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TISSUE-SPECIFIC STEM CELLS

Insulin-Like Growth Factor-Binding Protein 2 Secreted by a Tumorigenic Cell Line Supports Ex Vivo Expansion of Mouse Hematopoietic Stem Cells

Departments of ^aPhysiology and
^bDevelopmental Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA;
^dWhitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA;
^cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Key Words. Ex vivo expansion • Hematopoietic stem cells • Cell culture • Cytokines • Flow cytometry • Growth factors • Hematopoietic stem cell transplantation

Correspondence: Correspondence: Cheng Cheng Zhang, Ph.D., Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA. Telephone: 214-645-6320; Fax: 214-648-1960; e-mail: Alec.Zhang{at}UTSouthwestern.edu

Received on January 20, 2008; accepted for publication on March 19, 2008.

First published online in STEM CELLS EXPRESS  March 27, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC-supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin-like growth factor-binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serum-free culture system for mouse HSCs, containing SCF, thrombopoietin, fibroblast growth factor 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Hematopoietic stem cell (HSC) therapy is used to treat patients with leukemia, lymphoma, other cancers, and genetic defects such as sickle cell anemia and thalassemia [1]. HSCs are also a promising cell target for gene therapies [2]. One of the most important parameters for successful HSC transplantation is the dose of transplanted HSCs. Expansion of HSCs in culture will improve the survival rates of cancer patients who receive HSC transplantation and will also allow amplification of umbilical cord blood HSCs for use by adult patients. Furthermore, methods that facilitate ex vivo expansion of HSCs will greatly boost the development of gene therapy by allowing selection of transduced HSCs in which the desired genes are introduced into the appropriate DNA location.

Numerous attempts have been made to increase the number of long-term (LT)-HSCs in culture [3, 4]. Some such systems involve unknown factors that allow the stem cells to survive and multiply in number. The unknown factors can be supplied by coculturing the stem cells with feeder cells, which secrete an undefined panel of factors, or they can be supplied by adding undefined serum products to the growth medium. In the mouse model system, the use of stromal cell lines or combinations of cytokines resulted in significant self-renewal of HSCs assayed 4–6 weeks post-transplant and has led to as much as a sixfold expansion of murine LT-HSC activity in culture [5–9]. The introduction of exogenous transcription factors can expand HSCs more dramatically [10–13], although gene transduction of HSCs may cause undesired outcome for patients in clinical settings [2]. There remains a need for methods and compositions that allow the in vitro and/or ex vivo propagation of HSCs in a chemically defined medium while maintaining the potency of the propagated cells.

The fate of HSCs—self-renewal, differentiation, apoptosis, or quiescence—is regulated by components of their in vivo niche or microenvironment [14, 15]. We previously showed that insulin-like growth factor 2 (IGF-2), as well as several angiopoietin-like proteins (Angptls), a group of growth factors secreted by a fetal liver HSC-supportive cell population, supported ex vivo expansion of murine HSCs [16–18]. Here, we demonstrate that insulin-like growth factor-binding protein 2 (IGFBP2), secreted by a cultured tumorigenic cell line, stimulated ex vivo expansion of mouse HSCs.

Furthermore, we established a completely defined serum-free culture system for murine HSCs, which includes growth factors stem cell factor (SCF), thrombopoietin (TPO), fibroblast growth factor 1 (FGF-1), Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 3 weeks of culture. To our knowledge, this is the highest level of ex vivo expansion of HSCs yet achieved in a defined culture. This is also the first demonstration that IGFBP2 stimulates the expansion of murine stem cells. Our finding suggests that other cancer cells may secrete IGFBP2 or other HSC-stimulating proteins.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Mice
C57 BL/6 CD45.2 and CD45.1 mice were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) or the National Cancer Institute and were maintained at the Whitehead Institute or the University of Texas Southwestern Medical Center animal facility. All animal experiments were performed with the approval of Massachusetts Institute of Technology (MIT) or the University of Texas Southwestern Committee on Animal Care.

Culture Medium
STIF medium is defined as StemSpan serum-free medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10 µg/ml heparin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml mouse IGF-2 (all from R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and 10 ng/ml human FGF-1 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). SCF, thrombopoietin, and fibroblast growth factor-1 (STF) medium was the same medium but without IGF-2. The indicated amounts of purified mouse Angptl3 (a gift from R&D Systems) or human IGFBP2 (R&D Systems) were added. Conditioned medium was collected from confluent 293T or 3T3 cells after overnight culture.

Mouse HSC Culture
Twenty bone marrow (BM) side population (SP) Sca-1⁺CD45⁺ cells isolated from 8–10-week-old C57BL/6 CD45.2 mice were plated in one well of a U-bottomed 96-well plate (catalog number 3799; Corning Enterprises, Corning, NY, http://www.corning.com) with 160 µl of the indicated medium. Cells were cultured at 37°C in 5% CO₂ and the indicated levels of O₂. For the purpose of competitive transplantation, we pooled cells from 12 culture wells and mixed them with competitor/supportive cells before the indicated numbers of cells were transplanted into each mouse. For Western blotting, bone marrow Lin^– cells isolated by AutoMacs (Miltenyi Biotec Inc., Auburn, CA, http://www.miltenyibiotec.com) or by fluorescence-activated cell sorting (FACS) were cultured overnight in STF medium, followed by starvation for 4 hours in serum-free medium (containing 0.5% bovine serum albumin) and treatment with 500 ng/ml IGFBP2. For quantitative reverse transcription (RT)-polymerase chain reaction (PCR), Lin^–Sca-1⁺Kit⁺Flk-2^– cells were cultured in STF medium for 3 days, followed by replacement with fresh STF medium. One hour later, 500 ng/ml IGFBP2 was added as indicated.

Flow Cytometry
Donor bone marrow cells were isolated from 8–10-week-old C57BL/6 CD45.2 mice. SP Sca-1⁺CD45⁺ cells were isolated as described [18]. Lin^–Sca-1⁺Kit⁺Flk-2^– cells were isolated by staining with a Biotinylated lineage cocktail (anti-CD3, anti-CD5, anti-B220, anti-Mac-1, anti-Gr-1, anti-Ter119, and anti-7–4; StemCell Technologies) followed by streptavidin-PE/Cy5.5, anti-Sca-1-fluorescein isothiocyanate (FITC), anti-Kit-allophycocyanin (APC), and anti-Flk-2-phycoerythrin (PE).

For analyzing repopulation of mouse HSCs, peripheral blood cells of recipient CD45.1 mice were collected by retro-orbital bleeding, followed by lysis of red blood cells and staining with anti-CD45.2-FITC, anti-CD45.1-PE, anti-Thy1.2-PE (for T-lymphoid lineage), anti-B220-PE (for B-lymphoid lineage), anti-Mac-1-PE, anti-Gr-1-PE (cells costaining with anti-Mac-1 and anti-Gr-1 were deemed to be of the myeloid lineage), or anti-Ter119-PE (for erythroid lineage) monoclonal antibody (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). The percentage repopulation shown in all figures was based on the staining results of anti-CD45.2-FITC and anti-CD45.1-PE. In all cases, FACS analysis of the above-listed lineages was also performed to confirm multilineage reconstitution.

Competitive Reconstitution Analysis
The indicated numbers of mouse CD45.2 donor cells were mixed with 1 x 10⁵ freshly isolated CD45.1 competitor bone marrow cells, and the mixture was injected intravenously via the retro-orbital route into each of a group of 6–9-week-old CD45.1 mice previously irradiated with a total dose of 10 Gy. Bone marrow cells (10⁶ cells) collected from primary recipients were used for secondary transplantation. To measure reconstitution of transplanted mice, peripheral blood was collected at the indicated times post-transplant, and the presence of CD45.1⁺ and CD45.2⁺ cells in lymphoid and myeloid compartments was measured as described [16–19]. Calculation of competitive repopulating units (CRUs) in limiting dilution experiments was conducted using L-Calc software (StemCell Technologies) [19].

Western Blots
Purified proteins or crude proteins in conditioned medium were analyzed by electrophoresis on 4%–12% NuPage Bis-Tris polyacrylamide gels (Invitrogen), and proteins were electroblotted onto nitrocellulose membranes. To detect IGFBP2, the membranes were probed with anti-IGFBP2 polyclonal antibody (AF674; R&D Systems) at 0.1 µg/ml, followed with the horseradish peroxidase-conjugated donkey anti-goat antibody, and detected by a chemiluminescence kit (Pierce, Rockford, IL, http://www.piercenet.com). Total mitogen-activated protein kinase (MAPK) p42/44 and their phosphorylated forms were detected using rabbit anti-mouse p42/44 or pp42/44 antibodies (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com).

Mass Spectrometry
The conditioned medium was resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Protein "bands" ranging from 10 to 70 kDa were excised, and samples were analyzed by the MIT mass spectrometry core facility. Trypsin digestion was performed, peptide mixtures were loaded onto a triphasic liquid chromatography (LC)/LC column, and tandem mass spectra were analyzed.

Quantitative RT-PCR
Total RNA was isolated from bone marrow Lin^–Sca-1⁺Kit⁺Flk-2^– cells. First-strand cDNA was synthesized using SuperScript II RT (Invitrogen). Samples were analyzed in triplicate 25-µl reactions (300 nM primers; 12.5 µl of Master Mix); this procedure was adapted from the standard protocol provided in SyBR Green PCR Master Mix and RT-PCR protocols provided by Applied Biosystems (Foster City, CA, http://www.appliedbiosystems.com). Primers were purchased from Qiagen (Hilden, Germany, http://www1.qiagen.com) or Sigma-Aldrich. The default PCR protocol was used on an Applied Biosystems Prism 7000 Sequence Detection System. The mRNA level in each population was normalized to the level of β-actin RNA transcripts present in the same sample, as described [18, 20].

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Conditioned Medium Collected from Nontransfected 293T Cells Stimulates Ex Vivo Expansion of HSCs
Recently, we identified Angptls as a group of new growth factors for HSCs [18]. We found that recombinant Angptls, present in the serum-free medium of transiently transfected 293T cells, supported ex vivo expansion of mouse bone marrow HSCs [18]. In the course of several control experiments, we found, surprisingly, that serum-free conditioned medium collected from nontransfected 293T cells also supported expansion of HSCs, albeit much less well than the medium from cells transduced with Angptl genes. In the experiment shown in Figure 1A, 20 freshly isolated CD45.2 bone marrow SP Sca-1⁺CD45⁺ cells, a highly enriched HSC population, were cultured for 10 days in fresh serum-free medium supplemented with SCF, TPO, IGF-2, and FGF-1 (STIF medium); these had an average of 10.4% engraftment in recipients, as measured by competitive reconstitution analysis at 5 months post-transplant (Fig. 1A, bar 1). By contrast, the same HSCs cultured in conditioned medium collected from 293T cells that was later supplemented with SCF, TPO, IGF-2, and FGF-1 had a much higher engraftment of 50.5% (Fig. 1A, bar 2). This suggests that an HSC-stimulating activity resides in 293T-conditioned medium. This activity, however, was depleted after freeze/thaw of the conditioned medium (Fig. 1A, bar 3). The expanded cells were able to repopulate both lymphoid and myeloid lineages (Fig. 1B), demonstrating expansion of repopulating HSCs. In another experiment, the conditioned medium collected from nontransfected 293T cells was supplemented with SCF, TPO, and FGF-1 (without IGF-2); this medium also stimulated HSC expansion, whereas the conditioned medium from another cell line, 3T3 cells, did not support HSC expansion (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 1. Serum-free conditioned medium collected from 293T cells stimulates ex vivo expansion of hematopoietic stem cells. (A): Twenty freshly isolated CD45.2 bone marrow side population Sca-1+CD45+ cells were cultured for 10 days in serum-free Iscove's modified Dulbecco's medium supplemented with 10 ng/ml SCF, 20 ng/ml thrombopoietin, 20 ng/ml insulin-like growth factor-2, and 10 ng/ml FGF-1 (STIF medium; bar 1), in freshly collected serum-free conditioned medium from 293T cells that was supplemented with the same growth factors (conditioned STIF medium; bar 2), or in the same conditioned medium that had undergone freeze/thaw (bar 3). Then, the entirety of the cultures was cotransplanted with 1 x 105 CD45.1 total bone marrow cells into CD45.1 recipients (n= 5–6). Engraftment at 5 months post-transplant is shown. *, Significantly different from bar 1 and bar 3 values. Student's t test, p < .005. (B): Multilineage contribution of cultured cells at conditions represented by bar 2 at 5 months post-transplant (n= 6). Data shown in the top panel are representative fluorescence-activated cell sorting plots of peripheral blood mononuclear cells from one mouse at 5 months post-transplant (bar 2 [A]) Percentages of cells in each quadrant are listed. The summary of data from mice in bar 2 of (A) is plotted in the bottom panel of (B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Insulin-like growth factor-binding protein 2 (IGFBP2) is the factor in serum-free 293T-conditioned medium that stimulates ex vivo expansion of hematopoietic stem cells. (A): Western blot analysis of purified human IGFBP2 (positive control; lane 1), serum-free 3T3 cell-conditioned medium (negative control; lane 2), and serum-free 293T cell-conditioned medium (lane 3) detected by an anti-IGFBP2 polyclonal antibody. (B): Twenty freshly isolated CD45.2 bone marrow side population Sca-1+CD45+ cells were cultured for 10 days in 160 µl of serum-free conditioned medium of 293T cells (bars 1 and 3) supplemented with SCF, thrombopoietin, and fibroblast growth factor-1, or in the same medium containing 10 µg/ml anti-IGFBP2 antibody (bars 2 and 4). Together with 1 x 105 competitor CD45.1 bone marrow cells, the total culture was injected into CD45.1 recipients (n= 4–5). Peripheral blood cells from transplanted mice were analyzed for the presence of CD45.2+ cells in lymphoid and myeloid compartments at 6 weeks (bars 1 and 2) or 7 months (bars 3 and 4) after transplant. *, Significantly different from the value of bar 1 or bar 3; p < .05. Abbreviations: kD, kilodalton; MW, molecular weight.

Purified Recombinant IGFBP-2 Stimulates Ex Vivo Expansion of HSCs
We tried to add purified IGFBP2 to the HSC culture medium, in the absence or presence of other factors. As with other known HSC growth factors, IGFBP2 alone could not support HSC expansion; the inclusion of IGFBP2 in our serum-free medium supplemented with SCF, TPO, FGF-1, and IGF-2 supported expansion of HSCs (data not shown). Because IGFBP2 can bind and modulate the biological effects of IGFs [21], we next tested whether IGFBP2 stimulated HSC expansion if we did not add IGF-2 to the culture. Figure 3 shows that culture of HSCs in the presence of STF medium supplemented with IGFBP2 resulted in dramatically increased repopulating activities compared with freshly isolated HSCs. In one experiment (Fig. 3A), 20 freshly isolated SP Sca-1⁺CD45⁺ cells supported an average of 1.0% engraftment (Fig. 3A, bar 1). The cultured progenies of the same number of cells after 10 days in STF medium had an increased engraftment of 9.8% (Fig. 3A, bar 2). Again, culture of the same number of purified HSCs in 293T-conditioned medium supplemented with SCF, TPO, and FGF-1 (293T-conditioned STF medium) resulted in a dramatic increase of repopulating HSC activity, to 39.3% (Fig. 3A, bar 3). The addition of 100 ng/ml recombinant IGFBP2 to STF medium also resulted in a large increase in HSC expansion (Fig. 3A, bar 4) relative to freshly isolated cells or cells cultured in STF medium (Fig. 3A, bars 1 and 2). Consistent with the results of Figure 2B, treatment of 293T-conditioned STF medium with 10 µg/ml anti-IGFBP2 neutralizing antibody again abrogated the HSC-stimulating effect of IGFBP2 (Fig. 3A, bar 5); the control isotype antibody did not show any inhibitory effect (Fig. 3A, bar 6).

View larger version (23K): [in this window] [in a new window]   Figure 3. Purified insulin-like growth factor-binding protein 2 (IGFBP2) stimulates ex vivo expansion of hematopoietic stem cells. (A): Shown are competitive repopulation at 4 months post-transplant from 20 freshly isolated side population (SP) Sca-1+CD45+ cells (bar 1); the progenies of the same number of cells cultured for 10 days in SCF, thrombopoietin, and fibroblast growth factor-1 (STF) medium (bar 2); in 293T-conditioned medium supplemented with SCF, thrombopoietin (TPO), and fibroblast growth factor-1 (293T-conditioned STF medium) (bar 3); in STF medium with 100 ng/ml IGFBP2 (bar 4); in 293T-conditioned STF medium pretreated with 10 µg/ml anti-IGFBP2 antibody (bar 5); and in 293T-conditioned STF medium pretreated with 10 µg/ml control antibody (bar 6); (n= 5). * and **, Significantly different from the values of bar 1 and bars 2 and 5, respectively; p < .05. (B): Twenty CD45.2 bone marrow SP Sca-1+CD45+ cells were cultured for 10 days in serum-free medium with 10 ng/ml SCF, 20 ng/ml TPO, and 10 ng/ml fibroblast growth factor-1 (STF medium) (bars 1 and 4); in STF medium containing 500 ng/ml IGFBP2 (bars 2 and 5); and in STF medium containing both 500 ng/ml IGFBP2 and 100 ng/ml angiopoietin-like protein 3 (bars 3 and 6). The cells were then cotransplanted with 1 x 105 CD45.1 total bone marrow cells into CD45.1 recipients (n= 6–7). Engraftments at 1 and 4 months post-transplant are shown. * and **, Significantly different from bar 4 and bar 5 values, respectively. Student's t test, p < .05. (C, D): Bone marrow of three mice at conditions represented by bar 6 of (B) were pooled at 4 months post-transplant and transplanted into secondary recipients. Total hematopoietic and multilineage engraftments of secondary transplanted mice (n= 5) are shown.

To ensure that the LT-HSCs indeed were expanded during culture, we pooled the bone marrow from primary recipients and transplanted them into secondary recipients. The originally cultured donor cells repopulated lymphoid and myeloid lineages at 1.5, 4, and 7 months after the secondary transplant (Fig. 3C, 3D).

In an additional experiment that demonstrated multilineage reconstitution, we cultured 100 CD45.1 bone marrow SP Sca-1⁺CD45⁺ cells for 10 days in serum-free STF medium supplemented with 100 ng/ml IGFBP2. The cells were then cotransplanted with 1 x 10⁶ CD45.2 bone marrow Sca-1^– cells into CD45.2 recipients. At 6 months post-transplant, those recipient mice fully reconstituted with CD45.1 cultured donor cells were selected for peripheral blood analysis (n= 3). A comparison of the peripheral blood cell counts with those of normal nonreconstituted mice showed normal numbers of erythrocytes and platelets and normal hematocrits and hemoglobin levels (Table 2). These experiments reinforce our conclusion that IGFBP2 supports ex vivo expansion of HSCs with an effect independent of that of other known HSC growth factors, including Angptls.

View larger version (13K): [in this window] [in a new window]   Figure 4. Limiting dilution analysis of the repopulating ability of hematopoietic stem cells before and after culture. Adult BM side population (SP) CD45+Sca-1+ cells were directly transplanted or cultured for 21 days in serum-free conditioned SCF, thrombopoietin, and fibroblast growth factor-1 medium containing 100 ng/ml purified angiopoietin-like protein 3 and 500 ng/ml insulin-like growth factor-binding protein 2. Irradiated CD45.1 congenic mice were injected with 1 x 105 CD45.1 BM competitor cells and 1, 5, 25, or 100 freshly isolated SP CD45+Sca-1+ cells (left; n= 24) or the cultured progenies of 0.2, 1, 5, or 10 SP CD45+Sca-1+ cells (right; n= 26). One hundred freshly isolated SP Sca-1+CD45+ cells and the cultured progeny of 5 or 10 input cells repopulated all recipients, and these data points are not plotted. Plotted is the percentage of recipient mice containing more than 1% CD45.2 myeloid and lymphoid cells in nucleated peripheral blood cells 4 months after transplant versus the number of input-equivalent cells injected. Abbreviations: BM, bone marrow; CRU, competitive repopulating unit.

View larger version (18K): [in this window] [in a new window]   Figure 5. Insulin-like growth factor-binding protein 2 (IGFBP2) induced rapid p42/44 MAPK activation and expression of several Hox genes. (A): Bone marrow Lin– cells were starved for 4 hours in serum-free Iscove's modified Dulbecco's medium containing 0.5% bovine serum albumin and then subjected to treatment with 500 ng/ml recombinant IGFBP2 for 0, 5, 15, and 30 minutes. Cells were directly lysed in RIPA buffer. Protein lysates from equal numbers of cells (30 µl of lysate, equivalent to 2 x 105 cells per lane) were subjected to Western blotting analysis. Levels of phosphorylated and total p42/44 MAPK proteins are shown. (B): Bone marrow Lin–Sca-1+Kit+Flk-2– cells were cultured in SCF, thrombopoietin, and fibroblast growth factor-1 medium treated with or without 100 ng/ml IGFBP2 for 3 hours before being collected for analysis. The gene expression in samples untreated by IGFBP2 was normalized to 1. The numbers of replicated experiments are shown. Each experiment contained three real-time polymerase chain reactions (PCRs). Shown are the results of averages of all real-time PCRs. *, Significantly different from the values of untreated samples; p < .05.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Previously, we showed that IGF-2 and Angptls independently stimulated expansion ex vivo of hematopoietic stem cells [16–18]. Here, we establish that IGFBP2 is an additional HSC-supportive factor secreted by tumorigenic 293T cells. IGFBPs are a family of six circulating proteins that bind IGF-1 and IGF-2 with an affinity equal or greater than that of the three IGF receptors. IGFBPs modulate the biological effects of IGFs by controlling IGF distribution, function, and activity [21]. IGFBP2 preferentially binds IGF-2 over IGF-1. IGFBP2 is expressed in the fetus and in a number of adult tissues and biological fluids [23]. It is overexpressed in many tumors, and in some cases its expression level correlates with the grade of malignancy [24–26]. The expression of IGFBP2 is controlled by a number of hormones, growth factors, and transcription factors, including growth hormone, insulin, IGF-1, IGF-2, transforming growth factor-β, interleukin-1, chorionic gonadotropin, follicle-stimulating hormone, estrogen, glucocorticoids, SP1, activating enhancer binding protein 4, and nuclear factor-B (reviewed in [26]), as well as p53 [27].

Our finding that IGFBP2 promotes the expansion of HSCs was unexpected in light of the IGF-dependent inhibitory effects that IGFBP2 has on normal somatic growth [28]. Nevertheless, consistent with our result, IGFBP2-deficient mice showed decreased spleen weight and total splenic lymphocyte numbers [29]. Recently, several studies suggested that in addition to modulating IGF activities, IGFBP2 also has intrinsic bioactivities that are independent of IGF1 or IGF2. For example, IGFBP2 binds to the cell surface [24, 30], and its binding to integrin 5 [24, 31, 32] or v [33] influences cell mobility [24, 31, 32] and proliferation [25, 26]. IGFBP2 was shown to stimulate telomerase activity [25], modulate MAPK and phosphoinositide 3-kinase activities [25], and activate matrix metalloproteinase 2 [34] and the Akt pathway [31]. In addition, it was shown that oxidative stress leads to the uptake of IGFBP2 into the cell cytosol after 12–24 hours [26, 35].

Here we showed that IGFBP2 stimulated ex vivo expansion of HSCs even when IGF-2 was not included in the culture medium. It also has additive or synergistic effects with Angptl3 and other HSC cytokines, including SCF, TPO, and FGF-1. Consistently, in bone marrow Lin^– cells, a population enriched in HSCs and hematopoietic progenitors, IGFBP2 stimulated activation of MAPK within 5 minutes, suggesting that IGFBP2 binds to an as yet unidentified receptor on the cell surface and induces a signal transduction pathway that is important for HSC proliferation [36]. Future studies will be conducted to clarify whether IGFBP2 directly stimulates signal transduction pathways in purified HSCs and whether IGF-initiated signaling is involved.

We also showed that within 3 hours of treatment, IGFBP2 induced expression of several Hox genes in bone marrow Lin^–Sca-1⁺Kit⁺Flk2^– cells, a cell population highly enriched in HSCs. Hox proteins are an evolutionary preserved family characterized by a 60-amino acid DNA-binding homeodomain. Hox genes of the A, B, and C clusters, but not the D cluster, are transcribed during normal hematopoiesis [37]. Importantly, ectopic expression of HoxB4 supports HSC self-renewal in culture [12]. These results support our preliminary hypothesis that IGFBP2 supports HSC expansion partially through upregulation of the expression of several Hox genes. Additional studies are needed to investigate the detailed mechanism for IGFBP2's function on HSCs.

It is surprising that IGFBP2 was identified as a protein secreted by a tumorigenic cell line that supports stem cells. It is known that bone marrow hematopoietic progenitors can be recruited to solid tumor sites in vivo [38]. We plan to test whether other cancer cells are enriched in HSC-stimulating activities, such as IGFBP2. This may open a new avenue for the study of stem cell niches in pathological conditions. It will also be interesting to determine whether the role of IGFBP2 in solid cancer development has any connection with its ability to expand HSCs in the tumor microenvironment. Our finding that IGFBP2 stimulates the expansion of HSCs suggests that IGFBP2 may be a growth promoter for certain normal and cancer stem cells.

In summary, we isolated IGFBP2 as an HSC-supportive secreted factor from the conditioned medium of tumorigenic 293T cells. The ability of IGFBP2 to support HSC expansion is independent of the addition of previously identified factors, including IGF-2 and Angptls. Added together to a serum-free defined medium, IGFBP2, Angptl3, SCF, TPO, and FGF-1 supported an 48-fold increase of repopulating HSCs in culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. The culture conditions that we have defined for expansion of HSCs will be useful for a variety of applications, including HSC transplantation, gene delivery, and drug discovery. Although the work described in this paper is confined to mouse HSCs, we recently showed that a serum-free culture containing SCF, TPO, FGF-1, Angiopoietin-like 5, and IGFBP2 supports an 14–20-fold net expansion of repopulating human cord blood HSCs [39].

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Support to H.F.L. is from NIH Grant R01-DK067356. Support to C.C.Z. is from NIH Grant K01-CA120099-01 and the Michael L. Rosenberg Endowed Scholar Fund from University of Texas Southwestern Medical Center.

	FOOTNOTES

 
Author contributions: H.H. and O.K.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; S.I.: collection and/or assembly of data, manuscript writing; M.K. and J.Z.: collection and/or assembly of data, data analysis and interpretation; H.F.L.: financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; C.C.Z.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

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