HMGA2 overexpression increases engraftment levels of CB CD34⁺in serially transplanted immunodeficient mice. CB CD34⁺ cells were transduced with a lentiviral vector overexpressing HMGA2 and assayed in vitro and in vivo by transplantation into NSG mice. (A) Relative numbers of CD34⁺CD90⁺ cells over 21 days of culture following shRNA transduction. Numbers are normalized to the control cells. (B-C) Relative numbers of CFU-GM (B) and BFU-E (C) after 2, 7, and 14 days of culture following shRNA transduction. Numbers are normalized to the control cells. (D) Overview of in vivo transplantation assay for HMGA2-overexpressing cells. (E) Chimerism of huCD45⁺ZsGreen⁺ cells in primary recipient mice transplanted with HMGA2-overexpressing cells as analyzed in peripheral blood after 6 to 7 and 14 weeks and (F) in BM after 14 weeks. (G) Levels of HSC engraftment (CD34⁺CD38⁻ cells) measured in BM of primary recipients 14 weeks after transplantation. Representative FACS plots gated on huCD45⁺ZsGreen⁺ cells and the accumulated data are shown. (H) Unfractionated BM equivalent of one-third of femur from primary mice were transplanted in secondary mice. Chimerism of huCD45⁺ZsGreen⁺ cells in BM of secondary NSG recipients. Numbers on the top show the positively engrafted mice (CD45⁺ZsGreen⁺ cells ≥0.1%). *P < .05; **P < .01; ***P < .001.