Application Note: Clonal Analysis Of Mouse ES Cells Self-Renewal

Embryonic stem (ES) cells are pluripotent cells that can differentiate into almost any specialized cell type and also have the ability to generate new, undifferentiated stem cells (Niwa, 2000). The latter process is called self-renewal. ES cell self-renewal is a key process that is studied using standard procedures in laboratories throughout the world. Generating cultures of mouse or human ES cells that remain in a proliferating, undifferentiated state is a multistep process. The culture dishes used to grow the cells contain a "feeder" layer of non-dividing cells and/or are treated with leukemia inhibitory factor (LIF). The proliferating colonies of cells are removed and dispersed into new culture dishes, each of which also contains a murine embryonic fibroblast (MEF) feeder layer. When seeded at low density, each ES cell grows out to form a colony. Depending on the state of the cell and the culture conditions, these colonies consist of self-renewing, non-differentiated cells, while cells may be differentiating in other colonies. At this point, the cultures are scored manually using a microscope for the number of differentiating and non-differentiating colonies. This procedure is useful to detemine the effects of various factors on the self-renewal of ES cells. (Smith, 2001; Jones,1998).