Induced Pluripotent Stem Cells Form Limbal-Like Stem Cells

Limbal epithelial stem cells or LESCs are found at the periphery of the cornea and they continuously renew the corneal epithelium. Loss of this stem cell population can cause loss of corneal transparency and eventual loss of vision.

Genetic conditions can cause LESC deficiency, such as congenital aniridia, Stevens-Johnson syndrome or Ocular cicatricial pemphigoid. Other causes of LESC deficiency include chemical or thermal burns to the eye, microbial infections, extended contact lens wear, sulfur mustard gas poisoning, or chronic inflammation of the eye,

Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman's layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.
Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman’s layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.

Treatments of LESC deficiency include limbal stem cell grafts from one eye to another, but these grafts have a 3-5-year graft survival of only 30%-45%. If LESCs are expanded in culture on human amniotic membrane, then 76% of the grafts will successfully take 1-3 years after grafting. This procedure is not standardized. If LESCs are grafted from a cadaver, their survival is low.

Given these less than optimal treatments for LESC deficiencies, Alexander Ljubimov and his team from UCLA have used induced pluripotent stem cells (iPSCs) to make cultured LESCs. Ljubimov and his coworkers derived iPSCs from the skin cells of volunteers with non-integrating plasmids. Then they grew these cells on corneas that have been stripped of their cells and human amniotic membranes and these cells differentiated into LESC-like cells.

Ljubimov and others also made iPSCs from human LESCs, and when they cultured these iPSCs derived from LESCs on human amniotic membranes for two weeks, the cells differentiated into LESCs that made LESC-specific genes, and had the epigenetic characteristics of LESCs.

These experiments show that the cell source for iPSC derivation can greatly influence the epigenetic characteristics of the iPSC line. Also these experiments show that iPSCs can be used to make LESCs that can potentially be used for therapeutic purposes.

A New Technique to Fix Damaged Eyes With Stem Cells

Engineers at the University of Sheffield have invented a new delivery technique for delivering stem cells to eyes. They have high hopes that this technique will help repair the eyes of those patients who have suffered damage to their eyes.

The front of the eye is bordered by the transparent cornea, which transmits light to the lens. The cornea is exposed to the outside world and if there is an accident that affects the eye, the cornea is usually the part that takes a beating. The cornea undergoes constant turnover as dead cells are constantly sloughed from the cornea during blinking. At the junction between the cornea and the sclera is an area called the limbus. Located at the limbus is a population of limbal epithelial stem cells or LESCs. LESCs have many features commonly observed in other stem cells, such as small size, high nuclear to cytoplasmic ratio, and they lack expression of molecules commonly found in mature corneal cells, such as cytokeratins 3 and 12.

Human Limbus

LESCs are slow-growing, but in the event of injury they can become highly proliferative (See Lavker R.M, Sun T.T. Epithelial stem cells: the eye provides a vision. Eye. 2003;17:937–942. DOI: 10.1038/sj.eye.6700575).

LESC deficiency can result from chemical or thermal burns to the eye or as a result of certain inherited diseases. Partial or full LESC deficiency causes abnormal corneal wound healing and surface integrity. Also LESC deficiency causes the conjunctiva to grow over the cornea, and this is disastrous for the eye because the cornea is devoid of blood vessels, which is the reason why it is transparent. However the conjunctiva (the white of the eye) is filled with blood vessels and is not transparent. Thus chronic inflammation, recurrent erosion, ulceration and stromal scarring can occur and cause painful vision loss

Long term restoration of visual function requires renewal of the corneal epithelium, and this requires the placement of a new stem cell population by means of a limbus graft. From where do you get a new limbus for transplantation? Autografts use limbal cells from the good eye, but this runs the risk of scarring the cornea of the other eye.procedure is the use limbal cells from cadavers (limbal allografts). Also, making sure that the graft adheres to the requires the use of sutures, but these sutures can cause substantial amounts of irritation. Therefore, the Sheffield research group designed a new technique.

With this new technique, a disk made of biodegradable material is loaded with limbal stem cells and then placed over the eye. This disc has an outer ring pockmarked with small niches for stem cells can hide. The material in the center of the disc is thinner than that on the edges, and therefore, the center of the disc biodegrades faster. This releases the stem cells in center of the disc into the cornea where they can grow and help repair it.

Because these small niches in the disc resemble the stem cells niches found in the limbus, these discs do an excellent job of nurturing the limbal stem cells and distributing them to the cornea. Limbal grafts are either done with amniotic membrane as a carrier, but this procedure leads to increased inflammation in the eye and there is a chance that the grafts will not integrate into the limbus. The biodegradable disc groups the limbal stem cells into clusters that are more likely to ingrate into the limbus.

According to Professor Sheila MacNeil, “Laboratory tests have shown that the membranes will support cell growth, so the next stage is to trial this in patients in India, working with our colleagues in the LV Prasad Eye Institute in Hyderabad. One advantage of our design is that we have made the disc from materials already in use as biodegradable sutures in the eye so we know they won’t cause a problem in the body. This means that, subject to the necessary safety studies and approval from Indian Regulatory Authorities, we should be able to move to early stage clinical trials fairly quickly.”

In the developing world, corneal blindness is rather common in some professions and treating it is a rather pressing problem. High instances of chemical burns to the eye or accidental damage to the eye are common, but complex treatment strategies such as amniotic membrane grafts are not available to the general public.

This technique also possibilities in more developed countries, since current techniques use donor tissue to deliver the cultured cells, and this requires a tissue bank to which some people do not have access. Also, the use of the cell-impregnated disk will reduce the risk of disease transmission with grafts.