In Japan, genetic testing can diagnose Retinitis Pigmentosa (RP) in approximately 40% to 50% of cases. RP is recognized as a rare disease, affecting one in every 4,000 to 8,000 people. This translates to an estimated 30,000 patients in Japan, and close to 3 million people affected globally.

The first sign of this disease is usually night blindness, making it hard to see in dim light. This is typically followed by a slow narrowing of vision, which starts from the outer field and moves inwards. As the disease progresses further, it often results in reduced sharpness of vision and difficulties in distinguishing colors. The pace at which these symptoms progress can vary widely among patients, and the sequence in which they appear can also be different. For instance, some patients may first notice a decrease in their vision clarity or color discrimination abilities before they experience night blindness.

At present, there is no definitive cure for RP. Treatment options are typically symptomatic, including the use of sunglasses to reduce glare, and the administration of Vitamin A or circulation-enhancing medications. Gene therapy and artificial retinas are beginning to be utilized, but as of now, these treatments are not widely available or accessible. We are developing regenerative medicine that aims to restore visual function by transplanting retinal sheets, including visual cells created from iPS cells, into the retinas of patients whose visual cells have degenerated. Additionally, when the causal gene is related to the retinal pigment epithelium (RPE), we are also planning treatments that can maintain visual function by transplanting RPE.

RPE plays various vital roles in maintaining our vision, and it’s known that genetic abnormalities expressed in RPE, along with pathological changes due to aging, such as oxidative stress and abnormal accumulation of waste, can lead to RPE dysfunction. This dysfunction can cause a group of inherited diseases known as retinal pigment degeneration and Age-Related Macular Degeneration (AMD), which has been the focus of our previous clinical trials on RPE cell transplantation. In fact, several retinal degenerative diseases exist for which RPE dysfunction is the primary cause. Consequently, these retinal degenerative diseases are collectively referred to as RPE dysfunction [Reference 1]. This umbrella term includes diseases such as crystalline retinopathy, an allied disease of retinal pigment degeneration, and those retinal pigment degeneration disorders accompanied by RPE-related genetic abnormalities (abnormalities in genes such as RPE65, RDH5, MERTK). These rare inherited retinal degenerative diseases, some of which are listed as specific intractable diseases, along with AMD, a common cause of blindness in the elderly, are all encompassed under RPE dysfunction. AMD affects about 700,000 people in Japan and approximately 300 million worldwide. However, the number of patients with other diseases categorized under RPE dysfunction is not known. Symptoms of RPE dysfunction are identified when visual acuity decreases, vision becomes impaired in dim light, or color perception becomes difficult. The age of onset and the type and progression speed of symptoms vary with the disease type and individual differences among patients. Age-Related Macular Degeneration is a typical example of RPE dysfunction.

RPE dysfunction, which encompasses various retinal degenerative conditions like crystalline retinopathy, Best’s disease, Stargardt disease, retinitis pigmentosa (with related genetic defects like MERTK or RPE65), high myopia, multifocal retinal pigment epitheliopathy, and pigmentary striae, currently doesn’t have any definitive cure.

Nevertheless, there’s a form of gene therapy, targeting RPE65 gene mutations associated with protein deficiency in retinitis pigmentosa. It was approved in the U.S in 2017, and in Japan in 2023. This treatment shows promise, but it’s important to note that it’s effective only if administered early in the disease progression. So, for patients with advanced stages of the disease, this therapy might not be applicable.

For patients with AMD, the current approach is routine medical management which involves the use of antioxidant supplements and anti-VEGF agents. Supplements including vitamins, lutein, and zeaxanthin are utilized to shield retinal cells from oxidative stress. Anti-VEGF agents, on the other hand, are used to treat wet AMD. They work by blocking VEGF’s interaction with its receptors, thereby preventing abnormal blood vessel growth. Despite these interventions, since they don’t address the underlying cause of the disease, stopping the treatment can often lead to high rates of disease recurrence.

There has been some progress in treating dry AMD, with the recent approval of an anti-complement drug. This drug is anticipated to slow down the progression of Geographic Atrophy (GA), a condition associated with dry AMD. However, it’s not capable of reversing already lost retinal cells.
So, the current state of treatment for RPE dysfunction is such that there are only a handful of targeted gene therapies for specific types of RPE dysfunction. There’s still no cure for the common problem of RPE cell degeneration and loss that’s seen in both inherited and non-inherited forms of the disease. Some research groups, including ours, are investigating regenerative treatments, like cell transplantation, as potential new approaches to treating these conditions [see references 1 and 2 for details].

Induced pluripotent stem (iPS) cells are a type of stem cell that is reprogrammed from adult cells to return to an embryonic-like state, similar to embryonic stem cells (ES cells). This process enables these cells to acquire the capability to differentiate into nearly all types of cells in the body.

iPS cells were first reported in 2006 by a team led by Professor Shinya Yamanaka of Kyoto University, who succeeded in establishing iPS cells from mouse skin cells for the first time in the world. Initially, Yamanaka’s team identified 24 genes believed to be key in maintaining cellular pluripotency, which is the ability of a cell to differentiate into any type of cell. These genes were introduced into skin cells. Then, by employing a process of elimination, they discovered that only four genes were necessary to reprogram these adult cells back to an embryonic-like state [Reference 1]. These genes – Oct3/4, Sox2, c-Myc, and Klf4 – are now known as the Yamanaka factors.

This revolutionary finding implied the potential to convert any cell in the body into a cell similar to pluripotent stem cells derived from an embryo, thus opening up new frontiers in regenerative medicine. The ability to create iPS cells from a patient’s own cells could reduce the risk of rejection during transplantation, paving the way for personalized medicine. Moreover, these cells can be used for drug development and modeling diseases, leading to an explosive expansion in research in this field.

For this groundbreaking work, Professor Yamanaka, along with Dr. John Gurdon, who had conducted pioneering research on cellular reprogramming, was awarded the 2012 Nobel Prize in Physiology or Medicine. Since then, Yamanaka has been recognized with numerous other awards for his exceptional contributions to the field.

Cells utilized in regenerative medicine, such as somatic stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), are characterized by their self-replicating capabilities and pluripotency. ES cells are stem cells established from a cluster of cells within a blastocyst following fertilization, and they can differentiate into almost all types of cells (tissues). iPS cells were induced from mouse fibroblasts using four transcription factors (Oct3/4, Flk1, Sox2, c-Myc) by Professor Shinya Yamanaka of Kyoto University, serving as ES cell-like pluripotent stem cells. The following year, the same group succeeded in creating human iPS cells, which are now widely used not only in medical fields such as regenerative medicine and drug development support, but also in basic research such as rejuvenation, aging processes, and elucidation of cell differentiation mechanisms. On the other hand, adult somatic stem cells exist in living tissues, are maintained in a special niche microenvironment, and are believed to be involved in tissue regeneration by replacing cells lost during development and tissue damage.

Initially, iPS cells were created from skin cells, but subsequent studies have shown that they can be generated from various types of cells. Nowadays, it’s more common to derive them from blood cells, making the process as simple as drawing blood. The genes used to induce iPS cells have also been modified, and the methods used to introduce these genes have evolved from retroviruses to plasmids and Sendai viruses. Today, iPS cells can even be created using mRNA. However, it’s important to note that iPS cells form a heterogeneous group, meaning they can greatly differ from each other. Even when generated using the same process, individual variations can lead to significant differences in gene expression levels. Moreover, the same cell line can behave differently depending on the cell culture techniques used. Even when following the same protocol, the cells can change differently during maintenance culture and passage, emphasizing the complexity of working with these cells.

During our pioneering work with iPS cells, we leveraged insights from the broader field of stem cell research to guide the transformation of these iPS cells into Retinal Pigment Epithelial (RPE) cells. While iPS cells are heterogenous and exhibit significant differences even within the same line, when they are differentiated into RPE cells, these differences become insignificant. Historically, there are no instances of these RPE cells turning cancerous, making them a safe choice for our work. We devised a robust method that ensures the creation of consistently safe RPE cells with identical functionalities from each patient’s unique iPS cells [Reference 2, Figure 2]. This strategic approach made it possible for autologous transplants, effectively bypassing potential issues of immune rejection.

Subsequently, utilizing the iPS cell stock created by the Center for iPS Cell Research and Application (CiRA) at Kyoto University *that matched the 6-locus HLA of a patient1 , we derived RPE cells and successfully transplanted them into the retina of an elderly patient with age-related macular degeneration. Notably, this procedure was carried out without the use of systemic immunosuppressive drugs [Reference 3]. The findings suggest that, when there is a precise HLA match, potential rejection reactions can be efficiently controlled through local steroid administration alone.

Exploiting the insights gained from these clinical trials, we are currently developing treatments that could allow us to transplant cells into any patient without requiring immunosuppressive drugs. This involves creating RPE cells from iPS cells that have undergone genetic alterations to partially disable specific HLA genes, thereby creating a universal cell line applicable to all patients.

(*) The production of HLA homozygous iPS cells has been taken over by the CiRA Foundation since April 2020.

In 2014, our forerunner, the RIKEN Laboratory for Retinal Regeneration, led a landmark clinical application of induced pluripotent stem (iPS) cells. We carried out the world’s first subretinal transplantation of Retinal Pigment Epithelium (RPE) cells derived from iPS cells for treating neovascular age-related macular degeneration[Reference 1,2]. This achievement marked the first application of human iPS cells since their discovery in 2006. To ensure safety and efficacy in this pioneering therapy, rigorous quality control tests, including whole-genome and exome analyses, were conducted. We managed to overcome numerous regulatory challenges, thus making a significant stride in the field.

The treatment involved procedures for neovascular removal and transplantation of iPS cell-derived RPE sheets. Throughout the process, no adverse events related to the surgical technique were observed. A year-long follow-up revealed no indications of abnormal proliferation or rejection of the transplanted cells. Furthermore, the grafted retinal structure was well maintained[1]. Impressively, even seven years post-surgery, the transplanted RPE sheet, derived from iPS cells, remained intact without signs of tumorigenesis or abnormal cell proliferation.

The patient, a 78-year-old at the time of surgery, had previously received 13 intravitreal injections of anti-VEGF agents to suppress neovascularization due to exudative age-related macular degeneration. Remarkably, for over seven years post-surgery, the patient required no further intravitreal injections and maintained preoperative visual acuity.

RPE cells function as a sheet-like tissue under the retina, and it is safer for the surgery if the incisions in the eyeball and retina are as small as possible. While having a sheet from the beginning allows for accurate graft placement, the creation of an RPE cell sheet is time-consuming and labor-intensive. Additionally, this approach involves significant disturbance to the retina during transplantation. On the other hand, administering RPE cells as a cell suspension allows for easier preparation and storage and requires only a small surgical incision. However, it is challenging to ensure the precise number of cells are delivered to the transplantation site.

To overcome the disadvantages of both cell sheet and cell suspension methods, while leveraging their benefits, we have developed the RPE strip [2]. By culturing RPE cells, differentiated from iPS cells, in narrow grooves for two days, we can form a strip-like aggregate, approximately 200μm in diameter and about 2cm in length. Once this strip is placed on a culture dish, we observe RPE cells starting to grow from it the next day. As the cultivation continues, these cells expand into a sheet while expressing mature RPE markers, such as BEST1 and ZO-1. Interestingly, it’s also possible to preserve the strip in its aggregated form.

In November 2022, we initiated clinical trials using the RPE strip, demonstrating its potential to be transplanted to targeted areas underneath the retina with minimal invasion, thereby complementing the shortcomings of both cell sheet and cell suspension methods. Furthermore, a few weeks post-transplantation, it was observed that the strip expands into a monolayer sheet, effectively integrating into the retina. Currently, we are considering the logistics, including transportation methods, to provide this strip formulation in a ready-to-use form for domestic and international use.

One significant factor to consider in iPS cell transplantation is the recipient’s immune response. Our first clinical trial in 2014 employed autologous iPS cells, i.e., those derived from the patient’s own body, to minimize the risk of rejection. This method leverages the Human leukocyte antigen (HLA), a cell surface molecule instrumental in distinguishing self from non-self. The immune system eliminates cells bearing non-self HLA, ensuring the body’s protection. Therefore, in an autologous transplant, there’s no need for immunosuppressants to manage HLA-mismatch-induced rejection responses. However, this approach has its drawbacks; it requires extensive preparation time, labor, and substantial cost due to the need to establish iPS cells for each patient.

To overcome these challenges, the Center for iPS Cell Research and Application (CiRA) at Kyoto University launched the iPS Cell Stock Project for Regenerative Medicine. The project provides iPS cells from donors homozygous for the six major HLA loci, thereby reducing the likelihood of rejection in patients who share at least one pair of each of the six major HLA loci with the donor.

Our 2017 clinical trial used these HLA-matched RPE cells for transplantation. We demonstrated that such cells could be safely administered without necessitating systemic immunosuppressant use [Reference 3].

Despite the advantages of HLA-matched cells, this method is still limited to patients whose HLA type aligns with the six major HLA homozygous iPS cells provided by CiRA. As a solution, we established iPS cells using gene editing to remove part of the six major HLA loci and differentiated these into RPE cells [Reference 4]. The eye exhibits stronger immunosuppressive properties than other organs, which further lowers the risk of rejection. In vivo and in vitro validations revealed no rejection responses to these modified RPE cells. By retaining some HLA, we maintain natural immunity against tumors and resistance to viruses, combining both safety and efficacy in suppressing the rejection response for intraocular transplantation.

Retinal organoids are three-dimensional constructs that replicate the structure of in vivo retinas. They can be produced in vitro from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, facilitated by specific soluble factors that induce differentiation. Interestingly, these organoids mimic the natural progression of retinal development, establishing a complex three-layered structure comprising the outer nuclear layer, inner nuclear layer, and ganglion cell layer. During this process, all cell types, including photoreceptor cells, differentiate from retinal progenitor cells.

Retinal organoids can be produced utilizing different soluble factors. A popular strategy involves guiding differentiation from ES/iPS cells to the retina via the neural ectoderm. Our research employs a method utilizing Bone Morphogenetic Protein 4 (BMP4), part of the Transforming Growth Factor-beta (TGFβ) family. We use the Serum-free Floating culture of Embryoid Body-like aggregates with quick reaggregation (SFEBq) technique, where ES/iPS cell aggregates are suspended, and BMP4 is applied to induce differentiation towards retinal cells. This approach has enabled the production of high-quality retinal organoids, and it was instrumental in the world’s first transplantation of an iPS cell-derived retinal sheet into a patient with retinitis pigmentosa in 2020 at Kobe Eye Center Hospital. Our ongoing efforts are centered around using this technique to develop larger retinal organoids for transplantation.

Furthermore, retinal organoids differentiated from iPS cells of patients with retinal degenerative diseases offer potential tools for pathology analysis and for developing treatments to halt disease progression. Our aim is to leverage retinal organoids as transplant tissues, targeting the restoration of lost vision and visual fields.

Our transplantation process primarily utilizes retinal and photoreceptor precursor cells, which undergo post-transplantation maturation. This process establishes a light signal transmission pathway. Notably, electron microscopy reveals the formation of specialized light detection compartments, known as rod outer segments, after this maturation. Simultaneously, the cells express essential functional molecules intrinsic to photoreceptor cells, such as opsin and transducin. Our primate-based transplant trials further demonstrate the robust integration and enduring stability of transplanted photoreceptor cells, confirmed up to two years post-transplantation.

For the restoration of visual function, it is essential that transplanted photoreceptor cells are not only responsive to light stimulation, but also capable of transmitting light information to downstream secondary neurons. This involves the formation of ribbon synapses, a specialized structure that facilitates the transmission of graded light signals from photoreceptor cells to second-order neurons, such as bipolar cells. This process is critical for conveying light information. Beyond the evidence from immunohistochemistry, we have utilized electrophysiological techniques to demonstrate the light signal transmission capability of transplanted photoreceptors. By transplanting sheets derived from human retinal organoids into rats with immunodeficient retinal degeneration, we recorded the light response of the host retina using a multi-electrode array (MEA) system. These findings indicate that transplanted retinas are able to respond to light and transmit that information to host ganglion cells.

We have further assessed the restoration of vision by conducting behavioral experiments. In one experiment, retinal organoids derived from mouse iPS cells were transplanted into mice with end-stage photoreceptor degeneration. The transplant recipients exhibited a response to light in shuttle avoidance tests, reflecting a successful integration of the transplanted photoreceptor cells and their physiological response to light. In a separate study, human retinal organoid-derived sheet transplants were applied to monkeys with artificially induced photoreceptor degeneration. The results from visually-guided saccades (VGS) tests suggested noticeable improvements in the visual field, further affirming the potential of our transplant approach in restoring light-induced behavior.

At present, our transplantation technique involves the use of retinal sheets that are derived from induced pluripotent stem (iPS) cell-generated retinal organoids. During transplantation, these sheets are primarily composed of retinal and photoreceptor precursor cells. Post-transplantation, these cells mature into photoreceptors and differentiate into secondary neurons such as bipolar and horizontal cells, as well as Müller glial cells. Our method of transplanting retinal sheets, which maintains the polarity of the immature tissue, has been found to offer consistent long-term integration and stability of the transplanted photoreceptor cells, a feature that distinguishes it from transplantation methods using purified photoreceptor cells in suspension.

However, there is a potential challenge that photoreceptor cells and secondary neurons differentiated within the transplanted retina may establish synaptic connections, thus inhibiting the formation of synapses between transplanted photoreceptor cells and the host’s secondary neurons. In response to this, we’re actively improving our transplantation strategies to maximize therapeutic outcomes.
One of these enhancements involves the development of next-generation transplantation retinae, where gene-editing technologies are employed to regulate gene expression within the retina. By modifying the differentiation and maturation trajectory of the transplanted retina, our goal is to optimize the efficiency of synaptic connections between the host and transplanted retina.

Until now, our team, in collaboration with the Robotic Biology Institute Inc., developers of the general-purpose humanoid robot LabDroid “Maholo”, and Epistra Inc., developers of AI and other information technologies, has been dedicated to establishing next-generation biological experimentation using robotics and AI. Maholo, a humanoid robot, carries out experiments using identical apparatus and tools as a human, thanks to its two arms [Figure 1].

In research reported by RIKEN, this system successfully cultivated cells under 143 conditions, chosen from 200 million potential parameter combinations, over 111 days [Reference 2,3]. The results demonstrated a production of iPSC-RPE that was 88% more efficient compared to pre-optimized cultures, as assessed by pigment appearance scores (RPE cell ratios). Additionally, we’ve developed a system in which Mahoro autonomously passaged cells at the predicted time when the cell culture would become 80% confluent [Reference 3]. This demonstrates that using an autonomous robot AI system dramatically expedites the systematic, unbiased exploration of experimental possibilities, with great promise for application in medical and research settings.

Our company is propelling the techniques developed in the realm of basic research, undertaking the task of automating cell manufacturing through the use of robotics and AI. We are striving to devise processes that consistently generate high-quality cells suitable for clinical use. We’ve already achieved approval from the Committee for Designated Regenerative Medicine and the Health Sciences Council for clinical trials at Kobe Eye Center Hospital, where Maholo assists in part of the iPS-RPE production process.This innovative incorporation of robotics in clinical cell production has led to a more stable manufacturing process. By optimizing cell culturing techniques without altering human culture and protocols, we’ve been able to seamlessly transition this approach into clinical cell production.

Additionally, our cell culture processing facility, known as the Facility for iPS derived retinal stem cell therapy (FiRst, approximately 357 m²), received a “Special Cell Processing Product Manufacturing License” from the Kinki Bureau of Health and Welfare of the Ministry of Health, Labour and Welfare on October 20, 2022. FiRst is designed with two independent cell processing rooms, allowing for ongoing manufacturing in one room while updating equipment in the other, thereby ensuring smooth automation implementation and methodological enhancements. It is set to carry out cell manufacturing for future clinical trials.