Lecture 8 (Corneal Stem Cell Therapy) Flashcards
Why are conventional corneal transplants useless against limbal stem cell deficiencies?
Conventional corneal transplants are of no use in these instances since conventional corneal transplantation or keratoplasty involves the transplantation of the central part of the cornea (usually about 5mm in diameter). The limbal region (containing the epithelial stem cells) is not transplanted so without a source of stem cell the corneal epithelial cell sheet quickly dies.
What are the four requirements for the successful stem cell therapy of the corneal epithelium?
- A substitute basal lamina (to grow the cells on)
- A system for isolating the stem cells
- A culture system which will allow the ex vivo expansion of stem cells and promotes the formation of tight cell junctions.
- A surgical technique for transplantation
Describe step 1 in corneal transplantation: the substitution of the basal lamina.
This is a demanding objective and requires an in depth understanding of the cell interactions within the tissue, namely the cell to cell junctions, the cell to basal lamina interactions, the composition of the basal lamina, the mechanism of cell migration and the differences between stem cells and differentiated cells in the tissue. The amniotic membrane of the placenta can be used as an ex vivo basal lamina to expand limbal epithelial SCs in culture, and has been described as ‘the perfect substitute basal lamina’ due to the fact that the composition of its extracellular matrix is similar to that of the limbal basal lamina. This approach was derived from the clinical use of human amniotic membrane to repair corneal perforations. Many properties make the amniotic membrane ideal for use during the stem cell therapy of the corneal epithelium, including it’s transparency, it’s ability to suppress inflammation, scarring and angiogenesis, and the fact that it is cheap, with an endless supply.
Describe step 2 in corneal transplantation: the isolation of the stem cells.
The digestion of the explant, followed by the suspension of the stem cells, is considered to be the best system for stem cell isolation. The suspension culture system is designed to remove all stem cells from the explant and seed them onto the amniotic membrane. Suspension culture is preferable to explant culture because stem cells would not migrate from the explant to the amniotic membrane - this is why digestion is required.
Describe step 3 in corneal transplantation: the ex vivo culture system.
Its hard to get adult stem cells to grow well ex vivo in culture. To circumvent this problem, one common approach is to cultivate them on a feeder layer made primarily of growth-arrested feeder cells. These provide essential growth factors for the stem cells. It works well, and was first pioneered by Rheinwald and Green in 1975.
We also use the patients own serum rather than bovine serum in the culture media.
Airlifting is used to promote tight junction formation, a structure which is essential for corneal integrity. The culture medium level is lowered until it just covers the cells – just like the tear film on the surface of your cornea. Without airlifting, tight junctions do not form and the engineered epithelium is vulnerable to infection.
Without tight junctions, the tear film will penetrate into the corneal stroma, causing stromal swelling and opacity.
Describe step 4 in corneal transplantation: a surgical method for transplantation.
Basically very simple; just involves scraping off the diseased conjunctiva cells and suturing on the amniotic membrane with the stem cells on top.
This gradually disappears over several months, leaving the stem cells on the surface.
What is the source of allogenic corneas, and what are the problems associated with this approach to the treatment of corneal disease?
Allogenic corneas are taken from donated cadavers, however, as they are not autogenic tissue, their use in transplantation surgery will need to be followed with the long-term administration of immunosuppresive drugs, which can present serious health risks.
Describe ‘stem cell plasticity’.
Traditionally, adult stem cells have been viewed as
committed to a particular cell fate to produce cells
from the tissue of origin, but not cells of non-related
tissues. For example, neural stem cells
(NSCs) give rise to the three main types of nerve
cells present in the adult brain, haematopoietic stem
cells (HSCs) produce blood, etc. Various reports
over the last six years challenge this central dogma
by demonstrating that adult stem cells, under
certain microenvironmental conditions, give rise to
cell types besides the cell type in the tissue of
origin possibly indicating that they can switch cell
fate. For example, HSCs, besides forming blood
cells, have been reported to give rise to liver cells,
and NSC may not only give rise to nerve cells but
also give rise to early haematopoietic precursors.
These observations have been termed “stem cell
plasticity”.
Why, in the absence of functional corneal stem cells, are oral mucosal stem cells used as an alternative?
Oral mucosal stem cells are a potential substitute for the corneal epithelium due to their reasonable degree of similarity in structure and function. Under the right culture conditions, oral mucosal stem cells can transdifferentiate into cells which function as corneal stem cells.
This is an autogenic method since the patient can supply their own cells – so there is no need for potentially harmful immunosuppressive medication.
What are the clinical results when oral mucosal stem cells are used as a substitute stem cell source during corneal transplantation?
Mucosal cells become more ‘corneal-like’ during ex vivo expansion on the amniotic membrane, with fewer cell layers, smaller cells, and a strong attachment formed to the membrane. The clinical results have proved to be excellent – and the technique is now gaining acceptance around the world as a highly effective treatment.
The only problem associated with the therapy is the slight vascularisation of the edge of the cornea – this is fairly easy to control.
Why have corneal transplants proved so successful?
- Cornea is immunologically privileged
- Corneal epithelial stem cells easy to isolate
- Amniotic membrane replicates the stem cell basal lamina almost perfectly
- Surgery very simple – on outside of body
- Easy to monitor 24/7
- Medication can be given topically
- Easy to replace if it fails first time
- No ethical problems and low risk – so no endless waiting for approval.
