Cell-derived Retinal Pigment Epithelial Cells Research Articles

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.

SummaryHuman embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

Differential gene expression analysis using RNA sequencing: retinal pigment epithelial cells after exposure to continuous-wave and subthreshold micropulse laser.

Subthreshold micropulse laser (SMPL) is more clinically efficient for the treatment of diabetic macular edema (DME) than the conventional continuous-wave (CW) laser. We aimed to characterize transcriptome changes after the application of these lasers and to compare the transcripts. Human pluripotent stem cell-derived retinal pigment epithelial cells were exposed to laser irradiation. Differentially expressed genes (DEGs), distribution of heat shock protein (Hsp) family, gene expression profile, and gene ontology (GO) enrichment analysis based on RNA sequencing data were investigated at 3 h and 24 h after irradiation. CW laser induced more DEGs than SMPL (1771 vs. 520 genes). The expression of the Hsp family was confirmed in both groups: however, the induction patterns was different for different genes. GO enrichment analysis revealed that CW laser upregulated the expression of DEGs involved in vasculature development (GO: 0001944), related to apoptosis and repair after cell injury whereas SMPL upregulated the expression of DEGs involved in photoreceptor cell maintenance (GO: 0045494), photoreceptor cell development (GO: 0042461), and sensory perception of light stimuli (GO: 0050953). The results provide insights into the genetic responses and may contribute to the understanding of the molecular mechanisms of laser-induced thermal effects.

Cell maturation influences the ability of hESC-RPE to tolerate cellular stress

BackgroundTransplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. The transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. Yet, no comparison of the ability of hESC-RPE at different maturity levels to tolerate cellular stress has been reported.MethodsHere, we analyzed the ability of the hESC-RPE populations with early (3 weeks) and late (12 weeks) maturation status to tolerate cellular stress caused by chemical cell stressors protease inhibitor (MG132) or hydrogen peroxide (H2O2). After the treatments, the functionality of the RPE cells was studied by transepithelial resistance, immunostainings of key RPE proteins, phagocytosis, mitochondrial membrane potential, Ca2+ signaling, and cytokine secretion.ResultsThe hESC-RPE population with late maturation status consistently showed improved tolerance to cellular stress in comparison to the population with early maturity. After the treatments, the early maturation status of hESC-RPE monolayer showed impaired barrier properties. The hESC-RPE with early maturity status also exhibited reduced phagocytic and Ca2+ signaling properties, especially after MG132 treatment.ConclusionsOur results suggest that due to better tolerance to cellular stress, the late maturation status of hESC-RPE population is superior compared to monolayers with early maturation status in the transplantation therapy settings.

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin.

To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N). hES-RPE cells were used in the experiment. The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence. The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose-dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 µg/cm2, respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature. VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.

PEX6 Mutations in Peroxisomal Biogenesis Disorders: An Usher Syndrome Mimic.

PurposePeroxisomal biogenesis disorders (PBDs) represent a spectrum of conditions that result in vision loss, sensorineural hearing loss, neurologic dysfunction, and other abnormalities resulting from aberrant peroxisomal function caused by mutations in PEX genes. With no treatments currently available, we sought to investigate the disease mechanism in a patient with a PBD caused by defects in PEX6 and to probe whether overexpression of PEX6 could restore peroxisome function and potentially offer therapeutic benefit.DesignLaboratory-based study.ParticipantsA 12-year-old boy sought treatment with hearing loss and retinopathy. After negative results in an Usher syndrome panel, targeted genetic testing revealed compound heterozygous mutations in PEX6. These included a 14-nucleotide deletion (c.802_815del: p.(Asp268Cysfs∗8)) and a milder missense variant (c.35T→C:(p.Phe12Ser)).MethodsPatient-derived skin fibroblasts were cultured, and a PEX6 knockout cell line was developed using clustered regularly interspaced short palindromic repeats and Cas9 technology in HEK293T cells to emulate a more severe disease phenotype. Immunoblot analysis of whole cell lysates was performed to assess peroxisome number. Immunofluorescence studies used antibodies against components of the peroxisomal protein import pathway to interrogate the effects of mutations in PEX6 on protein trafficking.Main Outcome MeasuresPrimary outcome measures were peroxisome abundance and matrix protein import.ResultsPeroxisome number was not significantly different between control fibroblasts and patient fibroblasts; however, fewer peroxisomes were observed in PEX6 knockout cells compared with wild-type cells (P = 0.04). Analysis by immunofluorescent microscopy showed significantly impaired peroxisomal targeting signal 1- and peroxisomal targeting signal 2-mediated matrix protein import in both patient fibroblasts and PEX6 knockout cells. Overexpressing PEX6 resulted in improved matrix protein import in PEX6 knockout cells.ConclusionsMutations in PEX6 were responsible for combined hearing loss and retinopathy in our patient. The primary peroxisomal defect in our patient’s skin fibroblasts was impaired peroxisomal protein import as opposed to reduction in the number of peroxisomes. Genetic strategies that introduce wild-type PEX6 into cells deficient in PEX6 protein show promise in restoring peroxisome function. Future studies of patient-specific induced pluripotent stem cell-derived retinal pigment epithelium cells may clarify the role of PEX6 in the retina and the potential for gene therapy in these patients.

Trends of Stem Cell Therapies in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a highly prevalent irreversible impairment in the elderly population worldwide. Stem cell therapies have been considered potentially viable for treating AMD through the direct replacement of degenerated cells or secretion of trophic factors that facilitate the survival of existing cells. Among them, the safety of pluripotent stem cell-derived retinal pigment epithelial (RPE) cell transplantation against AMD, and some hereditary retinal degenerative diseases, has been discussed to a certain extent in clinical studies of RPE cell transplantation. Preparations are in progress for its clinical application. On the other hand, clinical trials using somatic stem cells are also being conducted, though these had controversial outcomes. Retinal regenerative medicine using stem cells is expected to make steady progress toward practical use while new technologies are incorporated from various fields, thereby making the role of ophthalmologists in this field increasingly important.

Deterioration of phagocytosis in induced pluripotent stem cell-derived retinal pigment epithelial cells established from patients with retinitis pigmentosa carrying Mer tyrosine kinase mutations

Retinitis pigmentosa (RP) is an incurable retinal degenerative disease with an unknown mechanism of disease progression. Mer tyrosine kinase (MERTK), which encodes a receptor of the Tyro3/Axl/Mer family of tyrosine kinases, is one of the causal genes of RP. MERTK is reportedly expressed in the retinal pigment epithelium (RPE) and is essential for phagocytosis of the photoreceptor outer segment. Here, we established induced pluripotent stem cells (iPSC) from patients with RP having homozygous or compound heterozygous mutations in MERTK, and from healthy subjects; the RP patient- and healthy control-derived iPSCs were differentiated into RPE cells. Although cytoskeleton staining suggested that polarity may have been disturbed mildly, there were no apparent morphological differences between the diseased and normal RPE cells. The internalization of photoreceptor outer segments in diseased iPSC-RPE cells was significantly lower than that in normal iPSC-RPE cells. This in vitro disease model may be useful for elucidating the mechanisms of disease progression and screening treatments for the disease.

Immunological aspects of RPE cell transplantation.

Retinal pigment epithelial (RPE) cells have several functions, including support of the neural retina and choroid in the eye and immunosuppression. Cultured human RPE cells directly suppress inflammatory immune cells. For instance, they directly suppress the activation of T cells in vitro. In contrast, transplanted allogeneic human RPE cells are rejected by bystander immune cells such as T cells in vivo. Recently, human embryonic stem cell-derived RPE cells have been used in several clinical trials, and human induced pluripotent stem cell (iPSC)-RPE cells have also been tested in our clinical study in patients with retinal degeneration. Major safety concerns after stem cell-based transplantation surgery include hyper-proliferation, tumorigenicity, or ectopic tissue formation, but these events have currently not been seen in any of these patients. However, if RPE cells are allogeneic, there are concerns about immune rejection issues that have been raised in previous clinical trials. We therefore performed a preclinical study of allogeneic iPSC-RPE cell transplantation in animal rejection models. We then conducted autogenic or allogeneic iPSC-RPE cell transplantation in clinical studies of patients with age-related macular degeneration. In this review, we focus on immunological studies of RPE cells, including iPSC-derived cells. iPSC-RPE cells have unique inflammatory (immunosuppressive and immunogenic) characteristics like primary cultured RPE cells. The purpose of this review is to summarize the current findings obtained from preclinical (basic research) and clinical studies in iPSC-RPE cell transplantation, especially the immunological aspects.

A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

SummaryRecent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

Evaluating BEST1 mutations in pluripotent stem cell-derived retinal pigment epithelial cells.

Bestrophin-1 (BEST1) is a calcium-activated chloride channel (CaCC) predominantly expressed at the basolateral membrane of the retinal pigment epithelium (RPE). Over 250 mutations in the BEST1 gene have been documented to cause at least five retinal degenerative disorders, commonly termed bestrophinopathies, to which no treatment is currently available. Therefore, understanding the influences of BEST1 disease-causing mutations on the physiological function of BEST1 in RPE is critical for deciphering the pathology of bestrophinopathies and developing therapeutic strategies for patients. However, this task has been impeded by the rarity of BEST1 mutations and limited accessibility to native human RPE cells. Here, we describe a pluripotent stem cell (PSC)-based pipeline for reproducibly generating RPE cells expressing endogenous or exogenous mutant BEST1, which provides us with a powerful "disease-in-a-dish" approach for studying BEST1 mutations in physiological environments.

Human Stem Cell-Derived Retinal Pigment Epithelial Cells as a Model for Drug Screening and Pre-Clinical Assays Compared to ARPE-19 Cell Line

Background and ObjectivesEye diseases have a high socioeconomic impact on society and may be one of the fields in which most stem cell-related scientific accomplishments have been achieved recently. In this context, human Pluripotent Stem Cell (hPSC) technology arises as an important tool to produce and study human Embryonic Stem cell derived-Retinal Pigmented Epithelial Cells (hES-RPE) for several applications, such as cell therapy, disease modeling, and drug screening. The use of this technology in pre-clinical phases attends to the overall population desire for animal-free product development. Here, we aimed to compare hES-RPE cells with ARPE-19, one of the most commonly used retinal pigmented epithelial immortalized cell lines.Methods and ResultsFunctional, cellular and molecular data obtained suggest that hES-RPE cells more closely resembles native RPEs compared to ARPE-19. Furthermore, hES-RPE revealed an interesting robustness when cultured on human Bruch’s membrane explants and after exposure to Cyclosporine (CSA), Sirolimus (SRL), Tacrolimus (TAC), Leflunomide (LEF) and Teriflunomide (TER). On these conditions, hES-RPE cells were able to survive at higher drug concentrations, while ARPE-19 cell line was more susceptible to cell death.ConclusionsTherefore, hES-RPEs seem to have the ability to incur a broader range of RPE functions than ARPE-19 and should be more thoroughly explored for drug screening.

Improving cell survival and engraftment in vivo via layer-by-layer nanocoating of hESC-derived RPE cells

BackgroundHuman embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of hESC-RPE cells is a great challenge.MethodsHere, we report hESC-RPE cells that are embedded with polyelectrolytes gelatin and alginate by layer-by-layer (LbL) self-assembly technique, based on the opposite charge of alternate layers. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo.ResultsThis strategy obviously decreased the immunogenicity of hESC-RPE cells without affecting its activity. LbL-RPE cell transplants into the subretinal space of Royal College of Surgeons (RCS) rats optimized cell engraftment and decreased immunogenicity compared to untreated RPE cell transplants (immunosuppression was not used during the 21-week study). Visual-functional assay with electroretinogram recordings (ERGs) also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants.ConclusionsWe demonstrate that transplanted LbL-RPE cells have better viability and grafting efficiency, optimized immunogenicity, and visual function. Therefore, LbL engineering is a promising method to increase the efficacy of hESC-RPE cell transplantation.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro

ABSTRACT Purpose Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). Methods ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin’s effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. Results Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. Conclusions This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin’s viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Preclinical safety studies of human embryonic stem cell-derived retinal pigment epithelial cells for the treatment of age-related macular degeneration.

As pluripotent stem cell (PSC)‐based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age‐related macular degeneration is one of the first that has reached patients. We have previously established a xeno‐free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)‐derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole‐genome sequencing (WGS) to assess genome stability, single‐cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC‐based therapies and the data support safety of the hESC‐RPE cells generated through our in vitro differentiation methodology.

Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide.

SummaryEpithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-β and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.

Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II

SummaryHuman embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Annexin A8 regulates Wnt signaling to maintain the phenotypic plasticity of retinal pigment epithelial cells

Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and β-catenin, which was reduced in the presence of activated TGF-β1. TGF-β1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced β-catenin translocation and GSK-3β phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration.

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.