Fig. 6. The schematic illustrates the assay by which the frequency of symmetric proliferative cycles that give rise to two stem cells was evaluated in a given ANSCs population. Emx2 loss-of-function ANSCs were established from animals in which Emx2 expression was knocked out; conversely, Emx2 gain-of-function ANSCs were obtained by isolation and expansion from mice constitutively overexpressing Emx2, as well as by infection with a retrovirus carrying the Emx2 sense sequence. Both types of ANSCs were compared with their controls. Single clones were subjected to a clonogenic assay in which the number of secondary clonogenic cells was expressed as percentage of the total cell number in the clone. In the absence of differential cell death – which was not observed in any of these cultures (Table 3) – this value indicates the ratio between symmetric proliferative cycles (generating two stem cells) and symmetric differentiative cycles (generating two more differentiated cells) that contributes to the formation of the clone. If the frequency of the proliferative symmetric divisions exceeds that of the differentiative ones, the number of stem cells in the clone is greater than one and the stem cell population increases. As expected in this culture system, this was observed under all the conditions tested. However, in the Emx2 KO-derived cells, the relative frequency of symmetric proliferative divisions was far greater than in wild-type animals, as shown by the higher frequency of secondary clones generated, whereas this frequency decreased in gain-of-function cells. Asymmetric cycles that may also have occurred during clone formation generated a stem and a more differentiated cell at each division. Thus, alterations in their frequency did not influence the outcome of this assay.
