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In this study six versions of recombinant human hoxb4 proteins were produced and their effectiveness evaluated in expanding human haematopoietic stem and progenitor cells in vitro and in vivo. An N-terminal-tat and C-terminal histidine-tagged version of hoxb4 (T-hoxb4-H) showed the highest activity in expanding colony forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) when used at 50 nmol/l concentration in cell culture. Human cord blood CD34+ cells cultured with 50 nmol/l T-hoxb4-H showed a significant increase in severe-combined immunodeficient mouse-repopulating cells (SRCs). In a mouse model of immune-mediated bone marrow (BM) failure, T-hoxb4-H showed an additive effect with cyclosporine in alleviating pancytopenia. In addition, T-hoxb4-H expanded CFC and LTC-IC on BM samples from patients with refractory severe aplastic anaemia and myelodysplastic syndromes: after culturing with 50 nmol/l T-hoxb4-H for 4 d, BM cells from 10 of the 11 patients showed increases in CFC and LTC-IC, and the increase in LTC-IC was statistically significant in samples from four patients. Recombinant human hoxb4 could be a promising therapeutic agent for BM failure.
Deficiency in long-term haematopoietic stem cells (HSCs) and short-term progenitor cells is characteristic of human bone marrow (BM) failure syndromes, such as aplastic anaemia (AA) and hypocellular myelodysplastic syndromes (MDS), and leads to the clinical manifestation of severe BM aplasia and fatal pancytopenia (Young et al, 2006; Nakao, 2008). CD34+ cell number, and haematopoietic progenitor cell colony formation as reflected in quantitation of haematopoietic progenitor cells using the long-term culture initiating cell (LTC-IC) assay, are markedly decreased in AA and MDS (Maciejewski et al, 1994, 1996; Sato et al, 1998). Immunosuppressive therapy (IST) is effective in improving blood counts in 60–70% of AA patients. However, low blood counts often persist and relapse is frequent, requiring repeated treatment (Scheinberg et al, 2006). While growth factors have been used in addition to IST to support these patients, responses are usually limited to a single cell lineage, and in many cases, patients are not responsive to the treatment (Young & Maciejewski, 1997). Stem cell replacement therapy provides a definitive cure, but difficulties in obtaining well-matched donors and transplantation-associated complications limit this option to only a minority of AA patients (Young et al, 2006).
An agent that could expand patient residual HSCs would be useful in the treatment of AA and other BM failure syndromes. Many attempts have been made to expand HSCs in vitro using different combinations of cytokines, with disappointing outcomes despite maintenance of long-term repopulating stem cells can be achieved in the best circumstances (Conneally et al, 1997; Tisdale et al, 1998; Ueda et al, 2000; Gammaitoni et al, 2003). Thus, attention has shifted to transcription factors that govern stem and progenitor cell fate decisions. One well-studied factor is the homo-box B4 gene (HOXB4), a member of the homo-box family of transcription factors. Retroviral expression of HOXB4 in mice significantly improved HSC regeneration in vivo, with three log increases of HSCs in both primary and secondary recipients (Sauvageau et al, 1995; Thorsteinsdottir et al, 1999; Antonchuk et al, 2001), without affecting normal differentiation or inducing cell transformation (Sauvageau et al, 1995). Similarly, retroviral expressed hoxb4 expanded mouse HSCs by more than 1000-fold in vitro with the expanded HSCs remaining multipotent (B, T, and myeloid) and competitive in repopulating primary and secondary recipients (Antonchuk et al, 2002). In one report, retroviral-expressed human hoxb4 expanded human HSCs in vitro, but the effect was much less than that observed in the mouse (Buske et al, 2002). Recombinant hoxb4 has been produced in order to avoid the potential toxicities of retroviral vectors. A recombinant hoxb4, tat-hoxb4, expanded mouse HSCs (Krosl et al, 2003). However, the effect of recombinant human hoxb4 on HSCs from normal human donors and BM failure patients is unknown.
In this study, we produced six versions of recombinant human hoxb4 with the purification tag, six histidines, and the cell permealizaition tag, tat, at different locations relative to hoxb4, and tested their effects on human HSCs using colony-forming-cell (CFC) and LTC-IC assays. We selected one version of hoxb4 with the tat tag at the N-terminal and the histidine tag at the C-terminal (T-hoxb4-H) that had the highest HSC-expansion activity, determined its optimum working concentration, and tested its effectiveness in expanding severe-combined immunodeficient (SCID) mouse-repopulating cells (SRC) in human cord blood CD34+ cells. T-hoxb4-H was also tested in vivo in a mouse model of BM failure in conjunction with the immunosuppressive agent, cyclosporine. Further, the effectiveness of T-hoxb4-H was examined in ex vivo expansion of CFC and LTC-IC from normal volunteers, as well as from AA and MDS patients. Our results showed that recombinant hoxb4 expanded HSCs and could be a potential therapeutic agent for BM failure syndromes.
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- Materials and methods
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Primitive HSCs are the ultimate source of haematopoietic progenitor cells that sustain lifelong production of mature blood cells. In human BM failure diseases, such as AA and hypocellular MDS, immune attack or intrinsic cellular defects cause a severe HSC deficiency, resulting in marrow hypoplasia and pancytopenia, which is fatal if not treated (Maciejewski et al, 1994, 1996; Yamaguchi et al, 2003). Counteracting autoimmunity and providing normal functional HSCs are used to treat patients with BM failure.
Our current study aimed to explore a novel treatment strategy for BM failure syndromes by expanding residual HSCs using recombinant human hoxb4, a transcription factor that has been extensively studied for its role in promoting HSC self-renewal (Sauvageau et al, 1995; Antonchuk et al, 2002; Amsellem et al, 2003; Krosl et al, 2003). The choice of hoxb4 was based on previous studies in mouse models, in which the transduction of HSCs with a hoxb4-containing retroviral vector resulted in marked HSC expansion (Antonchuk et al, 2002; Sorrentino, 2004). In a mouse HSC transplantation model, lethally-irradiated recipients reconstituted with BM cells that had been transduced with a control vector had only 5–10% of normal number HSCs regenerated, whereas recipients reconstituted with BM cells transduced with a retroviral vector expressing hoxb4 had 100% of normal number of HSCs regenerated (Sauvageau et al, 1995). Expansion of HSCs did not continue after normal numbers of HSCs were regenerated, suggesting the effects of hoxb4 were subjected to normal homeostatic controls. When mouse HSCs were transduced with hoxb4 and cultured in vitro for 14 d, the number of HSCs expanded 40-fold from the initial numbers, or 2000-fold relative to control cultures with cytokines only (Antonchuk et al, 2002).
The mechanism of hoxb4-mediated HSC expansion is not well understood. Gene expression profiling in combination with subsequent functional analysis with enriched adult murine HSCs has suggested that hoxb4 modulated the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal (Schiedlmeier et al, 2007). Hoxb4 might protect adult HSCs from detrimental effects mediated by the pro-inflammatory cytokine TNF-α. This protection also probably contributes to the competitive repopulation advantage of hoxb4-expressing HSCs observed in vivo (Schiedlmeier et al, 2007).
The high level of hoxb4 expression achieved with viral vectors provided the possibility of using hoxb4 for HSC expansion ex vivo for clinical use. However, one major concern is that uncontrolled high levels of hoxb4 expression, achieved by using viral vectors with strong promoters, have been associated with leukaemias in animal models, and abnormalities in myeloid differentiation in cell cultures (Schiedlmeier et al, 2003; Brun et al, 2004; Larochelle & Dunbar, 2008; Zhang et al, 2008). To avoid the use of viral vectors, recombinant hoxb4 fusion protein that contains a plasma-membrane permeabilization sequence ‘tat’ to allow the entrance of hoxb4 protein into cell cytoplasm was shown to stimulate HSC-expansion (Krosl et al, 2003).
We reasoned that the position of the purification and permeabilization tags could affect hoxb4 activity. Therefore, we generated six versions of recombinant hoxb4, varying in the locations of the histidine and tat tags. Indeed, one version of recombinant hoxb4 could not be purified. Of the five versions of purified human hoxb4, including version V in which the histindine and tat tag at the N-terminal of the protein was identical with the one already reported (Krosl et al, 2003), version II that had an N-terminal tat and a c-terminal histidines, showed the highest activity in expanding CFC (10-fold) and LTC-IC (15-fold) in fresh human BM cells in a 4-d culture at 50 nmol/l concentration. In a study using lentiviral HOXB4-transduced feeder layers, passively diffused hoxb4 induced about 20-fold expansion of LTC-IC and 2·5-fold expansion of SRC in human cord blood cells when cells were cultured for 5 weeks (Amsellem et al, 2003); we achieved comparable levels of LTC-IC and SRC expansion on fresh human BM cells and human cord blood CD34+ cells by culturing with the version II T-hoxb4-H protein in 4 d. In our experiments, there were no significant differences in total cell numbers between T-hoxb4-H- and BSA-treated fresh human BM cells, cord blood CD34+ cells, or patient BM cells, yet T-hoxb4-H orchestrated significant CFC, LTC-IC and SRC expansion, with the expansion of LTC-IC being much greater than that of CFC. These data are consistent with the previous report that hoxb4 exerts its activity on more primitive HSCs (Sauvageau et al, 1995).
Adding to the previously published data in murine models of marrow failure, in which IST with anti-thymocyte globulin, cyclosporine, or regulatory T cells ameliorated BM destruction (Bloom et al, 2004; Chen et al, 2007), recombinant hoxb4 demonstrated additional protection in conjunction with cyclosporine in preventing BM failure, possibly by expanding HSCs in vivo (It was not surprising the HSC expansion could not be achieved in this situation with hoxb4 alone without immunosuppression, as any gain in HSCs would be eliminated by active immune attack).
Using frozen BM cells, we found that recombinant hoxb4 expanded HSCs ex vivo from patients with AA and MDS. These patients were not candidates for haematopoietic stem cell transplant and had been refractory to IST. They could benefit from treatment to expand residual HSCs. Although the number of patient samples tested was small, the in vitro results are promising, because 90% of the samples responded positively to hoxb4 treatment, as assayed by CFC and LTC-IC. Cells from both MDS patients responded favourably to hoxb4 stimulation, with significant LTC-IC expansion, and cells from three of nine AA patients also showed significant LTC-IC expansion. Together with in vivo data from the mouse model, we hypothesize that hoxb4 might be useful in treating BM failure patients when combined with IST, and patients with higher number of residual HSCs would be more likely to benefit.