High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Abstract

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34 ؉ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34 ؉ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34 ؉ subpopulation (P ‫؍‬ .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P ‫؍‬ .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical

Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice

“…As previously shown, this model allows us to functionally study the contribution of the different sources of human hematopoietic cells to the xenogeneic engraftment in the recipient mice, both in terms of timing and percentage. [10][11][12] The results that we report here support the concept that MPB cells contribute to early engraftment, not interfering with the engraftment of the transplant of a fixed number of CB cells, except at high MPB/CB cell ratios. …”

Gatekeeper pathways and cellular background in the pathogenesis and therapy of AML

“…85 Previously, the Wnt pathway (to which b-catenin belongs) has been linked to stem cell renewal and functions probably through the activation of the HoxB4 protein that was shown to expand the stem cell pool when overexpressed in human and murine stem cells. 52,[86][87][88] Using oligonucleotide arrays, g-catenin (also called plakoglobin) was recently identified as a target of different leukemic fusion proteins and overexpression of the gene led to an AML. 89,90 Unfortunately, most of the identified genes linked to the acquired self-renewal of the leukemic progenitor cells are also involved in normal stem cell self-renewal, which makes their potential use for the therapy of AML questionable.…”

HOXB4 confers a constant rate of in vitro proliferation to transduced bone marrow cells

“…Viruses pseudotyped with the VSV-G envelope protein were produced by transient calcium phosphate cotransfection of amphotrophic Phoenix cells in a 10 cm dish with 12 mg retroviral vector DNA together with 12 mg plasmid DNA for gag-pol expression and 4 mg of a third plasmid encoding the VSV-G envelope protein (Schiedlmeier et al, 2002). Cell culture supernatants were harvested 24, 36, 48, 60 and 72 h after transfection and stored in aliquots at À701C until use.…”

“…As a consequence, temporal overexpression of HOXB4 and/or corresponding HOXB4 target genes could have enormous therapeutical potential for human HSCs. Indeed, high level HOXB4 expression conferred a profound in vivo competitive advantage on human cord blood CD34 þ cells (Schiedlmeier et al, 2002). Moreover, recently, a more application-oriented approach revealed human and murine HSC expansion in vitro upon administering recombinant HOXB4 protein (Amsellem et al, 2003;Krosl et al, 2003).…”

“…Moreover, recently, a more application-oriented approach revealed human and murine HSC expansion in vitro upon administering recombinant HOXB4 protein (Amsellem et al, 2003;Krosl et al, 2003). However, HOXB4-mediated proliferation is dose dependent and high HOXB4 expression levels substantially impair differentiation of myelo/erythroid and B lineage cells (Buske et al, 2002;Schiedlmeier et al, 2002;Brun et al, 2003).…”