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Slow T‐cell reconstitution is a major clinical concern after transplantation of cord blood (CB)‐derived hematopoietic stem cells. Adoptive transfer of in vitro‐generated T‐cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T‐cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta‐like‐4 (DL‐4). Culture of human CD34 +  CB cells in this new DL‐4 system enabled the in vitro generation of large amounts of T‐cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc −/−  (NSG) mice, DL‐4 primed T‐cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T‐cell reconstitution. T‐cell reconstitution was even faster and more robust when ex vivo‐manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro‐generated human T‐cell progenitors to accelerate T‐cell reconstitution and also introduces a feeder‐cell‐free culture technique with the potential for rapid, safe transfer to a clinical setting. S TEM  C ELLS  2012;30:1771–1780

Human T‐Lymphoid Progenitors Generated in a Feeder‐Cell‐Free Delta‐Like‐4 Culture System Promote T‐Cell Reconstitution in NOD/SCID/γc −/− Mice