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. 2013 Aug 22;24(10):824–839. doi: 10.1089/hum.2012.202

FIG. 2.

FIG. 2.

Myeloid in vitro differentiation of modified human HSPC. (a) Cell proliferation during in vitro differentiation cultures of myeloid cells from human umbilical cord blood CD34-positive HSPCs, comparing cells differentiated in the presence of rhuG-CSF or rhuGM-CSF, nontransduced (NT) or transduced with EGFP, CD19R, or CD19RCD28 (arithmetic means of four experiments). Values in y-axis are represented in logarithmic scale. (b) Distribution of CFU counts for CFU assay with human CD34-positive HSPCs, nontransduced or transduced with lentiviral vectors carrying EGFP, CD19R, or CD19RCD28. CFU-E, CFU-erythroid (p=0.68); BFU-E, burst-forming unit-erythroid (p=0.99); CFU-GEMM, CFU-granulocyte, erythroid, macrophage, megakaryocyte (p=0.88); CFU-GM, CFU-granulocyte, macrophage, 50–100 cell colonies (p=0.21), 100–500 cell colonies (p=0.28), 500–1,000 cell colonies (p=0.12). Error bars represent means of triplicates+SD. (c) Expression of human cell surface makers CD34 (white columns) (p=0.1166), CD33 (gray columns) (p=0.4936), and CD14 (black columns) (p=0.1741) on in vitro-differentiated myeloid cells, nontransduced (NT) or transduced with CD19R, CD19RCD28, or EGFP, distributed by day of culture. Values represent arithmetic means of results from three experiments and error bars represent mean+SEM. (d) Expression of human cell surface markers on in vitro-differentiated myeloid cells, nontransduced (NT) or transduced with CD19R, CD19RCD28, or EGFP, distributed by culture conditions in rhuG-CSF and rhuGM-CSF and vector transduction, defined as CD34-positive (black columns) (p=0.2479), CD33-positive/CD14-positive (gray columns) (p=0.0059), and CD33-positive/CD14-negative (white columns) (p=0.02). Values represent arithmetic means of results from three experiments and error bars represent mean+SEM. (e) Expression of CAR in myeloid (black circles) and NK cells (black triangles) differentiated in vitro from human CD34-positive HSPCs, nontransduced (NT) or transduced with CD19R or CD19RCD28 (n=18). (f) Dihydrorhodamine 123 assay of myeloid cells differentiated in vitro with or without stimulation with phorbol myristate acetate, distributed by culture conditions in G-CSF (G) and GM-CSF (GM) and vector transduction (p=0.8634). Values represent arithmetic means of results from three experiments and error bars represent mean+SEM. CFU, colony-forming unit; HSPC, hematopoietic stem/progenitor cells; NK, natural killer.