Stem cells for treating retinal degeneration

RPE transplantation

The retinal pigment epithelium (RPE) consists of a single cell layer that separates the retina from the choroid. RPE cells have an essential function in maintaining vision [1]. They surround the outer segments of the photoreceptors to control retinal photo-stimulation and to isomerize and thereby recharge the chromophore retinal. In addition, the RPE ingests parts of the outer segments via phagocytosis, which contributes to the daily photoreceptor maintenance. Transport across the epithelium allows the exchange of nutrients, ions, and growth factors between the retina and choroidal capillaries. In addition, pigmented RPE can absorb scattered light and thus reduce photooxidative stress. An intact RPE also contributes to the immune-privileged function of the eye by demarcating it from the blood circulation [1]. In summary, RPE cells are essential for photoreceptor function and their dysfunction or degeneration directly affects photoreceptors and thereby vision. The idea of RPE cell replacement therapy was developed almost 40 years ago and just over 30 years ago, fetal RPE cells were already transplanted into an AMD patient for the first time [2]. Since then, an abundance of research has been constantly contributing to the improvement of this method, leading, among others, to the use of non-fetal RPE cells. In particular, the use of human stem cells brought a major breakthrough and provided a variety of protocols for RPE cell differentiation. In order to avoid allogeneic rejection, immunological interplay between the host and the transplanted cells has to be considered in the development of RPE cell replacement therapy [3]. Various clinical trials with RPE cells differentiated from human stem cells could be used for the treatment of AMD and Stargardt disease [4, 5]. Partial improvement of vision was observed in these approaches. There are already first clinical studies on stem cell-derived RPE cell replacement therapy. Essentially, two methods are distinguished: RPE cells are either injected as cell suspension or transplanted into the retina as RPE choroid sheets [6]. Both techniques require treatment with immunosuppressants for a prolonged period of time, since the cell material is foreign to the body. The first clinical trials showed great success in 18 patients [7]. Here, RPE cells were derived from human embryonic stem cells and transplanted. In addition to the improvement or constant visual acuity, the risks of undesired cell growth and rejection reactions could be excluded. Thus, the mid- to long-term safety of this method could be confirmed. In another clinical study with 12 patients, the number of cells to be transplanted was investigated in more detail, as well as the integration into the patient’s retina [8]. In this study, there was focus on cell numbers, using up to 200,000 RPE cells for transplantation. Structural and especially functional integration of the cells is particularly important to provide patients with a safe and successful treatment. A three-year follow-up study with three patients could also show excellent results in Stargardt patients after transplantation with RPE cells derived from human embryonic stem cells [9]. The long-term tolerability, safety and success of the method could be confirmed once again.

Transplantation of RPE single-cell layers could also be successfully validated in clinical trials in two AMD patients. In particular, the successful approach of the transplantation of a RPE single-cell layer on an artificial membrane into the retina give hope for a broad clinical application. The main challenge with this method is the precise placement of the membrane into the retina [4]. Several clinical trials are currently underway, the results of which may lead to new therapeutic approaches. In addition, further research is being conducted focusing on integration and functionality, and thus preservation and improvement of vision in patients. For ethical reasons, the use of human induced pluripotent stem cells is more favorable. This has already been successfully tested in one patient [5]. Of note, using hESC or hiPSC-derived RPE cells, it is important that no hyperproliferating cells, which may lead to tumors, are transplanted into the retina of patients. Therefore, long-term clinical studies with a focus on safety are essential for further clinical study designs.

Photoreceptor cell replacement

Transplantation of photoreceptors into the retina to restore vision is similarly promising [10, 11]. However, photoreceptor transplantation is particularly challenging because the synaptic connection of photoreceptors to downstream bipolar cells must occur in order to relay light information. It has been previously shown that for successful transplantation, the developmental stage of photoreceptors is enormously important for their survival and synaptic integration [12]. It has also been shown that primary photoreceptor progenitor cells, which continue to mature after transplantation into the subretinal space, are particularly suitable and can improve visual function [11]. The critical steps of preparation and purification, surgical intervention, and immunomodulation after transplantation [13] are further investigated and improved in animal models. In addition, combined methods are being developed to transplant photoreceptors together with RPE cells [14]. Several years ago, some groups independently discovered that material transfer occurs when mouse photoreceptors are transplanted into mice that still have intrinsic but dysfunctional photoreceptors left [15], [16], [17]. This transfer, presumably via nanotube-like processes [18], supports and reactivates the remaining photoreceptors in the recipient. As long as nonfunctional photoreceptors are present in the recipient, the donor cells can support them by material exchange, which is also an exciting method for restoring vision.

The required cell numbers and quality for widespread clinical application of photoreceptor cell replacement therapy are the major limitations. In addition, the use of primary photoreceptor cells derived from fetal tissues is ethically, legally, and technically difficult. Therefore, alternative methods for obtaining photoreceptors have been developed, for example, from fibroblasts or pluripotent stem cells. Fibroblasts can be differentiated into photoreceptors by using transcription factors [19] or chemical agents [20]. Similarly, human stem cells have been used to grow photoreceptors in the Petri dish [21]. However, the number of photoreceptors produced by these methods has been insufficient for transplantation.

The discovery to produce whole retinas, so-called retinal organoids, from pluripotent stem cells significantly advanced the field [22]. These retinal organoids are a prolific source of human photoreceptors, which can be used to further explore the efficacy and safety of cell replacement therapy. However, this method also requires overcoming important technological and economic challenges. Cell production for medical use requires protocols that are compliant with “Good Manufacturing Practices” (GMP). This includes the complete elimination of all animal components, as well as standardized, automated production of the cells, including efficient quality control. Some progress has already been made in the field of GMP-certified photoreceptor production [23]. However, all available protocols involve extremely long cultivation phases, requiring many manual steps by trained personnel and a huge consumption of complex media. New protocols for simplified production of retinal organoids are already being developed [24, 25], but it remains to be seen whether they are feasible for efficient and economical harvesting of therapeutic cell material. Unlike foreign donor cells, autologous cells reduce the risk of immune rejection due to human leukocyte antigen (HLA) proteins [26]. De novo reprogramming of patient stem cells and subsequent correction of the mutated gene, requires personalized medicine. This is technically feasible, but it is not economical for a general therapeutic approach. Cells from HLA “super donors” that are homozygous for an HLA allele and thus compatible with a larger patient population may provide a remedy [26]. However, these special stem cell lines have yet to be verified for the production of photoreceptor-rich retinal organoids. In principle, photoreceptor cell replacement therapy looks promising in preclinical trials, but there are still some technical hurdles to overcome for broad therapeutic application.

Conclusions

Human stem cells are important tools for experimental and clinical ophthalmology. Stem cell-derived retinal cell types [27] and organoids [28] serve as a human testbed facilitating meaningful disease modeling. Protocols for generating RPE cells are very advanced and corresponding photoreceptor protocols are emerging. There are still many technical and administrative road blocks ahead of us but they will be solved by joint efforts from the field. RPE cell therapy looks extremely promising within clinical trials and photoreceptor replacement will soon leave the preclinical stage [11]. In summary, we are in an exciting era in which stem cell technology is key for the next breakthroughs in ophthalmology.