Human CD34⁺ CBs cocultured with ECs give rise to multilineage engraftment in NSG mice. (A) Percentage of engrafted human CD45⁺ hematopoietic cells at 3 and 9 weeks after transplantation. (B) Total number of engrafted phenotypically marked human HPCs. (C) Total number of terminally differentiated mature cells per femur. (D) Percentage of human CD45 engraftment in secondary recipients transplanted with human CD45⁺ hematopoietic cells from primary recipients in panels A through C (N = 10 for each condition; 9 of 10 in the E4⁺EC group and 5 of 10 in the cytokines-alone group had significant human CD45 engraftment: > 1%). All human cells analyzed with the cell population that was gated on cells that were negative for mouse Ter119 and CD45. (E) A total of 50 000 CD45⁺CD34⁺ hCB cells were cultured with or without feeder layers in serum-free conditions and supplemented with 50 ng/mL SCF, 50 ng/mL TPO, and 50 ng/mL Flt3L for 12 days. CD45⁺CD34⁺ cells were isolated and transplanted into NSG mice with the corresponding cell doses. Unmanipulated CD45⁺CD34⁺ were used as controls. At 3 and 9 weeks after transplantation, mice were analyzed for human CD45⁺ cells in bone marrow. Mice with > 1% engraftment were considered positive. Mice that were positive at 3 weeks remained positive at 9 weeks, with higher levels of human CD45⁺ engraftment at 9 weeks. SCID-repopulating cells were determined using L-Calc Version 1.1 software. ***P = .0001. *P = .05. Reference group: E4⁺ECs + cytokines, analyzed 9 weeks after transplantation. (F) Representative contour plots of human HPC engraftment 9 weeks after transplantation. (G) Representative contour plots of multilineage human hematopoietic cell engraftment 9 weeks after transplantation. Note that human hematopoietic cells cocultured with E4⁺ECs have greater engraftment potential and give rise to a higher number of multilineage human hematopoietic progenitors and long-term engraftment in secondary recipients. Results were analyzed using the Student t test, and P < .05 was considered significant. Date are mean ± SD.