Effects of HOXB4 Overexpression on Ex Vivo Expansion and Immortalization of Hematopoietic Cells from Different Species

¹ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
² Department of Radiation Oncology, University of Washington School of Medicine, Seattle, WA, USA
³ Terry Fox Laboratory, British Columbia Cancer Agency, and Department of Medicine, University of British Columbia, Vancouver, BC, Canada
⁴ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA

^* To whom correspondence should be addressed. E-mail: hkiem{at}fhcrc.org.

	Abstract

Overexpression of the human HOXB4 has been shown to induce the expansion and self-renewal of murine hematopoietic stem cells. In preparation for clinical studies we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus we transduced CD34⁺ cells from nonhuman primates, dogs and humans with a HOXB4-expressing gammaretroviral vector and a yellow fluorescent protein (YFP)-expressing control vector. Compared to the control vector, HOXB4 overexpression resulted in a much larger increase in colony-forming cells (CFCs) in dog cells (28-fold) compared to human peripheral blood, human cord blood, and baboon cells (2, 4, and 5-fold respectively). Furthermore, we found that HOXB4 overexpression resulted in immortalization with sustained growth (>12 months) of primitive hematopoietic cells from mice and dogs but not from monkeys and humans. This difference correlated with increased levels of retrovirally overexpressed HOXB4 in dog and mouse cells compared to human and nonhuman primate cells. The immortalized cells did not show any evidence of insertional mutagenesis or chromosomal abnormalities. Competitive congenic transplantation experiments showed that HOXB4-expanded mouse cells engrafted well after 1 or 3 months of expansion, and no leukemia was observed in mice. Our findings suggest that the growth promoting effects of HOXB4 are critically dependent on HOXB4 expression levels and that this can result in important species-specific differences in potency.

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