**2.1 Conjunctiva**

*Regenerative Medicine*

cells (MSCs).

a 1-year follow-up.

genetic instability shown by these cells.

As for the anterior segment, the most used cells are limbal epithelial stem cells (LESCs), found at the level of the Vogt palisades. As regards the posterior segment, the stem cells used for the treatment of retinal degenerative diseases are embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and mesenchymal stem

corneal repair activity after severe damage of corneal surface.

LESCs: Also known as corneal epithelial stem cells, they are located in the basal epithelial layer of the corneal limbus. They form the border between the cornea and the sclera and are implied in the regular corneal renewal. They are also implied in

ESCs: The first ESCs were obtained from mouse embryos and immediately showed their ability to express neural markers and to migrate into the retina when applied intravitreally. Also, they seemed to be able to integrate into the retinal layers and act as neuroprotective factors. Clinical trials conducted in the human eyes have demonstrated that the subretinal application of these cells shows no signs of rejection, ectopic tissue development, negative proliferation, or tumor formation in

IPSCs: These cells are obtained from reprogramming adult somatic fibroblasts through retroviruses or lentiviruses. Compared with ESCs, they show less risk of rejection and less need for immunosuppressive therapy. However, further studies have suggested that IPSCs can stimulate oncogenes/suppress tumor suppressor genes, resulting in gene mutations and malignant transformation. The many molecular passages required for their production also seem to act as a trigger for the

MSCs: These cells are derived from many different tissues (peripheral blood, bone marrow, adipose tissue, cord blood, teeth, central nervous system, and liver). Once acquired, MSCs can be expanded in cell cultures maintaining their stemness. They can differentiate into various cells (mesodermal, ectodermal, and endodermal cells), including neuron-like cells. Since they are capable of secreting neurotrophic factors, repairing neural connections, and stimulating the formation of synapses, MSCs are also appreciated for their "structural" function. Moreover, they have shown a strong immunosuppressive action inhibiting the release of pro-inflammatory cytokines; therefore they allow both autologous and allogenic transplantation. Finally, their use does not seem to be related to tumor formation. For these reasons, researchers look at stem cells as a promising therapeutic option for degenerative retinal diseases. Nevertheless, it must be said that various ocular complications

related with the use of these cells have been described (see Section 3).

**2. Regenerative medicine in the anterior segment of the eye**

Stem cells are unspecialized cells that have been a focal point of the field of regenerative medicine, frequently considered as the future of medicine. The first medical science branch which directly benefits from stem cells for regenerative treatment was ophthalmology. The triumph of regenerative medicine in ophthalmology can be attributed to its accessibility, ease of follow-up, and the eye being an immune-privileged organ. Two key characteristic attributes of stem cells are pluripotency, the capacity to differentiate into multiple lineages, and proliferation. These cells have the ability to replace damaged or diseased cells under certain specific circumstances. Stem cell-based therapy has now reached a state where ocular tissues damaged by disease or injury can be repaired and/or regenerated. The eye is an ideal organ for studying regenerative medicine thanks to the ease of access for the therapeutic procedure as well as its status of being an immune-privileged organ. Such therapy involves various techniques in which stem cells are injected into both

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The conjunctiva, apart from being a protection against pathogenic entry, is a connective tissue provided by a high vascularization that offers channels for proper flow of nutrients and fluids. From the anatomical point of view, the conjunctiva is an unkeratinized stratified squamous epithelium, in which goblet cells are also present, that covers the exposed scleral surface (bulbar conjunctiva) and the interior part of the eyelids (tarsal conjunctiva). Conjunctival cells undertake renewal similar to the corneal epithelium, but with a still elusive source of stem cells. Conjunctival stem cells undergo a differentiation pathway that can take them to become either mucin-producing goblet cells or epithelial cells. The dividing basal cell migration starts from the bulbar conjunctiva and takes it to the corneal surface before differentiation. Conjunctival epithelial cells are negative for CK3 and CK12 but positive for CK19. As shown in clonal culture assays, the stem cells located in the fornical niche can differentiate into epithelial cells as well as goblet cells. This provides important evidence that the stem cell pool supporting conjunctival renewal is located in the fornix region. Commitment to differentiate into goblet cells occurs relatively late; in fact goblet cells are generated by stem cell-derived transient amplifying cells. The decision of a conjunctival keratinocyte to differentiate into a goblet cell appears to be dependent upon an intrinsic "cell doubling clock." Ocular processes that affect the cornea also affect the conjunctiva; some examples are conjunctival scarring, cicatricial pemphigoid, thickening, dry eye, or mucin. In order to treat conjunctival stem cell deficiency and scarring, conjunctival autografts, oral mucous membrane grafts, nasal turbinate mucosa grafts, and amniotic membrane are often used. Conjunctival cells cultured on amniotic membrane have been used for cell transplantation in patients with limbal stem cell deficiency (LSCD). Recent patient follow-up reports have shown that transplantation of autologous conjunctival epithelial cells improved the clinical parameters of total LSCD with respect to vision acuity, impression cytology, and in vivo confocal analysis. These cells were cultivated ex vivo on amniotic membrane with the presence of epidermal growth factor, insulin, cholera toxin, and hydrocortisone to produce the corneal lineage; the cells were transplanted after 2 weeks of culture. Ultrathin polymembrane substrate has also been shown to support conjunctival epithelial cell proliferation.

#### **2.2 Cornea**

The cornea is at the outermost surface of the eye, and its fundamental characteristic is transparency, which is crucial for vision. It is a clear lens that determines the

majority part of the dioptric power of the eye (about 43D). Its normal thickness is between 520 and 540 μm and is composed of five layers which are from the outside to the inside: corneal epithelium, Bowman's layer, corneal stroma, Descemet's membrane, and corneal endothelium. Forty-five million people worldwide are bilaterally blind, and another 135 million have a severe impaired vision defect in both eyes because of loss of corneal transparency. In order to correct this kind of problems, therapies ranging from local medications to corneal transplants, and more recently to stem cell therapy, could be applied. The corneal epithelium is a squamous epithelium that has a constant renewal activity, with a vertical turnover of 7–14 days. The corneal stem cell pool is located in the limbus, at the periphery of the cornea, and these cells are called limbal epithelial stem cells (LESCs). The corneal epithelium has a renewal process which is performed by cells generated at the limbus and, migrating from there, in opposition to other squamous epithelia in which each stem cell has the role of regenerating a limited area of epithelium. In the corneal epithelium, stem cells are located at the corneal periphery in the basal layer of the limbal region, called the palisades of Vogt. These are visualized in small clusters and are strictly associated with the stromal matrix and the basal membrane, thereby assisting in cell-cell, cell extracellular matrix, and paracrine signaling communication. The corneal epithelial basal layer is composed mostly of TAC at various stages of maturity, and this could be demonstrated by their elevated expression of a specific isoform of the transcription factor p63 along with a high nuclear to cytoplasmic ratio. The positivity of ATP-binding cassette subfamily G member 2 (ABCG2) has been detected in LESCs as well as in several other cells located in the suprabasal region of the limbus, and these markers could be used to identify the LESC pool. Some reports also indicate that an RNA binding protein called Musashi-1 can be used to stain LESCs. Corneal stem cells also express some other specific markers, enolase, cytokeratin (CK)19, and vimentin, but do not express CK3, CK12, or connexin 43, which are present only in mature corneal epithelial cells. Stromal multipotent stem cells have been identified and expanded to neurospheres in cultures. Corneal stromal stem cells are located in the anterior region of the stroma adjacent to the basal side of the palisades of Vogt and were identified as a side population using the DNA-binding dye Hoechst 33342. These cells expressed genes encoding ABCG2, Bmi1, CD166, c-kit, Pax6, Six2, and Notch1 similarly as mesenchymal stem cell and corneal early development markers. Corneal stromal stem cells, when differentiated, express keratocyte markers like keratocan, ALDH3A1, CXADR, PTDGS, and PDK4. LESC deficiency, either partial or complete, is pathological and is caused by either chemical or mechanical injury or thermal burns or acquired by diseases like aniridia and Stevens-Johnson syndrome. Treatment of such conditions involves LESCs transplantation therapy. In unilateral cases of ocular disease, LESCs from the healthy eye are expanded ex vivo for therapeutic purposes using specific protocols which involve amniotic membrane or fibrin in the presence or absence of growth-arrested 3T3 fibroblast feeder layers. Taking in consideration non-limbal cell types, cultured oral mucosal cells and conjunctival epithelial cells have been transplanted with success to treat LSCD in humans. The peripheral cornea has been proven to contain a higher density of keratocyte precursors with high proliferative capacity. Restoration of corneal transparency, stromal thickness, and collagen fibril defects have been demonstrated as solvable through the injection of corneal stromal stem cells in mice. If it will be shown as successful, such therapy would eliminate the shortage of corneas from donors needed for transplantations. Although stem cell transplantation is performed worldwide, standardized protocols need to be established because of variability in clinical outcomes. An application example of LESCs transplant could be in patients with LSCD who are suffering from a severe loss of vision and annoying irritation, being also poor candidates for conventional

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*Regenerative Medicine and Eye Diseases DOI: http://dx.doi.org/10.5772/intechopen.92749*

keratolimbal allografts.

**2.3 Trabecular meshwork**

corneal transplant. Hence, new surgical strategies have been devised by transplanting LESCs from an autologous or allogeneic source. When in total LSCD only one eye is involved, the reconstruction of the damaged corneal surface can be effectively performed by the application of conjunctival limbal autograft. Although conjunctival limbal autograft has high success rates, if transplantation is carried out at the acute stage of chemical burns when inflammation remains in "active" stage, the surgical outcome is not satisfactory; this notion has been verified in a rabbit model. The potential risk to the patient's donor eye could be reduced with the application of different techniques: the first alternative is to perform LESCs allograft, in which an allogeneic source of LESCs is derived from either living donors matched with HLA or not matching cadavers. Systemic immunosuppression with cyclosporin A or other agents is necessary because the donor tissue is allogeneic, but this solution is potentially toxic. The success rate of limbal allografts declines with time even with systemic application of cyclosporin A. Elements implicated as factors contributing to the poor prognosis for keratolimbal allografts are keratinization, severe dry eye, chronic inflammation, uncorrected lid, and lid margin abnormalities. A combined immunosuppressive regimen together with a meticulous restoration of the ocular surface defense has been shown to further improve the long-term visual outcome of

The trabecular meshwork is a tissue included between the cornea and the iris in the anterior region that has the role of draining the aqueous fluid. It is divided into three parts which have their characteristic ultrastructures: inner uveal meshwork, corneoscleral meshwork, and juxtacanalicular tissue. Intraocular pressure is determined by the correct balance between aqueous production and outflow; a malfunction in this mechanism is a possible risk factor for the development of glaucoma. Trabecular meshwork cells also are implied in the removal of debris in the circulating aqueous humor. Trabecular meshwork cellular markers are vimentin, non-muscle actin, aquaporin-1, acetylated and acetoacetylated alpha-2 adrenergic receptor, matrix GLA protein, and chitinase-3-like-1. Recently, the isolation, characterization, and specific markers of trabecular meshwork cells have been widely studied. These studies suggest that trabecular meshwork cellular population has properties similar to stem cells, expressing mesenchymal cell-associated markers such as CD73, CD90, and CD105, and they have also the ability to differentiate into adipocytes, osteocytes, and chondrocytes. Moreover, further studies showed that trabecular meshwork cells with mesenchymal phenotype are isolated as a side population or as clones expressing specific stem cell markers, not present in mature cells, such as Notch1, OCT-3/OCT-4, ABCG2, AnkG, and MUC1. These stem cells have the ability to differentiate into the trabecular meshwork lineage expressing CHI3L1, AQP1, and TIMP3 markers that underlies to a phagocytic function. Lowering the intraocular pressure is the aim of treatments for glaucoma. The idea for this came primarily from the observation that trabecular meshwork cell division increased after argon laser trabeculoplasty. Current first-line treatments are topical and oral drugs, argon laser trabeculoplasty, and some surgical approaches. Stem cells isolated from human trabecular meshwork and expanded in vitro showed evidence of the ability to home to mouse trabecular meshwork and differentiate into trabecular meshwork cells in vivo according to recent studies. The expanded trabecular meshwork stem cells expressed the stem cell markers Notch1, ABCG2, and MUC1 and were expressing also the trabecular meshwork marker protein CHI3L1. These trabecular meshwork cells were multipotent and had phagocytic properties. Some groups are working on transplanting trabecular

#### *Regenerative Medicine and Eye Diseases DOI: http://dx.doi.org/10.5772/intechopen.92749*

*Regenerative Medicine*

majority part of the dioptric power of the eye (about 43D). Its normal thickness is between 520 and 540 μm and is composed of five layers which are from the outside to the inside: corneal epithelium, Bowman's layer, corneal stroma, Descemet's membrane, and corneal endothelium. Forty-five million people worldwide are bilaterally blind, and another 135 million have a severe impaired vision defect in both eyes because of loss of corneal transparency. In order to correct this kind of problems, therapies ranging from local medications to corneal transplants, and more recently to stem cell therapy, could be applied. The corneal epithelium is a squamous epithelium that has a constant renewal activity, with a vertical turnover of 7–14 days. The corneal stem cell pool is located in the limbus, at the periphery of the cornea, and these cells are called limbal epithelial stem cells (LESCs). The corneal epithelium has a renewal process which is performed by cells generated at the limbus and, migrating from there, in opposition to other squamous epithelia in which each stem cell has the role of regenerating a limited area of epithelium. In the corneal epithelium, stem cells are located at the corneal periphery in the basal layer of the limbal region, called the palisades of Vogt. These are visualized in small clusters and are strictly associated with the stromal matrix and the basal membrane, thereby assisting in cell-cell, cell extracellular matrix, and paracrine signaling communication. The corneal epithelial basal layer is composed mostly of TAC at various stages of maturity, and this could be demonstrated by their elevated expression of a specific isoform of the transcription factor p63 along with a high nuclear to cytoplasmic ratio. The positivity of ATP-binding cassette subfamily G member 2 (ABCG2) has been detected in LESCs as well as in several other cells located in the suprabasal region of the limbus, and these markers could be used to identify the LESC pool. Some reports also indicate that an RNA binding protein called Musashi-1 can be used to stain LESCs. Corneal stem cells also express some other specific markers, enolase, cytokeratin (CK)19, and vimentin, but do not express CK3, CK12, or connexin 43, which are present only in mature corneal epithelial cells. Stromal multipotent stem cells have been identified and expanded to neurospheres in cultures. Corneal stromal stem cells are located in the anterior region of the stroma adjacent to the basal side of the palisades of Vogt and were identified as a side population using the DNA-binding dye Hoechst 33342. These cells expressed genes encoding ABCG2, Bmi1, CD166, c-kit, Pax6, Six2, and Notch1 similarly as mesenchymal stem cell and corneal early development markers. Corneal stromal stem cells, when differentiated, express keratocyte markers like keratocan, ALDH3A1, CXADR, PTDGS, and PDK4. LESC deficiency, either partial or complete, is pathological and is caused by either chemical or mechanical injury or thermal burns or acquired by diseases like aniridia and Stevens-Johnson syndrome. Treatment of such conditions involves LESCs transplantation therapy. In unilateral cases of ocular disease, LESCs from the healthy eye are expanded ex vivo for therapeutic purposes using specific protocols which involve amniotic membrane or fibrin in the presence or absence of growth-arrested 3T3 fibroblast feeder layers. Taking in consideration non-limbal cell types, cultured oral mucosal cells and conjunctival epithelial cells have been transplanted with success to treat LSCD in humans. The peripheral cornea has been proven to contain a higher density of keratocyte precursors with high proliferative capacity. Restoration of corneal transparency, stromal thickness, and collagen fibril defects have been demonstrated as solvable through the injection of corneal stromal stem cells in mice. If it will be shown as successful, such therapy would eliminate the shortage of corneas from donors needed for transplantations. Although stem cell transplantation is performed worldwide, standardized protocols need to be established because of variability in clinical outcomes. An application example of LESCs transplant could be in patients with LSCD who are suffering from a severe loss of vision and annoying irritation, being also poor candidates for conventional

**96**

corneal transplant. Hence, new surgical strategies have been devised by transplanting LESCs from an autologous or allogeneic source. When in total LSCD only one eye is involved, the reconstruction of the damaged corneal surface can be effectively performed by the application of conjunctival limbal autograft. Although conjunctival limbal autograft has high success rates, if transplantation is carried out at the acute stage of chemical burns when inflammation remains in "active" stage, the surgical outcome is not satisfactory; this notion has been verified in a rabbit model. The potential risk to the patient's donor eye could be reduced with the application of different techniques: the first alternative is to perform LESCs allograft, in which an allogeneic source of LESCs is derived from either living donors matched with HLA or not matching cadavers. Systemic immunosuppression with cyclosporin A or other agents is necessary because the donor tissue is allogeneic, but this solution is potentially toxic. The success rate of limbal allografts declines with time even with systemic application of cyclosporin A. Elements implicated as factors contributing to the poor prognosis for keratolimbal allografts are keratinization, severe dry eye, chronic inflammation, uncorrected lid, and lid margin abnormalities. A combined immunosuppressive regimen together with a meticulous restoration of the ocular surface defense has been shown to further improve the long-term visual outcome of keratolimbal allografts.
