2.1. Use of iPS-Derived RPE Cells for Cell Therapy
The eye is an ideal site for stem cell therapies. First, it is considered an immune privileged organ since the inflammatory responses of the eye differ significantly from those in other tissues. Second, the eye allows for easy accessibility for monitoring and imaging. Third, in the case of serious complications, the eye as a unit can be removed, due to its relative isolation from other body systems. Stem cells in turn are an appealing option for retinal cell replacement due to their pluripotency and potentially unlimited capacity for self-renewal. Currently, there are two leading options for stem cells in retinal transplantation: (i) embryonic stem cells (ESCs), which can be isolated from developing embryos four to five days after fertilization; and (ii) induced pluripotent stem cells (iPSCs), which can be created from adult cells by the viral transduction of transcription factors [
16]. However, due to the ethical and technical concerns with using ESCs, iPSCs have largely been favored for retinal transplantation.
iPSCs in particular offer a compelling alternative approach for stem cell therapy. When derived from the transplant recipient, autologous iPS-derived cells reduce the risk of post-transplant rejection and obviate the need for immunosuppression after transplantation. The well-described iPSC culture standards also aid in the development of functional testing and optimization studies. Likewise, RPE transplantation into the retina poses fewer challenges than other kinds of cell transplantation since routine culture of RPE cells has been well described [
17,
18]. RPE monolayers exist in an easily identifiable hexagonal structure and can be isolated and transferred to a variety of substrates without the need for synaptic integration. Subsequently, studies on RPE replacement therapies using pluripotent stem cells have progressed rapidly. A multicenter trial focusing on the treatment of dry macular degeneration and Stargardt macular dystrophy showed that purified human ESC-derived RPE can be subretinally injected into patients with good results [
5]. This is also possible since the retina normally enjoys relative immune privilege, due to the blood-retinal barrier. This barrier consists of non-fenestrated retinal vasculature ensheathed by pericyte and astrocyte processes on the inner aspect and by tight junctions between RPE on the outer aspect. In a healthy state, this blood-retinal barrier provides protection to transplanted cells beneath the retina from the systemic immune system. However, in a diseased RPE state, the monolayer is disrupted due to faulty tight junctions and the retina may also become much more pro-inflammatory [
19,
20]. Therefore, cells transplanted into a diseased retina are likely to be at a higher risk for rejection, so autologous iPSC transplantation represents the best stem cell approach for curing degenerative retinal diseases. In fact, hiPSC-derived RPE has recently been approved in Japan for use in patient safety trials for treatment of AMD [
21].
Currently, human iPS-derived RPE (iPS-RPE) experiments are largely confined to animal models. In 2009, Carr
et al. performed subretinal injections of dissociated human iPS-RPE into Royal College of Surgeons (RCS) rats and observed restoration of RPE phagocytotic function, as measured by intracellular RHO staining, and long-term preservation of visual function, as measured by optokinetic head-tracking [
22]. Another model is the RPE-specific protein 65 kDA (RPE65) mutant mouse model, which is used to study Leber congenital amaurosis (LCA) and RP since the RPE 65 defect leads to a faulty isomerase which can no longer convert the chromophore necessary for rhodopsin to detect light [
23]. In 2012, Li
et al. injected dissociated human iPS-RPE into the subretinal space of the RPE65 mutant mouse model and showed integration of the transplant with host RPE, as well as a modest improvement of visual function as measured by electroretinogram (ERG) [
10]. The
Mfrprd6/
Mfrprd6 (rd6) mouse, which has a deletion in the Membrane Frizzled-Related Protein (
Mfrp) gene, is another widely used model. The resulting MFRP protein, an RPE-specific membrane receptor of unknown function, is abnormal and the mice exhibit progressive retinal degeneration, making the model a preclinical and progressive model of RP [
24]. In a recent study, subretinal injections of AAV-packaged wild-type
Mfrp into rd6 mice showed improvement in visual function and RPE cell layer thickness [
25].
The most advantageous aspect of iPSC based therapy is the potential of autologous transplantation, which intends to address the problem of immune rejection. Despite the assumption that these autologous cells should not provoke an immune response in the recipient from whom the cells were derived, there have been conflicting reports that raise some concern of the immunogenicity of iPSCs. In a recent study, teratomas originating from subcutaneous injection of murine derived iPSCs were found to have abnormal gene expression in some cells, which elicited a T-cell dependent immune response in syngeneic mice [
26]. However, when Guha
et al. transplanted various types of murine iPS-derived cells to a site under the kidney capsule of B6 mice, they found no evidence of immune response to the iPSCs, no increased T cell proliferation
in vitro, no rejection of syngeneic iPSC-derived cells after transplantation, and no antigen-specific secondary immune response [
27]. Findings by Liu
et al. in 2013 suggests that iPSC immunogenicity increases with
in vivo differentiation, as the authors observed immune responses after transplantation of differentiated iPS-derived cardiomyocytes but no response when transplanting undifferentiated iPSCs [
28]. In contrast, Morizane
et al. performed a direct comparison between autologous and allogeneic transplantation of iPS-derived neural cells in brains of non-human primates and found that the autologous transplantation of iPS-derived neurons caused only a minimal immune response in the brain, while the allografts elicited an acquired immune response [
29]. Moreover, a higher number of dopaminergic neurons survived in autografted iPS-derived cells, which further support their use. Taken together, these findings reveals that different cell types derived from iPSCs might have distinctive immunogenicities in their syngeneic hosts. For the development of human iPS-based cell therapy, there remains still a challenge to evaluate the immunogenicity of human iPS-derived cells in an autologous human immune system.
2.2. Progress of RPE Disease Modeling Using iPSCs
Human iPS cells are useful for modeling RPE disorders since they can be isolated, expanded, re-seeded, and closely monitored both morphologically and functionally prior to testing [
30]. Phenotypes of patient-specific iPS cells may differ from those from a mouse model with the same mutation [
25], underscoring the necessity for multiple models of human genetic diseases. Since differences in phenotypic expression can be observed among species with the same genetic mutation, it is important to study patient-specific cell lines as a complement to mouse models.
The first retinal disease modeled with patient-specific iPS cells is Best vitelliform macular dystrophy (BVMD) [
13]. Caused by a defect in the RPE gene BEST1, which results in the subretinal accumulation of photoreceptor waste products, BVMD is characterized by central vision loss due to photoreceptor death. Singh
et al. created iPS-RPE from affected patients and compared them with those created from unaffected siblings. From their model, they concluded that the pathophysiology of the disease included delayed rhodopsin degradation after photoreceptor outer segment feeding, as evidenced by disrupted fluid flux and increased accumulation of autofluorescent material [
13]. This hiPSC model of BVMD possessed functional deficiencies consistent with the clinical features of the disease and was used to characterize clinically relevant disease phenotypes for BVMD.
iPS-derived RPE cells have also recently been used to model and study the pathophysiology of AMD. While genome-wide association studies (GWAS) have identified risk alleles for the disease, such as the ARMS2 and HTRA1 genes, how these alleles lead to pathology is still unclear. There is currently a lack of appropriate models for AMD; autopsy eyes from end-stage patients already possess terminal changes and cannot be used to determine how abnormal gene expression can lead to RPE pathology, and mice do not have maculae. To bypass these obstacles, Yang
et al. created a model for AMD by obtaining patient-specific iPS-derived RPE and pharmacologically accelerating the aging process with treatment of bisretinoid N-retinylidine-N-ethanolamine (A2E) and blue light [
12]. From a proteome screen of multiple A2E-aged patient-specific iPS-RPE lines, impaired superoxide dismutase 2 (SOD2) function was identified as a high risk factor for developing AMD. Using their iPS model, the researchers concluded that the ARMS2/HTRA1 risk alleles decreased SOD2 defense, making RPE more susceptible to oxidative damage and thus contributing to AMD pathogenesis.