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

Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34 super(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003; 101:2924). In contrast to murine CD34 super(-) Kit super(+)Sca-1 super(+)Lineage super(-) (KSL) cells, human CB-derived Lin super(-)CD34 super(-) 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 super(-) SRCs. Both CD34 super(+)flt3 super(+/-) cells showed SRC activity. In the CD34 super(-) cell fraction, only CD34 super(-)flt3 super(-) cells showed distinct SRC activity by IBMI. Although CD34 super(+)flt3 super(+) cells showed a rather weak secondary repopulating activity, CD34 super(+)flt3 super(-) cells repopulated many more secondary recipient mice. However, CD34 super(-)flt3 super(-) cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34 super(-)flt3 super(-) cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34 super(+)flt3 super(+/-) cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34 super(-)flt3 super(-) cells produced CD34 super(+)flt3 super(-) as well as CD34 super(+)flt3 super(+) SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34 super(-) SRCs, like murine CD34 super(-) 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 super(-)CD34 super(-)c-kit super(-)flt3 super(-). Disclosure of potential conflicts of interest is found at the end of this article.