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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.

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.

Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone-crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.

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.

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).

The breath figure (BF) method is an easy, low-cost method to prepare films with a highly organized honeycomb-like porous surface. The particular surface topography and porous nature of these materials makes them valuable substrates for studying the complex effects of topography on cell fate, and to produce biomimetic materials with high performance in tissue engineering. Numerous researchers over the last two decades have studied the effects of the honeycomb topography on a variety of primary and immortalized cell lines, and drew important conclusions that can be translated to the construction of optimal biomaterials for cell culture. The literature also encouragingly shows the potential of honeycomb films to induce differentiation of stem cells down a specific lineage without the need for biochemical stimuli. Here, we review the main studies where BF honeycomb films are used as substrates for tissue engineering applications. Furthermore, we highlight the numerous advantages of the porous nature of the films, such as the enhanced, spatially controlled adsorption of proteins, the topographical cues influencing cellular behavior, and the enhanced permeability which is essential both in vitro and in vivo. Finally, this review highlights the elegant use of honeycomb films as drug-eluting biomaterials or as reservoirs for distinct drug delivery systems. Statement of Significance Combining biocompatible surfaces and 3D nano/microscale topographies, such as pores or grooves, is an effective strategy for manufacturing tissue engineering scaffolds. The breath figure (BF) method is an easy technique to prepare cell culture substrates with an organized, honeycomb-like porous surface. These surface features make these scaffolds valuable for studying how the cells interact with the biomaterials. Their unique surface topography can also resemble the natural environment of the tissues in the human body. For that reason, numerous studies, using different cell types, have shown that honeycomb films can constitute high performance substrates for cell culture. Here, we review those studies, we highlight the advantages of honeycomb films in tissue engineering and we discuss their potential as unique drug-eluting systems.

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.

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.

Purpose: Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. Methods: In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration. Results: In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. Conclusion: In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.

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

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

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.

Purpose Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique has potential in the treatment of patients with severe ocular surface injuries. Currently, this technique is mainly based on utilization of xenogenic/allogenic components such as murine feeders, serum and amniotic membrane. The use of animal-derived materials possesses risk of pathogen transmission, immune reactions and graft rejection. Methods The formation of stratified sheets by human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor concentration was examined. The integrity of the epithelium was measured by transepithelial electrical resistance. The tissue-engineered cell sheets were also studied for histology and immunohistochemical markers for epithelial keratins (K), cell proliferation and cell adhesion. Results In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue positive for keratins K3/12, K4, and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. Conclusion Tight multi-layered epithelium with proliferative potential can be produced from human oral mucosal epithelial cells under defined culture conditions and without the use of serum.

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.

Purpose Corneal epithelial reconstruction by using cultivated oral mucosal epithelial transplantation (COMET) technique is a viable treatment option for severe ocular surface disorders. So far this has been based on utilization of xenogenic/allogenic components such as 3T3 mouse feeder cells, serum or amniotic membrane. In this study, we present a new defined and serum-free method to obtain oral mucosal epithelium for COMET. Methods Epithelial cells of oral mucosal biopsies were isolated and seeded on collagen IV inserts in a defined progenitor cell targeted (PCT) medium. After 90% confluency the medium was switched to a defined non-PCT medium, and differentiation was induced by increased calcium concentration and airlifting for 6 days. No serum was added to the cultures. The integrity of the epithelium was measured by transepithelial electrical resistance (TEER) before embedding in paraffin. 5 μm sections were studied for histology and immunohistochemical markers for epithelial keratins (K), cell proliferation and cell adhesion. Results The cultivated oral epithelial cells formed a stratified tissue of 4 to 15 cell layers. The cell sheets showed normal keratin expression profiles and were positive for K15 and p63, indicating the presence of putative progenitor cells. The cultivated epithelium also exhibited excellent TEER values, high integrity and ZO1 expression. Conclusion We have developed a novel method to produce tight multi-layered epithelium with proliferative potential for COMET under defined, serum-free conditions, potentially suitable for clinical applications.

Autoimmune regulator (AIRE) is a transcriptional regulator involved in establishing immunological self-tolerance. Mutations in the AIRE gene lead to the development of the autosomal, recessively inherited, organ-specific autoimmune disease, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). The AIRE protein is mainly localized in the cell nucleus where it is associated with nuclear bodies. The N-terminal part of AIRE has been previously shown to mediate nuclear localization of the protein. However, the functional nuclear localization signal (NLS) and nuclear import mechanisms of AIRE have not been identified. We show that, although the amino-acid sequence of AIRE contains a potential bipartite NLS consisting of amino acids 110-114 and 131-133, only the latter part constitutes a functional NLS. Furthermore, we show by in vitro binding assays that AIRE interacts with multiple members of the nuclear transport receptor importin alpha family, mainly alpha1, alpha3, and alpha5, and that these interactions depend on the intactness of the Arg-Lys-rich NLS of AIRE. In addition, we found that AIRE binds to the 'minor' NLS-binding site of importin alpha3 and alpha5 proteins consisting of the C-terminal armadillo repeats 7-9. Our findings strongly suggest that the nuclear import of AIRE is mediated by the classical importin alpha/beta pathway through binding to several importin alpha family members.