AMD and Stem Cell-Based Therapies

Abstract

Age-related macular degeneration (AMD) is a prevalent and complex disease leading to severe vision loss. Stem cells offer promising prospects for AMD treatment as they can be differentiated into critical retinal cell types that could replace lost host retinal cells or provide trophic support to promote host retinal cell survival. However, challenges such as immune rejection, concerns regarding tumorigenicity, and genomic integrity must be addressed. Clinical trials with stem cell-derived retinal pigment epithelial cells have shown preliminary safety in treating dry AMD, but improvements in manufacturing and surgical techniques cell delivery are needed. Late-stage AMD poses additional hurdles, possibly requiring multi-layered grafts. Advancements in automation technologies and gene correction strategies show potential to enhance iPSC-based therapies. Stem cell-based treatments offer hope for AMD management, but further research and optimization are essential for successful clinical implementation.

Introduction

Age-related macular degeneration (AMD) is a complex disease characterized by the loss of the choroidal endothelial cells with subsequent degeneration of the retinal pigment epithelium (RPE) and photoreceptors¹. While the exact cause of AMD is not fully understood, oxidative stress, chronic inflammation, genetic factors, and mitochondrial dysfunction are believed to play a role^2–4.

Current management options for AMD are limited. In dry AMD, there are emerging treatments, such as pegcetacoplan and avacincaptad pegol and a focus on dietary supplementation to prevent disease progression^5–7. Wet AMD can be treated with intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs, which help limit the growth of abnormal blood vessels in the retina⁸. Newer drugs include faricimab which targets both VEGF and Ang-2. To date, there is no curative treatment⁹.

Cell-based therapies using stem cells offer a promising approach for treating AMD¹⁰. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cells lost in AMD such as RPE cells, choroidal endothelial cells (CECs), and photoreceptor cells^11,12. Several protocols have been developed to differentiate iPSCs into photoreceptor precursor cells, RPE cells, and CECs involving the use of growth factors, chemical molecules, and specific signaling pathways^13,14.

Challenges in iPSC-based regenerative medicine include immune rejection, concerns regarding tumorigenicity, genomic integrity, cross-contamination, and sterility^15–18. Researchers are addressing these challenges by using immunologically matched donor cells, ensuring the absence of undifferentiated pluripotent stem cells, conducting thorough genetic and karyotypic analyses, and implementing rigorous quality control measures^13,19.

Early data from clinical trials have demonstrated promising results, but further research is needed to optimize manufacturing processes, delivery methods, and long-term integration of transplanted cells. While iPSC-based therapies show promise for treating AMD, the treatment of late-stage dry AMD remains a significant challenge due to the loss of both RPE and photoreceptors likely requiring the use of multi-layered grafts to restore visual function²⁰.

AMD background

AMD is the leading cause of blindness in people over 55 in developed countries²¹ that can lead to severe vision loss. It is estimated that the number of AMD patients will increase from 196 million in 2020 to 288 million in 2040²². The disease manifests in two primary forms: dry (atrophic) and wet (neovascular) AMD^23,24. The early stages of dry AMD involve pigmentary changes and drusen formation within the macula, the central region of the retina. As dry AMD advances, it can result in geographic atrophy (GA), where loss of photoreceptor cells, RPE cells, and CECs occurs with features visible upon fundoscopic examination^25,26. Wet AMD, on the other hand, involves the development of abnormal subretinal choroidal vessels referred to as choroidal neovascular membrane (CNVM). The resulting neovascularization can lead to fluid leakage which is toxic to the retina²⁷. Ultimately, both pathways may lead to the development of a scotoma, or blind spot, in the central visual field sparing the periphery visual field²⁸.

The outer retina, which includes photoreceptor cells and RPE cells, and the choriocapillaris, plays a critical role in vision^29,30. Photoreceptor cells convert light into electrical signals, while RPE cells support photoreceptors by providing nutrients and recycling visual pigments. Bruch’s membrane (BrM) serves as a filtering for molecules passing between the choriocapillaris and RPE as well as a barrier to abnormal cellular migration³¹. The choriocapillaris, as part of the choroid layer beneath the BrM, supplies oxygen and nutrients to the outer retina. In AMD, these layers can be detrimentally affected, with wet AMD involving abnormal blood vessel growth from the choriocapillaris and dry AMD leading to atrophy of the retinal layers, further emphasizing the importance of these structures for maintaining visual health³².

AMD is a complex disease which demonstrates a multifactorial inheritance pattern. The most significant risk factor is age, with individuals over 60 being most susceptible, and the risk increases dramatically with advancing age^33,34. Ethnicity also plays a role, with Caucasians at higher risk than African Americans, while global prevalence varies among different ethnic groups³⁵. Cigarette smoking is a notable environmental risk, increasing the odds of AMD development by a factor of almost two compared to non-smokers³⁶. Alcohol use, obesity, and western diets also appear to be modifiable risk factors³⁷. Genetics contribute significantly to AMD risk, with approximately 40 genes associated with the disease, including those involved in the complement cascade^38–40. Polymorphisms in complement genes and those in the ARMS2/HTRA1 locus are strongly linked to increased AMD risk⁴¹. Understanding these risk factors is crucial for developing targeted interventions and preventative measures for AMD.

Stem cell introduction

Since the advent of induced pluripotent stem cells^42,43, stem cell technology, particularly induced pluripotent stem cells (iPSCs), has emerged as a powerful tool for investigating disease. Through patient-specific iPSCs derived with known genetic variation, researchers can gain crucial insights into the mechanism of diseases. This holds especially true for studying human tissues which are otherwise often inaccessible for basic research such as the retina. Moreover, generations of patient-specific cell populations further allow researchers to study the effects of genetic variations in vitro and develop potential targeted therapeutic strategies. To accomplish this, directed differentiation methods aid in generating specific cellular populations, and these cells can be genetically edited to assess the causality of identified variants, distinguishing between necessary and sufficient genetic factors in disease development. For example, iPSC technology has proven effective in understanding monogenic inherited retinal degeneration, leading to the discovery of novel gene variants contributing to retinitis pigmentosa and Usher syndrome^44–46. Correcting pathogenic variants in iPSCs using gene editing technologies, such as CRISPR-Cas9, has shown promising results, demonstrating the potential for future therapeutic interventions in retinal disease^47–51.

Stem cell technology provides a novel avenue for regenerative medicine. Three sources of stem cells exist for treatment of retinal disease: ESCs, iPSCs, and adult stem cells. Both ESCs and iPSCs are pluripotent and therefore able to differentiate into any cell lineage⁵² whereas adult stem cells have limited differentiation potential within a specific lineage⁵³. While ESCs have traditionally been used for cell replacement therapy, their use has been subject to both ethical and immunologic concerns. iPSCs and adult stem cells are a viable alternative and, like ESCs, allow for the generation of cells to replace damaged tissue. Multiple past and current clinical trials have undertaken these studies with retinal cells sourced from ESCs, iPSCs, and adult stem cells.

Stem cells to retinal organoids

In one groundbreaking study, Sasai and his collaborators developed a protocol for obtaining eyecup-like structures in vitro⁵⁴. These studies successfully recreated various aspects of retinal development using retinal organoids (ROs), including the formation of multiple neuronal cell types including, bipolar cells, ganglion cells, horizontal cells, amacrine cells, and Muller cells. Subsequent reports expanded on the utility of the organoid system by identifying stages of organoid maturation and confirming the similarity of RO cell types to those found in fetal and adult retinas through single-cell RNA sequencing analysis^46,55–57.

Stem cells to RPE

Methods for deriving RPE cells from stem cells, particularly iPSCs, have been developed to harness their potential for cell-based therapies in AMD. These methods aim to generate functional RPE cells for two reasons: studying disease states in vitro and transplantation into the retina with the goal of restoring visual function^10,58.

Differentiation protocols involve the use of specific growth factors, chemical molecules, and signaling pathway inhibitors to guide the iPSCs toward an RPE cell fate. Common factors and molecules utilized include fibroblast growth factor-2 (FGF-2), noggin, activin A, insulin-like growth factor-1 (IGF-1), nicotinamide, WNT antagonists (Dkk-1), bone morphogenetic protein (BMP) antagonists, and small chemical compounds like SB431542 and CHIR99021^59–64.

Various differentiation methods have been developed, including spontaneous differentiation, and directed differentiation. Spontaneous differentiation involves changing the culture medium to create an environment conducive to RPE development, although it often yields low efficiency⁶⁵. Directed differentiation methods use specific combinations of factors and molecules to drive iPSCs directly into the RPE lineage, resulting in more efficient and rapid differentiation⁶⁶. Validation of RPE differentiation involves evaluating the cells through histological assessment, gene expression analysis, immunofluorescence, and functional assays⁶⁷.

Despite considerable progress, challenges remain in optimizing the differentiation efficiency, scalability, and functionality of iPSC-derived RPE cells. Additionally, ensuring the safety, sterility, and absence of tumorigenic potential in the derived cells is crucial before considering clinical-grade applications^16,68,69.

Stem cells to choroidal endothelial cell

Researchers successfully developed a protocol for generating CECs from iPSCs⁷⁰. This stepwise protocol employed a lentiviral reporter to track endothelial cell differentiation, transitioning from mTESRTM1 to an endothelial cell medium supplemented with BMP-4, activin A, and FGF-2. Further differentiation into CECs was promoted by VEGF and connective tissue growth factor (CTGF). The research also provided two novel methods of isolating iPSC-derived CECs, leveraging the zeocin resistance offered by the reporter lentivirus versus using anti-CD31 MACS beads.

Importantly, the generated iPSC-derived CECs demonstrated positive staining for CA4 and RGCC, choroid-specific markers, indicating successful differentiation into CECs of the choriocapillaris. Additionally, the cells successfully formed 3-dimensional tube networks – an essential characteristic of mature CECs. In a significant step, these cells were also shown to possess the ability to recellularize decellularized human donor choroidal extracellular matrix.

Autologous vs allogenic

The eye possesses partial immune privilege, attributed to factors such as the blood-ocular barrier and tolerogenic microenvironments^71–73. However, breaches in this privilege can occur because of several factors, compromising the success of cellular therapies for eye diseases. Both allogeneic and autologous approaches are being explored for treating age-related macular degeneration, but it remains uncertain which method is most effective and how long transplanted cells can survive in the potentially hostile eye environment⁷⁴. Studies indicate that short-term systemic immunosuppression may contribute to the long-term survival of allogenic cells, while rejection rates are high without immune suppression. In contrast, immune suppression is unlikely to be needed with autologous cells.

Challenges to autologous iPSCs

Most clinical trials to date have used ESCs due to the significant barriers to iPSC-generated RPE cells. The use of patient-derived iPSCs for autologous cell replacement in clinical trials poses challenges due to the lack of a suitable clinical manufacturing process for simultaneous production and differentiation of multiple patient cell lines⁷⁴. There have been concerns that reprogramming of somatic cells to iPSCs can introduce genetic alterations in oncogenes and tumor suppressor genes⁷⁵. While efforts are underway to automate iPSC generation and differentiation, current trials will likely require labor-intensive manual processes, limiting patient enrollment⁷⁶. Patient selection based on clinical data is critical, but predicting the retinal differentiation capacity of iPSC lines before enrollment is difficult. A recent study identified a gene panel that can predict retinal differentiation capacity of iPSC lines, aiding in cell line selection⁷⁷. Incorporating assays such as this into the validation pipeline could enhance patient selection and facilitate future interventions to improve retinal differentiation capacity.

Autologous iPSCs are increasingly becoming a viable option for treating retinal diseases as they offer potential advantages over allogenic therapies by bypassing immune response issues. However, a major challenge is the need to correct disease-causing genes in patient-derived cells before transplantation to prevent disease reoccurrence. CRISPR-Cas9 technology shows promise for gene correction, but concerns about unintended genetic modifications remain⁷⁸. As of current clinical trial data, the necessity of genome correction for known high-risk variants in treating AMD with autologous transplantation remains uncertain. Moreover, off-target effects in genome editing raise safety concerns. To ensure the genetic integrity of donor iPSCs during reprogramming and expansion, single-cell genomics prove valuable for monitoring purposes.

To overcome issues with production capacity, development of automated platforms like CompacT SelecT and CellX, capable of automating iPSC generation and differentiation is vital^74,76,79. These systems integrate robotics and liquid handling devices to maintain numerous cell lines in parallel. Utilizing image analysis algorithms in these automated systems could potentially allow for monitoring and validation of cells at every stage of development.

AMD clinical trials

Early cell replacement approaches for treating AMD involved relocating cells from a healthy area of the retina to the site of damage using techniques such as ‘retinal translocation’ ^80,81. However, this method posed significant health burdens and surgical risks⁸². To improve outcomes, researchers explored using RPE allografts in the subretinal space⁸³. Initial trials with cultured fetal RPE patches and cell suspensions showed promise, but emphasized the need for intact Bruch’s membrane and correct RPE orientation for stable engraftment, leading to the development of patch-based cell replacement techniques⁸⁴. Limited availability of suitable fetal tissue and concerns about tumorigenicity from other cell sources, like aRPE-19, were challenges at the time. Ultimately, this issue was resolved with the advent of stem cell technology.

Multiple clinical trials using stem cells are currently under way (NCT01691261, NCT04339764, NCT03167203, NCT02286089, jRCTa050200027, NCT02590692, NCT01674829). Clinical trials to-date have overall demonstrated safety of transplanting stem cell-derived RPE. In a phase 1/2a study, the safety and preliminary efficacy of human embryonic stem cell (hESC)-derived RPE cells on an ultrathin parylene substrate were evaluated⁸⁵. Of 15 subjects who received the subretinal implant, the surgery was well-tolerated with manageable adverse events and no implant migration. While underpowered for significance, patients treated trended towards visual improvement. Moreover, the study demonstrated the safety of stem cell-derived RPE and provided encouraging preliminary efficacy data for the treatment of geographic atrophy, leading the authors to conclude that these findings support future recruitment of patients in early-stage disease where there is a greater potential to prevent loss of vision. The first autologous iPSC clinical trial used autologous iPSC-RPE cell sheet in subjects with exudative AMD⁸⁶. iPSCs were derived from skin fibroblasts and were differentiated into RPE cells. When transplanted into immunodeficient mice, no tumorgenicity was observed. The 1-year results of this trial demonstrated safety without any evidence of visual worsening and overall stability of the transplanted sheets. Lastly, At University of Michigan, we have an ongoing first-of-its-kind clinical trial utilizing adult stem cell-derived RPE cells (NCT04627428). While the results are pending, this source of cells provides another potential avenue for treatment.

Surgical considerations

Central to visual function is the three-neuron circuit consisting of the photoreceptor cell, the bipolar cell, and the ganglion cell, all of which are essential for the detection of light. In the pathology of AMD, the end-stage disease is characterized by the loss of photoreceptor cells, disrupting this crucial circuit. Fortunately, various surgical solutions are available. Two primary surgical approaches exist for accessing the required anatomical space: utilizing either a subretinal bleb or a subretinal incision⁸⁷. In practice, a bleb creates a localized detachment allowing access to this potential space. At the University of Michigan and in discussions with surgeons involved in similar clinic trials (personal communication), there have been observations that large areas of geographic atrophy can impede subretinal bleb formation due to strong adhesive forces of the overlying, atrophic retina and BrM. Cell delivery can be achieved either as a suspension or through a scaffold. When using suspensions, factors such as the gauge of the delivery system must be considered, as they can affect shearing forces and thus cell viability. Scaffolds may contain one or multiple cell types and might necessitate specialized delivery mechanisms and involve large macular incisions to accommodate cell-scaffold transplants which can lead to hemorrhage and proliferative vitreoretinopathy. RPE replacement may suffice for early-stage disease, although one consideration is that choroidal endothelial cell loss precedes RPE loss in AMD. Late-stage disease presents additional challenges due to photoreceptor cell loss, meaning that treatment for this stage may necessitate a layer of photoreceptor precursor cells to restore visual function. Additionally, the connections between bipolar and ganglion cells must be intact.

Multiple technical challenges exist. First, visualization of the potential space between the RPE and neural retina may be technically challenging during surgery which may be further hindered using non-pigmented RPE cellular suspensions. Intraoperative OCT presents a solution to this hurdle. Other risks include submacular hemorrhage which may present a barrier to transplantation viability, especially those interventions involving large cell-scaffold transplants. Epiretinal membranes have been reported as adverse events presumably due to loss of RPE cells from the transplantation site as well as proliferative vitreoretinopathy⁸⁸. Lastly, when assessing for efficacy, classic visual acuity metrics may not be sensitive enough to capture meaningful improvements and surrogate measurements may be required such as fundus autofluorescence and microperimetry.

Conclusion

Stem cell-based therapies offer a promising approach for treating AMD. Early clinical trial data indicates safety and potential efficacy. However, further research is needed to optimize manufacturing processes, delivery methods, and long-term integration of transplanted cells. The success of these therapies relies on addressing challenges related to immune rejection, tumorigenicity, and surgical considerations. With ongoing advancements in stem cell technology and surgical techniques, stem cell-based therapies have the potential to revolutionize the treatment of AMD and restore visual function for affected individuals.