Day-12 cobblestone areas in MS-5 cocultures of W51⁺ cells. Nonsorted, transduced CB-CD34⁺ cells expressing W51 were plated onto a monolayer of the MS-5 stroma. The photograph shows a representative early cobblestone area (CA). All early CAs were strongly EGFP⁺ (A). (B) The frequency of early week-2 CA formation depends upon the interval of cytokine prestimulation of CB-CD34⁺ cells. CB-CD34⁺ cells were prestimulated for either 0, 1, 2, 3, 4, and 5 days and plated (1 × 104/T12.5 in triplicate) onto an MS-5 monolayer, with enumeration of week-2 early CAs.