Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell‐derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage‐gated Ca2+ (CaV) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)‐derived RPE. Whole‐cell patch‐clamp recordings from these cells revealed slowly inactivating L‐type currents comparable to freshly isolated mouse RPE. Some hESC‐RPE cells also carried fast transient T‐type resembling currents. These findings were confirmed by immunostainings from both hESC‐ and mouse RPE that showed the presence of the L‐type Ca2+ channels CaV1.2 and CaV1.3 as well as the T‐type Ca2+ channels CaV3.1 and CaV3.2. The localization of the major subtype, CaV1.3, changed during hESC‐RPE maturation co‐localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L‐type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC‐RPE. Overall, this study demonstrates that a functional machinery of voltage‐gated Ca2+ channels is present in mature hESC‐RPE, which is promising for the success of transplantation therapies. stem cells translational medicine 2019;8:179&15

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

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.

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), Ca 2+ and its ATP-dependent signaling pathways play important roles in the retina maintenance functions. Changes in intracellular Ca 2+ concentration can be measured from living cells by Ca 2+ imaging. Combining these measurements with quantitative analysis of Ca 2+ 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 Ca 2+ response data recorded from large fields of intact RPE monolayers. The analysis revealed significant heterogeneity in ATP-induced Ca 2+ 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.

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.