variations in the procedure which will be discussed: removal of the explant from the culture versus limbal positioning of explant; the use of a bandage amniotic membrane (as part of a sandwich technique) versus bandage contact lens; and post-operative lid closure using a botulinum toxin induced ptosis or central tarsorrhaphy.
The results from published cases and case series will be discussed (Baylis et al 2011 Journal of Cell Biochem). The overall success rate of cultured limbal epithelial cell transplantation is 76% (77% for autografts and 73% for allografts).
5) The regulation of cultured limbal epithelial cell therapy
The culture expansion of human limbal epithelium and its clinical application as part of a clinical trial is highly regulated (for example EMA and MHRA). The actual culture process has to be performed in accordance with good manufacturing practise in order to produce transplant grade cultures. The culture process therefore has to be performed in specialised “clean” laboratories and the culture has to be monitored for sterility and quality.
The success of cultured limbal epithelial cell transplants is difficult to determine as no formal clinical trial data is available to date. It is envisaged that EMA or MHRA approved clinical trials will be conducted to determine the true efficacy of this treatment modality for limbal stem cell deficiency.
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