Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

Abstract

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells. rES cells could be cultured through at least 22 passages on laminin in medium supplemented with serum replacement (SR), basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGFβ1), and maintained stable proliferation rates and normal karyotypes, while displaying all the embryonic stem cell characteristics including morphology, alkaline phosphatase (AKP), Oct-4, cell surface markers SSEA-3, SSEA-4, TRA-1–60 and TRA-1–81, and formed cystic embryoid bodies in vitro. In addition, the studies showed that TGFβ1, bFGF and laminin are necessary for maintaining the undifferentiated growth of rES cells in long-term culture. Moreover, withdrawal of TGFβ1 increased the differentiation rate by decreasing the expression of integrins. Therefore, this system would provide a well-defined culture system for rES cells, and would facilitate research into self-renewal and differentiation mechanisms of rES cells.