HEMATOPOIESIS AND STEM CELLS Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL 4-mediated degradation of HOXB 4

Hematopoietic stem cells (HSCs) are pluripotent, asymetrically selfrenewing cells that give rise to all mature blood cells through successive rounds of differentiation. The signals that govern the selfrenewal process have been intensively pursued, as it may be possible to promote HSC expansion by transiently enforcing proliferation pathways or blocking differentiation cues, which would be highly desirable as a means to augment the number of HSCs for transplantation. Currently, HSC transplantation is used to treat hematologic diseases, such as leukemia and bone marrow failure. HSCs are derived from 3 sources: umbilical cord blood (UCB), adult bone marrow, and peripheral blood after treatment with recombinant human granulocyte colony-stimulating factor (G-CSF), a cytokine that mobilizes HSCs from the bone marrow and allows their harvest through peripheral blood apheresis. Ex vivo expansion of HSCs from these sources would improve transplantation outcomes and help meet the demand for stem cell transplants by permitting the use of samples of limited quantity (eg, cord blood) or with low total numbers of HSCs (eg, poor HSC mobilizers). Ex vivo expansion of HSCs and progenitor cells has been the subject of considerable investigation with the use of combinations of hematopoietic growth factors, synthetic small molecular weight compounds identified by high-throughput screening, and the use of supportive bone marrow stroma. In human studies, adult HSCs can undergo repeated rounds of asymmetric self-renewal with maintenance of the stem cell pool but with little or no expansion. In contrast, fetal and neonatal stem cells can be maintained in culture for 2 to 3 months with absolute increases in the number of HSCs. Ex vivo expansions of sevenfold to ninefold in SCID-repopulating HSCs have been reported over 12 to 14 days in a number of studies. Homeobox (HOX) genes encode transcription factors that regulate patterning during embryonic development and hematopoiesis, both prenatally and post-natally. In early development, HOX gene expression is both temporally and spatially regulated, as reflected by the sequential order of transcription with respect to their 39 to 59 chromosomal position. However, the spatiotemporal regulation ofHOX gene expression is not observed in hematopoiesis, but instead assumes a complex, overlapping expression pattern that is not lineage-specific. Northern blotting analysis revealed that most HOX genes, excluding HOXD, are highly expressed in HSCs and progenitor cells, and are generally down-regulated as cells terminally differentiate into mature, lineage-specific blood cells. To date, the mechanisms underlying transcriptional regulation of HOX genes during hematopoiesis remain largely unknown. The identification of HOX genes that are highly expressed in CD34 HSCs and early progenitors led to several overexpression studies that revealed the effects of specific HOX genes on HSC proliferation. In particular, sustainedHOXB4 expression by retroviral transduction promoted the selective expansion of murine HSCs in cell culture and after bone marrow transplantation. Moreover, the enhanced proliferation of HOXB4-transduced HSCs was not