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.
Highlights
Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration
To address the potential importance of extracellular matrix (ECM) coatings in the orchestration of cellular functions and responses of human embryonic stem cell (hESC)-RPE, we investigated the role of the key RPE basement membrane proteins collagen type IV (Col-IV), LN, and Nid-1, to the in vitro maturation and functionality of the hESC-RPE after cryopreservation, focusing on barrier properties, phagocytosis and C a2+ signalling
We examined the adhesion and morphology of cryopreserved hESC-RPE (Table 1) 24 h after thawing and seeding on coverslips dip-coated with Col-IV, LN, Col-IV + LN, or Col-IV + LN + Nid-1
Summary
Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. We and others have previously shown that the choice of protein composition for coating cell culture surfaces has major effects on RPE differentiation efficiency, as well as RPE structure, basal lamina production and barrier properties of hPSC-RPE in fresh cultures[16,20]. The effects of the cell culture surface protein composition on the maturation of hPSC-RPE tight junctions during differentiation have not been reported before. Another essential aspect for RPE physiology is calcium ( Ca2+) signalling, as several critical RPE functions rely on Ca2+-dependent regulatory m echanisms[24]. The effects of different ECM protein coatings on hPSC-RPE Ca2+ signalling have not been previously studied
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