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The Journal of Immunology, 2006, 177: 2294-2303.
Copyright © 2006 by The American Association of Immunologists

Constitutively Active beta-Catenin Promotes Expansion of Multipotent Hematopoietic Progenitors in Culture1

Yoshihiro Baba*, Takafumi Yokota{dagger}, Hergen Spits{ddagger}, Karla P. Garrett*, Shin-Ichi Hayashi§ and Paul W. Kincade2,*

* Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; {dagger} Department of Hematology and Oncology, Osaka University, Suita, Osaka, Japan; {ddagger} Department of Cell Biology and Histology of the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and § Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-KitlowSca-1low/–CD19– CD11b/Mac-1–Flk-2–CD43+AA4.1+NK1.1–CD3–CD11c–Gr-1–CD45R/B220+ cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The hemopoiesis process is the sequential development from hemopoietic stem cells (HSC)3 to mature blood cells, in which the fate of various cellular intermediates and lineages is determined by a combination of factors such as cytokines, adhesion molecules, and transcription factors (1, 2, 3). The defining characteristics of HSC are their ability to extensively self-renew while maintaining the potential to give rise to all specialized blood cell types. The relative balance between self-renewal and differentiation is regulated by both intrinsic and extrinsic signals (4, 5, 6). HSC are thought to detach from specialized niches near the bone surface before gradually giving rise to committed lymphoid, myeloid, and erythroid progenitors (7). One recent report indicates that Wnt is an important component of the HSC niches (8).

Embryonic stem cells, epidermal stem cells, epithelial stem cells, and HSC all seem to be responsive to, or dependent on, Wnt receptor signaling for self-renewal (9, 10, 11, 12, 13, 14). Although many of the 19 known Wnt proteins, 10 Frizzled family receptors, and two low-density lipoprotein receptor-related protein coreceptors are expressed in bone marrow, their importance and mechanisms of action are far from clear (15). For example, Wnt may be produced by hemopoietic cells as well as components of the marrow niche, opening the possibility of autocrine or paracrine stimulation. Furthermore, Wnt proteins target stromal cells as well as hemopoietic cells, making it difficult to know whether stem cell responses are direct (16). HSC expanded in culture under the influence of purified Wnt 3A and thrived in irradiated recipients treated with Wnt 5A, but the two Wnt proteins can have opposing actions (9, 10, 17). Wnt 3A signals through Frizzled family receptors, leading to inactivation of glycogen synthase kinase 3beta and stabilization of beta-catenin in an unphosphorylated state. Accumulated beta-catenin in the cytosol translocates into the nucleus, leading to activation of target genes through association with LEF/TCF transcription factors (18, 19). Wnt 5A does not use and may in fact antagonize this canonical beta-catenin-dependent signaling pathway (20). In addition to beta-catenin stabilization, Wnt ligation of Frizzled receptors can cause activation of JNK, or an intracellular increase in Ca2+ (21). Conditional targeting of beta-catenin in adult HSC did not compromise their function, (22) but the closely related plakoglobin ({gamma}-catenin) protein might substitute for beta-catenin under physiological conditions (23, 24, 25). These findings imply that multiple independent pathways control stem cell self-renewal, and redundancy between the pathways or with different pathways is necessary under physiological circumstances. In addition, Notch receptors may deliver complementary signals that regulate stem cell behavior (8).

The complexity of this problem has been reduced in several studies by introduction of stabilized beta-catenin to hemopoietic cells. For example, Reya et al. (9) demonstrated that enforced retroviral expression of stable beta-catenin promoted self-renewal of HSC derived from Bcl-2-transgenic mice. Although overproduction of Bcl-2 was useful in allowing stem cells to be propagated in stromal cell-free cultures, it is possible this anti-apoptosis protein also influenced cell cycle status (26). We recently found that introduction of stable beta-catenin restored a surprising degree of multipotency to committed lymphoid or myeloid progenitors (27). Similarly, transduction of human CD34+ cells with stable beta-catenin slowed their differentiation in short-term cytokine stimulated cultures (28). These observations strongly suggest that canonical Wnt pathway signaling can slow, and possibly even reverse the loss of stem cell differentiation options.

Although active beta-catenin signaling can immortalize various tissue types and may be important in malignancy (29, 30, 31, 32), we are unaware of any studies in which this effect has been explored with early hemopoietic cells from normal animals. A key question is whether it would alone be sufficient to sustain stem cell properties for prolonged periods. Therefore, we have used retrovirus-mediated transduction to achieve constitutive activation of beta-catenin in lineage marker negative (Lin–) c-Kithigh Sca-1+ murine bone marrow cells. This category is highly enriched for long-term repopulating stem cells (HSC) as well as multipotent progenitors and the most primitive of lymphoid progenitors (33). The treatment dramatically influenced the ability of these cells to expand in culture while maintaining many primitive characteristics and responsiveness to normal signals. Multipotential cell lines prepared in this way may be important experimental tools for probing stem cell behavior and mechanisms responsible for lineage fate decisions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

C57BL/6 (CD45.2 alloantigen) and NOD/SCID mice were purchased from The Jackson Laboratory and maintained in our laboratory animal facility. The murine stromal cell lines OP9 and OP9-DL1 were generously provided by Dr. J. C. Zúñiga-Pflücker (University of Toronto, Toronto, Canada). Multipotent cell line EMLC1 was obtained from American Type Culture Collection.

Antibodies

Anti-CD45RA (14.8) mAb developed in our laboratory and the anti-CD11b/Mac-1 (M1/70) mAb were used as culture supernatants of the respective hybridomas. Purified anti-erythroid (Ter-119) and anti-Gr-1 (Ly-6G, RB6-8C5) Abs, FITC-conjugated anti-Ter-119, anti-Gr-1, anti-CD11b/Mac-1, anti-CD45R/B220 (RA3/6B2), anti-CD19 (ID3), anti-CD2 (LFA-2), anti-CD3 (145-2C11), anti-CD34 (RAM34) and anti-CD8{alpha} (53-6.7) Abs, PE-conjugated anti-IL-7R {alpha}-chain (SB/199), anti-Sca-1 (Ly6A/E, E13-161.7), anti-CD19, anti-Gr-1, anti-CD11c (HL3), anti-CD45R/B220 (RA3/6B2), anti-pan NK cell (DX5/CD49), anti-NK1.1 (PK136), anti-TCR-beta (H57-597), anti-TCR {gamma}{delta} (GL3), anti-CD43 (S7), anti-C1qRp (AA4.1), anti-CD135 (Flk2/Flt3, Ly-72), anti-CD4 (L3T4), biotin-conjugated anti-Sca-1, anti-VCAM-1 (429 MVCAM.A), allophycocyanin-conjugated anti-c-Kit (2B8), anti-CD11b/Mac-1 (M1/70), anti-CD3 (145-2C11), and anti-CD8{alpha} (53-6.7) mAb were all purchased from BD Pharmingen. Allophycocyanin-conjugated anti-F4/80 was purchased from eBioscience. A PE-Texas Red tandem-conjugated streptavidin was purchased from Caltag Laboratories. For immunoblotting, we purchased Abs specific to hemagglutinin (HA) or actin (Santa Cruz Biotechnology) or beta-catenin (BD Transduction Laboratories).

Cell sorting

Bone marrow cells were harvested and enriched for Lin– cells by incubation with Abs to lineage markers, Gr-1 and CD11b/Mac-1 for myeloid cells, CD45RA for B lineage cells, and Ter-119 for erythroid cells, followed by negative selection using the MACS cell separation system (Miltenyi Biotec). Sorting of the HSC/multipotent progenitor-enriched fraction was done as described elsewhere (34). Briefly, these partially lineage-depleted cells were further stained with FITC anti-lineage markers Gr-1, CD11b/Mac-1, CD2, CD19, CD3, CD8{alpha}, Ter-119, CD45RA, and PE anti-IL-7R {alpha}-chain, allophycocyanin anti-c-Kit, and biotin anti-Sca-1 Abs followed by streptavidin-PE-Texas Red. The Lin–IL-7R{alpha}–c-KithighScaI+ subset was then isolated to high purity on a MoFlo (DakoCytomation).

Retrovirus production and infection

A cDNA encoding HA-tagged stable beta-catenin, which has substitute mutations of S33A, S37A, T41A, and S45A to prevent phosphorylation, was cloned by PCR into XhoI/NotI sites of the LZRS-IRES-GFP retroviral vector (see Fig. 1). The retrovirus vector were transfected into EcoPack2 (BD Clontech) by FuGENE 6 (Roche) and transfected cells were selected by 2 µg/ml puromycin (Sigma-Aldrich). Supernatants were harvested 24 h after changing fresh media and immediately used for infection.


Figure 1
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  		FIGURE 1. Retroviral transduction of stable beta-catenin in Lin–c-Kithigh Sca-1+ hemopoietic cells. A, Schematic representation of the retroviral vector, encoding stable beta-catenin linked by an internal ribosome entry site (IRES) to a cDNA encoding enhanced GFP. B, The sorting gate used to prepare starting populations of adult bone marrow is shown. C, GFP expression is shown for sorted cells that were transduced for 48 h with control vector or stable beta-catenin containing viruses. Transduction efficiencies typically ranged from 30 to 60% as shown. LTR, Long terminal repeat.

 
For infection, a sorted HSC-enriched fraction was deposited into a single well of a 24-well dish at 1 x 104–5 x 105 cells per well. Cells were prestimulated in X-VIVO 15 medium (BioWhittaker) containing 1% detoxified BSA (StemCell Technologies), 2 mM L-glutamine, 5 x 10–5 M 2-ME, 100 U/ml penicillin, and 100 mg/ml streptomycin in the presence of 20 ng/ml recombinant mouse stem cell factor (SCF), 100 ng/ml Flk2/Flt3 ligand (FL), and 20 ng/ml thrombopoietin for 16–18 h. Subsequently, an equal volume of virus supernatant containing growth factor and 8 µg/ml polybrene (Sigma-Aldrich) were added. Spin infection was conducted for 2 h at 2000 rpm in a centrifuge at 32°C and then incubated at 37°C for 5 h. Culture medium was replaced with fresh medium containing fresh virus supernatant, growth factor, and polybrene, and then spin infection was performed under the same condition. After infection, cells were incubated at 37°C overnight. The next day, culture medium was replaced with fresh medium containing growth factor for an additional 24 h. After transduction, GFP-positive cells were purified by MoFlo. All cytokines were purchased from R&D Systems.

Culture assay and flow cytometry

To examine myelo-erythroid potential, 250 cells of each sorted fraction were cultured in IMDM-based methylcellulose medium supplemented with 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6, and 3 U/ml recombinant human erythropoietin (MethoCult GF 3434; StemCell Technologies). After 9 days, colonies were enumerated and classified according to shape and color under an inverted microscope. To evaluate B lineage and myeloid lineage differentiation, 1000 cells of each sorted fraction were cocultured with OP9 or MS-5 stromal cells in single wells of 24-well plates in the presence of SCF (20 ng/ml), FL (100 ng/ml), and IL-7 (1 ng/ml) for indicated periods. At the end of culture, cells were counted excluding stromal cells and then subjected to flow cytometry. A biotinylated anti-VCAM-1 mAb was used to exclude potential contamination of VCAM-1+ stromal cells in the analyzed populations and 7-aminoactinomycin D was used to exclude dead cells. Flow cytometry was performed on a BD FACSCalibur (BD Biosciences), and the data were analyzed with FlowJo software (Tree Star).

Stromal cell-free long-term culture

Highly enriched HSC populations transduced with control vector or stable beta-catenin were cultured in IMDM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin in the presence of 20 ng/ml SCF, 20 ng/ml IL-6, 100 ng/ml FL, and 20 ng/ml thrombopoietin (R&D Systems). Nonadherent cells were continuously passaged and long-term cultured cells were maintained in IMDM supplemented with 10% FCS with 20 ng/ml SCF and 20 ng/ml IL-6.

Adoptive transplantation of cultured hemopoietic cells transduced with beta-catenin

The HSC-enriched fraction of bone marrow was transduced with control vector or stable beta-catenin before being cultured on OP9 stromal cells in the presence of SCF, FL, and IL-7 for 10 days. A total of 1 x 106 cultured cells was transferred i.v. into sublethally irradiated (200 rad) NOD/SCID mice. Bone marrow, spleen, and thymi were harvested from these mice and analyzed by flow cytometry 5 wk postinjection. CD45 and GFP were used to distinguish host cells from donor cells.

Immunoblotting

Sorted cells were lysed in buffer containing 1% Triton X-100, 0.05% SDS, 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF), and the cleared lysates were boiled with 2x SDS sample buffer. Samples representing 5 x 105 cells were electrophoretically transferred to nitrocellulose membranes, blotted with appropriate Abs, and visualized with an ECL system (Pierce). Images were captured on a Roche LumiImager. Where necessary, the bands were quantified using ImageQuant software.

Ig gene rearrangement assay

Genomic DNA was isolated from sorted cells with DNeasy Tissue kit (Qiagen). PCR was conducted as described elsewhere (33). DHL(5') and J3(3') primers were used to detect DH-JH rearrangement with {alpha}-actin as a positive control.

Semiquantitative RT-PCR analysis of gene expression

The mRNAs were isolated from sorted cells using MicroPoly(A) Pure (Ambion). cDNA was prepared from DNase I-treated mRNA using oligo(dT) and Moloney murine leukemia virus reverse transcriptase. PCR was conducted in buffer containing 200 µM dATP, dGTP, dTTP, 100 µM dCTP, and 0.5 µCi [{alpha}-32P]dCTP. Aliquots were removed at cycle 25, 28, and 31 for beta-actin and cycle 32, 35, and 38 for all others to ensure that PCR remained within the exponential range of amplifications. Aliquots (5 µl) were denatured in a formamide-loading buffer and applied to a 6% polyacrylamide gel containing 7 M urea. Incorporation of [{alpha}-32P]dCTP into PCR product bands was quantified by PhosphorImager (Molecular Dynamics). Primer sequences and amplification conditions are available from the authors on request.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Constitutive activation of beta-catenin induces the expansion of uncommitted progenitors in vitro

Stable beta-catenin expression in HSC from Bcl-2-transgenic mice enhanced their self-renewal in culture (9). In addition, beta-catenin appears to be activated in HSC, but not progenitor cells (9). We hypothesized that down-regulation of beta-catenin may be a requirement for normal HSC differentiation and exit from a self-renewing condition. To test this concept, a HSC-enriched fraction of bone marrow was transduced with control vector or constitutive active beta-catenin (Fig. 1) before being placed in methylcellulose cultures with recombinant cytokines (Fig. 2A). Transduction of HSC-enriched cells with beta-catenin dramatically decreased numbers of CFU. The same cells were also held in OP9 cocultures for 10 days in the presence of SCF, FL, and IL-7. HSC transduced with the control vector gave rise to CD19+ B cells and CD11b/Mac-1+ myeloid cells including CD11b/Mac-1+Gr-1+ granulocytes, CD11b/Mac-1+Gr-1– monocytes/macrophages, and CD11b/Mac-1+CD11c+ dendritic cells (data not shown). In contrast, B cell and myeloid cell production from HSC transduced with constitutive active beta-catenin was strongly inhibited (Fig. 2, B and C). The active beta-catenin resulted in expansion of a primitive CD19–CD11b/Mac-1– population, suggesting that HSC may have been maintained in an immature state. Very similar results were obtained when MS5 stromal cells were used instead of OP9 (data not shown). Also, the results were comparable when the HSC-enriched fraction (Lin–c-KithighSca-1+) of E15 fetal liver was used instead of adult bone marrow (data not shown).


Figure 2
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  		FIGURE 2. The expression of stable beta-catenin in an HSC-rich fraction inhibits B cell and myeloid cell progression while causing accumulation of CD19–Mac-1– uncommitted progenitors. A, Sorted GFP+ cells transduced with control vector or stable beta-catenin were evaluated in methylcellulose assays (250 cells/dish) to determine their potential for clonal growth as CFU-GM, BFU-E, or CFU-GEM(M). The results are representative of three independent experiments (mean ± SD). Similar results were obtained in six independent experiments. B, GFP+ cells transduced with control vector or stable beta-catenin were sorted and cocultured on OP9 stromal cells in the presence of SCF, FL, and IL-7 for 10 days. B lineage (CD19) or myeloid lineage (Mac-1) differentiation was assessed by flow cytometry. C, Data indicate calculated yields, i.e., numbers of cells recovered per transduced input cell. Similar results were obtained in six independent experiments. Significant differences from control vector are indicated. *, p < 0.05) or **, p < 0.01, determined by Student’s t test.

 
Longer-term consequences of active beta-catenin expression in hemopoietic cells were then explored by placing 10,000 cells harvested from the first 10-day cultures described onto fresh OP9 stromal cells with the same cytokines. Stable beta-catenin-transduced cells had a dramatic growth advantage and consistently expanded 105- to 106-fold within 40 days (Fig. 3A). In contrast, HSC transduced with the control vector could not be expanded for longer than 2 wk. The majority of beta-catenin-transduced cells in these long-term cultures had primitive characteristics. That is, they were c-KitlowSca-1low/–CD19–CD11b/Mac-1–Flk-2–CD43+AA4.1+NK1.1–DX5–CD3–CD11c–Gr-1–CD8{alpha}– (Fig. 3C and data not shown). As one exception to this pattern, the cells uniformly expressed high levels of CD45R/B220. May-Grünwald-Giemsa staining of sorted CD19–CD11b/Mac-1– cells revealed a lymphoid morphology with a high nucleus to cytoplasmic ratio (Fig. 3C).


Figure 3
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  		FIGURE 3. The expression of stable beta-catenin in primitive cells provides a growth advantage in culture. A, GFP+ cells transduced with control vector or stable beta-catenin were sorted and cultured on OP9 stromal cells in the presence of SCF, FL, and IL-7. After the first 10 days, and at 10-day intervals, cells were harvested and 10,000 were replated on fresh OP9 stromal cells. Duplicate cultures were prepared in three independent experiments and the results were pooled for analysis. Significant difference from control vector are indicated. *, p < 0.01 was determined through Student’s t test. B, B lineage (CD19) or myeloid cell (Mac-1) differentiation was assessed by flow cytometry at each time point. C, CD19–Mac-1– cells in 10-day primary cultures initiated with stable beta-catenin were sorted for further analysis. May-Grünwald-Giemsa staining and flow cytometry results are shown. Thick line histograms represent staining obtained with Abs specific for the indicated markers, whereas dotted histograms show isotype-matched negative control staining.

 
Small numbers of myeloid and lymphoid cells were produced in beta-catenin-expressing long-term cultures (Fig. 3B). Interestingly, cells bearing lineage markers, and especially CD19, tended to have low amounts of GFP fluorescence (Fig. 4A). Western blot analysis of HA-tagged epitope protein revealed that levels of stable beta-catenin in GFPhigh cells were 6-fold more than those in GFPlow cells (Fig. 4B). Surprisingly, endogenous beta-catenin levels paralleled expression of the introduced cassette in these samples. It may be that sorting for GFPlow cells selects for differentiated progenitors that would have reduced Wnt signaling. As another possibility, Wnt pathway signaling could increase endogenous beta-catenin expression. Finally, stable transduction of the multipotential EMLC1 cell line (35) with active beta-catenin caused a 70% reduction of IL-3-induced myelopoiesis (CD11b/Mac-1+Gr-1+ cell production) compared with EMLC1 cells transduced with the control vector (data not shown). All of these results suggest that active beta-catenin promotes survival and/or expansion of primitive cells while blocking their progression in any given lineage. Lineage markers were acquired on small numbers of cells that may have spontaneously down-regulated expression of the transduced cassette.


Figure 4
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  		FIGURE 4. Down-regulation of stable beta-catenin correlates with differentiation of primitive cells. A, Cells transduced with stable beta-catenin were held on OP9 stromal cells for a total of 20 days before flow cytometry to evaluate GFP expression. Cells were gated as indicated (far left) before analysis with lineage-specific markers as shown (middle and right). B, The same gates were then used for sorting GFPhigh or GFPlow populations as shown. C, The fractions were then lysed, fractionated by SDS-PAGE, and immunoblotted (IB) with Abs specific for the HA-tagged beta-catenin or actin. Whole bone marrow (WBM) cells were used as controls. Endogenous beta-catenin protein (*) is marked, and HA-tagged beta-catenin bands are indicated with arrowheads. Molecular mass standards are shown in kDa.

 
Cells with stable beta-catenin retain significant differentiation potential

These experiments suggested that although stable beta-catenin permits a substantial amount of expansion from primitive stem/progenitor cells, at least some of the transduced population had differentiation potential. Because SCF is known to be important for stem cell self-renewal, we withdrew this cytokine and maintained the cells on OP9 stromal cells with just FL and IL-7. Although cell numbers 10 days later were comparable to cultures with SCF, we observed dramatic acquisition of lineage markers (CD19, CD11b/Mac-1, Gr-1, CD11c, CD3{epsilon}, CD4, CD8{alpha}, and Ter-119) in SCF-deficient cultures (Fig. 5A). Although the OP9 cells may make some SCF, it appears insufficient to maintain an undifferentiated state.


Figure 5
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  		FIGURE 5. SCF helps to maintain an undifferentiated condition in beta-catenin-expressing cells that retain multiple lineage options. A, One group of transduced cells were held for 10 days on OP9 stromal cells with FL and IL-7, while a second set of cultures contained SCF. The black line histogram shows expression of lineage markers (CD19, CD11b/Mac-1, Gr-1, CD11c, CD3{epsilon}, CD4, CD8{alpha}, and Ter-119) on cells held with SCF. The gray line histogram depicts cells similarly cultured, but in the absence of SCF. B, Another subgroup of transduced cells was held for 10 days on OP9 stromal cells with FL, SCF, and IL-7. Primitive CD19–Mac-1– cells were isolated at that time by sorting and tested for differentiation potential. Cells subcultured onto OP9 with FL and IL-7 generated both CD19+ B lineage and CD11b/Mac-1+ lineage cells (left two panels). In contrast, primitive cells produced neither of these lineages when subcultured onto OP9 stromal cells transduced with the Delta-like-1 Notch ligand. CD3{epsilon}+ T lineage cells emerged within 3 wk under those conditions. Similar results were obtained in four independent experiments. C, CD19+ cells were recovered and sorted from the cultures illustrated in B. Their genomic DNA (lane 3) was evaluated for Ig DH-JH rearrangements in comparison to DNA from freshly isolated CD19+ cells (lane 1) or water (lane 2). DNA standards are shown in lane M.

 
More rigorous tests of differentiation potential involved sorting of Lin– cells from 10-day cultures of transduced bone marrow cells and then replating them without SCF on OP9 or Delta-like-1-transduced OP9 stromal cells (OP9-DL1; Fig. 5B). This step was done because the Delta-like-1 Notch ligand is known to promote T lymphopoiesis in this context (36). Substantial numbers of CD19+ B lineage and CD11b/Mac-1+ myeloid cells were produced on OP9. In contrast, B lineage cells were suppressed and CD3+ T lineage cells emerged on OP9-DL1 (Fig. 5B). Lymphocytes with {gamma}{delta}+ TCR receptors predominated over those with {alpha}beta receptors in this circumstance (data not shown). As with normal hemopoietic progenitors (37) production of myeloid cells was suppressed by Notch receptor signaling (Fig. 5B). Because artificial elevation of beta-catenin bypasses the normal requirement for TCR signaling in the thymus (38), Ig gene rearrangements were assessed in CD19+ cells recovered from OP9 cocultures. Ig DH-JH rearrangements in beta-catenin-transduced cells were indistinguishable from CD19+ cells freshly isolated from normal bone marrow (Fig. 5C).

Parallel experiments assessed the ability of expanded beta-catenin-expressing cells to home and differentiate in a more natural environment. Ten days after transduction and culture on OP9, cells were transplanted to sublethally irradiated NOD/SCID mice and then evaluated 5 wk later. Small numbers of donor-type myeloid and B lineage cells, but not T cells were found in the bone marrow and spleens of animals that received active beta-catenin-containing cells (Fig. 6). T lineage potential was also not observed in the thymus. In contrast, control vector-transduced cells had no measurable ability to differentiate in recipient mice (Fig. 6A). All donor-type CD45.2+ cells within the marrow of three recipients continued to express GFP, whereas progression to CD19+ lymphocytes corresponded to loss of GFP in one mouse (Fig. 6B).


Figure 6
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  		FIGURE 6. Primitive cells transduced with active beta-catenin and propagated on OP9 stromal cells retained some potential for differentiation in immunodeficient mice. Cells transduced with the control vector or beta-catenin were cocultured on OP9 stromal cells in the presence of SCF, FL, and IL-7. After 10 days, 1 x 106 cell s were injected into sublethally irradiated NOD/SCID mice. After 5 wk, cells were isolated from spleen and thymus (A) and from bone marrow (B) before analysis by flow cytometry. Three of the recipients of beta-catenin-transduced cells had profiles (bottom left) as shown, and results from an exceptional animal are shown (bottom right). In each case, donor-type cells were identified according to the CD45.2 marker with or without GFP. BM, Bone marrow.

 
Therefore, primitive cells with active beta-catenin differentiated when SCF was reduced and produced myeloid, B, and T lineage cells in response to appropriate environmental cues. Although there was some potential for differentiation in sublethally irradiated immunodeficient mice, transduced cells cultured in this way may have lost the potential for thymic reconstitution.

Environmental signals required for maintenance of active beta-catenin-expressing cells

Stromal cells make at least some SCF, as well as other cytokines, and we wanted to learn whether cells with active beta-catenin could be maintained under defined conditions. Therefore, the HSC-enriched fraction was transduced with active beta-catenin or control vector. Two days later, GFP+ cells were sorted and placed in stromal cell-free liquid cultures containing SCF, thrombopoietin, FL, and IL-6. Cells transduced with the control vector produced granulocytes and did not expand beyond 2 wk (data not shown). In contrast, beta-catenin-transduced marrow cells retained their undifferentiated appearance and continued to proliferate for longer than 5 mo. The cells continued to proliferate on withdrawal of both thrombopoietin and FL, but quickly died when either SCF or IL-6 was removed (data not shown). Although other lines of transduced cells could be established and maintained without IL-6, we found that they had less differentiation potential than those maintained with the two cytokines combined (data not shown).

This long-term, stromal cell-independent line was subsequently maintained with just 100 ng/ml SCF and 20 ng/ml IL-6. The cells expressed very high levels of the stem/progenitor markers c-Kit and Sca-1 (Fig. 7A). They were negative for most lineage-specific markers (Gr-1, CD19, Ter119, CD3, CD4, CD8, CD11c, NK1.1, DX5) with the exception of CD45R/B220 and CD11b/Mac-1. Levels of Sca-1 and c-Kit were higher, and CD11b/Mac-1 was more uniformly positive than was the case with the stromal cell-propagated, beta-catenin-transduced cells already described. Long-term propagated cells maintained a mononuclear morphology, with a small amount of basophilic cytoplasm (Fig. 7B). Light scatter properties were consistent with an average size of 25 µm (data not shown). Western blot analysis and flow cytometry revealed that HA-tagged beta-catenin and GFP fluorescence expression were maintained. Therefore, active beta-catenin-expressing cells with primitive features could be propagated indefinitely with just two recombinant cytokines.


Figure 7
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  		FIGURE 7. Constitutively active beta-catenin allows propagation of long-term stromal cell-independent hemopoietic cells. A, Flow cytometry analysis was performed on transduced, long-term cultured cells. Solid line histograms represent staining obtained with Abs specific for the indicated cell surface markers. Dotted line histograms show staining obtained with isotype-matched control Abs and the results are representative of at least three analyses. B, A May-Grünwald-Giemsa-stained cytospin preparation is also shown.

 
Long-term, stromal cell-independent cells retain some normal differentiation potential

The experiments described show that cells transduced for short periods with constitutively active beta-catenin could generate both lymphoid and myeloid cells (Fig. 5B). A key question was whether these properties would be stable in a line maintained under defined conditions. Surprisingly, cells propagated for more than 5 mo responded to recombinant M-CSF and generated pure macrophages (Fig. 8A). Homogenous populations of polymorphonuclear granulocytes were made in response to G-CSF, whereas macrophages and granulocytes resulted from addition of GM-CSF (Fig. 8, B and C). IL-6 was withdrawn, but SCF was left in culture for these experiments. B lineage potential was also retained, inasmuch as the long-term line produced CD19+ cells when transferred to OP9 stromal cells plus FL and IL-7 (Fig. 8D). Note that some hemopoietic cells migrated beneath stromal cells to produce cobblestone-like foci, and medium-sized lymphocytes were produced (Fig. 8D). GFP levels in these lymphocytes were dramatically reduced relative to parent cultures (data not shown). In contrast to stromal cell-propagated, beta-catenin-transduced cells (Fig. 5B), this line did not generate CD3+ T lineage cells when transferred to OP9-DL1 stromal cells along with FL and IL-7 (data not shown). In fact, the cultured cells did not survive more than 1 wk under these conditions.


Figure 8
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  		FIGURE 8. Long-term propagated hemopoietic cells can respond to normal differentiation cues. Flow cytometry results are shown for long-term (longer than 5 mo) cultured beta-catenin-transduced cells following stimulation with 100 ng/ml SCF and 20 ng/ml M-CSF (A), 100 ng/ml SCF and 20 ng/ml G-CSF (B), and 100 ng/ml SCF and 20 ng/ml GM-CSF (C). Each May-Grünwald-Giemsa-stained cytospin preparation is shown on the right. Similar results were obtained in two independent differentiation assays. D, Long-term cultured cells produced CD19+ B lineage cells when placed on OP9 stromal cells in the presence of FL and IL-7 for 2 wk. Typical results (left) from three independent experiments are shown. Cobblestone area formation (middle) beneath OP9 stromal cells is illustrated. The May-Grünwald-Giemsa staining of cytospin preparations is shown on the right.

 
Long-term propagated cells were also transplanted to lethally irradiated congenic recipients plus recipient-type bone marrow as well as to sublethally irradiated NOD/SCID or RAG-2–/– mice. Donor-type cells were not found in any of these recipients and there was no sign of tumor formation 6 wk after the injections.

We conclude that active beta-catenin maintains multipotential progenitors in an undifferentiated condition. Remarkably, the ability to generate most, but not all, cell types in culture was retained during months of subculture in simple conditions.

Gene expression patterns were remarkably stable following short-term Wnt pathway signaling

Hemopoietic lineage-related genes are likely to be substantially influenced by artificial overexpression of stable beta-catenin. Of more interest would be genes known to be related to stem cell survival and self-renewal. We examined expression of a number of these 48 h after transduction, reasoning that early changes might be informative about how multipotency is retained (Fig. 9). Semiquantitative RT-PCR analyses revealed small changes associated with mock transduction and culture relative to freshly isolated stem/progenitor cells. With the possible exception of Id-1 that declined slightly and Notch-1 that increased relative to cultured control cells, we did not find a substantial influence of constitutive Wnt signaling on expression of this panel of genes.


Figure 9
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  		FIGURE 9. Gene expression patterns of stable beta-catenin-transduced cells. A HSC-enriched fraction of bone marrow was transduced with control vector (Control) or stable beta-catenin for 48 h before sorting for GFP+ cells. The freshly isolated HSC-enriched fraction was used as an additional control (Fresh). Semiquantitative RT-PCR was conducted to amplify transcripts for the indicated genes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Self-renewal is an essential property of stem cells and a key to some of the most important questions in biology. A growing body of information suggests that the Wnt family of proteins, receptors, downstream mediators, and target genes contribute to the self-renewal of normal and transformed stem cells. However, the complexity of these pathways has made it difficult to describe and implicate discrete mechanisms. Therefore, we manipulated one key mediator of Wnt signaling in a highly enriched population of murine stem/progenitor cells. The results support previous studies and demonstrate that active beta-catenin can trap primitive hemopoietic cells in a self-renewing state without overtly transforming them or blocking responsiveness to normal differentiation cues. However, this response was not sufficient under our culture conditions to retain all stem cell properties.

Our initial finding was that beta-catenin-transduced cells could be expanded in stromal cell cocultures that also contained SCF, FL, and IL-7. After just 10 days of culture, the protein diminished progression in B lymphoid and myeloid lineages, as well as expansion of committed myelo-erythroid progenitors. In contrast, cells transduced with a control vector failed to expand beyond 2 wk. When analyzed after 20 days of coculture, cells bearing CD19 or CD11b/Mac-1 tended to have low levels of GFP and stable beta-catenin expression. This interesting finding suggests that spontaneous down-regulation of beta-catenin may have permitted or been a consequence of differentiation in stromal cell cocultures.

The predominant population in cultures initiated with beta-catenin-transduced marrow cells lacked either CD19 or CD11b/Mac-1. Further characterization revealed them to be c-KitlowSca-1low/–Flk-2–CD43+AA4.1+NK1.1–DX-5–CD3–CD11c–Gr-1–CD8{alpha}– and the only lineage marker expressed was CD45R/B220. Although the latter Ag is frequently used as a B lineage marker, it is found on other cell types and was reportedly expressed by transformed, multipotential progenitors (39, 40, 41). Maintenance of these immature features required SCF; omission of the cytokine from the beginning favored expansion of CD19+ and CD11b/Mac-1+ cells. Consequently, we withdrew the cytokine in all subsequent experiments when the differentiation potential of cultured cells was being assessed.

The next goal was to learn whether constitutively active beta-catenin obviated the requirement for any normal survival signals. Stromal cells provide many environmental cues, but it was possible to generate a long-term line of stable beta-catenin-expressing cells in stromal cell-free conditions. SCF and IL-6 were essential and the cells were expanded for more than 5 mo with just these two cytokines. It is interesting that the same factors support long-term growth of hemopoietic cells transduced with either active Notch IC (42) or Lhx2 (43). Our stable beta-catenin-expressing cells require relatively high cell densities in culture and have not been successfully cloned. The same has been reported for Lhx2-transduced cells, (43), suggesting that production of juxtacrine/autocrine factors or physical contact may be important in these circumstances.

Despite the fact that beta-catenin-transduced cells had relatively simple growth requirements, injection into immunodeficient mice did not result in tumor formation. Nonetheless, expression of stable beta-catenin in human CD34+ cells arrested differentiation (28), and the Wnt pathway may be involved in some cases of leukemia (44, 45, 46, 47, 48, 49).

Therapeutic applications of stem cell propagation require that responsiveness to normal signals be retained. Therefore, we rigorously investigated the differentiation potential of long-term cultured beta-catenin-transduced cells. Remarkably pure populations of macrophages or granulocytes were made in response to M-CSF or G-CSF, respectively, whereas GM-CSF elicited formation of both cell types. Production of CD19+ B lineage cells was dramatic, but not quite as efficient when the cell line was transferred to OP9 stromal cells with FL and IL-7. OP9 probably makes some SCF, a factor that helps to maintain primitive features and may bias toward a nonlymphoid fate (Fig. 4 and data not shown).

Short-term cultured cells (10 days) retained some potential to generate myeloid and B lineage lymphoid cells following transplantation to immunodeficient recipients even though the experimental design was probably not optimal to achieve stem cell chimerism. Further investigation is needed to determine how long this activity is maintained in beta-catenin-transduced progenitors, but donor-type cells were not detected when the long-term stromal cell-free propagated line was transplanted. It was shown that HSC could be held and even expanded during 5 days of culture with purified Wnt 3A (10). Therefore, physiological levels of beta-catenin as well as particular combinations and concentrations of cytokines might be needed to retain stem cell properties for extended periods.

Requirements for maintenance of T lineage lymphoid differentiation potential also appear to be stringent. Cells expressing beta-catenin in short-term stromal cell cocultures generated CD3+ lymphocytes when transferred to Delta-like-1-transduced OP9 stromal cells. However, the ability to home to and colonize the thymus may have been compromised because we found only background numbers of donor-type lymphocytes when the same short-term cultured cells were transplanted into immunodeficient mice. Additional components of the bone marrow environment might be exploited to protect this property, because Bcl-2-transgenic cells with beta-catenin generated T cells in vivo (9). Long-term propagated cells with stable beta-catenin retained the potential for B and myeloid, but not T lineage lymphocyte potential. Artificial expression of beta-catenin from the DN3 thymocyte stage stimulated TCR-independent differentiation (38). Although we have not rigorously investigated this point, B lineage lymphocyte differentiation was not remarkable in beta-catenin-transduced progenitors. That is, we found normal patterns of DH-JH Ig gene rearrangements.

Our model demonstrates the ability of beta-catenin-mediated signals to renew primitive hemopoietic cells but begs the question of which target genes are responsible. Previous studies demonstrated that beta-catenin induces expression of HoxB4 and Notch-1 (9). Also, Wnt3a signals up-regulation of the Notch target gene Hes-1 in hemopoietic cells (8). Although Notch 1, HoxB4, Bmi-1, Gfi-1, p21, and C/EBP{alpha} could all be involved in stem cell self-renewal (50, 51, 52, 53, 54, 55), none were substantially elevated in stable beta-catenin-transduced cells as assessed by semiquantitative RT-PCR analysis (Fig. 9). Notch-1 levels were approximately equivalent to freshly isolated cells and slightly elevated relative to cultured control cells. Cell proliferation or apoptosis/survival-related genes such as c-myc, cyclin D, or Bcl-2 also were unaffected, although apoptosis of human cells in short-term cultures was reduced by beta-catenin (28, 45, 54, 55). The Id-1 transcriptional repressor appeared to be down-regulated by stable beta-catenin, but the significance requires further investigation. Additional Wnt target genes such as c-fos, c-jun, and Bcl-xL have been identified in other tissues (56, 57). Thus, expansion of the screen to include such genes and examination across different time intervals could be informative with regard to how stable beta-catenin sustains multipotent hemopoietic cells.

Previous studies strongly suggested that the Wnt/beta-catenin signaling pathway plays a critical role in physiological maintenance of stem and primitive progenitor cells (reviewed in Ref. 5). However, the extent to which this effect was dependent on beta-catenin and the importance of having a Bcl-2 transgene in the responding cells were unclear. Drugs that reversibly and specifically activate beta-catenin might prove useful for therapeutic stem cell manipulation, but much more needs to be learned about relationships among Wnt/beta-catenin, HoxB4, Bmi-1, and Notch family molecules (8, 9, 51, 52). Cells with artificially high levels of beta-catenin retained remarkable differentiation potential for many months, but those held under our conditions should not be regarded as true stem cells. In fact, the cells were compromised with respect to engraftment and this manipulation alone would not produce stem cells suitable for human therapy. However, beta-catenin is likely to be an important contributor to stem cell self-renewal and further investigation should be informative about such issues as stem cell plasticity and transcription factor hierarchies.


   Acknowledgments
 
We thank Viji Dandapani, Jacob Bass, and Diana Hamilton for expert technical assistance. In addition, we appreciate the secretarial help provided by Shelli Wasson.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AI 20069 and AI 58162 from the National Institutes of Health and Grant P20-RR15577 from the Center for Biomedical Research Excellence COBRE Program of the National Center for Research Resources. P.W.K. holds the William H. and Rita Bell Endowed Chair in Biomedical Research. Back

2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail address: Kincade{at}omrf.ouhsc.edu Back

3 Abbreviations used in this paper: HSC, hemopoietic stem cell; SCF, stem cell factor; HA, hemagglutinin; FL, Flk2/Flt3 ligand. Back

Received for publication November 11, 2005. Accepted for publication May 23, 2006.


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