HOXA4Induces Expansion of Hematopoietic Stem Cells In Vitro and Confers Enhancement of Pro-B-Cells In Vivo

Abstract

Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.

HOXA4Provides Stronger Engraftment Potential to Short-Term Repopulating Cells ThanHOXB4

“…This was accompanied with expansion of total bone marrow (BM) cultures by HOXA4 that were up to 100-fold more important than control after 3 weeks of culture [6], and were comparable to earlier reported expansions with HOXB4-overexpressing progenitor cells [7]. The capacity to expand primitive hematopoietic cells appeared to be attributed to all paralog four members as evidenced in an embryonic stem cell overexpression model [8].…”

“…Our group recently demonstrated that overexpression of HOXA4, which is highly homologous to HOXB4, also led to a net expansion of functional mouse HSC in vitro [6]. This was accompanied with expansion of total bone marrow (BM) cultures by HOXA4 that were up to 100-fold more important than control after 3 weeks of culture [6], and were comparable to earlier reported expansions with HOXB4-overexpressing progenitor cells [7].…”

“…All BM cultures underwent massive proliferation resulting in average expansions of 5 · 10 6 -fold over the initial numbers after 4 weeks for all three conditions (Fig. 1C), which is 100-fold over the expansion of control cultures as reported before [6]. Cooverexpression of HOXA4 and HOXB4 did not further enhance proliferation.…”

“…B6SJL mice were intravenously injected with 150 mg/kg of 5-fluorouracil (5-FU; Mayne Pharma (Canada), Inc.) to recruit HSC into cell cycle, as previously reported [6]. After 4 days, BM cells were isolated from these mice and cocultured for 2 days on confluent layers of the GP+E-86 packaging cell line stably producing MSCV-HOXA4-GFP, MSCV-HOXB4-GFP, MSCV-HOXA4-YFP, or MSCV-GFP retrovirus [17] in Dulbecco's modified Eagle's medium (DMEM; Wisent, Inc.) supplemented with 15% heat-inactivated fetal bovine serum (FBS; PAA Laboratories, Inc.), 6 ng/mL interleukin (IL)-3, 10 ng/mL IL-6, 100 ng/mL stem cell factor (SCF), 10 -5 M 2-mercaptoethanol (Mallinckrodt Baker, Inc.), 50 ng/mL gentamicin (Wisent, Inc.), 10 ng/mL ciprofloxacin (Wisent, Inc.), and 6 ng/mL polybrene (TekniScience, Inc.).…”

Searching for the Key to Expand Hematopoietic Stem Cells

“…HOXA4 overexpressing HSC expanded 6.6-fold after a week of culture. Although HOXA4 expressing HSC produced mature myeloid and lymphoid progeny in irradiated recipient mice, B-cell progenitors were preferentially expanded compared to myeloid progenitors (Fournier et al, 2011).…”