PUM1 and PUM2 contribute to human HSPC expansion. (A) RT-qPCR analysis of hPUM1 and hPUM2 transcripts in human stem/progenitor cell subpopulations: HSC (CD34⁺CD38^lowCD90⁺), MPP (CD34⁺CD38^lowCD90⁻), CMP (CD34⁺CD38⁺CD45RA⁻IL-3Rα^low), MEP (CD34⁺CD38⁺IL-3Rα⁻CD45RA⁺), and GMP (CD34⁺CD38⁺IL-3Rα⁺CD45RA⁺). Results are normalized to GAPDH expression and expressed relative to PUM expression in HSCs (n = 3). (B-F) Cord blood CD34⁺ cells were transduced or not (nt) with lentiviral vectors encoding shRNA targeting PUM1 (shPUM1-a, sequence #a in supplemental Table 8), PUM2 (shPUM2-a, sequence #a in supplemental Table 8), or luciferase as control (shCtrl), and were maintained in culture posttransduction. (B) Immunoblot analysis of the indicated proteins at day 4 posttransduction. Actin was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 4). (D) Cell cycle analysis upon propidium iodide labeling at day 3 posttransduction (n = 4). (E) Annexin V labeling at day 5 posttransduction (n = 4). (F) At day 7 posttransduction, shRNA/GFP⁺ output cells were seeded in methylcellulose medium and numbering of CFC was assessed after 2 weeks (n = 3); representative micrographs of colonies are presented on the right side (×20). BFU-E (burst forming unit-erythroid), GM-CFC (granulomacrophagic colony forming cell). (G) Cord blood CD34⁺ cells from the same batch were transduced with lentiviral vectors encoding shRNA targeting PUM1 (shPUM1-a), PUM2 (shPUM2-a), or luciferase as control (shCtrl). Three days posttransduction, sublethally irradiated NSG-immunodeficient mice received a 1:1 mixture of sorted shPUM/Tomato⁺ CD34⁺ cells and shCtrl/GFP⁺ CD34⁺ cells. Presence of GFP⁺ and Tom⁺ cells among human BM CD45⁺ cells of engrafted mice was scored 12 weeks later by FACS analysis. Each symbol represents data from a single chimeric mouse. Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).