Knockdown of CUX1 leads to increased engraftment of human hematopoietic cells in immunodeficient mice. (A) CUX1 is highly expressed in normal human hematopoietic stem cells. The CUX1 transcript expression profile was extracted from published microarray data.³³  Hematopoietic stem cells (HSC, lineage⁻, CD133⁺, CD34^dim, n = 10), common myeloid progenitors (CMP, lin⁻, CD34⁺, CD38⁺, IL-3R^lo, CD45RA⁻, n = 4), granulocytes (FSC^hi, SSC^hi, CD16⁺ CD11b⁺, n = 4), monocytes (FSC^hi, SSC^lo, CD14⁺, CD45^dim, n = 5), megakaryocytes (CD34⁻, CD41⁺, CD61⁺, CD45⁻, n = 6), and erythrocytes (CD34⁻, CD71⁻, GlyA⁺, n = 6). Data were normalized such that the mean of each gene, across all 38 hematopoietic cell types tested, is zero. (B) shRNA constructs targeting CUX1 lead to haploinsufficient CUX1 protein levels in K562 cells. Western blot was performed on lysates of K562 cells stably expressing CUX1 shRNA A, shRNA B, or control shRNA. Data represent the mean ± SD from 3 experiments. (C) Residual CUX1 transcript levels in human hematopoietic stem cells transduced with lentivirus expressing shRNA targeting CUX1. Lineage-depleted human cord blood cells were transduced with 2 different shRNA constructs targeting CUX1 (shRNA A or shRNA B), or a nonspecific control shRNA. RT-PCR was performed on sorted GFP⁺ cells on the day of transplantation. Each sample was performed in triplicate, and 1 of 2 experiments is shown. (D) Transduction efficiency was similar across the 3 groups of transplanted cord blood stem cells. Transduction efficiency was determined by flow cytometry for GFP⁺ cells before transplantation. (E) Knockdown of CUX1 leads to increased engraftment of human hematopoietic cells in NSG mice. Peripheral blood from mice transplanted with human xenografts was collected on week 10 after transplantation and tested for human CD45 expression (left bars) and coexpression of human dCD45 and GFP (right bars). The numbers of mice per group are indicated. Data are mean ± SEM. (F) Peripheral blood human CD45⁺/GFP⁺ cells from CUX1 shRNA A, CUX1 shRNA B, or control shRNA transduced xenografts were stained for the lineage markers CD13/CD33 (myeloid cells) and CD3 (T cells).