Many outer retinal degenerative diseases such as retinitis pigmentosa, age-related macular degeneration and Stargardt disease share a common
 
endpoint of photoreceptor and retinal pigment epithelium (RPE) loss that leads to severe vision loss. RPE replacement strategies may be feasible for many of these diseases, but AMD is the subject of particular interest because of its prevalence. In this article, we focus on AMD, recognizing that many stem-cell therapy advances for AMD may translate to other degenerative retinal diseases.

Anti-VEGF therapy has been very effective in disrupting the progression of neovascular AMD (nAMD) and even reversing vision loss in many cases. However, non-nAMD, characterized by regions of irreversible RPE cell loss, or geographic atrophy (GA), slowly results in severe and irreversible vision loss. The Age-Related Eye Disease Study (AREDS) showed progression from intermediate to non-nAMD takes about 2.5 to five years.1 To date, no effective treatment exists for non-nAMD.

The exact etiology of AMD is unknown, but a combination of genetic and environmental factors have been implicated.2,3 While the primary cause of acute vision loss in nAMD is choroidal neovascularization (CNV), RPE loss and geographic atrophy are final common endpoints for both nAMD and non-nAMD.

Questions Stem Cell-based Therapy Must Answer
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The RPE monolayer is essential both for the survival of photoreceptors and the underlying choriocapillaris. Among other functions, the RPE secretes pigment epithelial-derived factor (PEDF), vascular endothelial growth factor (VEGF) as well as the extracellular matrix which may have an antiangiogenic function.

For this reason, any stem cell-based therapy must answer several questions, among them:

-Will the donor RPE survive long enough to justify surgical risks?  
-Will the donor RPE maintain its polarity and function; that is, will donor RPE form functional synaptic connections with host photoreceptor neurons and facilitate the visual cycle?
-Can the donor RPE reverse or prevent further degeneration?
-Are sources of donor RPE ethically available?
-What is the best surgical technique?

While RPE
Focus for the Future Of Stem Cell Research

•  Development of novel, noninvasive
diagnostic tests to assay retinal pigment epithelium and retinal function at the molecular and cellular level.
•  Development of novel surgical tools and surgical methods for optimal delivery of RPE.
•  Advancement of stem-cell science for the purpose of understanding host retinal immune response.
•  Development of clinical-grade methods to genetically modify stem cell-derived RPE.
•  Development of rehabilitation strategies for training patients to use optimum fixation viewing techniques with transplanted stem cells for visually guided tasks to enhance function.
replacement strategies are not likely to benefit the acute vision loss associated with CNV in nAMD, the prevalence of GA as a final common endpoint in both nAMD and non-nAMD suggests that both forms of the disease may benefit from RPE replacement strategies at some point.  

Investigators demonstrated proof-of-principle decades ago that RPE transplantation could work in human subjects and animal models.4–6 These studies were complemented by macular surgeries that demonstrated that translocating the fovea over an apparently normal region of RPE allowed short-term visual gains.7 Unfortunately, long-term follow-up showed high GA recurrence rates in the new subfoveal RPE region.8,9

Other sources of cells have been used as donor RPE including homologous, heterologous or autologous adult RPE transplantation as well as fetal RPE transplantation.8,10-14

Attempts at human RPE transplantation in GA using autologous and allogeneic RPE have had limited success but strongly suggest that RPE replacement strategies can work if the limitations associated with the cell sources and surgical methods could be overcome.15-20
​
Sources for Stem Cells
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Investigators have reported several hundred RPE or stem cell-based grafts since the first human homologous and autologous RPE transplantation in 1991, and this number is growing.16,21,22 Some of these studies use non-RPE stem-cell populations, such as bone marrow-derived stem cells, delivered with either intravitreal or intravenous adminstration.23 The predominant mechanism of action of these studies is through nonspecific trophic effects of stem cells to support retinal and RPE function. Interestingly, evidence has shown RPE repopulation by systemic administration of some bone marrow-derived cell lines in animal models,24,25 but the safety and efficacy of systemic administration seems more problematic than with local delivery methods, which are also viable and very well developed.  

Among the numerous sources of donor RPE, induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESC) are the most feasible. Both stem cell forms are a source of potentially endless RPE donor cells that can be fully differentiated into RPE either as cell suspension or monolayers.

Take-home Point
The quest to restore sight from several blinding retinal diseases has shown promise through groundbreaking research emerging from stem cell-based therapies. Subretinal transplantation of autologous or allogeneic retinal pigment epithelium (RPE) over the past several decades has shown that stem cell-derived RPE can rescue photoreceptor function and improve some aspects of visual function in animal models and in human subjects with neovascular age-related macular degeneration and non-neovascular AMD. Thanks to advances in stem-cell biology and regenerative medicine, new methods of differentiating RPE from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) can provide a potentially unlimited supply of RPE cells. Parallel advances in noninvasive retinal imaging and vitreoretinal surgery now provide the tools to effectively identify relevant anatomic changes with functional correlations in vivo and deliver stem cell-based therapies. This review focuses on the potential and challenges of stem cell-based treatments for AMD.


• iPSC. Induced pluripotent stem cells are derived from fully differentiated adult somatic cells that are reprogrammed in vitro to differentiate into RPE.25 They perform phagocytic functions, demonstrate RPE-like gene expression profiles and promote photoreceptor survival.26–28 A Japanese study that used autologous iPSC RPE cells for AMD therapy was recently stopped due to unexpected mutations (three aberrations in DNA copy number),27,29 and was suspended because of concerns about the possible effects the deletions could have.30.31

• hESC. In contrast, human embryonic stem cells are derived from the inner cell mass of blastocysts and can also be programmed to differentiate into RPE.31 Manufacture of these cells can undergo strict
quality-control testing and avoid the complicated and high-risk process of surgically harvesting autologous or allogeneic grafts.

Lastly, both
 
Figure. Methods for delivering stem cells include: A) intraocular encapsulated cell technology via insert; B) intravitreal injection; C) pars plana vitrectomy with subretinal cell suspension injection via cannula; D) pars plana vitrectomy with subretinal graft placement via cannula; and E) subretinal delivery of stem-cell suspension.
hESC and iPSC may be amenable to genetic manipulation. This may allow manipulation of immunogenic properties or introduction of new genes to supplement function of the cells in vivo.
Regardless of the stem cell source, transplanted RPE cells will likely require some form of mechanical or physical substrate because RPE survival depends highly on extracellular substrates.32,33 For example, a healthy Bruch’s membrane has been shown to improve the survival, repopulation and confluence of RPE cells.34

Such a substrate must not only support RPE attachment and differentiation; it must also be amenable to surgical manipulation and be immunologically silent. While donor RPE in suspension may attach to exogenous Bruch’s membrane, they more commonly aggregate in multiple layers and assume an abnormal phenotype.35

In addition, dissociated hESC-derived RPE can dedifferentiate and may not redifferentiate appropriately. Groups have developed various scaffolds to support RPE survival and implantation, including biodegradable scaffolds and biologically inert but nondegradable scaffolds such as polyester membranes, plasma polymers, polyimide and parylene.20,36–39 Researchers have shown that subretinal implantation of human embryonic stem cell-derived RPE monolayers on a parylene substrate have improved survival compared to cell suspensions.40,41

Evaluating the Host Tissue
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Detailed assessment coupling macular anatomical and functional correlations are critical for assessing the viability of a subject’s retina for stem cell-based rehabilitation and for postoperative assessment of
success.

Spectral-domain
 
optical coherence tomography (SD-OCT) allows detailed assessment of photoreceptor structure and has shown that not all regions of GA are uniform.40,42 Autologous RPE transplantation suggests that subjects with recent loss of visual function may benefit most from RPE transplantation.43 It seems that the visual potential of neurosensory retina over areas of long-standing GA is poor.

Subjects with such severe and chronic anatomical changes may not show significant improvement in visual function under any circumstances. It is possible that RPE transplantation in this population may preserve the remaining RPE and retina at the borders of geographic atrophy lesions or slow the progression of disease.

It is exciting to speculate about stem-cell therapy as a replacement for neurosensory retina, specifically photoreceptor cells, either alone or in addition to RPE transplantation, although this is not currently in any clinical trial. Since most cases of severe non-nAMD ultimately involve loss of photoreceptors, it is likely that such a treatment will be necessary for severe disease.  

Interestingly, transplantation of human fetal retina into nude adult rat retina has resulted in histologically detectable synaptic connections.44 Adult
 
retinal transplantation in humans has been demonstrated to be safe in subjects with end-stage retinitis pigmentosa and AMD, but gains in visual function have not been demonstrated.45–48



Stem-Cell Delivery Techniques
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Regardless of the source of RPE, the delivery techniques include intravitreal injection, subretinal injection of cell suspension of RPE or subretinal placement of a sheet of tissue containing RPE (Figure D).49

Injection of cell suspensions into the subretinal space is advantageous because it does not require a large retinotomy and is relatively fast and simple.50 Major limitations of this method include the reflux of RPE cells into the vitreous, relatively poor adherence to Bruch’s membrane and failure to form an effective monolayer.36,51

Alternatively, the delivery of subretinal sheets containing RPE has also been demonstrated but requires larger retinotomies, takes longer and is prone to incorrect implant orientation and more postoperative complications.36,52

The main advantage of implanting RPE sheets with a scaffold is that the orientation, polarization and function of the RPE is more likely to consistently replicate that of the native RPE.

Despite the limitations of both methods, current clinical trials hold some promise.22,44,52,53 In some cases cells can be implanted in a self-contained and nonimmunogenic manner that allows the secretion of trophic factors but isolates the cells from the host environment.  RS

The authors acknowledge the California Institute for Regenerative Medicine (CIRM) and Research to Prevent Blindness (RPB) for grant support.

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