Corneal Endothelial Cell Sheet Bioengineering from Neural Crest Cell-Derived Adipose Stem Cells on Novel Thermo-Responsive Elastin-Mimetic Dendrimers Decorated with RGD

Abstract Due to the limitations in in vivo division of Human Corneal Endothelial Cells (HCECs), and a shortage of cornea donations, there is increasing focus on bioengineering corneal endothelium (CE) using alternative cells. Cell sheet engineering allows for the maintenance of the cellular junctions necessary for CE function. The aim of this study was to generate CE-like cell sheets on innovative thermo-responsive biopolymer, using hADSC-derived CE-like cells. Thermo-responsive biopolymers were fabricated by conjugating elastin-like polypeptide (ELP) functionalized with RGD to a G4 dendrimer as a nanoplatform, named elastin-mimetic dendrimer (EMD). The hADSCs were first reprogrammed into neurospheres on Poly-L-lysine, induced by small molecules. Then, neural crest cells (NCCs) were differentiated into CE-like cells on EMD, and cultured until the CE-like cell sheet formed. EMD exhibited excellent biocompatibility improving cell spreading, adhesion, proliferation and NCCs differentiation into CE-like cells, and lower critical solution temperature (LCST) near physiological temperature ideal for CE-like cell sheet harvesting. NCCs and CE-like cells markers were confirmed by qRT-PCR, immunocytochemistry, and western blot. After 18 days, cell sheets with cellular junctions were harvested by decreasing the temperature. This study thus represents the successful generation of CE-like cell sheets from hADSC-derived CE-like cells on EMD.