Induction of c-kit molecules on human CD34+/c-kit(<low) cells: Evidence for CD34+/c-kit(<low) cells as primitive hematopoietic stem cells

c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells, However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans, We purified CD34(+)/c-kit(<low) cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules, When the CD34(+)/c-kit(<low) cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h, The c-kit expression gradually increased until day 8, When CD34(+)/c-kit(low) or CD34(+)/c-kit(+) cells that had been induced from CD34(+)/c-kit(<low) cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34(+)/c-kit(low) or CD34(+)/c-kit(+) cells, respectively, Furthermore, CD34(+)/c-kit(<low) cells have a similar hematopoietic potential to CD34(+)/c-kit(low) cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures, These findings suggest that CD34(+)/c-kit(<low) cells mature into CD34(+)/c-kit(low) and CD34(+)/c-kit(+) cells, and acquire the reactivity to various humoral hematopoietic stimuli, Moreover, CD34(+)/c-kit(<low) cells showed a low level of rhodamine 123 retention, suggesting that CD34(+)/c-kit(<low) cells have multidrug resistance, Therefore, the CD34(+)/c-kit(<low) cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit(<low) cells is the first maturational step of hematopoiesis.