Adult human RPE for transplantation: renewing an old promise

Abstract

Considering the incidence of retinal pigment epithelium (RPE)-related blinding disease will grow to 200 million globally by 2020, the impact of restoring vision by successfully replacing failing or dying RPE is great. In spite of fervent efforts to use primary RPE as a source for transplantation for over 30 years, a clinical therapy has yet to be developed. Due to the progress of pluripotent stem cell technologies and development of RPE differentiation protocols, primary human RPE culture has largely been set aside as a source of RPE for transplantation, as human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived RPE have become the current popular source for transplantation. Recently, a series of seminal advances in human primary RPE culture has renewed an interest in their potential as a source for RPE transplantation. Primary RPE are better studied and understood than hESC/hiPSC-derived RPE, have an inherent lower risk of tumor formation, and can be Major Histocompatibility Complex (MHC) donor-matched, making them valuable candidates alongside pluripotent stem cells as sources for cell transplantation therapy for RPE-related eye diseases.

In context

Some of the most prevalent blinding diseases, including Age-related Macular Degeneration, Stargardt’s Disease, Retinitis Pigmentosa and others, affect a single epithelial layer in the back of the eye, called the retinal pigment epithelium (RPE). For over the past 40 years, much hope has rested in using adult RPE, for example isolated from cadaver donors, for transplantation, to replace the diseased RPE in affected patients. Critical barriers to this objective are 1. being able to isolate and grow RPE that maintain their physiological and morphological characteristics in vitro and 2. assure successful engraftment and survival of the transplanted cells. What we observed was that often, once dissected, RPE isolated from cadaver donor eyes would change their physiology and not maintain their RPE functions when cultured in vitro. Here we summarize new advances in using adult RPE, which have renewed their promise in treating RPE-related eye diseases.

• adult stem cell
• retinal pigment epithelium
• cell transplantation
• eye disease

The retinal pigment epithelium (RPE) is a single layer of cells, which performs many functions necessary for healthy phototransduction (1) and any dysfunction in these roles leads to vision loss. Cell transplantation of healthy RPE to replace dystrophic or lost RPE has been considered a potential therapy to preserve or even restore lost vision. Considering the number of individuals with RPE-related blinding disease to be over 200 million worldwide by 2020 (2), obtaining sufficient RPE for such a demand has been a challenge on which many in the RPE field are focused. Here, we cover some of the roadblocks, surgical successes, and recent developments that renew the sense that we are close to providing a transplantation therapy for patients suffering from RPE-related diseases.

Much hope rests on the idea that a simple ‘RPE-only’ transplantation can be developed, a method that can be scaled up to threat such a large population of patients.

The foundation of the hope rests on a handful of surgical successes. Macular translocation is the surgical rotation of the retina so that the retina is placed on a healthy RPE layer. While the success rate is variable due to the complexity of the surgery, when there are no complications, macular translocation has demonstrated dramatically improved vision (3, 4). Similarly, a peripheral RPE-choroid patch translocation underneath the macula also improves vision, yet the risks of complications are high (5). As a result, only a handful of retinal surgeons attempt either of these surgeries and few patients receive their benefit.

Current state of the RPE transplantation

Transplanting cells as a therapy for human disease is relatively new and limited primarily to blood-based diseases (6, 7), skin (8), and cornea (9), though much hope rides on its potential to be effective in treating diseases throughout the human body. RPE transplantation for RPE-related diseases is at the forefront of this movement, because these cells and their location possess many attractive attributes when developing a cell therapy. The RPE is a single layer and is therefore considered to be one of the more easily transplantable tissues, because elaborate or long-distant connections do not need to be re-established. The eye has a unique feature in providing a window, the cornea, through which progress of the transplanted tissue can be easily monitored. RPE are pigmented and distinguishable from the transparent retina, particularly in the regions where native RPE is lost. Sophisticated ophthalmological surgical techniques, imaging devices and visual acuity tests are additional benefits, making the eye the ideal location to pioneer the development and optimization of cell transplantation therapy.

Alongside the struggles of primary RPE culture was the development of human embryonic stem cell (hESC) (10), retinal neurospheres (11), induced pluripotent stem cell (hiPSC) cultures (12), and differentiation methods towards the RPE lineage (13, 14). These cell sources hold the promise of unlimited supply and, in the case of hiPSC, autologous transplantation potential. As a result, attention to and development of primary human RPE as a source for transplantation therapy have largely been set aside.

Many groups are moving swiftly into the clinic to test whether these seemingly unlimited sources of cells will be the next clinical revolution. A clinical trial, funded by Ocata Therapeutics, Inc. (formerly Advanced Cell Technology), is ongoing, where hESC-derived RPE are injected into the subretinal space as a single cell suspension. Preliminary reports indicate the treatment so far is safe, and has led to an improvement in vision in some patients (15, 16). The London Project to Cure Blindness led by Dr. Pete Coffey in collaboration with Pfizer plans to use hESC-derived RPE attached to a porous polyester scaffold to replace lost RPE (http://www.theguardian.com/science/2009/jan/30/stemcells-genetics).

Human iPSC-derived RPE are not far behind. A clinical trial is in its early stage at the RIKEN Institute in Kobe, Japan, led by stem cell biologist Dr. Masayo Takahashi, using iPSCs to establish patient-specific cells for transplantation to reduce immune rejection. According to a press release from the RIKEN Institute (http://www.riken-ibri.jp/AMD/english/index.html), a sheet of hiPSC-derived RPE was transplanted underneath a patient's existing retina as an informal clinical safety test. An exercise of caution is warranted as there have been some reports of immune attack on genetically matched iPSCs 17–(20) , tumorigenicity (21) and loss of RPE characteristics in later passages that reduce their expansion potential and increases muscle contraction markers, a risk factor for Proliferative Vitreoretinopathy (22). On the other hand, a safety study demonstrated that transplantation of 1.5×10⁴ iPSC-derived RPE in a collagen-lined sheet elicited no tumor formation after 15 months in an immunodeficient mouse (23).

Primary RPE culture as a source for transplantation

The first culture of RPE cells was done almost 100 years ago with the in vitro cultivation of the embryonic optic cup from 72-h chick embryos, demonstrating their continual development in the absence of neural connections or blood supply (24). Not until 1973 would a method of culturing RPE as a monolayer be developed (25). Clinical implications were clearly in the authors’ mind as they note that these cultures may ‘form valuable substrates for pharmaceutical research’. Interestingly, the observation of epithelial morphology loss was also acknowledged, which is still a dominant factor delaying RPE transplantation therapy. The year 2015 will be the 30th anniversary of the first publication on the transplantation of RPE (26): re-establishment of a pseudo-monolayer was observed within 24 h of transplant in the owl monkey. There was, however, indication of an inflammatory response as early as a few hours after transplantation, foreshadowing what many since have struggled to overcome 27–(33).

For over 30 years, focus has been directed towards primary adult human RPE cultures as a promising source of RPE transplantation. Adult RPE cultured from human cadaver donors have been examined but their transformation from a homogenous cobblestone monolayer to a mix of distinct morphologies in vitro was observed (34). Cultured RPE undergo an epithelial to mesenchymal transition (EMT), in which the cells lose their epithelial features, including tight junctional complexes, polarized membranes and shape, and gain mesenchymal characteristics, such as migration, morphological elongation and contractile-like properties (35, 36). The failure of attachment of cultured adult human RPE onto aged Bruch's membrane has been observed and can be a problem in transplantation, but can be reversed by Extracellular matrix (ECM) reconstitution (37).

More recently, methods in preventing EMT in primary cultures have been established. Porcine cultures have been shown to reverse initial EMT progress and were able to re-establish a cobblestone monolayer using Ca²⁺ as a trigger (38). Cell culture media allowing a much more homogenous culture of fetal RPE was also developed (39). An improved culture method and media were described, capable of maintaining fetal human RPE monolayers that displayed native RPE electrophysiology, protein polarization, apical-to-basal fluid transport and more (40). This was the most extensive demonstration of native RPE physiology from a human RPE culture and introduced human fetal RPE as a promising candidate for RPE transplantation. Nonetheless, limited tissue resources, potential ethical or political resistance reduced enthusiasm for this cell as a source for RPE transplantation.

The renewed promise of primary RPE

Deceased human donors have been considered a possible source of RPE for transplantation for over 30 years. Unfortunately, RPE freshly isolated from human donors do not reattach to the Bruch's membrane following detachment from the original host Bruch's membrane (41). RPE lose the integrins necessary for reattachment and cannot reproduce them quick enough to reestablish a functional relationship with the choriocapillaris before undergoing apoptosis. To reverse this, perhaps establishment of novel culture conditions in which cells will be stimulated, possibly by a cytokine provided in the media, will allow RPE to re-attach to the Bruch's membrane, survive and maintain RPE identity before EMT is irreversibly completed.

More recently, a stem cell has been identified in the adult human RPE layer (RPESC), which can divide to re-establish an RPE epithelial monolayer (42). A novel culture method has also been developed, which allows maintenance of the epithelial physiology of cultured adult human RPE without undergoing EMT (43). This method promotes cell–cell contact inhibition while fostering moderate levels of proliferation, therefore maintaining a delicate balance between expansion and dedifferentiation. 5×10⁶ RPE cells can be obtained from one donor. In order to cover the macula, some estimates have shown that as low as 5×10⁴ RPE are sufficient, therefore up to 100 patients could be treated with RPE cells isolated from one single donor. Moreover, these cultures can be expanded 100-fold, producing up to 5×10⁸ cobblestone RPE from one donor, sufficient to treat up to 10,000 patients. Once transplanted as a sheet attached on a polyester porous scaffold in a rabbit model, RPE not only survived for more than 1 month, but were also shown to remain a polarized epithelial monolayer (33). Recently a contract was granted by New York State NYSTEM agency to Dr. Sally Temple to develop a cell transplantation therapy using primary adult human RPE (http://stemcell.ny.gov/nystem-consortia). Taken together, these developments renew the adult human RPE as a cell source for cell transplantation therapy and may be considered alongside hESC- and hiPSC-derived RPE as promising technology for patients with RPE-related diseases.

Conflict of interest and funding

The authors have not received any funding or benefits from industry or elsewhere to conduct this study.