Schematic representation of the experimental strategy.

CD34⁺CD38^low cells labeled with CFSE were first incubated for 4 days in conditions with or without stromal cells (step 1). At this time, cells were analyzed for the expression of fluorescence. Each peak corresponds to a cell generation and was numbered by reference to the 0-division point defined by analyzing an aliquot of the cells cultured under the same conditions but in the presence of Colcemid to block mitosis. The additional CFSE-negative population, seen in the upper right graph, corresponds to the MS5 cells. Cells that had divided fewer than 2 times and 2, 3, and 4 times were sorted, and their LTC-IC activities were assessed. In some experiments, a 2-step procedure was followed. Two-division cells were sorted on day 4 and incubated for 2 more days with cytokines, with or without MS5 cells (step 2). After this period (6 days from day 0), cells that had achieved 2 more divisions (4 since day 0) were sorted, and their LTC-IC activity was assessed.