Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+ CD38- cells expressing Flk2/Flt3

In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34(+) haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34(+) cells and their immature subpopulations, CD34(+) CD33(-) and CD34(+) CD38(-) cells. Methycellulose clonal culture of sorted CD34(+) Flk2/ Flt3(+) and CD34(+) Flk2/Flt3(-) cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 x 10(4) lineage marker-negative (Lin(-))CD34(+)Flk2/Flt3(-) cells were transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45(+) cells in bone marrow 11-12 weeks after the transplantation. In contrast, all recipients transplanted with 1 x 10(4) Lin(-) CD34(+) Flk2/Flt3(+) cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin(-) CD34(+) CD38(-) Flk2/Flt3(+) cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long-term-repopulating haematopoietic stem cells.