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The use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT) is a promising technique that permits a degree of human leukocyte antigen mismatch between the graft and the host without the concomitant higher rate of graft‐versus‐host disease that would be observed between an adult marrow graft and a mismatched host. A disadvantage to the use of UCB for HSCT is that immune reconstitution may be significantly delayed because of the low stem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide a greater number of stem cells; however, there are persistent concerns that ex vivo‐expanded CD34 cells may lose pluripotency and the ability to contribute meaningfully to long‐term engraftment. To address this issue, we transduced CD34‐selected UCB cells with a lentiviral construct expressing luciferase, and determined homing and engraftment patterns in vivo by noninvasive bioluminescent imaging in sublethally irradiated NOD/SCID/IL‐2Rγ −/−  (NSG) mice. Graft contribution to multilineage commitment was also confirmed by analysis of primary and secondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that, other than a mild delay at the onset of engraftment, there were no significant differences in lineage repopulation or in long‐term or secondary engraftment between culture‐expanded and unexpanded UCB CD34‐selected cells. The results suggest that multipotent stem cells can be expanded ex vivo and can contribute meaningfully to long‐term hematopoietic engraftment. STEM CELLS 2009;27:1932–1940

Noninvasive Bioluminescent Imaging Demonstrates Long‐Term Multilineage Engraftment of Ex Vivo‐Expanded CD34‐Selected Umbilical Cord Blood Cells