Expansion of CB CD34⁺ cells in stromal coculture. Cells (20 000) were plated in T₂₅ flasks on OP9 mouse BM stroma transfected with an adenovirus-expressing TPO. (A) Nonadherent cell expansion from CB CD34⁺ cells. Total viable nucleated cells were counted using Trypan blue. (B) CFC expansion. Total numbers of CFCs were established by semisolid methylcellulose assays in triplicate dishes on the initial CD34⁺ population and on the suspension cells obtained after each weekly demi-depopulation. The results represent the mean ± SD. (C) Secondary week 5 CAFC expansion. Input CD34⁺ cells and weekly suspension cell populations were assayed for CAFCs by 5-week coculture on MS5 stroma. (D) Telomerase activity of input CD34⁺ cells and equal numbers of unseparated suspension cells obtained at weekly intervals. Activity was calculated from the TRAP signal and expressed as total product generated (TPG). The input activity corresponded to percent of the TRAP activity of an NB cell line extract used as a reference standard.