Introduction
Age-related macular degeneration (AMD) is a devastating neurodegenerative disease and leading cause of blindness in people over 55 years of age that affects a central nervous system tissue, the retinal pigmented epithelium (RPE)[
1]. More than 11 million Americans over the age of 50 are affected by AMD, and with an aging population, this number will almost double by 2050[
2]. AMD is a multifactorial disease and its pathogenesis remains largely unknown, implying a complex interaction of genetic, environmental, metabolic and functional factors[
3]. Clinically, AMD leads to the impairment of central visual acuity that is required for daily tasks such as reading, writing, driving, and recognizing faces, important for independent living. AMD occurs in two general forms, dry and wet. The dry form of AMD is characterized by polymorphic deposits called drusen that accumulate between the RPE and Bruch’s membrane[
4]. The wet form is accompanied by choroidal neovascularization with subsequent formation of a disciform scar. Affected individuals may lose vision in both atrophic (dry) and the neovascular (wet) forms of AMD, however dry AMD is significantly more common, accounting for some 90% of total reported cases[
5]. Dry AMD can transform into the wet form in approximately 10% of the patients, with devastating neovascularization-induced central vision loss[
5]. There is currently no curative treatment for patients affected with AMD, with the best attempts seeking to forestall further degeneration at the retina[
6]. Vitamin supplementation is recommended and is modestly beneficial for a small population of patients[
7]. For the wet form of AMD anti-vascular endothelial growth factor (VEGF) therapy is applicable, however, the therapy is often administrated after significant damage has already been induced to the retina[
8]. Consequently, the need for developing effective treatments to improve outcomes for patients with AMD is pressing[
9]. Since the development of human embryonic stem cell lines in 1998[
10] and the advent of induced pluripotent stem cells[
11,
12] there has been enthusiasm in the scientific community for the potential utility of these cells in the understanding and treatment of AMD[
13‐
18].
Stem cell biology can offer profound insight into the mechanisms of AMD[
19] and can provide new approaches for autologous cell-based therapy in AMD as supported by the recently FDA approved clinical trial (NCT01344993). Generation of RPE derived from patient-specific induced pluripotent stem (iPS) cells may offer the ability to recapitulate the disease state and screen new therapeutics, improving upon the limited treatment strategies currently available to afflicted patients. This review will examine the breakthroughs and limitations of utilizing stem cells for disease modeling and therapeutic application in age-related macular degeneration.
AMD: disease progression and etiology
Impairment in RPE functions in AMD induces loss of central vision at the macula as a result of photoreceptor degeneration[
1]. RPE comprises a monolayer of pigmented cells with the apical membrane facing the light-sensitive outer segments of photoreceptors and the basolateral membrane facing the fenestrated capillaries of the choroid[
20,
21]. It plays many crucial roles in the retina including formation of blood/retina barrier by tight junctions, transportation of nutrients such as glucose or vitamin A from blood to the photoreceptors, conveyance of water from subretinal space to the blood, establishment of immune privilege of the eye, maintenance of ion composition in the subretinal space, light absorption, isomerization of retinal in the visual cycle, secretion of growth factors, and phagocytosis of the outer segments of the photoreceptors[
22,
23]. Due to their high metabolic activity, RPE cells are constantly subjected to oxidative stress and high levels of peroxidized lipid membranes[
24]. Extended exposure to oxidative stress can disrupt RPE tight junctions, inducing the breakage of the blood barrier and producing abnormal membrane bleb structures[
25,
26]. Furthermore, impairment of RPE function in dry AMD can induce formation of abnormal extracellular deposits called drusen that accumulate between the RPE and Bruch’s membrane[
4].Drusen, the clinical hallmark of AMD, consist of pathological extracellular deposits of degenerative material[
4,
27‐
31]. Drusen contain lipid and carbohydrate deposits, and have shown to include elements from both intracellular and extracellular sources. For example, integrins, lipoproteins, ubiquitin, inhibitor of metalloproteinase 3, advanced glycation end products, beta amyloid, fibronectin, and vitronectin have been identified in drusen[
30,
32‐
34]. Extracellular products include amyloid components, apolipoprotein E, factor X, immunoglobulin lambda chains, complement components, like the C1-q complex, late stage-activated complement components such as C5b-9 complex, and major histocompatibility complex (MHC) class II antigens[
35]. Intracellular components are mainly derived from RPE and consist of cellular and basal lamina fragments, lipofuscin and melanin, organelles[
36]. Some of the components of drusen are found in non-occular diseases. Similarities are found with amyloidosis, elastosis, and glomerular basement membrane disease[
37]. Amyloid beta, a waste product that accumulates in the CNS with aging and Alzheimer’s Disease is a key component of drusen. Increased accumulation of amyloid beta with aging is found along Bruch’s membrane, blood vessels, and in the photoreceptor outer segment[
38].
Genetic factors are now considered as reliable biomarkers to predict the risk of developing AMD, potential for disease severity and likelihood of progression[
39]. Genetic studies of AMD determined by candidate gene approaches and genome wide association studies demonstrate the involvement of an inflammatory component[
40]. Polymorphisms on chromosome 1 in complement factor H (
CFH)[
41], complement 2 (
C2), complement factor B (
CFB), complement 3 (
C3), complement factor H-related gene (
CFHR1) and complement factor I (
CFI) are associated with increased risk of developing AMD[
42‐
48]. Furthermore, polymorphisms on chromosome 10 in
ARMS2 (Age-related Maculopathy Susceptibility 2)[
49] and the HTR1A serine peptidase 1 (
HTRA1) genes predispose to wet AMD[
49‐
52]. Polymorphisms in Apolipoprotein E (
APOE), a component of drusen and a gene involved in lipid metabolism, appear to increase susceptibility to AMD[
41,
53,
54]. Proteins with major roles in regulation of plasma lipids, such as hepatic triglyceride lipase (
HL) and the cholesteryl ester transfer protein (
CETP), as well as nearby markers of the inhibitor of metalloproteinase 3 (
TIMP3) gene are also associated with an increased risk of AMD[
40]. In addition, polymorphisms in
VEGFA, a factor involved in angiogenesis, were shown to increase the risk of AMD[
55]. Interestingly, there may also be a role for maternally inherited mitochondrial DNA (mtDNA) specifically the genes encoding for the various subunits involved in oxidative phosphorylation. Inherited variants located in the mtDNA
T2 haplogroup, characterized by 2 variants in the complex
I gene, have also been associated with advanced AMD[
56]. In addition, other variants associated with mitochondrial haplogroup
J,
T and
U have also been associated with AMD[
57,
58].
A genetic condition referred as Stargardt disease is caused by a mutation in the ABCA4 gene also recapitulates the symptoms of macular degeneration but presents with much earlier onset, resulting in severe visual impairment and loss of central vision before the age of 20[
59]. Stargardt disease points to a significant genetic component that likely plays a role in development of AMD given that patients may progress later in life depending on variable environmental factors[
3,
39,
59‐
61].
Aside from genetic factors, studies have shown that environmental and epigenetic factors also play an important role in the etiology of AMD. Gene expression during ocular development appears to be greatly impacted by the epigenetics, with respect to cell types in both the lens and retina, thus having implications ranging from early stages of disease to propensity for neovascularization during progression[
62]. Concordance studies with monozygotic twins have found that nutritional and behavioral factors that influence epigenetics, such as vitamin D intake and smoking history, confer greater likelihood of developing AMD[
63]. These environmental factors have been shown to significantly alter epigenetic regulation, such as methylation and acetylation, and therefore may confer a variable gene expression profile despite identical genetic information. Most recently, a study by Wei et al. showed that hypomethylation of
IL17RC increases levels of circulating gene products, mainly inflammatory chemokines and cytokines, implicating both epigenetics and certain immune mediators in the pathogenesis of AMD[
64] . Furthermore, a recent study showed that Glutathione S-transferase isoforms mu1 (
GSTM1) and mu5 (
GSTM5) undergo epigenetic repression in AMD RPE/choroid, which may increase susceptibility to oxidative stress in the retinas of AMD donors[
65]. Another study showed that epigenetic factors regulate clusterin/
APOJ expression, one of the proteins in drusen[
65,
66]. This continues to be an area of exploration, as the subject of epigenetics in AMD was recently thoroughly reviewed[
67] and the field will undoubtedly continue to expand.
Current procedures & ramifications
Current treatment options in AMD can only hope to slow the progression of disease, although a recent review of the literature suggests that the field of AMD therapy is dynamically changing and growing rapidly, with some strategies seeking to correct the damage of AMD[
72]. Most therapies that are currently utilized in the clinic have shown mild success in slowing degeneration of RPE and preventing the onset of neovascularization. Laser therapy has been shown to significantly reduce drusen accumulation in patients with dry AMD within a three-month period post-operation[
73]. However despite the overall reduction in drusen with this laser photocoagulation, the risk of later developing choroidal neovascularization (CNV), geographic atrophy, or loss of central vision is not reduced[
74]. In fact, studies have shown that patients given higher intensity laser therapy are at a higher risk of developing choroidal neovascularization[
75].
Anti-angiogenic therapies are currently FDA-approved for neovascular AMD, with clinical trials showing significant improvement in visual acuity and slowed progression of disease[
76]. It has been shown that patients with neovascularization demonstrate abnormally high levels of VEGF-A in the choroidal layer and vitreous humor and that this expression contributes greatly to the growth and proliferation of immature capillaries[
77,
78]. These vessels demonstrate abnormal capillary lumens and increased permeability, making them particularly susceptible to spontaneous hemorrhage, thereby causing significant macular damage[
77,
78] . The anti-VEGF treatment helps to decrease the formation of new vessels and prevent further infiltration of the choroidal layer into the nearby RPE. Numerous studies have shown clinical efficacy for ranibizumab and bevacizumab, monoclonal antibodies that specifically bind VEGF-A[
79,
80]. Both antibodies have demonstrated efficacy in slowing vision loss and improving visual acuity[
81,
82]. However, some serious side effects have been noted including macular hemorrhages and retinal detachment[
83].
A surgical technique has also been designed for treatment of AMD involving the partial or total translocation of the macula to area of less diseased RPE[
84,
85]. This approach has resulted in improved visual acuity for a percentage of patients, however it presents significant complications, including fibrosis and widespread failure of RPE survival on Bruch’s membrane despite minimal improvements in vision, bleeding, corneal astigmatism, and retinal detachment with proliferative vitreoretinopathy[
86‐
88]. Many patients also experience tilting of the visual image or diplopia after retinal rotation[
84]. Given the complications associated with the surgical procedures, retinal translocation efforts have been limited. However, the concept of utilizing a healthy RPE layer persists and has inspired the implantation of non-diseased RPE cells derived from donors and stem cell-based therapies for replacement of the disease cells in the retina.
Cellular transplant as therapy for AMD
AMD is initiated with the dysfunction and death of RPE, leading to photoreceptor loss and significant deficits in vision. Therefore, the key in successful cell-based therapy in AMD would be early replacement of the damaged RPE[
21]. Several studies have shown that transplanted RPE cells have the potential to rescue photoreceptors[
89‐
91]. To date, a number of studies have investigated various stem cell types as potential sources for retinal transplantation including ESCs, adult stem/progenitor cells and more recently induced pluripotent stem cells (iPSCs)[
92‐
94]. Use of stem cells for retinal repair offers enormous promise for generation of adequate and appropriate cell populations for transplantation. Subretinally transplanted RPE that were differentiated from ESCs have led to improvements in visual acuity in preclinical models of the disease[
16,
70] In addition, human iPSCs have been differentiated towards functional RPE cells, and we have demonstrated that human iPSC-derived RPE are functionally and phenotypically similar to native RPE[
18]. Unfortunately, the subretinal transplantation of RPE cell suspensions in the Royal College of Surgeons (RCS) rat model, a genetic model of RPE degeneration, has only resulted in short-term survival and maintenance of photoreceptors[
14]. Therefore, the efficiency of cell delivery and the degree of visual rescue often remain unsatisfactory, despite the apparently positive findings[
95‐
97]. This lack of efficacy may be due to a number of reasons: 1) RPE cells are adherent monolayer cells and therefore must attach to a compliant matrix following transplantation, 2) the basal lamina layer of Bruch’s membrane may be damaged or absent in advanced retinal disease, with age, or following macular surgery[
98], lacking the supportive structure upon which RPE cells are normally attached; thus, it is difficult for newly transplanted cells to attach in such a non-tolerant environment, 3) transplanted cells may clump together rather than forming appropriately polarized monolayer RPE[
99]. Furthermore, lack of cell-to-cell contact may also lead to transition of RPE cells to inappropriate phenotypes such as epithelial-mesenchymal transition[
97,
100].
Therefore, the gap between theory and clinical exploitation remains considerable[
101,
102]. In addition, safe and efficient tissue delivery needs to be considered, as do survival and integration of the transplanted cells within the host[
103‐
105]. Any transplanted material must also be capable of maintaining an appropriate state of differentiation. In addition, immune surveillance is a significant issue, and so the approach of autologous sources of cells for transplantation to negate problems with graft rejection would be ideal[
106].
Biomaterials and cell delivery scaffolds
It has been documented that cells injected as a suspension often fail to survive and to regain a fully differentiated phenotype[
90,
107]. In addition, the viability of RPE cells delivered to the subretinal space is often dependent on the integrity of the underlying substrate, the Bruch’s membrane[
108,
109]. Thus, transplantation of a polarized RPE monolayer as a sheet seems to be more promising. Studies have shown that scaffolds made of biodegradable polyester such as poly (L-lactic acid) (PLLA) and poly (D, L-lactic-co-glycolic acid) (PLGA) could improve cell survival and organization of retinal progenitor cells (RPCs) and promote differentiation of the RPCs towards mature retinal cell phenotypes[
110]. These polymers were selected, as they are biocompatible, relatively easy to process and have been successfully used for tissue engineering applications[
111,
112]. The degradation rate of these polymers can also be manipulated by changing properties such as molecular weight and the ratio of lactic to glycolic units. Thus, polymers can be designed to degrade over the most appropriate timescale for the desired application. Several other polymers and preparation techniques have also been investigated. Many factors such as surface chemistry, mechanical properties and surface topology can affect the practicability of different materials for cell attachment and survival. Examples of other polymers are: Poly (methyl methacrylate) (PMMA) that has been used to manufacture ultrathin, micro-machined scaffolds for RPCs[
113]. Similarly, poly (glycerol sebacate) (PGS)[
114‐
116] has been used to manufacture a porous, elastic scaffold and poly (ε-caprolactone) (PCL)[
117] to produce ultrathin nanowire scaffolds. These polymers have supported successful growth of murine retinal progenitor cells both
in vitro and
in vivo in degenerative mouse models.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HM: Wrote, edited, prepared the manuscript for publication. MC: Wrote, edited, prepared the manuscript for publication. KH: Wrote and edited the manuscript. AS: Wrote and edited the manuscript. NG: Wrote, critically revised and added additional intellectual content to the manuscript. All authors read and approved the final manuscript.