Figure 4. In serial passage, Wnt3A protein leads to continued expansion of colony numbers.

A. Lin⁻, CD24⁺,CD29^hi cells isolated from Actin-GFP mice were cultured in Matrigel, plus either Dkk1, Wnt vehicle control or Wnt3A protein. Numbers of primary colonies are similar (1 colony out of 15 plated cells in primary culture). However, the number of secondary colonies arising from re-plated single cells is strongly influenced by Wnt. In the presence of Dkk1, the secondary colony number dropped by 50%. In the presence of Wnt3A, the colony number was more than 7 fold higher. B, Colony numbers in vehicle and Wnt3A were followed over 10 passages. In vehicle, colony number reached a plateau in the 4^th passage (black line). However, in the presence of Wnt3A, colony numbers expanded continually in each passage (red line). Starting from 35 primary colonies, 10⁶ colonies were calculated to be present in the 10^th passage. C, The sizes of the secondary colonies were measured after one week in culture post passage. Under each condition, there was no discernable difference in the average colony size. D. Primary and secondary colony numbers of wild type and Axin2^lacZ/lacZ mutant cells grown over a range of Wnt3A protein concentration (X-axis). While primary colony formation efficiency remained unaffected, Axin2^lacZ/lacZ mutant cells gave rise to higher numbers of secondary colonies compared to wild type cells (Y-axis), especially at limiting Wnt concentrations. At higher Wnt concentrations, the number of colonies plateaued irrespective of the genotype of the cells.