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Recently, we have identified human cord blood (CB)‐derived CD34‐negative (CD34 − ) severe combined immunodeficiency (SCID)‐repopulating cells (SRCs) using the intra‐bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34 −  Kit + Sca‐1 + Lineage −  (KSL) cells, human CB‐derived Lin − CD34 −  cells did not express detectable levels of c‐kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB‐derived CD34 −  SRCs. Both CD34 + flt3 +/−  cells showed SRC activity. In the CD34 −  cell fraction, only CD34 − flt3 −  cells showed distinct SRC activity by IBMI. Although CD34 + flt3 +  cells showed a rather weak secondary repopulating activity, CD34 + flt3 −  cells repopulated many more secondary recipient mice. However, CD34 − flt3 −  cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34 − flt3 −  cells with the murine stromal cell line HESS‐5. After 1 week, significant numbers of CD34 + flt3 +/−  cells were generated, and they showed distinct SRC activity. These results indicated that CB‐derived CD34 − flt3 −  cells produced CD34 + flt3 −  as well as CD34 + flt3 +  SRCs in vitro. The present study has demonstrated for the first time that CB‐derived CD34 −  SRCs, like murine CD34 −  KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long‐term repopulating human hematopoietic stem cells is Lin − CD34 − c‐kit − flt3 − .  Disclosure of potential conflicts of interest is found at the end of this article.

stem cells

Identification of Long‐Term Repopulating Potential of Human Cord Blood‐Derived CD34 − flt3 −  Severe Combined Immunodeficiency‐Repopulating Cells by Intra‐Bone Marrow Injection