Mesenchymal Stem Cell Transplantation for Retinal Degenerations and Dystrophies: Present and Future

Corresponding Author: Guanfang Su, Eye Hospital, 2nd Teaching Hospital of Jilin University, Changchun, China Email: sugf2012@163.com Abstract: Retinal degenerations are the main causes of irreversible blindness in developed countries. Up to date the main pathological mechanisms of these diseases are not fully understood and consequently there is no complete treatment option for those diseases. In this aspect stem cells have drawn attention of many researchers and health care professionals. Considering ethical issues, safety and facile isolation Mesenchymal Stem Cells (MSCs) are more preferable for practical use. They have been used for several preclinical and clinical trials. In general the results were promising, however broader practical use should be preceded by resolving many problems and questions. In this review we will describe mesenchymal stem cells, especially those derived from Bone-Marrow (BMSC), their main features, privilege, mechanisms of action and their potential use for the treatment of retinal degenerations. We will also discuss the results of several pre-clinical and clinical trials.


Introduction
Retinal degenerations, which are mainly due to the genetic defects, diabetes, aging and environmental factors, are the main causes of irreversible blindness in developed countries. The final result of all types of retinal degenerations and dystrophies is the loss of photoreceptor cells, which leads to irreversible vision loss. Generally, all types of retinal degenerations progress through similar main mechanisms, with differences at the first step of the pathologic pathway. They all progress to the apoptosis of photoreceptors, total ablation of the Outer Nuclear Layer (ONL) of the retina and consequent neural remodeling of the inner neural retina (Marc et al., 2003). In the meanwhile, some types of retinal cells can escape apoptosis and preserve their main functional ability as latent cells, which made possible restoration of lost vision with electrical implants (Da Cruz et al., 2013). This gives hope that transplantation of new photoreceptors will reactivate retina and restore vision.

Stem Cells
Stem cells are undifferentiated cells with the ability of self-renewal and differentiation into more specialized cells.
The ideas about existence of stem cells came first after bombing in Hiroshima and Nagasaki in 1940 s.
Those who died over a prolonged period from lower doses of radiation had compromised hematopoietic systems that could not regenerate either sufficient white blood cells to protect against otherwise nonpathogenic infections or enough platelets to clot their blood. Later, it was demonstrated that mice that were given doses of whole body X-irradiation developed the same radiation syndromes; at the minimal lethal dose, the mice died from hematopoietic failure approximately two weeks after radiation exposure. Soon thereafter, using inbred strains of mice, scientists showed that whole-bodyirradiated mice could be rescued from otherwise fatal hematopoietic failure by injection of suspensions of cells from blood-forming organs such as the bone marrow. In 1961 the hematopoietic stem cells were isolated from bone marrow and since then are used in clinical practice for hematologic diseases. Friedenstein et al. (1976) described mesenchimal stem cells and called them fibroblast precursors. Later, Thomson et al. (1998) discovered embryonic stem cell, Yamanaka and Takahashi (2006) showed that they were able to transform typical fibroblast from adult skin into pluripotent stem cell and they named it induced Pluripotent Stem cell (iPS). Now three main types of stem cells are in use: Embryonic Stem Cells (ESC), Adult stem cells, Induced Pluripotent Stem cells (iPS). IPS and ESC grow faster, are being pluripotent and can differentiate into any kind of cell upon appropriate stimulation, but they have practical problems, halting their clinical use. They raise ethical issues and have high risks of immune rejection and tumorogenesis. In contrast to ESC and iPS, MSCs are more suitable for stem cell therapy,because of facile isolation and invitroculture,prolonged self-renewal ability, autotrasplantation, low risk of tumor formation (Caplan, 2009) and a lack of ethical issues. There have been many studies showing that these cells are safe and have good plasticity, without producing tumors (Herzog et al., 2003;Phinney et al., 2006;Zhang and Alexanian, 2014). As such, they are preferred for transplantation therapy.
Mesenchymal stem cells can be isolated from different organs and tissues: Bone-marrow, adipose tissue, teeth pulp and also amniotic fluid, umbilical cord, placenta, etc (Erices et al., 2000;Prusa et al., 2003). The most researched and used are Bonemarrow derived Mesenchymal Stem Cells (BMSC). Generally BMSC are found in bone-marrow niche where they make trophic basis for hematopoietic cells (Bianco et al., 2001). Several researchers suggest, that these BMSC are located around the vessels in the stroma of BM and they play rolesaspericytes, that can be mobilized from BM with special stimulus and migrate to the site of injury to perform their cellular functions (Wu et al., 2007;Caplan, 2008). The ratio of BMSC is decreasing with age: For newborns it's 1:104 of nucleated cells of BM; for teens it goes down to 1:105 and for adults it goes further down to 1:106 or less (Caplan, 2009). This is consistent with that regeneration is faster and more complete in children than in adults and degenerative diseases start to manifest mainly in aged people.

Bone Marrow-Derived Mesenchymal Stem Cells
Bone marrow-derived Mesenchymal Stem Cells (BMSCs) are described with several features: They are isolated from bone-marrow, adherent to the bottom of plastic culture dishes, obtain spindle shape like fibroblasts and express a combination of cell surface markers (CD105, CD90, CD29, etc and negative for CD31, CD45, CD11b, etc). Under appropriate conditions, they can give rise to muscles, adipose tissue or bone cells. Although above characteristics are well among scientists, more detailed characterizations show that MSC from different species, organs and tissues, despite having many common features, have differences in the expression of cell surface markers, cytokine production and growth factors production, in addition tochanging certain characteristics after ex-vivo culturing (Javazon et al., 2004;Martins et al., 2009;Bayati et al., 2013).
MSC are able to recover injured tissues and organs through following mechanism: Cellular transdifferentiation and cell replacement, paracrine trophic function (Crisostomo et al., 2008). They also can be used as non-viral, safe, prolonged acting vehicles for gene therapy or drug introduction (Arnhold et al., 2006;Park et al., 2012).
The beneficial action of transplanted MSCs is suggested to be due to the production of trophic factors (Uccelli et al., 2011), however the ability of integration and differentiation is still questionable (Harris et al., 2006;Xu and Liu, 2008).
MSC are preferred for auototransplantation. Recently researches showed that BMSC don't express MHC class II antigens, co-stimulatory molecules CD80, CD 84 or CD40 and only express low quantity of MHC class I antigens, so they can be invisible for recipient immune system. This makes MSCs universal also forallo-and even xeno-transplantations (Chiou et al., 2005). It was also shown that MSC itself suppresses immune system by suppressing T-cell, Bcell and NK activity (Tse et al., 2003;Ribeiro et al., 2013). This function was even used for clinical experiments, Graft Versus Host Disease treatment, etc (Ning et al., 2008;Puymirat et al., 2009;Baron et al., 2010). MSC derived from adipose tissue, umbilical cord, amniotic fluid and bone-marrow showed generally similar characteristics, so cells for transplantations can be obtained from medical wastes (liposuction, placenta, Umbilical Cord (UC), amniotic fluid after delivery) and used safely for allotransplantations (Oh et al., 2011;Tejaswi et al., 2013;Ribeiro et al., 2013).

MSCs in Retinal Degenerations
Retinitis Pigmentosa (RP) and Age-Related Macular Degeneration (AMD) are the most common cases of retinal degenerations. Many scientific groups focused on these diseases, aiming at revealing pathological mechanisms and final treatment options for them. They also serve as main models for stem cell transplantation for retinal degenerations.

Retinitis Pigmentosa
Retinitis Pigmentosa is described by the primary or secondary loss of photoreceptors due to gene abnormalities. Its rate is about 1:3000-1:7000. Usually, RP starts with the loss of rods and followed by the loss of cones. RPE cells detach from Bruch's membrane, migrate to inner retina and associate with abnormal vessels. Extracellular matrix deposits between RPE cells and endothelial cells of the vessels and closely resembles Bruch's membrane in situ. Further disease progress to the formation of bone spicules (Ehinger, 2000). RP manifests in early teens with poor night vision (nyctalopia), prolonged dark adaptation and gradually progresses to tunnel vision and total blindness by the age of 40-50 years. Nevertheless, some patients preserve their central vision (preservation of cones) even with lost visual fields. Nearly 180 gene mutations are known for RP, but they represent just 15% of phenotypic appearance of the disease. This makes gene engineering therapy quite difficult or impossible for most patients. No effective treatment is currently available for patients with RP. Under these circumstances, stem cell therapy may be a hope to those patients.
Different groups showed that with special growth factors or mediums they were able to differentiate BMSC into neural progenitor cells and further to retinal cells (Kicic et al., 2003;Yang et al., 2010;Zhang and Alexanian, 2014). This lead to experiments on animals and recently to several clinical trials for RP treatment.
For the retina transplantation there are three possible routes: Intravitreal injection, subretinal injection and intravenous injection. The first two are local and have their benefits and drawbacks: Particularly subretinal injection provides direct contact between BMSC, RPE layer and photoreceptors, but it is difficult to perform and risky.During degeneration Muller glial seal occupies the area and may prevent cell implantation and incorporation (Kicic et al., 2003;Arnhold et al., 2007;Inoue et al., 2007;Gong et al., 2008). Intravitreal injection is easy to perform, less risky, but it doesn't provide close contact with retinal and transplanted cells (Castanheira et al., 2008;Hill et al., 2009;Li et al., 2009;Wang et al., 2010a;Tsuruma et al., 2014). In addition Internal Limiting Membrane (ILM) may prevent cells from integration. It will be interesting to see whether intravenous injection can be a successful route of the cell therapy for the retina. Many works showed that after IV injection MSCs migrated and localized in injured liver, heart, brain and retina, but in this case more cells were required for injection (Xu et al., 2007;Jackson et al., 2010;Wang et al., 2010b). In fact BMSC express main chemokine receptors (CC, CXC, C and CX3C) and can be attracted by their ligands, especially SDF-1 and IL-8 (Ringe et al., 2007;Shi et al., 2007). It is known that production of SDF-1 is augmented after injuries, so BMSC migrate exactly to the injured tissue, remaining in very small quantities in other organs. But in all preclinical trials animal models with only one disease were used. Thisis not similar to human body, especially in diabetic or aged people, with many problems within body. In this case it's not clear, whether stem cells will be able to migrate to relatively small retina? Although many beneficial effects from BMSC transplantation have been shown, it's not clear whether BMSC can be used to treat diseases at multiple sites of the body with intravenous injection.
After choosing the best route the second problem is when BMSC should be transplanted? For ESC even final stages of disease are eligible for transplantation with normal transdifferentiation and functional recovery (Singh et al., 2013), unfortunately for BMSC transplantation results are not that promising. The problem is due to remodeled retina, Muller glial seal and not permissive extracellular matrix for transplanted cells survival. This was shown in the work of Johnson and coworkers, who showed that after peeling of ILM with Muller cells neurits better integration and transdifferentiation rates were observed (Johnson et al., 2009). In animal models, highest levels of integration were observed in the models of retina laser injuries, which disrupt ILM (Castanheira et al., 2008;Wang et al., 2010a). Also higher integration and transdifferentiation rates were reported, if treatment was started within 24-48 h after injury.
However, the published articles showed rescue of anatomical structure and function of the retina compared to control untreated groups, but none found the toxic reaction or tumor formation after an observational period of over 230d. This made the recruitment of clinical trials with MSC transplantation possible. Several groups have already reported onthe safety and feasibility of an intravitreal injection of stem cell (Jonas et al., 2010;Siqueira et al., 2011;Siqueira et al., 2013).
Several pre-clinical and clinical trials are summarized in Table 1.
But before wide use of stem cell transplantation in clinical practice many questions should be answered, particularly: • How many cells should be transplanted and from which passage of cells • Is it necessary to primarily induce cells in-vitro to specified lines or should they be induced with in-vivo • In which stage of disease it is more effective and safer to transplant • Which is the best transplantation route

Age-Related Macular Degeneration
Age-Related Macular Degeneration is a multifactorial disease caused by genetic predisposition and environmental factors, occurring with a rate of 18% at the age of 50-60 and over 30% at the age of 70 (Friedman et al., 2004). It's the leading cause of irreversible blindness in the western countries and the rate is continuously going up because of the rapid increasing of averaged age (Kolar, 2010). Generally, AMD is classified as dry or nonexudative AMD and wet or neovascular AMD, according to the presence or the absence of Neovascularization (NV). Two late forms of the disease (GA, CNV) are so different in their manifestation and in treatment options, that it is appropriate to consider them as two different diseases and it's also not clear, what is the switch point between GA and CNV, or in another word, what determines the disease to progress into GA rather than CNV or vice versa? There are several treatment strategies for AMD (AREDS formulations, laser photocoagulation, photodynamic therapy, anti-VEGF drugs, etc.), but none of them is able to stop the progression, not to mention to cure the disease. So for those patients stem cell therapy is believed to be the main choice, however, it is quite different for two forms of disease: In GA use of stem cells is quite similar to that of RP, but in neovascularizations (wet AMD, DR) mechanisms are more complicated and controversial. The main character of wet AMD is neovascularization. Neovascularization is connected with impaired proportion of pro-and anti-angiogenetic factors, with overexpression of the pro-ones, especially VEGF. MSCs produce VEGF in big quantities, which contribute to angiogenesis, so in eyes, stem cells may lead to neovascularization and have pathological effects. Supportively, many published works show that endogenous stem cells (especially Lin-HSC, EPC) participate in new vessel formation. Most studies used mice models with bone-marrow reconstituted by bonemarrow-derived stem cells from the Green Fluorescent Protein (GFP) transgenic mice and CNV was induced by laser spots (Espinosa-Heidmann et al., 2003;Sengupta et al., 2003;Takahashi et al., 2004;Tomita et al., 2004;Espinosa-Heidmann et al., 2005;Sengupta et al., 2005;Lecomte et al., 2011). Some studies showed that nearly 50% of cells in the new vessels were GFP positive, indicating they arise from bone-marrow. Hou et al. (2010) used external BMSC and showed their incorporation in new vessels without significant increase in CNV size. What is more important is that they used induced cells to produce Pigmented Epithelium-Derived Factor (PEDF), a strong antiangiogenic factor. Then they observed a decrease in CNV formation and vessels surroundedby RPE cells, which restricts neovascularization. They proposed to use stem cells only as a safe, prolonged source of anti-angiogenic factors (Hou et al., 2010). Later, blood examination from patients with active CNV showed higher level of BM derived stem cells in peripheral blood, but with low functional ability: There is no significant difference in the CFU-EC between patients with CNV and the control group; in case of bilateral CNV the levels of CFU-EC andSDF-1were significantly decreased (Yodoi et al., 2007;Machalinska et al., 2011). It seems that BM derived stem cells don't contributeto new vessel formation, rather, they contribute tovessel maturation and regulate growth. Endogenous quantity of BMSCs is not enough and they are not functionally active, so they are not able to maturatenewvessels. Consistently, research of Chung et al. (2011) shows that after laser injury in non irradiated mice, injection of extra bone marrow stemcells contribute to resolution of retinal detachment without proliferative component (Chung et al., 2011). In the regards, further experiments are needed to understand the roles of transplanted MSCs in CNV formation.

Conclusion
Being the leading causes of blindness in the western countries, retinal degenerations don't have a definitive treatment option, which is a significant problem for both the patient and the clinicians. In the last 30 years research was focused on stem cells and their possible application in the treatment of retinal degenerative disease.
Despite the extensive research done in the past three decades and many pre-clinical and several clinical trials conducted, many questions and problems are still not solved, preventing wide application of stem cells in clinical practice. But significant progress has been made toward both prevention and treatment of retinal degenerations and we envision the clinical application of stem cells will open a new era of treating those eye diseases.

Conflict of Interest
Authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.