Dedifferentiation of CD34+kdr- progenitors by FLK1 gene transfer: Suppression of clonogenic activity and rescue of engrafting capacity

The VEGF receptor 2 (KDR in humans, Flk1 in mice) is detected on #1% of CD34+ cells from post-natal hematopoietic tissues and is specifically expressed on hematopoietic stem cells (HSCs); conversely, uni-oligopotent hematopoietic progenitor cells (HPCs) are comprised in the CD34+KDR- subset (Ziegler et al., Science, 1999). To assess the functional role of KDR/Flk1 in the HSC gene program, CD34+KDR- cells from bone marrow, cord and adult peripheral blood were transduced by Flk1 gene in PINCO retroviral vector containing the GFP marker (De Maria et al., Nature, 1999). Flk1+ cells were assayed for HPC clonogenic activity and for HSC repopulating activity in 12-wk extended long-term culture (ELTC) and NOD-SCID mice, as compared to control GFP+ cells. The in vitro clonogenic activity of Flk1+ cells was 80–90% suppressed. Conversely, the HSC activity was partially rescued: (a) the frequency of 12-wk ELTC initiating cells (ELTC-ICs) was 10–20% in Flk1+cells vs0% in GFP+ cells; (b) injection of 10–200x103 Flk1+ cells in NOD-SCID mice induced a multilineage myeloid-lymphoid engraftment, whereas GFP+ control cells did not engraft. These data indicate that Flk1 gene transfer induces functional dedifferentiation of CD34+KDR- HPCs: specifically, exogenous Flk1 abolishes the clonogenic activity of the majority of HPCs, while it rescues the stem cell activity of a minority of HPCs, possibly represented by primitive HPCs. It is apparent, therefore, that KDR/Flk1 plays a key role in the functional gene program underlying stem cell activity.