Bioprinting of human pluripotent stem cell derived corneal endothelial cells with hydrazone crosslinked hyaluronic acid bioink.

BACKGROUND: Human corneal endothelial cells lack regenerative capacity through cell division in vivo. Consequently, in the case of trauma or dystrophy, the only available treatment modality is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor which faces a high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink. METHODS: Corneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas. RESULTS: The bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10 days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer. CONCLUSIONS: Our results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.

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

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.

Corneal epithelial differentiation of human pluripotent stem cells generates ABCB5+ and ∆Np63α+ cells with limbal cell characteristics and high wound healing capacity.

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.

Culture surface protein coatings affect the barrier properties and calcium signalling of hESC-RPE.

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.

Analysis of ATP-Induced Ca2+ Responses at Single Cell Level in Retinal Pigment Epithelium Monolayers.

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.

Functional Voltage-Gated Calcium Channels Are Present in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium.

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 CaV 1.2 and CaV 1.3 as well as the T-type Ca2+ channels CaV 3.1 and CaV 3.2. The localization of the major subtype, CaV 1.3, 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.