hFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.

Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.