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Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

Background Transplantation 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. Methods Here, 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. Results The 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. Conclusions Our 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.

Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract

Background Human pluripotent stem cells (hPSCs) provide a promising cell source for retinal cell replacement therapy but often lack standardized cell production and live-cell shipment logistics as well as rigorous analyses of surgical procedures for cell transplantation in the delicate macula area. We have previously established a xeno- and feeder cell-free production system for hPSC differentiated retinal pigment epithelial (RPE) cells, and herein, a novel immunosuppressed non-human primate (NHP) model with a disrupted ocular immune privilege is presented for transplanting human embryonic stem cell (hESC)-derived RPE on a scaffold, and the safety and submacular graft integration are assessed. Furthermore, the feasibility of intercontinental shipment of live hESC-RPE is examined. Methods Cynomolgus monkeys were systemically immunosuppressed and implanted with a hESC-RPE monolayer on a permeable polyester-terephthalate (PET) scaffold. Microscope-integrated intraoperative optical coherence tomography (miOCT)-guided surgery, postoperative follow-up incorporated scanning laser ophthalmoscopy, spectral domain (SD-) OCT, and full-field electroretinography (ERG) were used as outcome measures. In addition, histology was performed after a 28-day follow-up. Results Intercontinental cell shipment, which took >30 h from the manufacturing to the transplantation site, did not alter the hESC-RPE quality. The submacular hESC-RPE xenotransplantation was performed in 11 macaques. The miOCT typically revealed foveal disruption. ERG showed amplitude and peak time preservation in cases with favorable surgical outcomes. Histology confirmed photoreceptor preservation above the grafts and in vivo phagocytosis by hESC-RPE, albeit evidence of cytoplasmic redistribution of opsin in photoreceptors and glia hypertrophy. The immunosuppression protocol efficiently suppressed retinal T cell infiltration and microglia activation. Conclusion These results suggest both structural and functional submacular integrations of hESC-RPE xenografts. It is anticipated that surgical technique refinement will further improve the engraftment of macular cell therapeutics with significant translational relevance to improve future clinical trials. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02395-6.

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method. publishedVersion

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.

Purpose: Delivery of Advanced Therapy Medicinal Products to the submacular space is increasingly evolving into a therapeutic modality. Cell replacement for age-related macular degeneration (AMD) and gene therapy for RPE65 are recent successful examples. Herein, a nonhuman primate (NHP) model was used to investigate surgical means to detach the macula. Methods: Sixteen eyes of 13 healthy macaques underwent a 25-gauge vitrectomy and subretinal injection of balanced salt solution monitored by microscope-integrated intra-operative optical coherence tomography (miOCT). The animals were followed with OCT and histology. Results: The miOCT monitoring allowed a more precise definition of surgical trauma ranging from an initial full-thickness foveal tear, or induction of a cystoid macular edema (CME), until no foveal defect was discernible, as the technique improved. However, as the subretinal fluid wave detached the fovea, the aforementioned lesions formed, whereas persistent retinal adhesion reproducibly proved to remain in the distal parafoveal semi-annulus. Measures to reduce foveal trauma during submacular fluid injection included reducing intraocular pressure, injection volume, and velocity, as well as the retinal location for bleb initiation, use of a vitreous tamponade, and a dual-bore subretinal cannula. Conclusions: A stable very low intraocular pressure and careful subretinal injection may avoid tangential macular stretching or mechanical CME formation, while vitreous tamponade may facilitate a more lamellar subretinal flow, all thereby reducing foveal trauma during submacular injection in NHP. Translational Relevance: These results can be relevant to any submacular surgery procedure used today, as they synergistically reduce the risk of compromising foveal integrity. publishedVersion

Age-related macular degeneration (AMD) is the leading cause of vision loss in senior citizens in the developed world. The disease is characterised by the degeneration of a specific cell layer at the back of the eye – the retinal pigment epithelium (RPE), which is essential in retinal function. The most promising therapeutic option to restore the lost vision is considered to be RPE cell transplantation. This work focuses on the development of biodegradable biomaterials with similar properties to the native Bruch’s membrane as carriers for RPE cells. In particular, the breath figure (BF) method was used to create semi-permeable microporous films, which were thereafter used as the substrate for the consecutive Langmuir-Schaefer (LS) deposition of highly organised layers of collagen type I and collagen type IV. The newly developed biomaterials were further characterised in terms of surface porosity, roughness, hydrophilicity, collagen distribution, diffusion properties and hydrolytic stability. Human embryonic stem cell-derived RPE cells (hESC-RPE) cultured on the biomaterials showed good adhesion, spreading and morphology, as well as the expression of specific protein markers. Cell function was additionally confirmed by the assessment of the phagocytic capacity of hESC-RPE. Throughout the study, microporous films consistently showed better results as cell culture materials for hESC-RPE than dip-coated controls. This work demonstrates the potential of the BF-LS combined technologies to create biomimetic prosthetic Bruch’s membranes for hESC-RPE transplantation. Statement of Significance Age-related macular degeneration (AMD) is a leading cause of central blindness in developed countries, associated with the degeneration of the retinal pigment epithelium (RPE), a specific cell layer at the back of the eye. Transplantation of RPE cells derived from stem cells is considered the best option to treat these patients. In this work, we developed a cell carrier for human embryonic stem cell-derived RPE that resembled the upper layers of the membrane that naturally supports the RPE cells in the retina. The new combination of technologies employed in this study resulted in very promising materials as confirmed by our studies on cell proliferation, morphology and function.

Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy.

Calcium is one of the most important second messengers in cells and thus involved in a variety of physiological processes. In retinal pigment epithelium (RPE), Ca2+ and its ATP-dependent signaling pathways play important roles in the retina maintenance functions. Changes in intracellular Ca2+ concentration can be measured from living cells by Ca2+ imaging. Combining these measurements with quantitative analysis of Ca2+ response properties enables studies of signaling pathways affecting RPE functions. However, robust tools for response analysis from large cell populations are lacking. We developed MATLAB-based analysis tools for single cell level Ca2+ response data recorded from large fields of intact RPE monolayers. The analysis revealed significant heterogeneity in ATP-induced Ca2+ responses inside cell populations regarding magnitude and response kinetics. Further analysis including response grouping and parameter correlations allowed us to characterize the populations at the level of single cells.

Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1646-1656, 2016.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

Purpose Retinopathy is an important manifestation of trifunctional protein (TFP) deficiencies but not of other defects of fatty acid oxidation. The common homozygous mutation in the TFP α-subunit gene HADHA (hydroxyacyl-CoA dehydrogenase), c.1528G>C, affects the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of TFP and blindness in infancy. The pathogenesis of the retinopathy is unknown. This study aimed to utilize human induced pluripotent stem cell (hiPSC) technology to create a disease model for the disorder, and to derive clues for retinopathy pathogenesis. Methods We implemented hiPSC technology to generate LCHAD deficiency (LCHADD) patient-specific retinal pigment epithelial (RPE) monolayers. These patient and control RPEs were extensively characterized for function and structure, as well as for lipid composition by mass spectrometry. Results The hiPSC-derived RPE monolayers of patients and controls were functional, as they both were able to phagocytose the photoreceptor outer segments in vitro. Interestingly, the patient RPEs had intense cytoplasmic neutral lipid accumulation, and lipidomic analysis revealed an increased triglyceride accumulation. Further, patient RPEs were small and irregular in shape, and their tight junctions were disorganized. Their ultrastructure showed decreased pigmentation, few melanosomes, and more melanolysosomes. Conclusions We demonstrate that the RPE cell model reveals novel early pathogenic changes in LCHADD retinopathy, with robust lipid accumulation, inefficient pigmentation that is evident soon after differentiation, and a defect in forming tight junctions inducing apoptosis. We propose that LCHADD-RPEs are an important model for mitochondrial TFP retinopathy, and that their early pathogenic changes contribute to infantile blindness of LCHADD.

Purpose To study immunogenic properties of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. Methods Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis in vitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE ± interferon-gamma (IFN-γ) stimulation (n = 1). Xeno-free hESC-RPE on PET with TER or PET alone were transplanted into 18 rabbits with short-term triamcinolone ± extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4 weeks, the eyes were processed for histology and transmission electron microscopy. Results Upon in vitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4 weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1 week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4 weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n = 1). Conclusions Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4 weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.

Summary Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide a promising cell source for studying retinal development, for disease modelling, and retinal cell replacement therapies. Several research groups, including ours, have demonstrated that hESC-RPE structure, function, and physiology resembles that of native RPE regarding cellular fine structure and expression of many RPE signature genes and proteins. To characterize the hESC-RPE proteome in larger scale, we have compared hESC-RPE protein expression to primary human RPE using isobaric tags for relative quantitation (iTRAQ) technology. The hESC-RPE proteome reflected that of native RPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No adverse signs such as increased stress, proliferation, or retinal degeneration-related changes were seen in hESC-RPE, while proteins involved in key RPE functions such as visual cycle and phagocytosis were detected. Proteomics provides valuable tools for validation of hESC-RPE, studying disease mechanisms, and discovery of new biomarkers and therapeutic targets.

Summary Human induced pluripotent stem cells (hiPSCs) offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF) in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation., Highlights • Small-molecule induction directs early stage differentiation • Subsequent maturation yields homogeneous populations of p63-positive cells • p63-positive progenitor cells are capable of terminal differentiation • The serum-free and feeder-free method can be upgraded to fully defined and xeno free, Skottman and colleagues describe an efficient and reproducible method for differentiation of corneal epithelial progenitor cells from human induced pluripotent stem cells. Early stage differentiation is directed with the help of two small-molecule inhibitors in combination with FGF, mimicking developmental mechanisms. The resulting p63-positive progenitor cells are capable of terminal differentiation toward corneal epithelial-like cells upon further maturation.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells and possess LESC-like characteristics.

Purpose Corneal epithelium is maintained by limbal stem cells, and their transplantation has been used to treat limbal stem cell deficiency (LSCD). However, this is only possible if enough healthy limbal tissue is available. Thus, novel cell sources for treating LSCD are needed. Human induced pluripotent stem cells (hiPSCs) provide unique opportunities for differentiation of limbal and corneal epithelial cells for cell transplantations. Methods In order to improve the efficiency and reproducibility of hiPSCs differentiation towards corneal epithelial progenitor cells, signaling cues active during ocular surface ectoderm development was replicated with two small-molecule inhibitors in combination with growth factors. The extent of differentiation was evaluated by following the expression of key markers using immunocytochemistry and qPCR at several time-points. Results Small-molecule induction down-regulated the expression of pluripotency marker OCT4 while up-regulating the eye-field transcription factor PAX6. Protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63-positive after five weeks of differentiation. Finally, after a total of six weeks in differentiation culture, the two markers specific to terminally differentiated corneal epithelium, cytokeratins 3 and 12, were expressed in an average of 35% and 71% of cells, respectively. Conclusion In contrast to all earlier studies, corneal epithelial cells were differentiated in serum-free culture conditions without the use of amniotic membrane or other undefined culture substrates. This highly efficient differentiation method could potentially be used for treating LSCD in the future. Commercial interest

Purpose Human pluripotent stem cell (hPSC) therapy is a potential approach for the replacement of degenerated retinal pigment epithelium (RPE) cells. For therapeutic use, safety and functionality of the RPE cells needs to be guaranteed. In addition to the basic cell and molecular biological characterizations it is vitally important to assess the differentiation status of acquired cells with functional tests. Methods We have previously shown that hPSC differentiate into RPE in xeno-free and defined conditions. Recent advances as a culture of hPSC-derived RPE cells sheets on clinically accepted material have led us to investigate the functionality of the RPE epithelium. The tightness of the epithelium was evaluated by measuring transepithelial electrical resistance and cell permeability. The phagocytic properties of hPSC-RPE cells were studied using rat retinal explants. Results Human PSC-derived putative RPE cells exhibits typical pigmented cobblestone-like morphology and express RPE specific markers at both mRNA and protein level. In addition, cultured cells form a polarized epithelium with high integrity. In addition, co-culturing hPSC-RPE monolayers with rat retinal explants demonstrated that rhodopsin is internalized by cells in vitro. Conclusion We have demonstrated that hPSC-RPE monolayers acquired RPE-like properties, including characteristic RPE phenotype, expression of RPE markers and barrier functions. In addition, cells were capable of binding and internalizing rat rhodopsin when co-cultured with rat retinal explants. Currently we investigate the correlations of the functions in the Royal College of Surgeons (RCS) rats.

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation. Public Library of Science open access

Purpose Stem cell therapy is a potential approach for the replacement of degenerated retinal pigment epithelial (RPE) cells essential for vision. For therapeutical use, the differentiation of RPE cells from human pluripotent stem cells (hPSC) should be performed in a culture environment fulfilling clinical quality requirements. For example the use of xeno-products should be avoided as this bears the danger of interspecies transfer of viruses and incorporation of immunogenic molecules. Results Animal-derived substances, such as fetal bovine serum, mouse fibroblasts, and Matrigel are widely used in the differentiation and culture of hPSC-RPE cells. Recently, some protocols utilizing human feeders in the culture of pluripotent cells and small molecules in the differentiation process have been published. In our studies, we have also successfully differentiated pigmented cells from hPSC lines without the use of animal cells or serum. These cells show characteristics of RPE cells such as morphology, polarity, expression of genes and proteins typical for RPE cells, and phagocytosis of photoreceptor outer segments. Conclusion Tissue engineering holds a great promise for treating retinal degenerative diseases. To date, RPE-like cells have been successfully produced from hPSCs and lately the protocols have been developed further towards xeno-free and defined culture systems enabling clinical grade cell production.

Purpose Defined differentiation of functional RPE cells from human embryonic (hESC) or induced pluripotent stem cells (iPSC) is a prerequisite for their use in individualised disease modelling, drug discovery and transplantation for retinal diseases. In this study we report differentiation of RPE cells from hESC and iPSC in condition enabling easy translation to clinical quality cell production. Methods Pluripotent stem cells were produced on human fibroblast feeder cells in serum-free medium. The differentiation of the cells was induced using bFGF and feeder cell removal approach under serum-free conditions. The pigmentation and RPE morphology of the cells were analysed and the expression of genes and proteins characteristics for RPE cells were studied. The in vitro functionality of the cells was analysed using ELISA measurements and phagocytosis of photoreceptor outer segments. The integrity of the generated RPE layer was analysed using transepithelial electric resistance (TEER) measurements. Results With our differentiation method, we were able to generate RPE cells with satisfying efficiency. The typical pigmented cobblestone-like morphology and expression of RPE specific markers were confirmed at gene and protein level. The differentiated cells were able to phagocytose and secreted growth factors typical for RPE cells. In addition, cells formed a well polarized epithelium with high integrity, exhibiting good TEER values. Conclusion We have developed progressive differentiation protocol for production of functional RPE cells from hESC and iPSC. The developed production method is currently translated to defined and animal component free conditions enabling clinical grade cell production.