Abstract
Purpose: :
Age-related macular degeneration (AMD) is characterized by the presence of degenerated and/or dysfunctional retinal pigment epithelial (RPE) cells. Human embryonic stem cells (hESCs) have shown promise in providing an unlimited source of RPE cells for cell replacement therapy. Our lab previously observed that the medium from polarized, hESC-derived RPE cell cultures (conditioned medium) could differentiate hESC into RPE cells more extensively than if the hESCs were cultured in regular medium. The purpose of this study is to confirm that the HES-3 cell line cultured in conditioned medium can promote more RPE differentiation than the HES-3 cells cultured in regular XVivo 10 medium and to identify the specific factors secreted in the conditioned medium responsible for this.
Methods: :
HES-3 cells were mechanically sliced into small cell aggregates, suspension cultured for 3 days to form embryoid bodies in neuronal differentiation medium, and cultured on Geltrex-coated dishes with conditioned medium and XVivo10 (1:1 ratio). Cells were harvested weekly up to 8 weeks. Differences between cells cultured in conditioned medium and XVivo 10 only were determined by Q-RT PCR of certain marker genes expressed during retinal development, including RPE-specific genes. To identify possible factors responsible for greater RPE cell appearance in cells grown in conditioned medium, protein arrays were performed on the conditioned and XVivo 10 media, and ELISAs were done to confirm and quantify the arrays.
Results: :
Q-RT PCR results indicated higher expression of RPE-specific genes (RPE65, PMEL, PEDF, and Bestrophin) in cells cultured in conditioned medium relative to the XVivo 10 only medium. A protein array indicated an upregulation of several proteins, including angiogenin, VEGF, PDGF-AA, MCP-1, and M-CSFR, in the conditioned medium.
Conclusions: :
These results indicate that RPE cell-conditioned medium contains factors that may be responsible for an increase in RPE cell differentiation as shown by the upregulation of RPE-specific/selective markers. The specific factors, or combination of factors, that are responsible for this effect need to be determined.
Keywords: retinal pigment epithelium • differentiation • development