**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 gradually leads to the death of retinal cells.

These mechanisms cover various biological aspects and can be summarized as follows:


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 mechanisms of cell apoptosis.

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

factors such as FGF and VEGF in order to counterbalance the insult, provided that it is transient [12]. In the case of cellular imbalance, for example, for genetic or inflammatory reasons, for reduction of the chorioretinal blood flow or when a large part of the cells has undergone apoptosis and death with consequent induction of a chronic para-inflammatory condition, the trigger of neuroretinal pathologies, or their progression, can occur. In our opinion, it is possible to apply a therapy aimed at reducing the impact and progression of the disease based on these mechanisms. The therapeutic aim is to slow down or prevent the death of residual retinal cells [13, 14], highlighting the possible efficacy of cell therapy on neurotrophic pathologies of the retina. Currently, in the presence of a dystrophic pathology responsible for a low vision condition, the patient can resort to visual rehabilitation using magnifying aids or filters to improve contrast. In a smaller number of centers, it is possible to benefit from therapies based on the neuromodulation of visual signals, in order to improve not only the image on the retina but also the perception of the same at the cortical level.

However, the progressive loss of photoreceptors contributes to reducing the performance obtained with visual rehabilitation, and the social impact of the progressive loss of functional autonomy should not be underestimated. New therapeutic approaches to neuroretinal degenerations for therapy include restoring defective genes, when the disease is caused by a genetic defect, and transplanting stem cells to replace or repair defective or dead cells, regardless of the cause [15, 16]. Gene therapy is a causal therapy but is currently not clinically available, and the therapeutic results obtained experimentally are still marginal in vivo. For this reason, the interest of the scientific community is also addressed to stem cell-based repair strategies, consisting in the systemic or local injection of stem/progenitor cells for the treatment of multiple chronic pathologies [15, 16]. Stem cells are undifferentiated cells that have the ability to self-renew and differentiate into mature cells. On this basis, cell replacement therapy has been evaluated in recent years as a viable alternative for various pathologies. This therapy hypothesizes the generation of retinal cells from stem cells to replace damaged cells in the diseased retina. This goal can be achieved by releasing embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) [17] in specific target positions of the eye. Stem cell therapy opens up the possibility of replacing or regenerating the cells now destroyed during the most common neurodystrophic diseases of the retina. However, ESC and iPCS have generated much controversy over ethical, immunological, and oncological issues. Instead, the use of MSC appears to be free from these concerns. The reparative therapy operated by the implanted cells aims to create better conditions for the viability of the residual cells, preventing or slowing their decline. We could therefore define cell therapy, or mediated cell therapy, or any therapeutic modality based on the use of cell grafts that aim not only at the neuroenhancement of compromised cells and the possible regeneration of some elements (such as receptors, mitochondrial components, connection fibers) but also to their integration with the above cells. It remains to be asked whether it is easier to preserve or promote the survival and function of diseased cells than to actively restore retinal cells after they have disappeared following the disease.

## **4.1 Mesenchymal stem cells as a therapeutic tool**

MSCs are characterized by a panel of superficial cell markers proposed by the International Society for Cellular Therapy in 2006. The MSC population is defined as positive over 95% for CD105, CD73, and CD90 and negative over 95% for CD45, CD34, CD14 or CD11b, CD79 or CD19, and HLA-DR [18]. Some molecules present on the surface of MSCs and endothelial cells, such as P-selectin and integrins,

**103**

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

tissues [19, 20].

let the MSCs themselves migrate to the lesion sites, following their intravascular administration. After joining the endothelium, the MSCs are able to cross it in a metal-dependent way. MSCs are obtainable from umbilical cord blood, peripheral blood, bone marrow, and adipose tissue. MSCs are multipotent: appropriate culture conditions associated with specific growth factors drive the differentiation of MSCs into specific cell types and can differentiate into various cell types, including osteocytes, adipocytes, vascular endothelial cells, cardiomyocytes, pancreatic beta cells, and hepatocytes. Therefore, MSCs play a key role in organogenesis, remodeling, and tissue repair. Experimental studies have also reported that MSCs have the potential to differentiate into retinal progenitor cells, photoreceptors, and retinal neuron-like cells. Furthermore, stem cells, in particular mesenchymal stem cells (MSCs), are able to perform multiple functions, such as immunoregulation, anti-apoptosis of neurons, and neurotrophin secretion, and the current opinion is that MSCs can exert neuroprotective and proregenerative effects, through the secretion of factors that act in a paracrine way. An increasing number of studies also report that MSCs are capable of giving rise to neuron-like cells. Not only are they able to differentiate into neurons for cell replacement therapy but to maintain and regulate the microenvironment through paracrine effects by modulating the plasticity of damaged host

Of all the MSC collection sites, adipose tissue is particularly interesting and rich in stem cells derived from fat, called ADSCs [21]. These cells are able to secrete neurotrophic growth factors and promote survival, restore the release of the synaptic transmitter, integrate into existing neural and synaptic networks, and re-establish functional connections [22]. ADSCs produce bFGF, vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), placental growth factor (PlGF), the transforming growth factor (TGFβ), hepatocyte growth factor (HG), insulin growth factor (IGF-1), interleukin (IL) and angiogenin, ciliary neurotrophic factor (CNTF), and the brain-derived neurotrophic factor (BDNF). Another type of mesenchymal tissue is represented by adipose tissue which, just like the bone marrow, contains a large population of stem cells within its stromal compartment. Stromal adipocytes or fat stromal cells secrete a series of hormones, factors, and protein signals, called adipokines, which are associated with the role of the adipocyte in energy homeostasis. Fat cells produce the base fibroblast growth factor (bFGF), the epidermal growth factor (EGF), the insulin-like growth factor-1 (IGF-1), the interleukin (IL), the transforming growth-β (TGFβ), the pigmented epitheliumderived factor (PEDF), and adiponectin. Another type of cell of mesenchymal origin is the platelet, originating from the subdivision of megakaryocytes. Platelets, normally known for their hemostatic action, also release substances that promote tissue repair, angiogenesis, and inflammation modulation. In addition, they induce cell migration and adhesion at angiogenesis sites, as well as the differentiation of endothelial progenitors into mature endothelial cells. Platelets produce plateletderived growth factor (PDGF), IGF-1, TGFβ, VEGF, bFGF, EGF, platelet-derived

angiogenesis factor (PDAF), and thrombospondin (TSP) [23].

The therapeutic potential of mesenchymal cells is based on the stabilizing effect against the retinal cells exerted by the cytokines and the growth factors released paracrinically when they are grafted. The binding of the growth factor to the specific surface receptor placed on the cytoplasmic membrane of the target cell is the initial step that triggers a cascade of events, activating particular second messengers that guarantee the signal transduction at the intracellular level. The ultimate goal is the regulation of enzyme activity or gene expression (**Figure 1**) [24, 25]. In particular, activated transcription factors, entering the nucleus and binding directly or indirectly to DNA, regulate the expression of various genes with

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

*Regenerative Medicine*

factors such as FGF and VEGF in order to counterbalance the insult, provided that it is transient [12]. In the case of cellular imbalance, for example, for genetic or inflammatory reasons, for reduction of the chorioretinal blood flow or when a large part of the cells has undergone apoptosis and death with consequent induction of a chronic para-inflammatory condition, the trigger of neuroretinal pathologies, or their progression, can occur. In our opinion, it is possible to apply a therapy aimed at reducing the impact and progression of the disease based on these mechanisms. The therapeutic aim is to slow down or prevent the death of residual retinal cells [13, 14], highlighting the possible efficacy of cell therapy on neurotrophic pathologies of the retina. Currently, in the presence of a dystrophic pathology responsible for a low vision condition, the patient can resort to visual rehabilitation using magnifying aids or filters to improve contrast. In a smaller number of centers, it is possible to benefit from therapies based on the neuromodulation of visual signals, in order to improve not only the image on the retina

However, the progressive loss of photoreceptors contributes to reducing the performance obtained with visual rehabilitation, and the social impact of the progressive loss of functional autonomy should not be underestimated. New therapeutic approaches to neuroretinal degenerations for therapy include restoring defective genes, when the disease is caused by a genetic defect, and transplanting stem cells to replace or repair defective or dead cells, regardless of the cause [15, 16]. Gene therapy is a causal therapy but is currently not clinically available, and the therapeutic results obtained experimentally are still marginal in vivo. For this reason, the interest of the scientific community is also addressed to stem cell-based repair strategies, consisting in the systemic or local injection of stem/progenitor cells for the treatment of multiple chronic pathologies [15, 16]. Stem cells are undifferentiated cells that have the ability to self-renew and differentiate into mature cells. On this basis, cell replacement therapy has been evaluated in recent years as a viable alternative for various pathologies. This therapy hypothesizes the generation of retinal cells from stem cells to replace damaged cells in the diseased retina. This goal can be achieved by releasing embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) [17] in specific target positions of the eye. Stem cell therapy opens up the possibility of replacing or regenerating the cells now destroyed during the most common neurodystrophic diseases of the retina. However, ESC and iPCS have generated much controversy over ethical, immunological, and oncological issues. Instead, the use of MSC appears to be free from these concerns. The reparative therapy operated by the implanted cells aims to create better conditions for the viability of the residual cells, preventing or slowing their decline. We could therefore define cell therapy, or mediated cell therapy, or any therapeutic modality based on the use of cell grafts that aim not only at the neuroenhancement of compromised cells and the possible regeneration of some elements (such as receptors, mitochondrial components, connection fibers) but also to their integration with the above cells. It remains to be asked whether it is easier to preserve or promote the survival and function of diseased cells than to actively

but also the perception of the same at the cortical level.

restore retinal cells after they have disappeared following the disease.

MSCs are characterized by a panel of superficial cell markers proposed by the International Society for Cellular Therapy in 2006. The MSC population is defined as positive over 95% for CD105, CD73, and CD90 and negative over 95% for CD45, CD34, CD14 or CD11b, CD79 or CD19, and HLA-DR [18]. Some molecules present on the surface of MSCs and endothelial cells, such as P-selectin and integrins,

**4.1 Mesenchymal stem cells as a therapeutic tool**

**102**

let the MSCs themselves migrate to the lesion sites, following their intravascular administration. After joining the endothelium, the MSCs are able to cross it in a metal-dependent way. MSCs are obtainable from umbilical cord blood, peripheral blood, bone marrow, and adipose tissue. MSCs are multipotent: appropriate culture conditions associated with specific growth factors drive the differentiation of MSCs into specific cell types and can differentiate into various cell types, including osteocytes, adipocytes, vascular endothelial cells, cardiomyocytes, pancreatic beta cells, and hepatocytes. Therefore, MSCs play a key role in organogenesis, remodeling, and tissue repair. Experimental studies have also reported that MSCs have the potential to differentiate into retinal progenitor cells, photoreceptors, and retinal neuron-like cells. Furthermore, stem cells, in particular mesenchymal stem cells (MSCs), are able to perform multiple functions, such as immunoregulation, anti-apoptosis of neurons, and neurotrophin secretion, and the current opinion is that MSCs can exert neuroprotective and proregenerative effects, through the secretion of factors that act in a paracrine way. An increasing number of studies also report that MSCs are capable of giving rise to neuron-like cells. Not only are they able to differentiate into neurons for cell replacement therapy but to maintain and regulate the microenvironment through paracrine effects by modulating the plasticity of damaged host tissues [19, 20].

Of all the MSC collection sites, adipose tissue is particularly interesting and rich in stem cells derived from fat, called ADSCs [21]. These cells are able to secrete neurotrophic growth factors and promote survival, restore the release of the synaptic transmitter, integrate into existing neural and synaptic networks, and re-establish functional connections [22]. ADSCs produce bFGF, vascular endothelial growth factor (VEGF), macrophage colony stimulating factor (M-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), placental growth factor (PlGF), the transforming growth factor (TGFβ), hepatocyte growth factor (HG), insulin growth factor (IGF-1), interleukin (IL) and angiogenin, ciliary neurotrophic factor (CNTF), and the brain-derived neurotrophic factor (BDNF). Another type of mesenchymal tissue is represented by adipose tissue which, just like the bone marrow, contains a large population of stem cells within its stromal compartment. Stromal adipocytes or fat stromal cells secrete a series of hormones, factors, and protein signals, called adipokines, which are associated with the role of the adipocyte in energy homeostasis. Fat cells produce the base fibroblast growth factor (bFGF), the epidermal growth factor (EGF), the insulin-like growth factor-1 (IGF-1), the interleukin (IL), the transforming growth-β (TGFβ), the pigmented epitheliumderived factor (PEDF), and adiponectin. Another type of cell of mesenchymal origin is the platelet, originating from the subdivision of megakaryocytes. Platelets, normally known for their hemostatic action, also release substances that promote tissue repair, angiogenesis, and inflammation modulation. In addition, they induce cell migration and adhesion at angiogenesis sites, as well as the differentiation of endothelial progenitors into mature endothelial cells. Platelets produce plateletderived growth factor (PDGF), IGF-1, TGFβ, VEGF, bFGF, EGF, platelet-derived angiogenesis factor (PDAF), and thrombospondin (TSP) [23].

The therapeutic potential of mesenchymal cells is based on the stabilizing effect against the retinal cells exerted by the cytokines and the growth factors released paracrinically when they are grafted. The binding of the growth factor to the specific surface receptor placed on the cytoplasmic membrane of the target cell is the initial step that triggers a cascade of events, activating particular second messengers that guarantee the signal transduction at the intracellular level. The ultimate goal is the regulation of enzyme activity or gene expression (**Figure 1**) [24, 25]. In particular, activated transcription factors, entering the nucleus and binding directly or indirectly to DNA, regulate the expression of various genes with

#### **Figure 1.**

*The growth factors produced by mesenchymal cells implanted in the suprachoroidal space can act both directly on the retinal cells and indirectly, through the mediation of Müller (MC) and RPE cells, generating angiotrophic, neurotrophic, anti-inflammatory, and antiapoptotic effects [26, 27]. Image courtesy of P. Limoli-Milan Low Vision Study Center.*

different mechanisms, promoting greater synthesis of proteins including enzymes and cytokines. These end products play a key role in cell survival, as assessed by the improvement in electrical activity recorded by ERG [27]. The growth factors are essential to trigger the cell transition from G0 or resting phase to G1 or growth phase. Furthermore these molecules stimulate a wide range of cellular processes, including mitosis, cell survival, migration, and cellular differentiation.

### **4.2 Pathophysiological co-factoriality and cell therapy**

The grafting of mesenchymal cells into the suprachoroidal space promotes a continuous paracrine increase in GF that can positively interfere with the evolution of retinal diseases in several ways.

Therapeutic activity can be classified into:


It is worth noting that the boundaries between these categories are not necessarily defined. The hemorheological activity and its increase help to restore an

**105**

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

effective retinal perfusion. Photoreceptor loss that occurs in retinal diseases has been identified as the cause of microvascular dysfunction due to the release of cellular waste secondary to apoptosis. In fact, there is a correlation between the extent of the blood flow and the evolutionary stage of the atrophic pathology, in a vicious circle that leads to the final loss of other photoreceptors. Several factors such as VEGF, bFGF, angiogenin, PDAF, PlGF, PDGF, EGF, and TGF-β have been shown to promote endothelial regeneration and therefore can contribute to the reperfusion of the choriocapillaris. Furthermore, others, including TSP and PEDF, inhibit pathological neovascular processes [28, 29]. Antioxidative activity prevents oxygen-induced photoreceptor cell death. One of the underlying causes of photoreceptor deterioration, which may explain the evolution of retinal degeneration, is hyperoxia which results in a more intense oxidation process and in the formation of reactive oxygen species (ROS). Excessive generation of reactive oxygen species causes damage to membrane lipoproteins and cellular DNA, thus leading to apoptosis and the death of photoreceptors [30]. The mechanism involved in hyperoxia can be illuminated by the excessive amount of oxygen in the choroid, similar to the arterial oxygen level, which results from the deterioration and death of the photoreceptor, in addition to foveal exposure to light and the concomitant lack of anti-enzyme oxidants, such as superoxide dismutase (SOD), glutathione-peroxidase, and catalases, normally expressed in the mitochondria of the internal segments of the cone and capable of catalyzing the decomposition of hydrogen peroxide into water and oxygen molecules [31]. The concentration of bFGF within photoreceptors has been shown to increase in response to stress in order to promote retinal cell survival and prevent oxygen-induced photoreceptor cell death [32, 33]. Anti-inflammatory activity can counteract the negative effects induced by microglial activation, which occurs as soon as the apoptotic processes induced by retinal degeneration begin [34, 35]. In turn, the apoptosis and death of photoreceptors are suggested by the ignition of an inflammatory microclimate that supports the chronicity and progression of a large number of neurodegenerative diseases. In particular, RPE performs a series of essential processes for homeostasis and retinal function and constitutes the front of the immune defense of the retina: RPE cells are able to secrete a diversified panel of pro-inflammatory cytokines, for example, IL-6, IL-8, chemoattractant monocyte protein-1 (MCP-1), and interferon-β (IFN-β), as well as anti-inflammatory factors, e.g., IL-11 and TGF-β. Intravitreal administration of MSC has been shown to exert a significant effect on the host's immune response by suppressing the production of pro-inflammatory cytokines, such as IFN-β and TNF-α through IL-1 receptor antagonist (IL-1RA) and prostaglandin E2 receptor (PGE2R) activation [36]. The therapeutic effect of MSCs is corroborated by the neurotrophic action of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF): in culturing retinal ganglion cells, under conditions of oxidative stress, MSC expels the last factor that helps reduce pro-inflammation cytokine release, e.g., tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) [33]. M-CSF, GM-CSF, and IL exercise an anti-inflammatory function and recruit macrophages by chemotaxis that help remove intraretinal cell debris [37]. The antiapoptotic activity is regulated by cytokines with an inhibiting

(antiapoptotic) or inducing apoptosis (pro-apoptotic) action [38].

Proteins of the Bcl-2 family are particularly known for their regulation of apoptosis by interacting with caspases, a family of cysteine-containing protease enzymes (proteinases or caspases specific to cysteine's aspartate). RPE and Müller cells produce a wide heterogeneity of factors, e.g., fibroblast growth factors (FGF-1, FGF-2, and FGF-5), transforming growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), some members of the Interleukin family, and the

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

*Regenerative Medicine*

**Figure 1.**

*Milan Low Vision Study Center.*

different mechanisms, promoting greater synthesis of proteins including enzymes and cytokines. These end products play a key role in cell survival, as assessed by the improvement in electrical activity recorded by ERG [27]. The growth factors are essential to trigger the cell transition from G0 or resting phase to G1 or growth phase. Furthermore these molecules stimulate a wide range of cellular processes,

*The growth factors produced by mesenchymal cells implanted in the suprachoroidal space can act both directly on the retinal cells and indirectly, through the mediation of Müller (MC) and RPE cells, generating angiotrophic, neurotrophic, anti-inflammatory, and antiapoptotic effects [26, 27]. Image courtesy of P. Limoli-*

The grafting of mesenchymal cells into the suprachoroidal space promotes a continuous paracrine increase in GF that can positively interfere with the evolution

It is worth noting that the boundaries between these categories are not necessarily defined. The hemorheological activity and its increase help to restore an

including mitosis, cell survival, migration, and cellular differentiation.

**4.2 Pathophysiological co-factoriality and cell therapy**

6.Therapeutic synergy with electrical stimulation (ES)

Therapeutic activity can be classified into:

of retinal diseases in several ways.

1.Hemorheological activity

3.Anti-inflammatory activity

2.Antioxidative activity

4.Antiapoptotic activity

5.Cytoprotective activity

**104**

effective retinal perfusion. Photoreceptor loss that occurs in retinal diseases has been identified as the cause of microvascular dysfunction due to the release of cellular waste secondary to apoptosis. In fact, there is a correlation between the extent of the blood flow and the evolutionary stage of the atrophic pathology, in a vicious circle that leads to the final loss of other photoreceptors. Several factors such as VEGF, bFGF, angiogenin, PDAF, PlGF, PDGF, EGF, and TGF-β have been shown to promote endothelial regeneration and therefore can contribute to the reperfusion of the choriocapillaris. Furthermore, others, including TSP and PEDF, inhibit pathological neovascular processes [28, 29]. Antioxidative activity prevents oxygen-induced photoreceptor cell death. One of the underlying causes of photoreceptor deterioration, which may explain the evolution of retinal degeneration, is hyperoxia which results in a more intense oxidation process and in the formation of reactive oxygen species (ROS). Excessive generation of reactive oxygen species causes damage to membrane lipoproteins and cellular DNA, thus leading to apoptosis and the death of photoreceptors [30]. The mechanism involved in hyperoxia can be illuminated by the excessive amount of oxygen in the choroid, similar to the arterial oxygen level, which results from the deterioration and death of the photoreceptor, in addition to foveal exposure to light and the concomitant lack of anti-enzyme oxidants, such as superoxide dismutase (SOD), glutathione-peroxidase, and catalases, normally expressed in the mitochondria of the internal segments of the cone and capable of catalyzing the decomposition of hydrogen peroxide into water and oxygen molecules [31]. The concentration of bFGF within photoreceptors has been shown to increase in response to stress in order to promote retinal cell survival and prevent oxygen-induced photoreceptor cell death [32, 33]. Anti-inflammatory activity can counteract the negative effects induced by microglial activation, which occurs as soon as the apoptotic processes induced by retinal degeneration begin [34, 35]. In turn, the apoptosis and death of photoreceptors are suggested by the ignition of an inflammatory microclimate that supports the chronicity and progression of a large number of neurodegenerative diseases. In particular, RPE performs a series of essential processes for homeostasis and retinal function and constitutes the front of the immune defense of the retina: RPE cells are able to secrete a diversified panel of pro-inflammatory cytokines, for example, IL-6, IL-8, chemoattractant monocyte protein-1 (MCP-1), and interferon-β (IFN-β), as well as anti-inflammatory factors, e.g., IL-11 and TGF-β. Intravitreal administration of MSC has been shown to exert a significant effect on the host's immune response by suppressing the production of pro-inflammatory cytokines, such as IFN-β and TNF-α through IL-1 receptor antagonist (IL-1RA) and prostaglandin E2 receptor (PGE2R) activation [36]. The therapeutic effect of MSCs is corroborated by the neurotrophic action of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF): in culturing retinal ganglion cells, under conditions of oxidative stress, MSC expels the last factor that helps reduce pro-inflammation cytokine release, e.g., tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) [33]. M-CSF, GM-CSF, and IL exercise an anti-inflammatory function and recruit macrophages by chemotaxis that help remove intraretinal cell debris [37]. The antiapoptotic activity is regulated by cytokines with an inhibiting (antiapoptotic) or inducing apoptosis (pro-apoptotic) action [38].

Proteins of the Bcl-2 family are particularly known for their regulation of apoptosis by interacting with caspases, a family of cysteine-containing protease enzymes (proteinases or caspases specific to cysteine's aspartate). RPE and Müller cells produce a wide heterogeneity of factors, e.g., fibroblast growth factors (FGF-1, FGF-2, and FGF-5), transforming growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), some members of the Interleukin family, and the

pigmented epithelium factor (PEDF). This multitude of growth factors, released in the retinal cytosol, is able to produce a wide trophic action on adjacent structures. As a consequence, the progressive loss of RPE and Müller cells hinders the incretion of these bioactive agents: their antiapoptotic action is therefore slowed down or completely blocked. The administration of mesenchymal cells can interfere with the apoptotic process involved in retinal degeneration. The growth factors excreted by the grafted mesenchymal cells perform a variety of functions; in particular they are able to facilitate the expression of the Bcl-2 gene in order to avoid the inexorable death of the cells, regardless of the root causes [17]. The cytoprotective activity of the GF contributes to neuroprotection by regulating the metabolic activity of the photoreceptors, which is widely compromised in diseases of the retina. Like bFGF, PEDF has been found to exert neurotrophic activity, inducing the overall survival of photoreceptors [39]. Significant data currently exist to suggest that certain factors such as EGF play a role in potentiating the neuroprotective action of Müller cells by stimulating their intracellular transcription and bFGF expression [40]. The VEGF released by the PRP has been shown to stimulate the proliferation of ADSCs which therefore promote the survival of grafted autologous fat and adipocytes [41]. BFGF is known to directly promote the survival of photoreceptors [42]. Synergy with electrical stimulation (ES) addresses four main aspects: survival of native cells, survival of transplanted cells, integration of transplanted cells, and functional formation of synapses/axon regeneration [43]. In recent years, the synergy between cell therapies and electrical stimulation has started to be considered as a possible treatment for degenerative diseases. Rat retinas treated with ES showed a reduction in apoptosis [44]. It has also been shown that in light-induced retinal degeneration models, stimulation with ES contains the death of photoreceptors and preserves the length of the external segment [45]. Consequently, it can be assumed that ES treatment can create a more balanced and less hostile environment by modifying the secretion of neurotrophic factors. ES affects the upregulation of neurotrophic factors in Müller cells normally involved in this protection mechanism. After ES, increased expression of in vivo beta fibroblast growth factor (b-FGF), insulin growth factor 1 (IGF-1), and brain-derived neurotrophic factor (BDNF) was observed. Conversely, ES reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and the pro-apoptotic gene Bax. The release of neurotrophic factors from the postsynaptic membrane made possible by neuromodulation, together with the enrichment of the same factors in the extracellular environment managed by autologous grafts, determines the formation of synapses at a presynaptic level, facilitating and strengthening neurotransmission [44, 45].

#### **4.3 Cell therapy and routes of administration**

The effectiveness of cellular treatments for atrophic pathologies of the retina, in order to stabilize and enhance the visual function, is based on two key elements: on the one hand, the surgical implant techniques and the cell lines used, and on the other by the quantity and quality of residual retinal cells, in other words from the earliness of the treatment. The technique must be simple, totally risk-free, and painless, and the exploited cells must not cause further damage to the residual retinal cell or to the person. Cytokines and growth factors, released paracrinically from the cells administered, must bind to membrane receptors to trigger the pathway of intracellular signal transduction. They still require retinal cells that are still alive.

From the studies carried out so far, it seems that the greater the number of residual cells, the greater the interaction between GF and chorioretinal cell membrane receptors, cellular activity, and, ultimately, the improvement of visual performance (VP) [27]. The release of growth factors in a retina with very low

**107**

**Figure 2.**

*courtesy of P. Limoli-Milan Low Vision Center.*

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

quantities.

bleeding are also possible.

cellularity hardly causes detectable neuroenhancement. To achieve this goal, different approaches have been explored by inserting these cells in the subtenonian space and in the intravitreal or subretinal space. But it seems that positioning the implant in the suprachoroidal space can satisfy efficacy and safety. In fact, the graft under the tenon, although it has therapeutic significance, does not allow the growth factors produced to reach the neuroretinal tissues inside the sclera in important

Intravitreal injections of cellular material are effective and simple to perform, but it is necessary to pierce the bulb and leave this material free in the vitreous chamber. Serious complications such as infection, vitreoretinal tractions, and

The release in the subretinal area seems to be the best for the possibility of a potential modification of cell lines due to the direct contact of MSCs with neuronal cells, but their grafting is even more dangerous when the retina is compromised by atrophic diseases [46]. The suprachoroidal graft maximizes the supply of growth factors that flow directly to the choroidal level and through the choroid to the entire retina without creating bulbar perforation. In our experience, in order to have the therapeutic action of growth factors in the retinal environment, we have explored the possibility of treating the dystrophic retina with the implantation of the cell types of mesenchymal origin mentioned above, in detail adipose stromal cells (ASC), stem cells derived from adipose tissue (ADSC) contained in the stromal-vascular fraction (SVF) of adipose tissue, and platelets (PLT) recovered in platelet-rich plasma (PRP) [47–49]. To this end, we used a surgical technique called Limoli retinal restoration technique (LRRT), described in previous works (**Figure 2**) [27, 50, 51]. The autotransplantation of ADSC, ACS, and PLT above the choroid plane improves the incretion of the bioactive factors produced in the choroidal flow and, consequently, promotes their widespread diffusion through the

*Autotransplantation of adipose tissue, ADSCs from vascular-stromal fraction, and PRP according to the Limoli retinal restoration technique (LRRT). The production of growth factors (GF), characteristic of these cells, is poured directly into the choroidal flow in paracrine mode, helping to maintain the trophism of the retinal cells. The GF, through the choroidal flux, have a direct action on the choroid, on the Müller cells, on the RPE cells with improvement of the physiology of the external segments (OS), on the rods, and on the cones. Image* 

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

*Regenerative Medicine*

pigmented epithelium factor (PEDF). This multitude of growth factors, released in the retinal cytosol, is able to produce a wide trophic action on adjacent structures. As a consequence, the progressive loss of RPE and Müller cells hinders the incretion of these bioactive agents: their antiapoptotic action is therefore slowed down or completely blocked. The administration of mesenchymal cells can interfere with the apoptotic process involved in retinal degeneration. The growth factors excreted by the grafted mesenchymal cells perform a variety of functions; in particular they are able to facilitate the expression of the Bcl-2 gene in order to avoid the inexorable death of the cells, regardless of the root causes [17]. The cytoprotective activity of the GF contributes to neuroprotection by regulating the metabolic activity of the photoreceptors, which is widely compromised in diseases of the retina. Like bFGF, PEDF has been found to exert neurotrophic activity, inducing the overall survival of photoreceptors [39]. Significant data currently exist to suggest that certain factors such as EGF play a role in potentiating the neuroprotective action of Müller cells by stimulating their intracellular transcription and bFGF expression [40]. The VEGF released by the PRP has been shown to stimulate the proliferation of ADSCs which therefore promote the survival of grafted autologous fat and adipocytes [41]. BFGF is known to directly promote the survival of photoreceptors [42]. Synergy with electrical stimulation (ES) addresses four main aspects: survival of native cells, survival of transplanted cells, integration of transplanted cells, and functional formation of synapses/axon regeneration [43]. In recent years, the synergy between cell therapies and electrical stimulation has started to be considered as a possible treatment for degenerative diseases. Rat retinas treated with ES showed a reduction in apoptosis [44]. It has also been shown that in light-induced retinal degeneration models, stimulation with ES contains the death of photoreceptors and preserves the length of the external segment [45]. Consequently, it can be assumed that ES treatment can create a more balanced and less hostile environment by modifying the secretion of neurotrophic factors. ES affects the upregulation of neurotrophic factors in Müller cells normally involved in this protection mechanism. After ES, increased expression of in vivo beta fibroblast growth factor (b-FGF), insulin growth factor 1 (IGF-1), and brain-derived neurotrophic factor (BDNF) was observed. Conversely, ES reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and the pro-apoptotic gene Bax. The release of neurotrophic factors from the postsynaptic membrane made possible by neuromodulation, together with the enrichment of the same factors in the extracellular environment managed by autologous grafts, determines the formation of synapses at a presynaptic level, facilitating

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and strengthening neurotransmission [44, 45].

**4.3 Cell therapy and routes of administration**

The effectiveness of cellular treatments for atrophic pathologies of the retina, in order to stabilize and enhance the visual function, is based on two key elements: on the one hand, the surgical implant techniques and the cell lines used, and on the other by the quantity and quality of residual retinal cells, in other words from the earliness of the treatment. The technique must be simple, totally risk-free, and painless, and the exploited cells must not cause further damage to the residual retinal cell or to the person. Cytokines and growth factors, released paracrinically from the cells administered, must bind to membrane receptors to trigger the pathway of intracellular signal transduction. They still require retinal cells that are still alive. From the studies carried out so far, it seems that the greater the number of residual cells, the greater the interaction between GF and chorioretinal cell membrane receptors, cellular activity, and, ultimately, the improvement of visual performance (VP) [27]. The release of growth factors in a retina with very low

cellularity hardly causes detectable neuroenhancement. To achieve this goal, different approaches have been explored by inserting these cells in the subtenonian space and in the intravitreal or subretinal space. But it seems that positioning the implant in the suprachoroidal space can satisfy efficacy and safety. In fact, the graft under the tenon, although it has therapeutic significance, does not allow the growth factors produced to reach the neuroretinal tissues inside the sclera in important quantities.

Intravitreal injections of cellular material are effective and simple to perform, but it is necessary to pierce the bulb and leave this material free in the vitreous chamber. Serious complications such as infection, vitreoretinal tractions, and bleeding are also possible.

The release in the subretinal area seems to be the best for the possibility of a potential modification of cell lines due to the direct contact of MSCs with neuronal cells, but their grafting is even more dangerous when the retina is compromised by atrophic diseases [46]. The suprachoroidal graft maximizes the supply of growth factors that flow directly to the choroidal level and through the choroid to the entire retina without creating bulbar perforation. In our experience, in order to have the therapeutic action of growth factors in the retinal environment, we have explored the possibility of treating the dystrophic retina with the implantation of the cell types of mesenchymal origin mentioned above, in detail adipose stromal cells (ASC), stem cells derived from adipose tissue (ADSC) contained in the stromal-vascular fraction (SVF) of adipose tissue, and platelets (PLT) recovered in platelet-rich plasma (PRP) [47–49]. To this end, we used a surgical technique called Limoli retinal restoration technique (LRRT), described in previous works (**Figure 2**) [27, 50, 51]. The autotransplantation of ADSC, ACS, and PLT above the choroid plane improves the incretion of the bioactive factors produced in the choroidal flow and, consequently, promotes their widespread diffusion through the

#### **Figure 2.**

*Autotransplantation of adipose tissue, ADSCs from vascular-stromal fraction, and PRP according to the Limoli retinal restoration technique (LRRT). The production of growth factors (GF), characteristic of these cells, is poured directly into the choroidal flow in paracrine mode, helping to maintain the trophism of the retinal cells. The GF, through the choroidal flux, have a direct action on the choroid, on the Müller cells, on the RPE cells with improvement of the physiology of the external segments (OS), on the rods, and on the cones. Image courtesy of P. Limoli-Milan Low Vision Center.*

### *Regenerative Medicine*

retinal tissue, finally exuding in the vitreous body. This action positively influences some functional parameters after interaction with the residual cells.

The relapses of cell therapy favor a better choroidal perfusion and a higher trophism of the photoreceptors, both directly (GF) and mediated by the RPE and Müller cells. It is therefore believed that the interaction between retinal cells and growth factors plays a crucial role in leading to an improvement in the prospects of degenerative retinopathy, to prevent and/or delay its progression.

The possible goals with cell therapy are schematically:


In our study, greater foveal or retinal thickness is associated with a better prognosis. On the other hand, the lack of cells cannot make interactions between growth factors and membrane receptors possible [27, 51]. For this reason, cell therapies

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**Figure 5.**

**Figure 4.**

must be proposed as soon as the disease starts to progress, when the cells are still numerous and the patient realizes the functional change. If a disease is stable and its impact on vision is accepted by the patient, it is not advisable to propose cellular

*The figure shows a case of dry AMD with a small atrophic area. An autologous suprachoroidal implant was performed and from the first month (bottom right) an improvement in visual performance was observed in terms of sensitivity, electrical activity, and visual acuity for far and near. Retinal neuroenhancement favored the re-centering and stabilization of fixations. Image courtesy of P. Limoli-Milan Low Vision Center.*

*Patient suffering from dry AMD with areolar evolution (retinography top left). The patient treated for 3 months with supplements did not show any increase in sensitivity even if we recorded a near viscous increase (picture below left). After suprachoroid implantation of autologous mesenchymal cells (T30 and T180), we observed an increase in sensitivity outside the atrophic area and a further improvement in the visual acuity. The near vision passes from 18 points of the initial evaluation to 10 points of the final evaluation which took* 

*place 9 months later. Image courtesy of P. Limoli-Milan Low Vision Center.*

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

#### **Figure 3.**

*The image shows the effect of a suprachoroidal implantation of autologous mesenchymal cells in a patient with retinitis pigmentosa. The wealth of cells in the foveal area, documented by OCT, and therefore the high number of interactions between growth factors produced and specific membrane receptors, has allowed (T180) an increase in visual performance (BCVA, dB, pts). Image courtesy of P. Limoli-Milan Studies Center.*

#### **Figure 4.**

*Regenerative Medicine*

retinal tissue, finally exuding in the vitreous body. This action positively influences

The relapses of cell therapy favor a better choroidal perfusion and a higher trophism of the photoreceptors, both directly (GF) and mediated by the RPE and Müller cells. It is therefore believed that the interaction between retinal cells and growth factors plays a crucial role in leading to an improvement in the prospects of

• The improvement of the reading performance by stabilizing the fixation

• The conservation of useful areas such as the fovea (when it is still present) or

In our study, greater foveal or retinal thickness is associated with a better prognosis. On the other hand, the lack of cells cannot make interactions between growth factors and membrane receptors possible [27, 51]. For this reason, cell therapies

*The image shows the effect of a suprachoroidal implantation of autologous mesenchymal cells in a patient with retinitis pigmentosa. The wealth of cells in the foveal area, documented by OCT, and therefore the high number of interactions between growth factors produced and specific membrane receptors, has allowed (T180) an increase in visual performance (BCVA, dB, pts). Image courtesy of P. Limoli-Milan Studies Center.*

some functional parameters after interaction with the residual cells.

degenerative retinopathy, to prevent and/or delay its progression. The possible goals with cell therapy are schematically:

• The partial reduction of the scotoma (**Figure 4**)

obtained with neuroenhancement (**Figure 5**)

the preferential reading field (**Figure 7**)

• The slowing down of retinal disease (**Figure 8**)

• The improvement of the choroidal flow (**Figure 6**)

• Restoration and neuroenhancement on residual cells (**Figure 3**)

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**Figure 3.**

*Patient suffering from dry AMD with areolar evolution (retinography top left). The patient treated for 3 months with supplements did not show any increase in sensitivity even if we recorded a near viscous increase (picture below left). After suprachoroid implantation of autologous mesenchymal cells (T30 and T180), we observed an increase in sensitivity outside the atrophic area and a further improvement in the visual acuity. The near vision passes from 18 points of the initial evaluation to 10 points of the final evaluation which took place 9 months later. Image courtesy of P. Limoli-Milan Low Vision Center.*

#### **Figure 5.**

*The figure shows a case of dry AMD with a small atrophic area. An autologous suprachoroidal implant was performed and from the first month (bottom right) an improvement in visual performance was observed in terms of sensitivity, electrical activity, and visual acuity for far and near. Retinal neuroenhancement favored the re-centering and stabilization of fixations. Image courtesy of P. Limoli-Milan Low Vision Center.*

must be proposed as soon as the disease starts to progress, when the cells are still numerous and the patient realizes the functional change. If a disease is stable and its impact on vision is accepted by the patient, it is not advisable to propose cellular

#### **Figure 6.**

*Patient suffering from dry AMD with a peripheral areolar evolution (blue arrows at the top left). Sensitivity appears to be compromised by a paracentral scotoma, but survival of the fovea allows for good visual ability (bottom left). After autologous suprachoroidal graft of mesenchymal cells (T180), we observed an increase in the thickness of the choroid (top right and center right). Despite the increase in the paracentral scotoma which has become more profound as an expression of a now dying area, the improvement of the choroidal circulation has contributed to the maintenance of the foveal area and the stabilization of visual performance. Image courtesy of P. Limoli-Milan Low Vision Center.*

surgery. Knowledge of the overall amount of retinal cells is of particular importance: the rehabilitator and surgeon should be aware of this as a precise predictor of outcome for patients treated with cell therapy.
