**3. Regenerative medicine in the posterior segment of the eye**

Considering the posterior segment, the main interest is focused on the retina, the target of regenerative medicine. Retinal anatomy is quite complex, and focusing to the microscopic structure, it can be divided into nine layers of nervous tissue that interfaces with the outermost layer of the pigmented epithelium. From external to internal, there are inner segment/outer segment layer, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.

Stem cells are immature, undifferentiated, highly proliferative cells which are capable of self-renewing and differentiating into many cell types [1]. Therefore, stem cells represent a potentially endless source of tissue renewal; that is why, in the modern era, stem cell therapy has been considered a valid approach for many different pathologies. Ophthalmologists and researchers were not slow to guess the potential applications of stem cell therapy in various degenerative retinal diseases such as retinitis pigmentosa, age-related macular degeneration, Stargardt's macular dystrophy, and other pathological conditions affecting the posterior pole of the eye, including retinal vascular occlusions [1, 2]. These pathologies are responsible for a progressive decline in visual acuity which, in the case of RP,

**99**

one of the patients.

within 4 weeks.

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

Stargardt's disease, and AMD, are due to a constant and irreversible loss of retinal photoreceptors and outer nuclear layers. With such premises, it is easy to imagine a therapeutic approach based on the use of stem cells to restore the lost retinal tissue. Stem cells have shown to be able to perform additional functions, such as nutritional support, apoptosis inhibition, synapse formation, immunoregulation, and neurotrophin secretion [1] and have increased even more the enthusiasm for their application in the ophthalmological field. Furthermore, the use of stem cells in the eye seems to offer numerous advantages: firstly, the amount of stem cells required is relatively low, which implies lower costs than those required for the treatment of other tissues of the human body; secondly, the surgical approach is quite easy and the transplanted cells can be easily monitored with the imaging methods currently used in clinical practice. Finally, the immune privilege of the eye allows avoiding long-term immunosuppressive treatment [1]. Several experimental studies conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), or mesenchymal stem cells (MSCs) have demonstrated that they tend to adapt to the retinal environment and can differentiate not only into photoreceptors and RPE cells but also into Müller, amacrine, bipolar, horizontal, and glial cells [1]. Retinitis pigmentosa represented one of the first targets of stem cell therapy in the ophthalmological area: pioneering animal studies have shown that pluripotent stem cells, when placed in murine retinitis pigmentosa models, are able to survive, multiplicate, differentiate, organize into, and function as photoreceptor cells, developing a retina-like organizational structure [3]; using mouse models, Singh et al. have established that at a stage when all host rod cells are lost, transplanted rod precursors can lead to the re-establishment of a proper, correctly polarized outer nuclear layer, indicating that stem cells may recreate a light-sensitive cell layer de novo and restore structurally damaged visual circuits. It has to be said that current methods for photoreceptor derivation from human pluripotent stem cells require long periods of culture and are often inefficient. Boucherie et al. [4] reported that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix can induce a rapid conversion into retinal progenitors, which are capable of subsequently differentiating into photoreceptor precursors in only 10 days and later acquire rod cell identity

Following such promising results, the first phase I/II clinical trials in humans were approved in the United States in 2010; hESC-derived retinal pigment epithelium cells were transplanted into the eyes of patients with Stargardt's macular dystrophy and dry age-related macular degeneration [5]. During differentiation, the stem cells "displayed typical RPE behavior and integrated into the host RPE layer forming mature quiescent monolayers"[5]; after surgery, structural exams showed that cells had attached and persisted during the study. An improvement in best corrected visual acuity was reported both in the patient affected by Stargardt's macular dystrophy and in the patient affected by dry AMD. And what is more important, in 4 months of follow-up, clinicians did not identify signs of hyperproliferation, abnormal growth, ectopic tissue development, or immune-mediated rejection [1, 5], which represent the main concern about stem cell therapy. These

Since they are autologous, IPSCs (obtained from reprogramming adult somatic fibroblast cells using retroviruses or lentiviruses) seem to have an even lower risk of rejection. However, because of their abnormal genetic composition, the risk of T cell-mediated immune response or oncogenesis should not be underestimated. In 2015, in fact, a Japanese study on IPSCs that was being conducted on human retinas was interrupted because of a new genetic mutation that occurred in the IPSCs of

findings support the safety of ESC-derived stem cells [1, 6].

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

*Regenerative Medicine*

process or other lens abnormalities.

**2.4 Lens**

**2.5 Iris**

meshwork cells or trabecular meshwork progenitor cells combined with argon laser

The lens is composed of the lens capsule, epithelium, and fibers and, like the cornea, is transparent. It is hypothesized that lens-specific stem cells reside in the lens capsule, although they have not yet been identified. The most confirmed hypothesis is that this cell pool comes from the ciliary body, which is anatomically close to the lens. It has been demonstrated that lens capsule regeneration occurs in lower vertebrates from cells residing in the ciliary body. According to this fact, the probability that lens stem cells might reside in the lens capsule is high. Lens progenitor cells have been derived from human ESCs as well as from induced pluripotent stem cells (iPSCs). Lens stem cells are presumed to have a significant role in the maintenance of the lens transparency and might be implied in cataractogenesis

The iris has the anatomical role of dividing the space between the cornea and lens into anterior and posterior halves. The microscopic structure consists of an anterior limiting layer that lines the anterior part of the iris stroma that contains muscles, nerves, and vessels and is posteriorly lined by a layer of pigmented and non-pigmented cells. The stroma and the vascular structure of the iris take embryological origin from the anterior region of the optic cup. Epithelial cells of the iris pigment have the ability to grow in spheres and express markers of neural stem/progenitor cells such as Msi, Nestin, and Pax6. It has been revealed by studies from the mouse iris that these cells can also differentiate both in neuronal and glial lineages and express markers such as Rho, Chx10, Otx2, and Olig2. The iris pigment epithelial cells have the potential to be used in cell-based therapy, but nevertheless not much work on validation and quality assessment has been done. Further studies

are needed before iris pigment epithelial cells can be used clinically.

**3. Regenerative medicine in the posterior segment of the eye**

ganglion cell layer, nerve fiber layer, and inner limiting membrane.

Considering the posterior segment, the main interest is focused on the retina, the target of regenerative medicine. Retinal anatomy is quite complex, and focusing to the microscopic structure, it can be divided into nine layers of nervous tissue that interfaces with the outermost layer of the pigmented epithelium. From external to internal, there are inner segment/outer segment layer, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer,

Stem cells are immature, undifferentiated, highly proliferative cells which are capable of self-renewing and differentiating into many cell types [1]. Therefore, stem cells represent a potentially endless source of tissue renewal; that is why, in the modern era, stem cell therapy has been considered a valid approach for many different pathologies. Ophthalmologists and researchers were not slow to guess the potential applications of stem cell therapy in various degenerative retinal diseases such as retinitis pigmentosa, age-related macular degeneration, Stargardt's macular dystrophy, and other pathological conditions affecting the posterior pole of the eye, including retinal vascular occlusions [1, 2]. These pathologies are responsible for a progressive decline in visual acuity which, in the case of RP,

trabeculoplasty as a novel cell-based therapy for glaucoma.

**98**

Stargardt's disease, and AMD, are due to a constant and irreversible loss of retinal photoreceptors and outer nuclear layers. With such premises, it is easy to imagine a therapeutic approach based on the use of stem cells to restore the lost retinal tissue. Stem cells have shown to be able to perform additional functions, such as nutritional support, apoptosis inhibition, synapse formation, immunoregulation, and neurotrophin secretion [1] and have increased even more the enthusiasm for their application in the ophthalmological field. Furthermore, the use of stem cells in the eye seems to offer numerous advantages: firstly, the amount of stem cells required is relatively low, which implies lower costs than those required for the treatment of other tissues of the human body; secondly, the surgical approach is quite easy and the transplanted cells can be easily monitored with the imaging methods currently used in clinical practice. Finally, the immune privilege of the eye allows avoiding long-term immunosuppressive treatment [1]. Several experimental studies conducted on embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), or mesenchymal stem cells (MSCs) have demonstrated that they tend to adapt to the retinal environment and can differentiate not only into photoreceptors and RPE cells but also into Müller, amacrine, bipolar, horizontal, and glial cells [1]. Retinitis pigmentosa represented one of the first targets of stem cell therapy in the ophthalmological area: pioneering animal studies have shown that pluripotent stem cells, when placed in murine retinitis pigmentosa models, are able to survive, multiplicate, differentiate, organize into, and function as photoreceptor cells, developing a retina-like organizational structure [3]; using mouse models, Singh et al. have established that at a stage when all host rod cells are lost, transplanted rod precursors can lead to the re-establishment of a proper, correctly polarized outer nuclear layer, indicating that stem cells may recreate a light-sensitive cell layer de novo and restore structurally damaged visual circuits. It has to be said that current methods for photoreceptor derivation from human pluripotent stem cells require long periods of culture and are often inefficient. Boucherie et al. [4] reported that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix can induce a rapid conversion into retinal progenitors, which are capable of subsequently differentiating into photoreceptor precursors in only 10 days and later acquire rod cell identity within 4 weeks.

Following such promising results, the first phase I/II clinical trials in humans were approved in the United States in 2010; hESC-derived retinal pigment epithelium cells were transplanted into the eyes of patients with Stargardt's macular dystrophy and dry age-related macular degeneration [5]. During differentiation, the stem cells "displayed typical RPE behavior and integrated into the host RPE layer forming mature quiescent monolayers"[5]; after surgery, structural exams showed that cells had attached and persisted during the study. An improvement in best corrected visual acuity was reported both in the patient affected by Stargardt's macular dystrophy and in the patient affected by dry AMD. And what is more important, in 4 months of follow-up, clinicians did not identify signs of hyperproliferation, abnormal growth, ectopic tissue development, or immune-mediated rejection [1, 5], which represent the main concern about stem cell therapy. These findings support the safety of ESC-derived stem cells [1, 6].

Since they are autologous, IPSCs (obtained from reprogramming adult somatic fibroblast cells using retroviruses or lentiviruses) seem to have an even lower risk of rejection. However, because of their abnormal genetic composition, the risk of T cell-mediated immune response or oncogenesis should not be underestimated. In 2015, in fact, a Japanese study on IPSCs that was being conducted on human retinas was interrupted because of a new genetic mutation that occurred in the IPSCs of one of the patients.

MSCs can differentiate into mesodermal, ectodermal, and endodermal cells and can be obtained from many different tissues, including cord and peripheral blood, teeth, central nervous system, liver, bone marrow, and adipose tissue [1]. Several studies have demonstrated that MSCs can easily turn into neuron-like cells and repair damaged cells through a paracrine action which results in a neuroprotective function. In rats, subretinal transplant of MSCs led to their differentiation into different retinal cell types. These results encouraged clinical trials on humans. In a study, Park et al. [2] isolated CD34+ cells from bone marrow and injected it intravitreally. They enrolled six patients affected by dry AMD, retinitis pigmentosa, or retinal vascular diseases. Follow-up included serial ophthalmic examinations, perimetry and/or microperimetry, fluorescein angiography, ERG, and OCT. After 6 months of follow-up, there was no evidence of worsening neither in BCVA nor in full-field ERG. No signs of intraocular inflammation were observed. Other studies on MSCs confirmed their safety in terms of hyperproliferation and systemic side effects. However, as reported, further MSC applications led to other sight-threatening intraocular complications such as elevated intraocular pressure, vitreous hemorrhages, tractional and rhegmatogenous retinal detachment, development of preretinal and vitreal fibrous tissue, and shallowing of the anterior chamber.

Retinal pigment epithelium replacement represents a promising evolution of stem cell therapy. The outer segments of photoreceptors have a very high metabolic demand and undergo a daily renewal; in the healthy retina, the apical processes of the RPE envelope the outer segments of rods and cones, which contain visual pigment, resulting in a diurnal outer segment recycling. Pathological conditions such as drusen deposits, accumulation of lipofuscin, or ischemic insult can result in a disruption of RPE, slowing photoreceptor metabolism and leading to cellular damage. RPE was one of the first tissues to be differentiated in vitro. Nowadays, there are many ongoing clinical trials for pluripotent stem cell-derived RPE replacement. The success of RPE replacement can be explained by various factors: for a start, RPE cell biology and phenotypes are precisely described and conserved among species [7]; the differentiation of embryonic stem cells into RPE cells follows default pathways that are well characterized; animal models of RPE dysfunction are easily available; the amount of RPE required to functionally restore affected retinas is relatively small compared with photoreceptors [7]; and the RPE layers within the retina can be easily visualized using optical coherence tomography, adaptive optics scanning laser ophthalmoscopy, and fundus image. Moreover, studies on animal models have established that sheet transplantation is much more beneficial and effective than single-cell suspension [7], making retinal patches a fascinating approach to degenerative retinal diseases. However, further studies have proven this technique unsuccessful in human models. Nevertheless, studies on retinal sheet transplantation are still ongoing.

Retinal tissue engineering is another intriguing idea for treating late-stage retinal conditions, but various technical and biological issues coming from lab-grown neuroretinal tissue design still need to be solved before it can work in clinics. The size of 3D retinal tissue derived from human pluripotent stem cells is much smaller than that required to obtain a significant clinical outcome, and the implantation of a single piece of retinal organoid may not result in an appreciable improvement in visual acuity in humans [8]. Because of their plasticity, human pluripotent stem cells make an extraordinary source for regenerative medicine. The current challenges of retinal tissue engineering include establishing reproducible protocols for the creation of retinal organoids from stem cells, producing larger pieces of retinal tissue from stem cells along with quality supporting biomaterials, improving surgical methods of delivering retinal organoids into subretinal space, and finding

**101**

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

ophthalmological field.

ally leads to the death of retinal cells.

• Inflammation and para-inflammation [10, 11]

• Neurotrophic aspects

• Oxidative aspects

• Vascular alterations

mechanisms of cell apoptosis.

• Apoptosis

follows:

grafts into the host's synaptic environment [8].

biomaterials to facilitate the survival and functional integration of hPSC-derived

**4. Cell therapy and atrophic retinal diseases: our experience**

Visually impaired patients are affected by a series of different neuroretinal diseases that can target nerve cells such as ganglion cells (RCG), photoreceptors, or support cells such as retinal pigment epithelium cells (RPE). The evolution of these pathologies leads to serious impairment of vision. There are many types of retinal degenerative diseases, including glaucoma, hereditary retinal dystrophy such as retinitis pigmentosa (RP) or Stargardt's disease, age-related macular degeneration (AMD), degenerative myopia, and diabetic retinopathy (DR). In each of these pathologies, regardless of its nature, a certain sequence of molecular events gradu-

These mechanisms cover various biological aspects and can be summarized as

The sequence can begin with oxidation, photooxidation, or photosensitivity. This is followed by the release of oxidizing substances and free radicals in the cellular environment which in turn causes lipid peroxidation, oxidation of the critical bonds in the protein chains and rupture in those of the DNA, activation of the endogenous nuclease, inhibition of the expression of the Bcl2 gene, and priming of

In physiological conditions, healthy retinal cells possess an arsenal of substances with protective action, including antioxidant systems (e.g., SOD) and enzymes, which serve to balance oxidants and free radicals, minimizing damage. One of the best known mechanisms to block or procrastinate apoptotic processes is the activation of the Bc12 gene by growth factors, thus avoiding the fate of death, regardless of the triggering cause. There are cells such as Müller cells or RPE cells, capable of producing, under hypoxic conditions, angiogenic and neurotrophic

In conclusion, pioneering studies conducted on animal models have provided hopeful evidence for the hypothesis that stem cell therapy is a valid approach to sight-threatening degenerative retinal diseases, including retinitis pigmentosa, Stargardt's disease, dry age-related macular degeneration, and vascular occlusions. A number of phase I/II clinical trials on humans seem to have confirmed the effectiveness of this method. We now know for sure that when placed in an appropriate tissue niche stem cells not only survive and proliferate but are capable of differentiating into proper retinal cells which exhibit functional characteristics of real photoreceptors, resulting in the development of a retina-like structure [9]. Further studies are needed to put such promising experimental data into clinical practice and establish standardized procedures for the application of stem cell therapy in the

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

*Regenerative Medicine*

anterior chamber.

tion are still ongoing.

MSCs can differentiate into mesodermal, ectodermal, and endodermal cells and can be obtained from many different tissues, including cord and peripheral blood, teeth, central nervous system, liver, bone marrow, and adipose tissue [1]. Several studies have demonstrated that MSCs can easily turn into neuron-like cells and repair damaged cells through a paracrine action which results in a neuroprotective function. In rats, subretinal transplant of MSCs led to their differentiation into different retinal cell types. These results encouraged clinical trials on humans. In a study, Park et al. [2] isolated CD34+ cells from bone marrow and injected it intravitreally. They enrolled six patients affected by dry AMD, retinitis pigmentosa, or retinal vascular diseases. Follow-up included serial ophthalmic examinations, perimetry and/or microperimetry, fluorescein angiography, ERG, and OCT. After 6 months of follow-up, there was no evidence of worsening neither in BCVA nor in full-field ERG. No signs of intraocular inflammation were observed. Other studies on MSCs confirmed their safety in terms of hyperproliferation and systemic side effects. However, as reported, further MSC applications led to other sight-threatening intraocular complications such as elevated intraocular pressure, vitreous hemorrhages, tractional and rhegmatogenous retinal detachment, development of preretinal and vitreal fibrous tissue, and shallowing of the

Retinal pigment epithelium replacement represents a promising evolution of stem cell therapy. The outer segments of photoreceptors have a very high metabolic demand and undergo a daily renewal; in the healthy retina, the apical processes of the RPE envelope the outer segments of rods and cones, which contain visual pigment, resulting in a diurnal outer segment recycling. Pathological conditions such as drusen deposits, accumulation of lipofuscin, or ischemic insult can result in a disruption of RPE, slowing photoreceptor metabolism and leading to cellular damage. RPE was one of the first tissues to be differentiated in vitro. Nowadays, there are many ongoing clinical trials for pluripotent stem cell-derived RPE replacement. The success of RPE replacement can be explained by various factors: for a start, RPE cell biology and phenotypes are precisely described and conserved among species [7]; the differentiation of embryonic stem cells into RPE cells follows default pathways that are well characterized; animal models of RPE dysfunction are easily available; the amount of RPE required to functionally restore affected retinas is relatively small compared with photoreceptors [7]; and the RPE layers within the retina can be easily visualized using optical coherence tomography, adaptive optics scanning laser ophthalmoscopy, and fundus image. Moreover, studies on animal models have established that sheet transplantation is much more beneficial and effective than single-cell suspension [7], making retinal patches a fascinating approach to degenerative retinal diseases. However, further studies have proven this technique unsuccessful in human models. Nevertheless, studies on retinal sheet transplanta-

Retinal tissue engineering is another intriguing idea for treating late-stage retinal conditions, but various technical and biological issues coming from lab-grown neuroretinal tissue design still need to be solved before it can work in clinics. The size of 3D retinal tissue derived from human pluripotent stem cells is much smaller than that required to obtain a significant clinical outcome, and the implantation of a single piece of retinal organoid may not result in an appreciable improvement in visual acuity in humans [8]. Because of their plasticity, human pluripotent stem cells make an extraordinary source for regenerative medicine. The current challenges of retinal tissue engineering include establishing reproducible protocols for the creation of retinal organoids from stem cells, producing larger pieces of retinal tissue from stem cells along with quality supporting biomaterials, improving surgical methods of delivering retinal organoids into subretinal space, and finding

**100**

biomaterials to facilitate the survival and functional integration of hPSC-derived grafts into the host's synaptic environment [8].

In conclusion, pioneering studies conducted on animal models have provided hopeful evidence for the hypothesis that stem cell therapy is a valid approach to sight-threatening degenerative retinal diseases, including retinitis pigmentosa, Stargardt's disease, dry age-related macular degeneration, and vascular occlusions. A number of phase I/II clinical trials on humans seem to have confirmed the effectiveness of this method. We now know for sure that when placed in an appropriate tissue niche stem cells not only survive and proliferate but are capable of differentiating into proper retinal cells which exhibit functional characteristics of real photoreceptors, resulting in the development of a retina-like structure [9]. Further studies are needed to put such promising experimental data into clinical practice and establish standardized procedures for the application of stem cell therapy in the ophthalmological field.
