Demonstration of multiple novel glycoforms of the stem cell survival factor CCg

We have investigated the presence of different glycoforms of cystatin C secreted by adult hippocampal rat-derived stem/progenitor cells (AHPs) into conditioned medium. A glycosylated form of cystatin C (CCg) has been identified previously in conditioned medium from AHPs as an autocrine/paracrine cofactor. Fibroblast growth factor-2 (FGF2) requires cooperation with CCg to support AHP survival at low density in vitro. The purpose of the present study was to investigate further if cystatin C consists of one glycoform or if several different glycoforms are secreted by AHPs in vitro. The presence of the glycoforms was studied using enzymatic deglycosylation in conjunction with gel electrophoresis and Western blotting. The glycoforms of cystatin C were isolated with a combination of gel electrophoresis and electroelution, yielding the intact glycoforms in liquid phase before enzymatic deglycosylation. Our results revealed several novel glycoforms, in contrast to previous publication. The results suggest that N- and O-linked glycans with sialic acid are attached to cystatin C. Furthermore, we have demonstrated that all glycoforms are present in conditioned medium after only 48 hr of culturing and that all nestin-positive AHPs are immunopositive against cystatin C. These findings suggest secretion of the glycoforms by cultured AHPs.