Apoptosis of mouse embryonic stem cells induced by single cell suspension

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES c ells was induced when the cells were dispersed as single cells, whereas thi s process was suppressed when they proliferated in aggregates. The apoptosi s of ES cells was repressed when the cells were cultured on feeders prepare d from STO cells, a cell line established from embryonic fibroblasts. Cultu re supernatants from STO cells did not block the apoptosis of ES cells, whi ch suggests that a direct interaction between ES cells and STO cells is req uired for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-y l)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS, Cellular ac tivity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restorati on. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leaka ge of substrates for oxidative phosphorylation from the mitochondria, and t hat these cells finally become committed to apoptosis. (C) 2000 Harcourt Pu blishers Ltd.