Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9(-/-) hematopoietic stem cells. Hoxa-9(-/-) marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-klt(+)flk-2(-)Sca-1(+) [KLFS] cells). However, sublethally irradiated Hoxa-9(-/-) mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9(-/-) cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1(hi)Mac-1(hi) cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9(-/-) marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.