Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.

An Automated Visual Psychophysics Method to Measure Visual Function in Swine Preclinical Animal Model.

Purpose: The aim of this study was to develop and validate a test to assess visual function in pigs using the visual psychophysics contrast sensitivity function. Methods: We utilized a touchscreen along with a pellet reward dispenser to train three Göttingen pigs on a visual psychophysics test and determined their contrast sensitivity function. Images with different contrast resolutions were used as visual stimuli and presented against a control image in a two-choice test. Following animals' acclimatization and the first phase of training, the system was arranged such that animals could self-run multiple consecutive trials without human intervention. Results: All animals were trained within a week and remembered the task with 1 day of reinforcement when tested 1 month after the last visual assessment. All trained animals performed well during the trial with minimal screen side bias, especially at contrast threshold above 40%. Conclusions: Göttingen pigs are trainable for a visual psychophysics test and able to self-run the trial without human intervention. Translational Relevance: Contrast sensitivity is one of the key parameters to assess visual function in humans. The possibility of measuring the same parameters in a large animal model allows for a better translation and understanding of drug safety and efficacy in preclinical ophthalmology.

Differentiation of monocytes and polarized M1/M2 macrophages from human induced pluripotent stem cells.

Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. "monocyte factory." We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. For complete details on the use and execution of this protocol, please refer to Karlson et al.1 and Panicker et al.2.

A biologically validated mathematical model for decoding epithelial apical, basolateral, and paracellular electrical properties.

Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances (n = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology.NEW & NOTEWORTHY This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.

Progress in Stem Cells-Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery.

Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell-based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE derivation, their delivery, and recent clinical studies related to pRPE transplantation in patients. We have also discussed a few limitations with in vitro RPE culture and potential solutions to overcome such problems which can be helpful in developing functional RPE tissue.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.

Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

Retinal Pigment Epithelium Cell Development: Extrapolating Basic Biology to Stem Cell Research.

The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells.

A platform of assays for the discovery of anti-Zika small-molecules with activity in a 3D-bioprinted outer-blood-retina model.

The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.

Long-Term Transplant Effects of iPSC-RPE Monolayer in Immunodeficient RCS Rats.

Retinal pigment epithelium (RPE) replacement therapy is evolving as a feasible approach to treat age-related macular degeneration (AMD). In many preclinical studies, RPE cells are transplanted as a cell suspension into immunosuppressed animal eyes and transplant effects have been monitored only short-term. We investigated the long-term effects of human Induced pluripotent stem-cell-derived RPE (iPSC-RPE) transplants in an immunodeficient Royal College of Surgeons (RCS) rat model, in which RPE dysfunction led to photoreceptor degeneration. iPSC-RPE cultured as a polarized monolayer on a nanoengineered ultrathin parylene C scaffold was transplanted into the subretinal space of 28-day-old immunodeficient RCS rat pups and evaluated after 1, 4, and 11 months. Assessment at early time points showed good iPSC-RPE survival. The transplants remained as a monolayer, expressed RPE-specific markers, performed phagocytic function, and contributed to vision preservation. At 11-months post-implantation, RPE survival was observed in only 50% of the eyes that were concomitant with vision preservation. Loss of RPE monolayer characteristics at the 11-month time point was associated with peri-membrane fibrosis, immune reaction through the activation of macrophages (CD 68 expression), and the transition of cell fate (expression of mesenchymal markers). The overall study outcome supports the therapeutic potential of RPE grafts despite the loss of some transplant benefits during long-term observations.

Cell-Based Therapies for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. The pathogenesis of AMD involves dysfunction and loss of the retinal pigment epithelium (RPE), a monolayer of cells that provide nourishment and functional support for the overlying photoreceptors. RPE cells in mammals are not known to divide, renew or regenerate in vivo, and in advanced AMD, RPE loss leads to degeneration of the photoreceptors and impairment of vision. One possible therapeutic approach would be to support and replace the failing RPE cells of affected patients, and indeed moderate success of surgical procedures in which relatively healthy autologous RPE from the peripheral retina of the same eye was transplanted under the retina in the macular area suggested that RPE replacement could be a means to attenuate photoreceptor cell loss. This prompted exploration of the possibility to use pluripotent stem cells (PSCs) as a potential source for "healthy and young" RPE cells for such cell-based therapy of AMD. Various approaches ranging from the use of allogeneic embryonic stem cells to autologous induced pluripotent stem cells are now being tested within early clinical trials. Such PSC-derived RPE cells are either injected into the subretinal space as a suspension, or transplanted as a monolayer patch upon scaffold support. Although most of these approaches are at early clinical stages, safety of the RPE product has been demonstrated by several of these studies. Here, we review the concept of cell-based therapy of AMD and provide an update on current progress in the field of RPE transplantation.

Regulatory considerations for developing a phase I investigational new drug application for autologous induced pluripotent stem cells-based therapy product.

Induced pluripotent stem cells (iPSC)-based therapies have been hailed as the future of regenerative medicine because of their potential to provide treatment options for most degenerative diseases. A key promise of iPSC-based therapies is the possibility of an autologous transplant that may engraft better in the longer-term due to its compatibility with the patient's immune system. Despite over a decade of research, clinical translation of autologous iPSC-based therapies has been slow-partly due to a lacking pre-defined regulatory path. Here, we outline regulatory considerations for developing an autologous iPSC-based product and challenges associated with the clinical manufacturing of autologous iPSCs and their derivatives. These challenges include donor tissue source, reprogramming methods, heterogeneity of differentiated cells, controls for the manufacturing process, and preclinical considerations. A robust manufacturing process with appropriate quality controls and well-informed, prospectively designed preclinical studies provide a path toward successful approval of autologous iPSC-based therapies.

CSF1R blockade induces macrophage ablation and results in mouse choroidal vascular atrophy and RPE disorganization.

The choroid, which provides vascular supply to the outer retina, demonstrates progressive degeneration in aging and age-related macular degeneration (AMD). However mechanisms that maintain or compromise choroidal homeostasis are obscure. We discovered that the ablation of choroidal macrophages via CSF1R blockade was associated with choroidal vascular atrophy and retinal pigment epithelial (RPE) changes including structural disruption, downregulation of visual cycle genes, and altered angiogenic factor expression. Suspending CSF1R blockade following ablation enabled spontaneous macrophage regeneration, which fully restored original macrophage distributions and morphologies. Macrophage regeneration was accompanied by arrested vascular degeneration and ameliorated pathological RPE alterations. These findings suggest that choroidal macrophages play a previously unappreciated trophic role in maintaining choroidal vasculature and RPE cells, implicating insufficiency in choroidal macrophage function as a factor in aging- and AMD-associated pathology. Modulating macrophage function may constitute a strategy for the therapeutic preservation of the choroid and RPE in age-related retinal disorders.

Retinal Pigment Epithelium Replacement Therapy for Age-Related Macular Degeneration: Are We There Yet?

Pluripotent stem cells (PSCs) are a potential replacement tissue source for degenerative diseases. Age-related macular degeneration (AMD) is a blinding disease triggered by degeneration of the retinal pigment epithelium (RPE), a monolayer tissue that functionally supports retinal photoreceptors. Recently published clinical and preclinical studies have tested PSC-derived RPE as a potential treatment for AMD. Multiple approaches have been used to manufacture RPE cells, to validate them functionally, to confirm their safety profile, and to deliver them to patients either as suspension or as a monolayer patch. Since most of these studies are at an early regulatory approval stage, the primary outcome has been to determine the safety of RPE transplants in patients. However, preliminary signs of efficacy were observed in a few patients. Here, we review the current progress in the PSC-derived RPE transplantation field and provide a comparative assessment of various approaches under development as potential therapeutics for AMD.

Retinal stem cell transplantation: Balancing safety and potential.

Stem cell transplantation holds great promise as a potential treatment for currently incurable retinal degenerative diseases that cause poor vision and blindness. Recently, safety data have emerged from several Phase I/II clinical trials of retinal stem cell transplantation. These clinical trials, usually run in partnership with academic institutions, are based on sound preclinical studies and are focused on patient safety. However, reports of serious adverse events arising from cell therapy in other poorly regulated centers have now emerged in the lay and scientific press. While progress in stem cell research for blindness has been greeted with great enthusiasm by patients, scientists, doctors and industry alike, these adverse events have raised concerns about the safety of retinal stem cell transplantation and whether patients are truly protected from undue harm. The aim of this review is to summarize and appraise the safety of human retinal stem cell transplantation in the context of its potential to be developed into an effective treatment for retinal degenerative diseases.

The Homeodomain Transcription Factors Vax1 and Six6 Are Required for SCN Development and Function.

The brain's primary circadian pacemaker, the suprachiasmatic nucleus (SCN), is required to translate day-length and circadian rhythms into neuronal, hormonal, and behavioral rhythms. Here, we identify the homeodomain transcription factor ventral anterior homeobox 1 (Vax1) as required for SCN development, vasoactive intestinal peptide expression, and SCN output. Previous work has shown that VAX1 is required for gonadotropin-releasing hormone (GnRH/LHRH) neuron development, a neuronal population controlling reproductive status. Surprisingly, the ectopic expression of a Gnrh-Cre allele (Gnrhcre) in the SCN confirmed the requirement of both VAX1 (Vax1flox/flox:Gnrhcre, Vax1Gnrh-cre) and sine oculis homeobox protein 6 (Six6flox/flox:Gnrhcre, Six6Gnrh-cre) in SCN function in adulthood. To dissociate the role of Vax1 and Six6 in GnRH neuron and SCN function, we used another Gnrh-cre allele that targets GnRH neurons, but not the SCN (Lhrhcre). Both Six6Lhrh-cre and Vax1Lhrh-cre were infertile, and in contrast to Vax1Gnrh-cre and Six6Gnrh-cre mice, Six6Lhrh-cre and Vax1Lhrh-cre had normal circadian behavior. Unexpectedly, ~ 1/4 of the Six6Gnrh-cre mice were unable to entrain to light, showing that ectopic expression of Gnrhcre impaired function of the retino-hypothalamic tract that relays light information to the brain. This study identifies VAX1, and confirms SIX6, as transcription factors required for SCN development and function and demonstrates the importance of understanding how ectopic CRE expression can impact the results.

Deep learning predicts function of live retinal pigment epithelium from quantitative microscopy.

Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and noninvasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to noninvasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine-learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate noninvasive cell therapy characterization can be achieved with QBAM and machine learning.

Longitudinal adaptive optics fluorescence microscopy reveals cellular mosaicism in patients.

The heterogeneity of individual cells in a tissue has been well characterized, largely using ex vivo approaches that do not permit longitudinal assessments of the same tissue over long periods of time. We demonstrate a potentially novel application of adaptive optics fluorescence microscopy to visualize and track the in situ mosaicism of retinal pigment epithelial (RPE) cells directly in the human eye. After a short, dynamic period during which RPE cells take up i.v.-administered indocyanine green (ICG) dye, we observed a remarkably stable heterogeneity in the fluorescent pattern that gradually disappeared over a period of days. This pattern could be robustly reproduced with a new injection and follow-up imaging in the same eye out to at least 12 months, which enabled longitudinal tracking of RPE cells. Investigation of ICG uptake in primary human RPE cells and in a mouse model of ICG uptake alongside human imaging corroborated our findings that the observed mosaicism is an intrinsic property of the RPE tissue. We demonstrate a potentially novel application of fluorescence microscopy to detect subclinical changes to the RPE, a technical advance that has direct implications for improving our understanding of diseases such as oculocutaneous albinism, late-onset retinal degeneration, and Bietti crystalline dystrophy.

Clinical-grade stem cell-derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs.

Considerable progress has been made in testing stem cell-derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.