Suppression of Homeobox Transcription Factor VentX Promotes Expansion of Human Hematopoietic Stem/Multipotent Progenitor Cells

Background: Difficulty in obtaining sufficient amounts of human hematopoietic stem/multipotent progenitor cells (HSC/MPPs) is a major hurdle that limits their application. Results: Down-regulation of VentX, a hematopoietic homeobox transcription factor, allows multilineage expansion of hematopoietic cells ex vivo and in vivo in SCID/NODγ2null mice. Conclusion: VentX is a novel regulator of human HSC/MPP expansion. Significance: VentX represents a novel target for mechanistic exploration and clinical application of human HSC/MPPs. Mechanisms that regulate proliferation and expansion of human hematopoietic stem/multipotent progenitor cells (HSC/MPPs) are targets of intensive investigations. Several cell intrinsic factors and signaling pathways have been implicated in the proliferation and differentiation of human HSC/MPPs. Nevertheless, expansion of human HSC/MPPs for clinical application remains a critical challenge. VentX is a human homeobox transcription factor that was recently identified as an anti-proliferation and pro-differentiation factor in human hematopoietic cells. Here, we report that VentX expression is up-regulated during ontogenesis of human hematopoietic cells. Strikingly, suppression of VentX expression led to significant expansion of HSC/MPPs ex vivo and a 20-fold increase in engraftment potential in the NOD/SCID/IL2Rγ2null mouse model. VentX suppression helped preserve the HSC/MPP pools and promote clonogenicity of hematopoietic progenitor cells. Mechanistically, we show that VentX regulates critical cell cycle regulators and Wnt downstream genes previously implicated in HSC/MPP proliferation and expansion.

Hematopoietic stem cells (HSCs) 3 give rise to multipotent progenitor cells (MPPs) and eventually mature cells of all lineages of blood and immune systems. HSC/MPPs play essential role in tissue homeostasis, regeneration, and host defense. HSC/MPP transplantation has shown great promise in the reconstitution of hematopoietic systems and treatment of malignancies. HSC/MPP-based gene therapy has revealed potentials for correcting hereditary metabolic and autoimmune diseases. The molecular mechanisms underlying HSC/MPP proliferation and differentiation have been explored extensively for effective expansion of human HSC/MPPs, which is required for clinical application of HSC/MPP transplantationbased therapy (1,2).
Over the past decades, several cell extrinsic signaling pathways and cell intrinsic factors of early embryogenesis have been shown to play important roles in the proliferation and differentiation of HSC/MPPs (3)(4)(5)(6)(7). In particular, using mouse models, it has been shown that murine HSCs can be expanded by Ͼ100fold by either enhanced Wnt signaling or overexpression of the homeobox protein HoxB4 (5,6). In comparison, clinically significant expansion of human HSC/MPPs remained a fundamental challenge. Despite a recent advance in HSC/MPP expansion by cytokine mixtures (8), neither enhanced Wnt signaling nor overexpression of HoxB4 or BMI1 was able to expand human HSC/MPPs by Ͼ2ϳ3-fold (9 -11). As such, safe and efficacious expansion of human HSC/MPPs remains a bottle neck that limits the clinical application of HSC/MPP-based therapy.
VentX is a unique human homeobox protein that is expressed restrictedly in human hematopoietic cells in a highly regulated manner. Initially cloned as a human homolog of the vertebrate Vent family of homeobox transcription factors, the function of VentX in cell proliferation and differentiation is being appreciated through reverse genetic modeling of the molecular mechanisms underlying dorsoventral axis formation during early vertebrate embryogenesis (12,13). As an human homolog of the vertebrate Xom/Vent2 homeobox genes, VentX was found to be a novel lymphoid enhancer factor/T-cell factor-associated antagonist of the canonical Wnt signaling and plays an important role in normal and malignant hematopoiesis (14 -16). Our recent investigations showed that, in addition to antagonizing canonical Wnt signaling, VentX is also an activator of the p53/p21 and p16 ink4a /Rb pathways and is able to induce senescence phenotypes in tumor cells (17).
Several lines of evidence suggest the potential involvement of VentX in controlling proliferation and differentiation of HSC/ MPPs. First, VentX expression is up-regulated during both myeloid and lymphoid ontogenesis and during early hematopoiesis (15,16,18). Second, VentX controls signaling pathways and cell cycle regulators that have been previously implicated in the proliferation and differentiation of hematopoietic cells (15)(16)(17). Third, VentX has been shown to control differentiation of primary human hematopoietic cells (16,18).
To further define a potential role of VentX in the proliferation and differentiation of human HSC/MPPs, we examined VentX expression during early hematopoiesis and explored the function of VentX in the expansion of CD34 ϩ HSC/MPPs through gain-of-function and loss-of-function approaches ex vivo and in vivo in the NOD/SCID␥2 null mouse model. We found that VentX inhibition helped preserve the stem cell pool of HSC/MPPs. Strikingly, we observed that knockdown of VentX in bone marrow CD34 ϩ HSC/MPPs resulted in a 20-fold increase in engraftment potential and multilineage development of hematopoietic cells in the NOD/SCID␥2 null mouse model. We found that VentX regulates the expression of several cell cycle regulators and Wnt downstream genes implicated in HSC/MPP proliferation and differentiation. Our data therefore reveal VentX as a novel regulator of HSC/MPP proliferation and differentiation and a potential druggable target for clinical application of HSC/MPP-based therapy.

EXPERIMENTAL PROCEDURES
CD34 ϩ Cell Isolation-Human bone marrow samples were obtained from discarded femoral head tissue after hip replacement surgery at Brigham and Women's Hospital, with Institutional Review Board approval. After Ficoll separation of mononuclear cells, the CD34 ϩ cells were enriched using a magnetically activated cell sorting CD34 ϩ progenitor kit (Miltenyi Biotec). The purity of the bone marrow CD34 ϩ cells was Ͼ95% as assessed by flow cytometry. Flow cytometry sorting of different subpopulations of CD34 ϩ cells (common myeloid progenitors, megakaryocyte/erythroid progenitors, and granulocyte/macrophage progenitors) was as described previously (11).
Lentiviral Vector Construct and Transduction of Bone Marrow CD34 ϩ Cells-The lentiviral vector pHAGE-CMV-eGFPW, which expresses a shRNA targeting VentX, was used for the transduction of bone marrow CD34 ϩ cells. The lentiviral vector contains an internal ribosome entry site that allows simultaneous expression of GFP monitoring the transduction rate or for cell sorting. The sequence of VentX shRNA was identical to that of the VentX siRNA described previously (15). A non-effective sequence targeting GFP was used as a control. Lentiviral packaging was carried out in the Dana-Farber/Harvard Cancer Center Vector Core Facility, and viral supernatants were stored at Ϫ70°C in aliquots until used. The CD34 ϩ cells were first cultured in Iscove's modified Dulbecco's medium containing FBS (10%), stem cell factor (100 ng/ml), Flt3 ligand (100 ng/ml), thrombopoietin (100 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml) (PeproTech) for 2 days and then transduced with a multiplicity of infection of 5 in the presence of 4 g/ml Polybrene. GFP-positive cells were sorted using a FACSAria high-speed sorter (BD Biosciences) at 24 h post-transduction at the Dana-Farber Cancer Institute Flow Cytometry Core Facility and used for all ex vivo experiments.
Overexpression of VentX in Bone Marrow CD34 ϩ Cells-Human bone marrow CD34 ϩ cells were transfected with pCS2-GFP or pCS2-GFP-VentX plasmid using a Nucleofector kit for human CD34 ϩ cells (Lonza) as described above. GFP-positive cells were sorted using a FACSAria high-speed sorter at 24 h post-transfection and used for subsequent experiments.
Colony-forming Cell (CFC) and Long-term Culture-initiating Cell (LTCIC) Assays-Assays for in vitro CFCs were performed by plating sorted GFP-positive CD34 ϩ cells (1 ϫ 10 4 /dish) in triplicates in complete methylcellulose medium (MethoCult GF H4434, STEMCELL Technologies Inc.), which contained a mixture of recombinant human cytokines (stem cell factor, IL-3, GM-CSF, and erythropoietin). Colonies were counted after 14 days of incubation at 37°C and classified according to standard criteria. For the LTCIC assay, transduced GFP-positive cells were sorted on M2-10B4 murine fibroblast cells in limiting dilutions from 30 to 900 cells/well in 96-well plates. Cultures were weekly fed with new medium. After 5 weeks of culture, all cells from the wells were transferred to a 35-mm Petri dish in complete methylcellulose medium as described above. After an additional 2 weeks of culture, wells were scored as positive or negative to yield the LTCIC frequency.
FACS Analysis-Phenotypic analysis of the CD34 ϩ cells and cells of different lineages was performed using flow cytometry after immunolabeling of cells with fluorescent dye-conjugated antibodies. Phycoerythrin (PE)-conjugated anti-human CD34 and CD45 and PE/Cy5-conjugated anti-human CD38 antibodies were obtained from eBioscience. The anti-cyclin D 1 primary antibody was purchased from Cell Signaling for intracellular staining. PE-conjugated rabbit anti-mouse IgG antibody was used for anti-cyclin D 1 secondary antibody staining. Staining of the cells was performed for 30 min on ice. Mouse bone marrow cells were blocked with anti-Fc antibody for 15 min at 4°C to avoid nonspecific binding. FACS analyses were performed on a FACScan (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Inc.).
Transplantation of NSG Mice-8-week-old NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice (commonly known as NOD scid gamma, or NSG) were purchased from The Jackson Laboratory and maintained under specific pathogen-free conditions. Before transplantations, mice were sublethally irradiated with 100 rads. 1 ϫ 10 6 CD34 ϩ cells were transduced with VentX shRNA or GFP shRNA lentivirus. At 24 h post-transduction, all cells (1 ϫ 10 6 , with ϳ20% GFP-positive cells) or positively transduced cells (2 ϫ 10 4 , with all GFP-positive cells) were injected into recipient mice. Human CD45 ϩ cells from the bone marrow of recipient mice were analyzed. For secondary transplantation, the bone marrow of chimeric primary recipient mice was injected into secondary recipient NSG mice without further purification of human cells.

VentX Expression Is Regulated during Hematopoiesis-
VentX is a homeobox transcription factor expressed in all lineages of hematopoietic cells. Our previous studies showed that VentX is a lymphoid enhancer factor/T-cell factor-associated Wnt repressor (15). Given the broad implications of Wnt signaling in hematopoiesis, we further explored the potential involvement of VentX in hematopoiesis by defining the expression pattern of VentX during early hematopoietic development. Through FACS analysis of different subpopulations of CD34 ϩ cells, followed by quantitative PCR analysis, we found that VentX expression was regulated during hematopoiesis ( Fig. 1). VentX expression was negligible in primitive CD34 ϩ CD38 Ϫ cells. Consistent with the findings of Rawat et al. (18), VentX expression levels increased significantly in progenitor cells of myeloid lineage (Fig. 1). There was no significant difference in VentX expression among different subgroups of myeloid lineages (Fig. 1).
VentX Regulates Expansion of Human HSCs ex Vivo-Our previous studies have shown that VentX regulates cell proliferation by controlling key pathways involved in the cell cycle (15)(16)(17). The finding that VentX expression is up-regulated during hematopoiesis prompted us to explore the potential role of VentX in the regulation of HSC/MPP expansion. To achieve this goal, VentX expression in human bone marrow CD34 ϩ cells was knocked down with a lentivirus-based shRNA approach. The transduction rates of pHAGE-shGFP (which encodes a non-effective sequence targeting GFP) and pHAGE-shVentX were ϳ20%. Using a quantitative PCR approach, the efficiency of VentX knockdown in pHAGE-shVentX-transduced CD34 ϩ cells was determined to be ϳ30% ( Fig. 2A). Positively transduced cells were plated in cytokine-supplemented stroma-free liquid cultures, and the cell numbers were counted weekly up to 4 weeks. Plotting of weekly cell counts of demidepopulated cultures showed that knockdown of VentX expression in CD34 ϩ cells led to a 3ϳ4-fold increase in total cell numbers compared with control cells from three independent experiments (Fig. 2B). To determine the potential effects of VentX on the clonogenicity and lineage development of CD34 ϩ cells, we performed CFC assays. As shown in Fig. 2 (C-F), knockdown of VentX resulted in an ϳ2-fold increase in the formation of BFU-E/CFU-E and CFU-GM colonies. The total number of progenitor cells increased by ϳ3-4-fold in the CFC assays (Fig. 2E). Knockdown of VentX promoted multilineage expansion of hematopoietic cells, especially erythroid lineage colony formation (Fig. 2F). To determine the effects of VentX knockdown on the HSC pools, we examined the proportion of CD34 ϩ cells in the ex vivo liquid culture and found that knockdown of VentX led to a 30% increase in the percentage of CD34 ϩ cells (Fig. 3, A and B) and an ϳ2.5-fold increase in the absolute cell numbers (Fig. 3C). A time course FACS analysis revealed that knockdown of VentX helped preserve the CD34 ϩ cell population for at least 2 weeks (Fig. 3D). In addition, knockdown of VentX also increased the percentage (Fig. 3, E and F) and absolute number (Fig. 3G) of primitive CD34 ϩ CD38 Ϫ cells. FACS staining showed that VentX knockdown did not change the lineage distribution of the differentiated progenitor cells (Fig. 3H). Furthermore, LTCIC frequency increased by ϳ3-fold in VentX shRNA-transduced CD34 ϩ cells (Fig. 3I). These results suggest that VentX knockdown may help maintain the CD34 ϩ stem cell pools, thereby promoting CD34 ϩ cell expansion.
Ectopic Expression of VentX Blocks CD34 ϩ Cell Proliferation and Expansion-To further determine the effects of VentX on the expansion of human bone marrow HSC/MPPs, we tested whether ectopic expression of VentX would prevent CD34 ϩ cell expansion. As shown in Fig. 4 (A-D), the colony formation of CD34 ϩ cells was largely abrogated by the ectopic expression of VentX, suggesting that VentX is a critical regulator of CD34 ϩ cell proliferation and expansion. A recent study demonstrated that an aryl hydrocarbon receptor antagonist, StemRegenin 1 (SR1), robustly promoted ex vivo expansion of human CD34 ϩ cells (19). To determine whether VentX plays an essential regulatory role in the expansion of human HSC/MPPs, we explored the effects of SR1 treatment on the expression levels of VentX and the effects of VentX on SR1-induced CD34 ϩ expansion. SR1 treatment accelerated the proliferation of CD34 ϩ cells (Fig. 4F, first two bars) and decreased the expression levels of VentX (Fig. 4E). Consistent with the notion that VentX plays an essential regulatory role in CD34 ϩ expansion, we found that ectopic expression of VentX prevented SR1-induced expansion of CD34 ϩ cells (Fig. 4F).
Our data indicate that down-regulation of VentX is a prerequisite for proliferation and expansion of CD34 ϩ cells.
VentX Regulates HSC/MPP Expansion in Vivo-We next sought to determine whether VentX knockdown promotes expansion of HSC/MPPs in vivo using the NOD/SCID/ IL2R␥2 null (NSG) mouse model. Human bone marrow CD34 ϩ cells were transduced with lentiviruses expressing either GFP or VentX shRNA. The unsorted transduced CD34 ϩ cells were then transplanted into three pairs of NSG mice. At 8 weeks post-transplantation, engraftment efficiency was determined by FACS analysis of human CD45 ϩ cells from bone marrow of recipient mice. We found that the human cell engraftment rate increased by ϳ1.5-2.5-fold in VentX shRNA-transduced CD34 ϩ cells compared with GFP shRNA-transduced CD34 ϩ cells (Fig. 5, A and B). Notably, knockdown of VentX allowed multilineage development of hematopoietic cells ( Table 1). Given that the lentiviral transduction rate of the CD34 ϩ cells was only ϳ20%, to further explore the effects of VentX knockdown on the expansion of HSC/MPPs, we sorted out the positively transduced CD34 ϩ cells and then transplanted the sorted CD34 ϩ cells into NSG mice. At 13 weeks post-transplantation, the percentage of human CD45 ϩ cells in the bone marrow of recipient mice was determined by FACS analysis. Strikingly, the percentage of human CD45 ϩ cells was consistently increased by 20-fold in VentX shRNA-transduced CD34 ϩ cells compared with GFP shRNA-transduced CD34 ϩ cells (Fig. 5, C and D). Supporting a potential role of VentX knockdown in the expansion of HSC/MPPs, we found that the number of human CD34 ϩ cells increased significantly in bone marrow of mice transplanted with VentX shRNA-transduced CD34 ϩ cells compared with control mice transplanted with GFP shRNAtransduced CD34 ϩ cells (45 Ϯ 7 and 720 Ϯ 13 per 10 5 total mononuclear cells for GFP shRNA-and VentX shRNA-transduced cells, respectively). To examine the effects of VentX on long-term engraftment of human CD34 ϩ cells, we performed secondary transplantation experiments. Whereas no detectable secondary engraftment was observed in mice transplanted with the control GFP shRNA-transduced CD34 ϩ cells, both of the two mice transplanted with the VentX shRNA-transduced CD34 ϩ cells showed positive human cell engraftment (0.25 and 0.28%, respectively) (Fig. 5E). were sorted by GFP and grown in stroma-free liquid cultures using a mixture of cytokines as described under "Experimental Procedures." The cells were counted weekly for 4 weeks, and the mean Ϯ S.D. of three independent experiments is shown. The number of cells at Day 0 represents the number of total transduced cells that were plated. The statistically significant difference (p Ͻ 0.05) is marked with asterisks. C-F, knockdown of VentX in CD34 ϩ cells increases the colony formation in semisolid culture. Positively transduced CD34 ϩ cells as described above were plated in triplicate in methylcellulose-based medium and allowed to form colonies for 2 weeks. C and D show the colony numbers of BFU-E/CFU-E and CFU-GM, respectively. E shows total progenitor cells from 2 ϫ 10 4 CD34 ϩ cells after culturing for 2 weeks. The significant difference between the two groups (three independent experiments for each group) was determined by Student's t test (p Ͻ 0.05). F, representative images of CFC assays showing the erythroid colonies in GFP shRNA-or VentX shRNA-transduced CD34 ϩ cells.

VentX Targets Cell Cycle Regulators in CD34 ϩ Cells-Our
recent studies have shown that VentX is a regulator of the Wnt signaling pathway and several components of the cell cycle machinery, such as cyclin D 1 , p21, and p16 ink4a (15,16). To explore the mechanisms underlying VentX-regulated HSC/ MPP expansion, we performed a targeted screen to determine the potential effects of VentX on these critical cell cycle regulators in CD34 ϩ cells. As shown in Fig. 6A, quantitative PCR demonstrated that overexpression of VentX increased the expression of p21 and p16 ink4a but down-regulated the expression of cyclin D 1 and c-myc in primary CD34 ϩ cells. The results were further confirmed by a loss-of-function approach in which knockdown of VentX by siRNA decreased the expression of p21 but increased the expression of cyclin D 1 and c-myc (Fig. 6B).
Cyclin D 1 has been implicated in CD34 ϩ cell proliferation (20 -22). The finding that the cyclin D 1 mRNA level was pronouncedly regulated by VentX prompted us to further examine whether the protein levels of cyclin D 1 are also subject to VentX regulation. As shown by FACS analysis of the intracellular staining of cyclin D 1 , VentX regulated the expression of cyclin D 1 at the protein level (Fig. 6, C and D).

DISCUSSION
Molecular mechanisms controlling HSC/MPP proliferation and differentiation are targets of extensive investigations for their role in the management of diseases involving HSCs, such as primary and secondary bone marrow failure syndrome, refractory anemia, and myelodysplastic syndromes. Difficulties . Knockdown of VentX in CD34 ؉ cells helps preserve stem cell pools of HSC/MPPs. Human CD34 ϩ cells transduced with either GFP shRNA (shGFP) or VentX shRNA (shVentX) were grown in liquid culture as described in the legend to Fig. 2B. The cells were harvested at the indicated time intervals and stained with PE-conjugated anti-CD34 and PE/Cy5-conjugated anti-CD38 antibodies for FACS analysis. A, representative FACS profile showing the percentage of CD34 ϩ cells from the GFP-positive populations at 2 weeks after plating. B, three independent experiments from A are shown. The significant difference between the two groups was determined by Student's t test (p Ͻ 0.05). C, absolute numbers of the CD34 ϩ cells in the two groups at 2 weeks after plating. D, GFP-positive CD34 ϩ cells were harvested at the indicated time points, and the percentage of the remaining CD34 ϩ cells was shown. The statistically significant difference (p Ͻ 0.05) is marked with asterisks. E, representative FACS profile showing the percentage of CD34 ϩ CD38 Ϫ cells from GFP-positive populations at 2 weeks after plating. F, three independent experiments from E are shown. The significant difference between the two groups was determined by Student's t test (p Ͻ 0.05). G, absolute numbers of CD34 ϩ CD38 Ϫ cells in the two groups at 2 weeks after plating. H, FACS analysis of the erythroid (glycophorin A) and myeloid (CD33) marker staining in the two groups at 2 weeks after plating. I, LTCIC assay of the GFP shRNA-and VentX shRNA-transduced CD34 ϩ cells. The statistically significant difference (p Ͻ 0.05) is marked with asterisks.
in obtaining sufficient numbers of HSC/MPPs also present a major hurdle for potential clinical applications of HSC/MPP transplantation. Multiple cell intrinsic factors and signaling pathways have been implicated in HSC/MPP expansion; nevertheless, the functions and mechanisms of these pathways in HSC/MPP expansion remain as topics of active debate (23)(24)(25). For example, although Wnt signaling was shown to be able to expand mouse HSCs, it seems that the levels of Wnt signaling play an important role in HSC/MPP expansion (26). In addition to the complex signaling events and their interpretations, there is growing appreciation of the species-specific property of HSC/MPPs (27). Evidently, although it is relatively easy to expand mouse HSCs, it remained a fundamental challenge to expand human HSC/MPPs by targeting cell intrinsic factors.
In this study, we have shown that VentX, a recently appreciated hematopoietic homeobox transcription factor, is a novel regulator of HSC/MPP expansion. We showed that suppression of VentX expression led to significant expansion of CD34 ϩ cells ex vivo and a Ͼ20-fold increase in engraftment potential in the NOD/SCID/IL2R␥2 null mouse model (Fig. 5). We showed that knockdown of VentX helped preserve the stem cell pools (Fig. 3) of HSC/MPPs, promoted clonogenicity, and allowed multilineage development of hematopoietic cells. The essential role of VentX in controlling expansion of CD34 ϩ cells was further illustrated by the finding that VentX expression was downregulated upon application of SR1, a recently identified small molecule that promotes expansion of CD34 ϩ cells, and by the finding that ectopic expression of VentX blocked SR1-induced CD34 ϩ cell expansion.
VentX represents the first cell intrinsic factor whose downregulation leads to significant expansion of human bone marrow CD34 ϩ cells. In comparison with other known cell intrinsic factors, such as HoxB4 and BMI1 (9 -11), VentX knockdown allowed more significant expansion of CD34 ϩ cells. In our current experimental system, a 30% knockdown of VentX led to a 20-fold increase in engraftment potential in NOD/SCID/ IL2R␥2 null mice (Fig. 5). Theoretically, the efficacy of HSC/ MPP expansion by VentX down-regulation could still be improved through more efficient knockdown of VentX. It should be noted, however, that we focused on the effects of VentX knockdown on the expansion of CD34 ϩ cells from human bone marrow. With the increasing application of cord blood in HSC transplantation and the notion that even modest HSC expansion would significantly increase the potential application of cord blood in adult HSC transplantation (28), the potential effects of VentX knockdown on the expansion of human cord blood CD34 ϩ cells would be of great interest for future exploration. The description of cell surface markers of bona fide human HSCs is constantly improving (29). In this study, we focused on CD34 ϩ HSC/MPPs. The clinical importance of these cells was reflected by the direct correlation with time to neutrophil recovery, an indicator of post-transplant mortality (27). The finding of VentX as the first cell intrinsic factor whose down-regulation promotes CD34 ϩ cell expansion  provides a novel therapeutic target for diseases involving HSC/ MPPs and HSC/MPP-based clinical therapy. We began to explore the mechanisms underlying VentX regulation of CD34 ϩ cell proliferation and differentiation. VentX expression is relatively low in early HSC/MPPs but increases dramatically during the proliferative differentiation of CD34 ϩ cells. Consistent with its role as a pro-differentiation and antiproliferation hematopoietic transcription factor, we found that knockdown of VentX promoted proliferation of CD34 ϩ cells (Fig. 2) and deferred their differentiation into lineage-specific progenitor cells (Fig. 3). As the stemness of HSC/MPPs decreases gradually during the proliferative differentiation of HSCs, our data suggest that VentX knockdown promotes the expansion of human HSC/MPPs by deferring differentiation of human HSC/MPPs. Although VentX knockdown promoted proliferation of CD34 ϩ cells, no obvious alteration of the cell cycle profile was observed by knockdown of VentX (data not shown).
At the molecular level, we found that VentX regulates the expression of genes downstream of canonical Wnt signaling, such as cyclin D 1 and c-myc, and other critical cell cycle regu-lators involved in HSC/MPP proliferation and differentiation, such as p21 (30 -32). Interestingly, we found that although overexpression of VentX increases the expression of p16, knockdown of VentX does not alter p16 expression significantly, suggesting that other factors are involved in controlling p16 expression in HSC/MPPs (Fig. 6, A and B). As such, the exact position of VentX in the molecular hierarchy of controlling HSC/MPP proliferation and differentiation remains to be further defined.
VentX was identified as an inhibitor of the oncogenic Wnt and as an activator of the p53/p21 and p16/pRb tumor suppressor pathways. Therefore, caution needs to be exercised for potential application of VentX knockdown in the expansion of human HSC/MPPs.