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.

Human cytomegalovirus interactions with the basement membrane protein nidogen 1

In 2000, we reported HCMV induced specific damage on chromosome 1. The virus' capacity to induce DNA breaks indicated potent interaction between viral protein/s and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12-24 hpi, and continuing throughout infection, steady state (ss) NID1 protein levels were decreased in whole cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor, MG132, to culture media stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found ss NID1 was also reduced in infected ECs and hypothesized virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1 knockout EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal CNS/PNS and is important for neuronal migration, neural network excitability and plasticity and regulates Schwann cell proliferation, migration and myelin production. We found NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus. SIGNIFICANCE: We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein, nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cells' (ECs) secretion of basement membrane proteins is critical for vascular wall integrity and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.