STAT5 imposes a long-term proliferative advantage on human HSCs but not progenitors in MS5 stromal cocultures. (A) Human cord blood CD34⁺ cells were transduced with control and STAT5A-ER retroviral vectors, and tNFGR-positive cells were sorted into HSC, CMP, GMP, and MEP populations. (B) RNA was extracted from each sorted population and used for real-time Q-PCR analysis. (C) Sorted HSCs (i), CMPs (ii), GMPs (iii), and MEPs (iv) were plated on MS5 stromal cells, stimulated with 4-OHT as indicated, and the cultures were demi-depopulated weekly for analysis. Weekly cumulative cell counts are shown for a representative experiment of 3 independent experiments. (D) Representative images from MS5 cocultures at week 2 as described in panel C. (E) MGG-stained cytospins from week-1 suspension cells from cocultures as described in panel C. (F) FACS analysis on week-1 suspension cells from MS5 cocultures as described in panel C. (G-H) Suspension cells from MS5 cocultures as described in panel C were analyzed for progenitor content by CFC assay. At week 2 (G) and week 3 (H), 2500 cells from each coculture were plated in a CFC assay in methylcellulose, and colonies were evaluated 2 weeks after plating. Total CFC numbers are shown. CFU-GM, colony-forming unit–granulocyte-macrophage; BFU-E, burst-forming unit–erythroid.