Induction of c‐kit Molecules on Human CD34 + /c‐kit Cells: Evidence for CD34 + /c‐kit Cells as Primitive Hematopoietic Stem Cells

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Abstract
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 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 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‐kitlow or CD34+/c‐kit+ cells that had been induced from CD34+/c‐kit cells were resorted and recultured using a methylcellulose culture system, they showed the same colony‐forming ability as the freshly isolated CD34+/c‐kitlow or CD34+/c‐kit+ cells, respectively. Furthermore, CD34+/c‐kit cells have a similar hematopoietic potential to CD34+/c‐kitlow 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 cells mature into CD34+/c‐kitlow and CD34+/c‐kit+ cells, and acquire the reactivity to various humoral hematopoietic stimuli. Moreover, CD34+/c‐kit cells showed a low level of rhodamine 123 retention, suggesting that CD34+/c‐kit cells have multidrug resistance. Therefore, the CD34+/c‐kit 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 cells is the first maturational step of hematopoiesis.