Protein profiling of human nonpigmented ciliary epithelium cell secretome: the differentiation factors characterization for retinal ganglion cell line

Abstract

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

Figure 1

Morphological changes in RGC-5 cells after treatment with HNPE conditioned SF-medium (40x) (a) before, and (b) after. The RGC-5 cells treated with HNPE conditioned SF-medium induced morphological changes, including longer axons and more neurite outgrowth (Figure 1(b)), compared to RGC-5 cells without treatment (Figure 1(a)).

Figure 2

Immunocytochemical analysis of Thy-1 and Brn-3b expression in RGC-5 cells differentiated by treatment with HNPE cell conditioned SF-medium. Staining with antibodies to the cell surface glycoprotein, Thy-1, have been commonly used as a marker to identify retinal ganglion cells. After cultivation with HNPE conditioned medium, RGC-5 cells have an enhanced Thy-1 expression, compared to the undifferentiated cells. RGC-5 cells without cultivation with HNPE conditioned medium express Brn-3b in a different pattern compared with treated RGC-5 cells. Specifically, Brn-3b has a nuclear localization in RGC-5 cells without cultivation with HNPE conditioned medium; however, upon treatment, RGC-5 cells express Brn-3b in a more punctate cytosolic manner.

Figure 3

1D SDS-PAGE image of HNPE conditioned SF-medium (5 μg/well, silver stained, left-hand side: molecular weight marker, kDa). The gel bands on the middle lane with serial numbers were analyzed by nano-HPLC-ESI-MS/MS. In the 30 bands, 132 proteins were identified. The gel bands on the right-hand side were the cell lysised proteins.