Abstract
Purpose:
Degeneration of the retinal pigment epithelium (RPE) is the main etiology of several retinal diseases. The use of stem/progenitor cells to replace the damaged tissue has been proposed recently. The aim of the study was to investigate whether immortalized human neuronal progenitor cells were able to differentiate towards RPE-like cells.
Methods:
ReNcells, human cortical neuronal progenitor cells (Millipore, Temecula, USA), were used for differentiation. The cells were incubated with RPE-conditioned medium (CM), pigment epithelium-derived factor (PEDF) or retinoic acid (RA) for up to 14 days. Gene and protein expression of stem (nestin), neuronal (βIII-tubulin), glial (GFAP) and RPE (RPE65, bestrophin) markers were analyzed by qRT-PCR and immunohistochemistry (IHC). Additional retinal genes (MITF, CRALBP, PAX-6, CHX-10) were investigated by qRT-PCR. RPE-related function after differentiation was tested by an in vitro phagocytosis assay.
Results:
In comparison to undifferentiated ReNcells, nestin was still slightly upregulated in all differentiated cells on the gene level but downregulated on the protein level. On the other hand, βIII-tubulin was upregulated on both levels under all treatment conditions. GFAP gene and protein levels were increased significantly in RA-treated cells only. RPE65 gene expression was upregulated in cells incubated with CM or RA. Gene expression of bestrophin was strongly upregulated under all conditions. IHC showed increased expression of these RPE markers in all differentiated cells. Gene expression of CHX-10, MITF and CRALBP was upregulated in all differentiated cells, whereas PAX-6 was upregulated only in CM & RA-treated samples. Importantly, gene expression of RPE-related genes was frequently higher in differentiated cells than in the RPE controls. In the phagocytosis assay ReNcells cultured with CM or PEDF showed significantly higher phagocytosis rate than undifferentiated ReNcells.
Conclusions:
The most efficient substance for differentiation towards RPE-like cells appeared to be RPE-CM. The results show that under the influence of growth factors neuronal progenitor cells can differentiate into RPE-like cells. Therefore, these cells might be a new source for regenerative treatment of degenerative retinal diseases.
Keywords: 721 stem cells •
701 retinal pigment epithelium •
500 differentiation