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STEM CELL GENETICS AND GENOMICS

HOXB4-Induced Self-Renewal of Hematopoietic Stem Cells Is Significantly Enhanced by p21 Deficiency

^a Molecular Medicine and Gene Therapy and Lund Strategic Center for Stem Cell Biology and Cell Therapy, Lund University Hospital, Lund, Sweden;
^b Center for Regenerative Medicine and Technology, AIDS Research Center and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

Key Words. Hematopoiesis • HOXB4 p21 • Stem cell expansion

Correspondence: Ann Brun, Ph.D., Molecular Medicine and Gene Therapy, Lund University Hospital, BMC A12, 221 84 Lund, Sweden. Telephone: +46 46 222 05 92; Fax: +46 46 222 05 68; e-mail: ann.brun{at}med.lu.se

Received July 18, 2005; accepted for publication September 29, 2005.

	ABSTRACT

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Enforced expression of the HOXB4 transcription factor and downregulation of p21^Cip1/Waf (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild-type (wt) and p21^–/– mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU-GM (colony-forming unit granulocyte-macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21^–/– BM (p21B4) cells compared with MIGB4-transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo, we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs.

	INTRODUCTION

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Hematopoietic stem cells (HSCs) must balance self-renewal and differentiation to provide sufficient primitive cells to sustain hematopoiesis throughout life and must simultaneously be capable of extensive proliferative expansion upon demand. A single HSC can reconstitute a lethally irradiated host and self-renew to give rise to mature progeny cells of all hematopoietic lineages [1, 2]. The balance between self-renewal and differentiation of HSCs is tightly regulated by a complex machinery of internal and external signals (represented by transcription factors, cell cycle regulators, growth factors, and adhesion molecules) that govern HSC fate options [3–6]. Because HSCs are important in clinical medicine for treatment of cancer and genetic disorders by blood and marrow transplantation, a variety of in vitro culture conditions using different combinations of cytokines have been described in attempts to expand the number of HSCs prior to transplantation. However, substantial expansion of HSCs cannot be accomplished using standard soluble cytokines. Usually, a considerable loss of HSCs ensues in cytokine cultures over time, although maintenance of long-term repopulating stem cells can be achieved in the best of circumstances [7–11].

Among the many transcription factors that govern stem and progenitor cell fate decisions, homeobox (Hox) transcription factors have emerged as important regulators throughout the hematopoietic hierarchy [12–19]. The Hox family of transcription factors has a highly conserved DNA-binding domain (the homeodomain) and exercises transcriptional regulation through interaction with many transcriptional co-factors (e.g., Pbx and Meis) to achieve specific transcriptional programming throughout hematopoietic development. Specific expression patterns of multiple Hox genes have been detected in normal and leukemic hematopoiesis, implying a specific role for the various Hox transcription factors at different stages of hematopoiesis [18–21]. HOXB4 has been considered an important regulator of primitive hematopoietic progenitors because it is expressed in primitive human CD34⁺ hematopoietic progenitors but is rapidly downregulated upon differentiation [22]. Using lack-of-function animal models, we have recently demonstrated a role for Hoxb4 as an HSC regulator [23, 24]. Hoxb4-deficient mice exhibit a hematopoietic phenotype that primarily affects the stem cell pool and results in reduced proliferative capacity of HSCs without affecting differentiation and lineage choice [23, 24]. In addition, gain-of-function models have shown that enforced expression of HOXB4 in vivo is a potent stimulus for murine HSC self-renewal and regenerative capacity after transplantation of irradiated recipients [25]. Importantly, recent findings demonstrate that an approximately 40-fold expansion of the murine repopulating HSC pool can be achieved by enforced expression of HOXB4 ex vivo for 10–14 days [26]. Because HOXB4 has been shown to expand HSCs by direct protein transfer [14, 27] and growth of human cord blood cells can be enhanced after overexpression of HOXB4, it may be possible to use HOXB4 to develop safe and effective HSC expansion for advanced stem cell therapeutics [17, 28].

The regulation of HSC self-renewal is governed by a balance between positive growth promoting signals and negative regulatory pathways that can promote quiescence or apoptosis. The negative regulatory pathways involved in the cell proliferation machinery include the cyclin-dependent kinase inhibitor p21^Cip1/Waf1 (p21), a G₁ checkpoint regulator. The p21 protein restricts HSC cycling and thereby maintains HSC quiescence and cell survival [29]. Complete deficiency of p21 leads to increased HSC proliferation and amplifies the size of the HSC pool under normal homeostatic conditions in the mouse [29]. Furthermore, downregulation of p21 in CD34⁺/CD38^– human cord blood cells resulted in an expanded stem cell number and improved stem cell function in vivo without affecting differentiation, indicating that controlled downmodulation of p21 may be used to amplify human HSCs for future cell and gene therapy [30].

Because enforced expression of HOXB4 and downregulation of p21 increase proliferation of HSCs and because our studies have shown that absence of Hoxb4 in hematopoietic progenitors from fetal liver significantly increases the expression of p21 [23], we asked whether the self-renewal effect generated by overexpression of HOXB4 in HSCs would be enhanced or decreased in p21-deficient HSCs. The findings show that p21 deficiency markedly enhances the HOXB4-mediated increase in self-renewal and regenerative capacity and indicate that the effect of HOXB4 is, at least in part, mediated independently of p21.

	MATERIALS AND METHODS

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Retroviral Vectors
The MSCV-IRES-GFP (MIG) retroviral control vector expressing GFP alone, and the MSCV-HOXB4-IRES-GFP (MIGB4) retroviral vector expressing both HOXB4 and GFP were kindly provided by Dr. R.K. Humphries (Terry Fox Laboratory, Vancouver, BC, Canada) [15].

Mice
Mice deficient in p21 were of the 129/Sv strain that express the cell surface marker Ly5.2 [29]. The congenic Bl6SJL (Ly5.1, competitor cells and recipient mice), 129/Sv (Ly5.2, wt control cells), and Bl6SJLxC57Bl/6 (expressing both Ly5.1/5.2, recipient mice) strains were bred in the barrier facility at the biomedical center, Lund University, Lund, Sweden. Mice were kept in ventilated racks and given autoclaved food and water. All animal experiments were approved by the Lund University Animal Ethical Committee.

Transduction of Primary Murine Bone Marrow Cells
Bone marrow (BM) cells were harvested from femurs and tibiae of wild-type 129/Sv (wt; p21^+/+) mice or p21^–/– mice treated with 5-flurouracil (5-FU; 150 mg/kg, Nycomed AB) 4 days prior to harvest. Harvested cells were pre-stimulated at a density of 0.5 x 10⁶ cells per ml for 48 hours in X-Vivo medium (X-Vivo 15; BioWhittaker, Walkersville, MD, http://biowhittaker.com) supplemented with 1% bovine serum albumin (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 10^–4 ß-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 2 mM L-Glutamine (Gibco-BRL, Paisley, U.K., http://www.gibcobrl.com), 100 units/ml Penicillin, 100 µg/ml streptomycin (Gibco-BRL), 50 ng/ml murine stem cell factor (mSCF; R&D Systems, Minneapolis, http://www.rndsystems.com), 10 ng/ml murine interleukin-3 (mIL-3; R&D Systems), and 50 ng/ml human interleukin-6 (hIL-6, a generous gift from Novartis International, Basel, Switzerland, http://www.novartis.com). For transduction, cells were centrifuged and re-suspended in 1 ml Iscove’s modified Dulbecco’s medium supplemented with 20% fetal calf serum (FCS; Gibco-BRL), 10^–4 M ß-mercaptoethanol, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and the above cytokines (complete medium) with the addition of 6 µg/ml of protamine sulphate (Sigma-Aldrich) and transferred to Retronectin (Takara Bio Inc., Otsu, Japan, http://www.takara-bio.com)-coated 24-well plates that had been preloaded with 1 ml supernatant of MIG or MIGB4 virus for 1 hour at 37°C. The transduction efficiency of p21^–/– BM (p21MIG) and wt p21^+/+BM (wtMIG) with the MIG vector was in the range of 30%–40%, whereas the MIGB4 transduction efficiency of p21^–/– BM (p21B4) and wt p21^+/+ BM (wtB4) was in the range of 20%–30%. Transduced cells were harvested after 48 hours and sorted for GFP-expressing cells.

In Vitro Culture of Transduced BM Cells
Two days after transduction, GFP-positive BM cells were sorted using a FACSVantage (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). GFP-positive (Ly5.2) BM cells (noncompetitive cultures), or 10% of sorted GFP-positive BM cells mixed with 90% nontransduced B6SJL (Ly5.1) BM cells (competitive cultures), were cultured in complete medium for 11 days. The total number of cells and percentage of GFP-positive cells were analyzed at day 4, 7, and 11 (Fig. 1A). Cells were split every 3–4 days to maintain a cell density of 1 x 10⁵ cells per 500 µl.

View larger version (23K): [in this window] [in a new window]   Figure 1. Proliferation of HOXB4-transduced cells is enhanced by p21 deficiency. (A): Design of in vitro experiments. After retroviral transduction, GFP-positive BM cells (wtMIG, wtB4, p21MIG, and p21B4) were sorted and expanded for 11 days in Iscove’s modified Dulbecco’s medium with 20% fetal calf serum, murine interleukin-3, human interleukin-6, and murine stem cell factor. (B): Sorted GFP-positive cells were plated in noncompetitive cultures and counted at days 4, 7, and 11. (C): In competitive cultures, 10% sorted GFP-positive cells and 90% competitor cells were mixed and cultured for 11 days. GFP-positive cells were examined by fluorescence-activated cell sorter on days 0, 4, 7, and 11. (D): The relative expansion of GFP-positive cells, compared with wtMIG-transduced cells, was examined on days 0, 4, 7, and 11 of the culture (n = 5–8 per group, *p < .05, **p < .001, vs. wtB4). (E): Quantitative polymerase chain reaction of cDNA derived from MIGB4 and MIG-transduced wt and p21–/– cells 0, 4, 7, and 11 days of culture post-transduction. The HOXB4 primers are specific for human HOXB4 and do not detect murine Hoxb4. Abbreviations: PS, pre-stimulation; TD, transduction.

Flow Cytometry
Peripheral blood (PB) samples were collected from the retro orbital plexus of recipient mice. The following antibodies were used for analysis of engraftment and lineage distribution: Rat antibodies against murine CD45.1 (Ly5.1), CD45.2 (Ly5.2), Mac1, Gr1, B220, and CD3 (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). Antibodies were conjugated with phycoerythrin, allophycocyanin (APC), or biotin. Biotinylated antibodies were detected by a secondary antibody conjugated with streptavidin APC (BD Pharmingen). Red blood cells were lyzed with ammonium chloride (NH₄Cl; StemCell Technologies), and dead cells were excluded by staining with 7-aminoactionmycine D (Sigma-Aldrich). Cells were analyzed on a FACSCalibur (Becton, Dickinson and Company), and results were analyzed using Cell Quest software (Becton, Dickinson and Company).

Quantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany, http://www1.qiagen.com), and cDNA was transcribed (Superscript II; GibcoBRL). Quantitative polymerase chain reaction (PCR) for human HOXB4 and murine p21 and hypoxanthine guanine phosphoribosyltransferase (HPRT) was performed using primers and TaqMan probes from Applied BioSystems (Foster City, CA, http://www.appliedbiosystems.com) and analyzed in an ABI Prism 7700 Sequence Detection System (Sequence Detector V1.7 software; PE Biosystems, Foster City, CA, http://appliedbiosystems.com). Threshold cycle (CT) values for the PCR products were normalized against HPRT, and relative intensity was generated using the comparative CT method according to the manufacturer.

Competitive Repopulation
After retroviral transduction, BM cells were cultured for 10 days. Transplantation was performed on days 0, 5, and 10 (Fig. 2). The expansion equivalent of 1.5 x 10⁵ unsorted BM cells (Ly5.2) were transplanted together with 8 x 10⁵ support cells (Ly5.1) into lethally irradiated (950 cGy) primary recipient mice (Ly5.1). Primary recipient mice were sacrificed 22 weeks after BM transplantation (BMT), and the equivalent of half a femur was transplanted into each lethally irradiated secondary recipient (Ly5.1).

View larger version (16K): [in this window] [in a new window]   Figure 2. Overexpression of HOXB4 in p21–/– HSCs leads to a greater engraftment than in wt HSCs. (A): Design of transplantation experiments. After retroviral transduction, BM cells (wtMIG, wtB4, p21MIG, and p21B4) were expanded for 0, 5, and 10 days, followed by transplantation of 1.5 x 105 unsorted Ly5.2 BM cells (or the expansion equivalent) together with 8 x 105 support cells (Ly5.1) into lethally irradiated recipient mice. (B): PB was analyzed at 16 weeks after BMT in primary recipients. The percentage of donor Ly5.2+ cells in PB is plotted. The percentage of PB donor cells was significantly increased in HOXB4-transduced p21-deficient cells after 10 days of culture (p < .03). (C): The relative repopulation ability of transduced cells that have (days 5 and 10), or have not (day 0), been expanded in vitro prior to transplantation into irradiated recipients. The percentage of Ly5.2 cells, relative to the percentage of Ly5.2 cells generated by wtMIG cells, is followed over time. The relative repopulation ability is shown at 3, 12, and 16 weeks after BMT (n = 3–6 per group). The relative repopulation ability is increased by a factor of two after 5 days of culture, and there is a 10-fold increase in the relative repopulation ability of HOXB4-transduced p21 cells compared with HOXB4-transduced wt cells (*p < .02). Abbreviations: BMT, bone marrow transplantation; HSC, hematopoietic stem cell; PB, peripheral blood; PS, pre-stimulation; TD, transduction; TP, transplantation.

Statistical Analysis
Results are expressed as mean ± SE of duplicate or triplicate data obtained from three or more experiments. Data were analyzed using the Mann-Whitney rank sum test (except for CRU assay; see above). p < .05 was considered statistically significant.

	RESULTS

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Proliferation of HOXB4-Transduced Cells Is Enhanced by p21 Deficiency
Murine repopulating HSCs can be expanded by enforced expression of HOXB4 by retroviral gene transfer [15, 26], and p21 deficiency results in increased proliferation and an augmentation in the stem cell pool under homeostatic conditions. Therefore, we asked whether enforced expression of HOXB4 was exerting its HSC effect through regulation of p21 or whether the p21 deficiency would generate an additive or synergistic effect, indicating alternative downstream HOXB4 targets, separate from p21. The in vitro expansion effect generated by retroviral-mediated overexpression of HOXB4 in p21-deficient BM cells was analyzed. BM cells were harvested from wt or p21^–/– mice after 5-FU treatment and cultured for 48 hours in the presence of serum and hematopoietic cytokines. The nontransduced cells were continuously cultured in the same media, while aliquots of wt and p21^–/– cells were transduced with the MIGB4 or MIG vector (Fig. 1A). GFP-positive cells were sorted and cultured at a density of 1 x 10⁵ cells per 500 µl. To characterize the consequence of HOXB4 overexpression in BM cells, we first determined the relative expansion rate of the cells in liquid culture over time. As shown in Figure 1B, the number of wtB4 and p21B4 cells on day 11 of expansion was increased by 2.9 ± 0.6 (p < .03)– and 5.1 ± 0.5 (p < .001)–fold, respectively, compared with wtMIG, and the HOXB4-transduced p21-deficient cells expanded two to three times more than wt cells (p < .05 p21B4 vs. wtB4).

To stringently test the competitiveness of HOXB4 overexpressing cells, 10% of sorted GFP-positive cells were mixed with 90% competitor cells (nontransduced cultured cells) to a total of 1 x 10⁵ cells per 500 µl and cultured for 11 days. Expansion of the transduced cells was followed over time using FACS to monitor the percentage of GFP-positive cells. The percentage of MIGB4-transduced cells increased over time in both p21^–/– and wt cultures compared with MIG-transduced control cells (Fig. 1C). Whereas no significant differences in the rate of expansion were seen between the wtMIG and p21MIG cells, wtB4 cells expanded 3.6 ± 0.5–fold and p21B4 cells expanded 9.5 ± 0.8–fold compared with wtMIG at day 11 (Fig. 1D). These findings show that the proliferative impact of HOXB4 in primitive p21-deficient hematopoietic cells increases threefold (p = .001, day 11).

We have previously shown that there is a good correlation between the expression level of HOXB4 protein and mRNA in cells overexpressing human HOXB4 [32]. Therefore, quantitative PCR of cDNA from cultured transduced wt cells was used to examine expression levels of murine p21 and human HOXB4 to determine whether enforced expression of HOXB4 down-regulates p21. Vector-derived HOXB4 expression levels were similar in both wt and p21^–/– cells transduced with the MIGB4 vector. When comparing the level of p21 expression in MIG-and MIGB4-transduced wt cells, a suppression of p21 expression was seen when HOXB4 was overexpressed. These findings indicate a connection between the HOXB4 and p21 regulatory pathways in controlling the proliferation of primitive hematopoietic cells (Fig. 1E).

Expansion of CFU-GM Progenitors by HOXB4 Is Enhanced by p21 Deficiency
Next, we evaluated the effects of the p21 deficiency on HOXB4-mediated progenitor cell expansion. Transduced cells were analyzed for their ability to generate CFU-GM in semisolid medium. Transduced GFP⁺ sorted cells from day 0 (input) and day 11 (output) of in vitro culture were plated in methylcellulose medium, and colonies were enumerated after 7 days. As expected, no difference in the number of colonies was detected between the different conditions at initiation of culture (Day 0, Fig. 3A). In contrast, 11 days of in vitro expansion led to significantly increased numbers of colonies derived from HOXB4-transduced wt and p21-deficient cells compared with wtMIG (17.9 ± 5.6–fold for wtB4, p < .05, 35.7 ± 5.2–fold for p21B4, p < .001) (Day 11, Fig. 3A). Furthermore, there was a significant increase in HOXB4-mediated expansion of p21-deficient progenitors (p < .03). Primary colonies were collected, reseeded, and scored for secondary colony formation 7 days later. Non-expanded MIGB4-transduced wt and p21-deficient cells showed increased reseeding efficiency when compared with MIG-transduced non-expanded cells, although this difference was not statistically significant (Fig. 3B). In addition, the size of the secondary colonies derived from both wt and p21-deficient HOXB4 overexpressing primary colonies were markedly larger than secondary colonies derived from primary MIG-transduced colonies. When cells had been expanded for 11 days prior to CFU-GM assay, no secondary colonies derived from MIG-transduced cells could be detected, whereas p21B4 colonies were increased by a factor of three compared with wtB4 secondary colonies (280 ± 148 vs. 90 ± 45, Fig. 3B, lower panel). Enforced expression of HOXB4 increased colony size, whereas p21 deficiency did not influence the size of colonies (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3. Expansion of colony-forming unit granulocyte-macrophage (CFU-GM) progenitors by HOXB4 is enhanced by p21 deficiency. (A): After transduction and sorting for GFP expression, the cells were plated in methylcellulose cultures as described in Materials and Methods, and CFU-GM colonies were enumerated at day 0 (input) and day 11 (output) (n = 5–8 per group; *p < .03 vs. wtB4 and p21MIG, p <.001 vs. wtMIG). (B): Ten percent (day 0, upper panel) or 20% (day 11, lower panel) of the cells in the methylcellulose cultures containing the primary colonies were replated, and secondary colonies were scored after an additional 7 days of culture. Because of experimental variation, the threefold enhancement in the p21-deficient cells of secondary colonies derived from day-11 primary cultures is not statistically significant.

HOXB4 Overexpression in p21-Deficient HSCs Leads to Increased Self-Renewal
To investigate the HOXB4 effect on self-renewal of HSCs, secondary transplantations were performed. Two of the primary mice transplanted with 5- and 10-day expanded cells were sacrificed at 22 weeks, and the equivalent of half a femur was transplanted into each secondary recipient. Analysis of PB from secondary recipients 17 weeks post-transplant showed robust reconstitution in recipients of p21B4 cells of both 5- and 10-day expanded cells whereas no measurable reconstitution could be demonstrated from the other cells (Fig. 4A). The p21B4 cells were able to give rise to both myeloid and lymphoid lineages as determined by FACS analysis (Fig. 4B), indicating an overall expansion of primitive cells without lineage restriction.

View larger version (36K): [in this window] [in a new window]   Figure 4. Increased reconstitution and multilineage engraftment of HOXB4 overexpressing p21–/– cells in secondary recipients. (A): The percentages of donor cells in peripheral blood (PB) of primary and secondary recipients are shown as average values for wtMIG, wtB4, p21MIG, and p21B4, respectively. PB was analyzed 22 weeks after bone marrow transplantation in primary recipients and 17 weeks after transplantation in secondary recipients. (B): Representative fluorescence-activated cell-sorting plots demonstrating multilineage engraftment (B, T, and myeloid cells) in PB of secondary recipients.

View larger version (16K): [in this window] [in a new window]   Figure 5. Increased in vitro expansion of HOXB4 overexpressing p21–/– hematopoietic stem cells. Fold change in CRUs of wtMIG, wtB4, p21MIG, and p21B4 after 0, 5, and 10 days of expansion. The graph shows fold change in CRU numbers at different time points relative to initial CRU numbers. Abbreviations: CRU, competitive repopulating unit; PS, pre-stimulation; TD, transduction.

	DISCUSSION

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It is common to use a 5-day expansion protocol to expand HSCs using enforced expression of HOXB4 [17, 22, 27]. Recently, Schmittwolf et al. reported that the engraftment potential of HOXB4-transduced cells was decreased after prolonged ex vivo culture [34]. It is not clear at present what the optimal length of ex vivo culture should be to achieve the highest long-term engraftment after HOXB4 expansion. We cultured HOXB4-transduced cells for 5 and 10 days, using a dilution of the transduced cells to adjust the proportion of transduced cells to 10% of total cells at the start of the culture, and analyzed the engraftment ability of these cells in lethally irradiated recipient mice. Engraftment of HOXB4-transduced wt and p21-deficient cells was increased compared with MIG-transduced control cells after both 5 and 10 days of expansion. In contrast, engraftment of MIG-transduced cells decreased over time in culture. HOXB4-transduced cells generated the highest engraftment when cultured for 5 days but showed a clear decline by day 10. These findings are compatible with our CRU experiments demonstrating that the numbers of CRUs per femur, after 5 days of expansion, were 1,303 in wtB4 and 4,445 in p21B4, whereas 10 days of expansion resulted in 309 CRUs per femur in wtB4 and 811 in p21B4. Antonchuk et al. reported that the number of HSCs increased 40-fold compared with the initial number using a 10-day expansion protocol [26]. The apparent discrepancy between these findings and our data may be explained by the different genetic backgrounds of the mice used in the two studies. Antonchuk et al. used C57Bl/6 or C57Bl/6-Pep3b as donor cells and sublethally irradiated C57Bl/6-W41/W41 as recipient mice, whereas our experimental design used 129/Sv donor cells and lethally irradiated Bl6SJL or C57Bl/6 x Bl6SJL for competitor cells and recipient mice. Other differences are culture conditions: We used serum-free pre-stimulation followed by transduction using virus-containing medium, whereas Antonchuk et al. used 15% serum throughout the culture time and co-culture with the virus-producing cell line for transduction. In addition, our transplantations were done with a dilution of the transduced cells to represent only 10% of the transplanted cells, thereby increasing the stringency of the assay.

At present, no reliable and efficient method for expansion of HSCs exists. Classical cytokine cultures, using cytokines that preferably act on primitive progenitor and stem cells, have proven ineffective [7–11]. It is important to develop efficient in vitro stem cell expansion protocols for BM transplantation, HSC expansion, and gene therapy. For efficient retroviral gene transfer, the target cells need to be in active cell cycle. Gene transfer efficiency into human HSCs using retroviral vectors has been rather low, resulting in relatively low numbers of transduced transplantable stem cells [35–38]. Unless the transduced cells have a selective advantage, the outcome of the gene therapy effort may be insufficient for an effective therapeutic response. One possible way of improving gene transfer efficiency may be to use lentiviral vectors because they can transduce nondividing cells; however, entry into the G₁ stage of the cell cycle is required for efficient gene transfer [39–42]. Therefore, lentiviral vector transgenesis will likely benefit from activation or growth stimulation of human HSCs in order to achieve high gene transfer efficiency.

The induction of symmetric HSC divisions to amplify the number of repopulating HSCs in vitro is challenging because self-renewal of HSCs frequently occurs by asymmetric cell divisions in which one daughter cell is a stem cell and the other is a more differentiated progenitor cell. It is interesting to note several reports that have employed retroviral-mediated gene transfer of HOXB4 to expand stem and progenitor cells without altered lineage decisions or induction of malignant transformation [12, 15, 25, 26, 43]. However, the issue may be more complicated because transcription factors used to expand stem cells may affect the fate of HSCs and progenitor cells in a concentration-dependent manner [44, 45]. This has also been demonstrated with Hox transcription factors. Carefully regulated levels of HOXA10 using the doxycyclin system have demonstrated that high levels of HOXA10 block erythroid differentiation whereas lower levels promote engraftment ability of HSCs [46, 47]. A similar effect has been demonstrated with HOXB4. High levels of HOXB4 expression, generated by adenoviral vector expression in CD34⁺ human hematopoietic cells, promoted myeloid differentiation and reduced the output of long-term culture initiating cell assay [32]. Similarly, retro-viral vectors expressing high levels of HOXB4 in a NOD/SCID (nonobese diabetic/severe combined immunodeficiency) transplantation model impaired the differentiation of hematopoietic progenitors to myeloid and B-cell lineages [28]. Carefully regulated expression of transcription factors, preferably combined with transient expression systems to avoid insertional mutagenesis [48, 49], would be required to expand stem cells effectively and safely. Alternate approaches could involve the use of the HOXB4 protein itself to expand HSCs ex vivo to avoid complications that may occur through genetic manipulation [14, 27]. Recently, interesting simple approaches have been reported to increase self-renewal of stem cells without affecting their pluripotency. Sato et al. employed a pharmacological inhibitor to activate Wnt signaling in murine and human embryonic stem cells [50]. The binding of Wnt proteins to a cognate receptor, Frizzled, leads to inhibition of glycogen synthase kinase-3 (GSK-3), which in turn leads to stabilization of ß-catenin and subsequent changes in gene transcription [51]. A pharmacological inhibitor of GSK-3 was successfully used to activate the canonical Wnt pathway to maintain the self-renewal ability of stem cells ex vivo. Analogous approaches have recently been employed to increase the self-renewal of HSCs by using histone deacetylase inhibitors (HDIs). Valproic acid, the potent HDI, was used to increase the proliferation and self-renewal of HSC through downregulation of p21. This was accompanied by up-regulation of Hoxb4, a target of Wnt signaling, and AC133 by hyperacetylation of the Hoxb4 and AC133 promoter regions [52, 53]. These findings indicate that careful genetic studies aimed at understanding what mechanisms govern HSC self-renewal can be used to design simple pharmacological approaches to amplify stem cells ex vivo.

	SUMMARY

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In this study, we have demonstrated that simultaneous modulation of two genetic pathways can generate a 10-fold increase in the number of repopulating HSCs after 5 days of ex vivo culture. These findings can be used to design effective and safe protocols for stem cell expansion, either by using carefully regulated transient gene expression systems to temporarily upregulate the production of HOXB4 and downregulate p21, or by using pharmacological reagents that can lead to similar alterations in these two regulatory circuits to increase HSC self-renewal ex vivo prior to transplantation.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Keith Humphries for kindly providing the retroviral vectors; Zhi Ma and Anna Fossum for skillful cell sorting; Eva Gynnstam, Mia Thagesson, and Åsa Nordfelt for excellent animal care; and Eva Nilsson for expert technical assistance. We also thank Jennifer Moody, Göran Karlsson, Ulrika Blank, and other members of the lab for comments on the content of this manuscript. This work was supported by grants to S.K. from the Swedish Cancer Society, the European Commission (INHERI-NET and CONSERT), the Swedish Gene Therapy Program, the Swedish Medical Research Council, the Swedish Children Cancer Foundation, a Clinical Research Award from Lund University Hospital, and the Joint Program on Stem Cell Research supported by the Juvenile Diabetes Research Foundation and the Swedish Medical Research Council. This work was supported also by a project grant from Lund University Hospital Donation Funds to A.C.M.B. The Lund Stem Cell Center is supported by a Center of Excellence grant in life sciences from the Swedish Foundation for Strategic Research. N.M. and A.C.M.B. contributed equally to this work.

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