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There is an unmet clinical need to treat especially bilateral limbal stem cell deficiency (LSCD). For this allogeneic source of limbal stem cells (LSCs) differentiated from human pluripotent stem cells (hPSC) could provide a solution. In our studies, we have developed efficient methods to produce an enriched population of ABCG2‐positive as well as later arising p63/ABCB5‐postive hiPSC‐LSCs and our current research hypothesis is that these cells including both quiescent and proliferative sub‐populations could represent high regenerative potential in future clinical applications. Towards that end, we have been conducting molecular and functional analyses of these different sub‐populations to increase the understanding of their identity and heterogeneity as comparison to native human corneal limbus. With this approach we wish to gather data‐driven strategy towards clinical applications. [ABSTRACT FROM AUTHOR]

Aims/Purpose: Dysfunctional protein clearance, oxidative stress, mitochondrial damage, and chronic inflammation cause retinal pigment epithelial (RPE) cell damage and the development of age‐related macular degeneration (AMD). PGC‐1α can alleviate oxidative stress, is a key player in mitochondrial quality control and could be considered as a treatment target in AMD. Here, we assessed the effect of PGC‐1α inhibition on induced pluripotent stem cell (iPSC)‐derived RPE cells exposed to hydroquinone‐induced oxidative stress. Methods: iPSC‐derived RPE cells from two neovascular AMD patients or one healthy age‐matched control were cultured for 8 weeks on transwell cell culture inserts until mature. Cells were exposed to the PGC‐1α inhibitor SR‐18292 for 1 h before an exposure to hydroquinone for an additional 4 h. Lactate dehydrogenase (LDH) was measured from collected medium samples to assess cellular viability. Cells were lysed in 3x Laemmli sample buffer for analysis by western blotting or subjected to RNA extraction, followed by RT‐PCR analysis. Results: Hydroquinone caused a robust increase in LDH release to the medium, which was not modulated by PGC‐1α inhibition. In neovascular AMD‐derived iPSC‐RPE cells, hydroquinone caused an accumulation of p62 and LC3‐II, an effect that was reversed by SR‐18292 pre‐treatment. In control iPSC‐RPE cells, neither hydroquinone nor SR‐18292 affected autophagic markers p62 or LC3. RT‐PCR analysis showed an increase in p62 mRNA levels following hydroquinone exposure in both control‐ and AMD‐derived iPSC‐RPE cells while no change was observed in the RNA levels of LC3B or mTOR. Conclusions: Increased oxidative stress caused autophagy dysregulation in AMD‐derived iPSC‐RPE cells but not in healthy age‐matched control cells. Interestingly, PGC‐1α inhibition promoted autophagy and the clearance of p62, but further studies are necessary to explore the underlying pathways and the disease‐specific differences observed between healthy and disease‐derived RPE cells. [ABSTRACT FROM AUTHOR]

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 < 200 Ω·cm2 > 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. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell‐derived retinal pigment epithelium (hPSC‐RPE) transplantation is currently under evaluation as treatment for macular degeneration. In our studies, we have shown that the culture substrate affects hPSC‐RPE properties in vitro, and our results also suggest that the choice of coating proteins may have implications to the functional properties of these cells also after important cryostorage cell banking. Importantly, transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. In our studies, we have shown that the maturation level of the cultured hPSC‐RPE cells influence for example their calcium signalling properties and for the ability to tolerate cellular stress. With our collaborators, we have also studied the role of ion channels and their involvement in diverse physiological functions of hPSC‐RPEs. Last but not least, we have transplanted hPSC‐RPE as cell sheets in a novel immunosuppressed non‐human primate (NHP) model with a disrupted ocular immune privilege to study safety and submacular graft integration. In transplantations, 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 to the histology performed after a 28‐day follow‐up. As a result, miOCT revealed foveal disruption and ERG showed amplitude and peak time preservation in cases with favourable surgical outcomes. Histology confirmed photoreceptor preservation above the grafts and in vivo phagocytosis by hPSC‐RPE, albeit evidence of cytoplasmic redistribution of opsin in photoreceptors and glia hypertrophy. The established immunosuppression protocol efficiently suppressed retinal T cell infiltration and microglia activation. As a conclusion, our results suggest the important role of biomimetic in vitro culture for the hPSC‐RPE maturation and for the functional transplantation outcome. [ABSTRACT FROM AUTHOR]

. 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. [ABSTRACT FROM AUTHOR]