In 1963 Canadian scientists Ernest A. McCulloch and James E. Till discovered and described a new cell type with unique self-renewing activities after bone marrow transplantation in mice; those cells were called stem cells. It is now known that stem cells are highly plastic cells with two essential characteristics: the ability to self-replicate and, to differentiate into all the other cell types. They can be found in almost all multi-cellular organisms. There are two major divisions of stem cells: embryonic SC and adult SC. Embryonic SC are present in the initial stages of embryonic development and have the ability to differentiate into all the tissues of a body; adult SC are found in a developed organism and are responsible for tissue repair and cell renewal. Depending on the ability to differentiate into specific cell types, stem cells can also be divided into several subgroups: omnipotent (totipotent), pluripotent, multipotent, oligopotent and unipotent. Omnipotent SCs are able to differentiate into any of the current known cell types. They present on the stage of the fertilized egg and remain during the first few divisions, after which they develop into pluripotent cells. Pluripotent SC can differentiate into a great number of cell types from all the three germ layers. The next category is multipotent SC, which can transfer into several cell types from closely related families of cells. Oligopotent SC can only transform into very limited number of cell types, for instance, lymphoid or myeloid stem cells. Finally, unipotent SC, such as muscle stem cells, can differentiate only into their own type of tissue, but continue to have the ability to replicate themselves. Some authors also distinguish quadripotential, tripotential and bipotential SC.
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Stem cells are able to divide in two different ways: symmetrical and asymmetrical. Symmetrical cell division leads to the formation of two stem cells and is common for the earlier stages of embryonic development. Asymmetrical division produces one stem cell and one specialized cell, or two different specialized cells (e.g. eosinophyl and erythrocyte from myeloid progenitor cells); this type of cell division is usually a property of "less potent" SC, such as hematopoietic stem cells.
Current modern medicine makes it possible to grow cultures of stem cells "in-vitro". The first work was done in 1980s using embryonic stem cells (ESC) in mice and other animals. Although it is possible to obtain human ESC under artificial conditions, there are still serious ethical concerns which prevent them from being properly studied and widely used. It was also shown that transplantation of ES cells in mice can cause tumors (teratomas), therefore careful and more long-term investigations are needed to insure safe use. Nevertheless, adult stem cells derived from the bone marrow have been used since 1980s for transplantation to cure immunodeficiency in humans and, more recently, autologous adult mesenchymal stem cells have been successfully used for cartilage regeneration in the human knee. Moreover, the research of potential embryonic stem cells applications is continuing at a rapid pace and many possibilities have already been considered, such as tissue and organ growth. To date, the treatments approved for use in humans, based on transplantation of adult stem cells, are the treatments for heart diseases, diabetes, corneal disorders, Parkinson's disease and other pathologies.
Despite of the abundance of stem cells in live organisms, this paper is more concerned with the current knowledge of stem cells in the human eye and their possible applications in practical ophthalmology. To date, the most studied stem cells in the eye are called limbal epithelial stem cells (LESCs), which are responsible for corneal epithelium healing and renewal; however there are other types of SC found in the eye, such as retinal SC and SC in the trabecular meshwork.
Limbal epithelial stem cells are adult SC with all the characteristic properties of the latter. LESCs are basically responsible for the maintenance of the corneal epithelium in a healthy state, epithelium regeneration in response to physiological wear and corneal wound healing processes. Discovered by Davanger and Evensen in 1971, LESC is considered to be the major source for corneal epithelium regeneration1, which explains the importance of limbal stem cell-related problems. The corneal epithelium itself is the outer layer of the cornea and usually consists of 5-7 layers of cells and comprises 10% of the total corneal thickness. The average time of complete epithelial renewal in healthy individuals after surgical or chemical removal is approximately 3-5 days. It was also noted that when removed, the corneal epithelium regenerates in a particular order: first of all cells move along the corneo-scleral limbus and only after the complete closure of the limbal area centripetal movement of the cells was observed, followed by the central corneal area healing. LESCs can be found in the limbal area, where the corneal epithelium connects to the conjunctiva, along the dashed line (Pic 1). In humans the distribution of LESCs is not even; the cells are more abundantly presented in the Picture 1 (Taken from the reference 1)
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inferior and superior regions compared to the temporal and nasal. This observation can be used by surgeons in order to reduce trauma to stem cells during eye operations, and thus reduce the level of postoperative LESCs deficiencies. In the limbus LESCs are located in a specific niche, which helps to maintain their undifferentiated state, and create palisades of Vogt, which protects them from shed forces. Palisades of Vogt are found in the sub-epithelial connective tissue and have a papilla-like appearance. Although the limbal area is believed to be the main source of stem cells in the eye, in animal models it has been found that transplantation of the central corneal epithelium has a similar effect to the transplantation of limbal grafts; however, further study is required to confirm this hypothesis in adult humans5.
Limbal epithelial stem cells have a number of distinguishable characteristics: slow cycling, high proliferative capacity, specific morphology, plasticity and asymmetric cell division. Phenotipically, limbal stem cells are cuboid in shape and smaller in comparison to the basal cells of the corneal epithelium, therefore it is possible to distinguish between different cell types. Another noticeable property of a stem cell is a high nucleus to cytoplasm ratio. Moreover, LESCs contain melanin for better ultraviolet protection. To date, there is no evidence that limbal stem cells are pluripotent, but it has been shown that LESCs can differentiate into neuronal cells. This property may be used to treat neurodegenerative diseases.
The ability of embryonic stem cells to divide unlimited number of times can be explained with the expression of telomerase in those cells. Telomerase is an enzyme which is capable of replacing of telomeric DNA lost when a cell divides, therefore preventing the cell from approaching the "Hayflick-limit". Hayflick-limit is a critical length of a DNA at which a cell can not divide any longer1. The same mechanism of achieving the unlimited capacity for self renewal is observed in cancer cells. It has been found that adult stem cells have lower proliferative capacity in comparison to embryonic stem cells because the level of telomerase expression does not appear to be enough for the prevention of the telomere loss. Possibly, this phenomena was developed during the evolution to prevent cancer, however it also accelerates aging processes1.
There were many attempts to find limbal epithelial stem cells markers which would be specific only for the stem cells in the eye, but currently there are no single identification marker which allow definitive recognition of a LESC. Nevertheless, several markers have been successfully used in combination with the absence of differentiation markers to identify LESCs5. Furthermore, for better reliability specific LESC morphology can be added to the combination. Among the most common stem cell markers are the ATP binding cassette protein (ABCG2), p63 (a transcription factor), notch 1 (transmembrane receptor), C/EBPd (CCAAT enhancer binding protein delta), vimentin and integin a9. Markers used for the identification of differentiation are involucrin, connexin 43, K12 and cytokeratin K3 1, 5.
Another important issue is the regulation of limbal stem cell differentiation. Interactions between stem cells and their microenvironment are believed to provide the control over LESC proliferation. The control over LESCs survival, proliferation and differentiation implies different factors, which can be classified as short and long range, and external and internal. For example, external factors include cell adhesion molecules, extracellular matrix, secreted factors and cell-to-cell interactions3. Cytokines are believed to play a significant role in the modulation of corneal wound healing. Recent studies have shown that there is a difference in the cell proliferation rate in the cornea, which has been described as the highest in the periphery and relatively low in the central and limbal areas of the cornea. Although there are some known facts about the regulations of stem cells functions, the elucidation of the exact mechanisms are incompletely and studies are still in progress.
As adult stem cells represent the only source for the epithelial regeneration and wound healing, the disorders of those cells lead to serious consequences for the vision, including blindness. The most common reasons for limbal epithelial stem cell deficiencies are alkali and acid burns, lens-induced keratopathy, Stevens Jonson syndrome, aniridia and ocular surgery. The first three are acquired and the last is hereditary. Less common diseases are immune-mediated mucous membrane pemphigoid, chronic limbitis. Usually limbal epithelial stem cell deficiencies manifest as corneal opacity, neovascularization, susceptibility to infections, and an unstable ocular surface which tends to breakdown. Consequently, patients often experience intense pain, severe photophobia and significant visual acuity reduction, which, without treatment, eventually leads to compete blindness. The diagnosis of stem cell's deficiencies is difficult because of the absence of a reliable LESC marker, so it remains symptomatic.
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Although standard corneal transplantation has not proved to be efficient in patients with LESC deficiencies, there are several possible treatments with the application of LESCs directly, which are successfully applied in such patients. In the case of one-sided lesion it is possible to transplant an auto graft harvested from the fellow healthy eye; if both eyes are affected, then it is possible to transplant an allograft from a cadaveric donor or from a living genetically relative person. However, there are a number of possible complications for both techniques: in the case of an autograft, the fellow eye can develop stem cell deficiency, especially if the graft has to be big; in the case of an allograft, the problem of tissue rejection is very common and also it is difficult to find a donor. To date, prolonged immunosuppressive therapy is required to reduce graft rejection level. Moreover, currently there is a lack of evidence of long term donor cell survival, which requires additional studies of the efficacy and safety of the method5.
In order to avoid some serious complications and to improve the results of graft transplantation, a series of modifications were developed. For instance, to reduce the risk of the development of LESC deficiency in the fellow eye in cases of unilateral lesion, it has been suggested by Pellegrini in 1997 to grow sheets of epithelium cells in artificial environment from a biopsy of a healthy autologus tissue not exceeding 2 mm2, prior to the transplantation. This technique has proved to be effective in patients after chemical burns, but still requires immunosuppressive therapy in cases of bilateral defects.
Another promising method implies the use of human amniotic membrane. Human amniotic membrane (HAM) comprises the innermost layer of the placental membranes. Usually it is 20-500 micrometers thick, has no blood or lymphatic vessels and no nerves. The membrane consists of a single layer of epithelial cells and a basement membrane. The main function of HAM is to protect the embryo during gestation. It also has anti-scarring, anti-angiogenic and anti-inflammatory properties. It was found that the reduction of scarring is explained by the suppression of TGF-b and myofibroblast differentiation. Moreover, HAM is capable of the production of growth factors, which can increase corneal epithelialisation5. Due to its unique properties, amniotic membrane is extensively used in ophthalmic surgery for the reconstructive operations on the cornea and conjunctiva. It was suggested by Tseng et all in 1998 to culture limbal tissue on the HAM for 2-3 weeks before the graft transplantation, which lead to a noticeable increase in corneal clarity and stability. In addition, Nakamura et all developed a fibrin coating which can be spread over the HAM to obviate the requirement for sutures. However, amniotic membrane is not absolutely transparent; therefore, the corneal clarity can only be improved to a certain degree. Moreover, it is difficult to maintain a consistent supply of membranes and, despite expensive screening tests, there is still a risk of the transmittion of viral infections from a HAM donor5.
All the described preclusions inspire scientists to look for alternative substrates, which would be beneficial for corneal epithelial transplantation. An alternative to an amniotic membrane could be artificial synthetic materials such as contact lenses or natural biopolymers, such as a fibrin substrate. The most common are collagen-based substrates due to their good biocompatibility, low levels of immune responses and relatively cheap price. One of its major drawbacks is its high water content, which leads to extreme weakness. This disadvantage can be partly avoided by a cross-linking of a collagen, but still does not solve the problem completely8. A group of Japanese scientists has suggested the use of a temperature responsive polymer as an alternative to plastic, in order to eliminate the requirement for enzymatic detachment of grown cells from the substrate. The mechanism of work of such a polymer is as follows: the surface of the substrate is hydrophobic under the normal conditions of 37*C and attaches itself to the cells; however, at temperatures below 32*C, the polymer becomes hydrophilic and detaches the cell culture by the formation of a hydration layer between the cells and the substrate.
An alternative source of stem cells obtained from the oral mucous membrane epithelium for corneal epithelium reconstruction has been tested on rabbits by Nakamura in 2003. This approach has the potential to eliminate the requirement of immunosuppressive therapy in patients with bilateral lesions; nonetheless, long-term investigation is indispensable for safe use of the technique in ophthalmic surgery3. It has also been found that mesenchymal stem cells, which can be obtained from adult's peripheral blood and skeletal muscle or foetal blood and liver, are capable of treating alkali burns after systemic administrations on rabbit corneas. This source of stem cells may also prove to be suitable for corneal re-epithelialisation in humans.
An interesting approach was introduced by a group of Japanese researchers in 2007, when they used Pax6 gene to induce mouse embryonic stem cells differentiation into epithelium like cells. The gist of the approach was to deliver Pax6 complementary DNA vector with a specific tag (green fluorescence protein) inside an embryonic stem cell by the process called electroporation (when an electrical field is used to increase dramatically cell membrane permeability by making membrane pores wide enough for a non-traumatic injection of large molecules, such as DNAs). After 14 days a colony of Pax6 transfected cells were observed to differentiate into neuronal cells, which were eliminated, and into corneal epithelium-like cells, which were used for the transplantation on chemically injured corneas. Epithelium-like cells were assessed by the presence of characteristic cells markers for the corneal epithelium, such as cytokeratin 12, E-cadherin, CD44, and integrin a4 and a close resemblance was established. Moreover, morphologically Pax6 transfected cells were very similar to native corneal epithelial cells. For the comparison, a different group of embryonic stem cells were cultured and transfected with an "empty vector" (without Pax6); this group appeared to be inapplicable for transplantation due to the inability to attach to the injured corneas. However, even after successful Pax6 transfection, substantial detachment of the grafted cells at 24 hours after transplantation was reported, probably due to mechanical forces (lid movements). Nevertheless, the idea of Pax6 transfection of embryonic stem cells proved to be feasible and has chances of becoming an unlimited source of corneal epithelial cells for the transplantation in humans6.
Another application for stem cells in eye surgery has been established after the discovery of retinal progenitor cells in the human ciliary marginal zone in 20003. In mammals the retinal pigment epithelium and the neural retina are developed in the early postnatal period and seem to lose the ability to regenerate in adulthood, in contrast to fish and amphibians. Postnatal growth of the eye results from strain of the retina rather than from new retinal cell's derivation. The discovery of retinal progenitor cells in humans may create an opportunity to use those cells for the treatment of retinal degenerative diseases, such as AMD (age related macular degeneration) and retinitis pigmentosa. Retinal progenitor cells introduce about 0,2% of pigmented cells in the ciliary margin. They have a significant resemblance to common stem cells, which is represented by the ability to proliferate, expression of nestin (neuroectodermal marker), the ability to self-renewal and multipotency3. The ability to form specific retinal cells, including intermediate neurons, photoreceptors and Muller cells has been shown "in vitro", however this does not usually happen "in vivo", therefore it is hypothesized that the mammal retinal progenitor cells reside in inhibitory conditions3. In theory, the most feasible approach to stem cells for the treatment of the retinal disorders is to regenerate retinal photoreceptors, because in order to implement their function they only need one connection to bipolar and horizontal cells; whereas, for successful restoration of inner retinal layers, such as amacrine cells and ganglion cells, it is necessary to create both functional "inputs" and "outputs"3.
A series of investigative trials has been undertaken by different groups of scientists throughout the world in order to study possible applications of stem cells for the treatment of retinal degenerative diseases. A research group in Taiwan has shown on a mouse model that RPE-like cells can be derived from specially designed mouse embryonic stem cells on Pax6 feeders, and improve visual function after the transplantation on the degenerated retina. The design of the study implies the induction of C2J mouse embryonic stem cells, labeled by yellow fluorescent protein, into retinal pigment epithelium (RPE) structures. The assessment of the obtained cells after 11 days of differentiation was managed by immunoblotting (western blot) and immunocytochemical techniques to identify RPE cell makers, including RPE65, bestrophin and ZO-19. It appeared that RPE-like cells expressed RPE65 and ZO-1, while the concentration of bestorphin was still low. After 7 days of culturing "in vitro", the obtained colonies of RPE like cells were transplanted into the subretinal space of P5 rd12 5 days old mice. Yellow fluorescent protein was used for the assessment of RPE-like cells survival in the retina after the injection. Out of 123 mice which received subretinal injections, in 76 mice retinal detachment and tumours were observed and documented during the 3-rd week, so only the remaining 47 were analysed by ERG (electroretinography). In only 12 out of 47 increased ERG response was observed, which comprises 25.5% of the functionally analysed mice and 9,7% of all the mice used for the experiment. Three control groups of mice, which received PBS (phosphate-buffered saline), mitomycin-C treated PA6 feeder cells, and mitomycin-C treated undifferentiated ES cells, were used to reduce the probability that the functional effect was caused by surgical trauma or other factors. Mitomycin-C was used because untreated non-differentiated embryonic stem cells were found to cause tumours. In the control groups there were no functional improvements, according to ERG.
This current paper (9) gives the first evidence that RPE-like cells can be used for the restoration of retinal functions, at the moment only applicable in the mouse model of retinitis pigmentosa. Moreover, the labelling of the cultured cells allows tracking of the fate of transplanted cells for a prolonged time (up to 7 months) in living organisms by non-invasive imaging. It was also shown that the Pax 6 stromal cell line can induce the expression of RPE-specific markers, such as RPE65, ZO-1 and bestrophin by the differentiated mouse embryonic stem cells. Nevertheless, there are serious complications of such a therapy, which include tumour formation, probably due to the pluripotency of the remaining after differentiation stem cells. In addition, functional improvement, detected by ERG, can manifest due to the changes only in a small area of the retina, where the injection was made, because ERG technique registers the summation of the retinal activity from the whole eye9. Therefore there may be direct correlation between the size of the injection and the functional improvement. Overall, the method described above seems to be both feasible and promising for the treatment of retinal degenerative diseases.
In the study described above embryonic cells were used to derive RPE-like cells; however, a number of other possible sources has been described in recent literature. For instance, neural stem cells, obtained from the adult mammalian brain tissue, including humans, have been used for the transplantation into the adult retina (in animal models). Neural stem cells were able to assimilate into the recipient's retina, but failed to fully transfer into retinal cell types. Retinal stem cells, which present in the early stages of mammalian development, have been shown to be capable of differentiation into different retinal cell types in animal models, but still would be difficult to transplant into humans because donor cells must come from a second trimester foetus. Foetal retinal cells could be another promising source of functional human retinal cells. These types of cells can be harvested from the foetal retina, before their intrinsic connections are not formed yet and the cells are still immature. In a number of studies foetal retinal cells were described to be viable and did not cause any major complications; however it is not clear whether the positive effect was observed because of the injected cells, or whether it was due to the trophic factors and the manipulation itself10.
Another challenge for the successful treatment using stem cells is the method of transplantation of the latter. Currently there are two techniques being commonly used: intravitreal and subretinal. Intravitreal injection is easier to perform and causes fewer complications, but requires cell migration to the outer retina, which can be an obstacle to the efficiency of the therapy. Subretinal delivery of stem cells is a significantly more demanding method, but delivers the cells immediately to the place of their application and limits their further migration to the space between the sensory retina and the retinal pigment epithelium10. To date, there is no strong evidence to prove one of the methods being significantly better than the other, and both are in use.
Although most attention in this paper has been paid to limbal epithelial stem cells and retinal stem cells, other ocular structures were also found to contain adult stem cells. A successful technique for the generation of lens progenitor cells from human embryonic stem cells was described in 2010, which can be beneficial for studies of lens differentiation and cataractogenesis. Stem cells were suggested for the treatment of glaucoma, as they were found in trabecular meshwork. It was noticed that after the laser treatment (trabeculoplasty) trabecular meshwork cell division increased dramatically (4 times as much as in a control group). Therefore, induction of those cells to differentiate into trabecular meshwork cells may be used for glaucoma treatment, and, possibly, substitute the requirement of laser surgery and drug use.
In conclusion, it should be mentioned that, although stem cells have great potential in ophthalmologic practice, there are still many problematic dilemmas and unanswered questions about their application in humans. Amongst such dilemmas is the problem of immune graft rejection in cases when non-autologous tissue is used, despite the immunopriviledged status of the eye. Another serious issue is tumour formation, which must be overcome prior to the use in humans. It is still difficult to determine with confidence which type of stem cells is the best for transplantation, which method of delivery is optimal, what is the most appropriate stage of stem cells differentiation for successful integration into the host retina and how to provide the longest graft survival after the implantation of the cell culture10. The mechanism of tissue repair by stem cells is not clear enough1. The identification of a specific stem cell marker would greatly facilitate future studies and help to elucidate mechanisms of stem cells functioning and differentiation. Long-term post-operative observation is required to assess safety and delayed efficacy of stem cell therapies3. Nevertheless, the investigations of possible ways to overcome all the difficulties mentioned above are in progress and there are many positive results documented. Therefore, maintaining the same rapid pace of development, in 10 years time stem therapy may become as common and effective as phacoemulsification of a cataract or laser refractive surgery.