SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection

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Abstract
Precise analysis of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood–derived CD34− severe combined immunodeficiency (SCID)– repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra–bone marrow injection (IBMI) technique. Lineage-negative (Lin−) CD34− cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4– mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34+ progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34+cells in vivo. These in vivo–generated CD34+ cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34− SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34− SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.