Apoptosis Induced by Single Cell Suspension Culture of Mouse Embryonic Stem Cells

Lee, Byung Min; Choi, Ook Hwan; Jung, Jin Sup

Abstract

OBJECTIVE
Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice and to study cell differentiation and early developmental processes in mice. This study was designated to examine apoptotic processes in ES cells according to culture conditions and to study roles of extracellular matrix on the process.
METHODS
Apoptosis was determined by DNA fragmentation and kinase activity during apoptotic process was measured.
RESULTS
The apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. Single cell suspension culture did not affect expression of bcl-2 and bax mRNA. Single cell suspension culture activated stress-activated protein kinase/c-jun-N-terminal kinase (SAPK/JNK), but not p38 kinase. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from mouse embryonic fibroblasts (MEF), or on the petri dishes coated with fibronectin or laminin, but not with collagen or poly-L-lysine. Culture supernatants from MEF cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and MEF cells is required for the suppression of apoptosis. Activation of SAPK/JNK by single cell suspension was protected by interaction of cells with laminin or fibronectin, but not with collagen or poly-L-lysine.
CONCLUSION
The suspension of ES cells as single cells causes serious damage and induces apoptosis, and the apoptotic process is mediated by the activation of SAPK/JNK and is inhibited by the interaction of ES cells with extracellular matrix.