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

Abstract

Abstract 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.