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Transient Disruption of Autocrine TGF- Signaling Leads to Enhanced Survival and Proliferation Potential in Single Primitive Human Hemopoietic Progenitor Cells¹

Xiaolong Fan^*, Gudrun Valdimarsdottir, Jonas Larsson^*, Ann Brun^*, Mattias Magnusson^*, Sten Eirik Jacobsen, Peter ten Dijke and Stefan Karlsson^*

Departments of ^* Molecular Medicine and Stem Cell Biology, Lund University, Lund, Sweden; and Division of Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF- negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF- type II receptor (TRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF- signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TRIIDN and green fluorescence protein in the same CD34⁺CD38^-Lin^- cells were constructed. Overexpression of TRIIDN specifically disrupted TGF--mediated signaling. Autocrine TGF- signaling in CD34⁺CD38^-Lin^- cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF- signaling in CD34⁺CD38^-Lin^- cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34⁺CD38^-Lin^- cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF--negative regulatory loop of primitive hemopoietic progenitor cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemopoietic stem cells (HSCs)³ are mostly maintained in quiescence in vivo (1, 2). At any given time point, only a small fraction of HSCs are activated and proliferated to sustain the daily need of new blood cells (1). To each individual HSC, the balance between positive and negative regulatory factors determines whether a given HSC remains in quiescence or proliferates. Ex vivo disruption of quiescence in HSCs is important for expanding HSCs and for successful gene delivery into HSCs, because dividing cells are required for oncoretroviral vector integration (3) and preferred for lentiviral vector integration (4). HSCs can be induced to proliferate by providing proliferative signals either through the synergistic effect of early acting cytokines or by disrupting the negative regulatory signals. In contrast to the usage of early cytokines for expanding HSCs (5, 6, 7), the possibility of disrupting the negative regulatory factors in the HSCs has not been explored in detail. A variety of studies using antisense oligonucleotides or neutralizing Abs against TGF- ligand or TGF- type II receptor have suggested that blockage of TGF- signaling can provide proliferative advantage to primitive hemopoietic progenitor cells that are otherwise quiescent and therefore difficult to be stimulated to proliferate (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). However, it is unclear which signal is precisely blocked in hemopoietic progenitor cells or HSCs.

TGF- signals via binding to type I (TRI) and type II (TRII) serine/threonine kinase receptors. TGF- first binds to TRII, which is a constitutively active kinase. Upon binding of TGF- ligand, TRI is recruited into the receptor-ligand complex and transphosphorylated by TRII. TRI, also known as ALK-5, will then phosphorylate Smad2 and Smad3 (18). Once activated, these receptor-regulated Smad proteins will then form complexes with the common partner Smad4, translocate into the nucleus and modulate target gene expression (18, 19, 20). The expression of Smad2, Smad4, and Smad5 has been demonstrated in CD34⁺ hemopoietic progenitor cells and treatment of CD34⁺ cells with antisense oligonucleotides against Smad5 mRNA partially reversed the TGF- inhibitory effect on the colony forming potentials of CD34⁺ cells (21).

In this study, we have used adenoviral vector-mediated gene delivery for transient overexpression of a dominant negatively acting mutant of TRII (TRIIDN) in primitive hemopoietic progenitor cells. Thus, the primitive hemopoietic progenitor cells can be rendered transiently unresponsive to both autocrine and paracrine TGF- signaling. We show that transient blockage of TGF- signaling enhances the survival and proliferation of primitive hemopoietic cells with a stem cell immunophenotype.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemopoietic growth factors and Abs

Recombinant megakaryocyte growth and development factor (MGDF) and c-kit ligand (KL) were generously provided by Amgen (Thousand Oaks, CA). rFlt3 ligand (FL) was provided by Dr. S. Lyman (Immunex, Seattle, WA). rIL-3 and IL-6 were generously provided by Novartis Pharmaceuticals (East Hanover, NJ). rG-CSF was purchased from Amgen. Human TGF-1 was purchased from R&D Systems (Minneapolis, MN) and reconstituted in 4 mM of sterile HCl containing 0.1% BSA. Human TGF-3 was obtained from Dr. K. Iwata (OSI Pharmaceuticals, Uniondale, NY), bone morphogenetic protein (BMP)-6 was provided by Dr. K. Sampath (Creative Biomolecules, Hopkinton, MA), and activin-A was obtained from Dr. Y. Eto (Ajinomoto, Kawasaki, Japan). All mAbs were purchased from BD Immunocytometry Systems (Mountain View, CA).

Generation of adenoviral vectors

The three adenoviral vectors shown in Fig. 1 have been used in this study. The adenovirus 5 (Ad5)-green fluorescence protein (GFP), in which the expression of the GFP gene is under the control of the murine phosphoglycerate kinase (PGK) 1 promoter and the human -globin intervening sequence 2 (IVS2) and polyadenylation signal, has been generated and described previously (22, 23). The Ad5-TRIIDN-internal ribosomal entry site (IRES) and Ad5-TRIIDN-block were generated by using the Ad-Easy system (kindly provided by Dr. Vogelstein, Baltimore, MD; Ref. 24). All the expression cassettes were inserted in the E1 region of adenoviral vectors. The cDNA encoding TRIIDN, a murine TRII mutant with a truncation of the kinase domain sequence, was kindly provided by Dr. M.-J. Goumans (Amsterdam, The Netherlands; Ref. 25). For generating Ad5-TRIIDN-IRES vector, a bicistronic expression cassette encoding the TRIIDN and GFP genes under the control of murine PGK promoter and rabbit -globin IVS2 and polyadenylation signal was constructed in a shuttle plasmid. To generate Ad5-TRIIDN-block, expression cassettes encoding the GFP and TRIIDN genes under the control of the PGK promoter and rabbit -globin IVS2 and polyadenylation were ligated into the shuttle plasmid. The plasmids encoding the recombinant adenoviral vector genome with these expression cassettes were generated in Escherichia coli BJ5183 by homologous recombination of these shuttle plasmids with plasmid pAd-Easy1 as described by He et al. (24). For generating corresponding adenoviral vectors, these adenoviral vector genomic plasmids were digested with PacI and transfected into 293 cells by using the standard CaPO₄ precipitation method. The adenoviral vectors were expanded in 293 cells, purified, and titered according to methods described previously (22).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Generation of adenoviral vectors encoding the TRIIDN gene. In the Ad5-GFP vector, the expression of the GFP gene is under the upstream control of the murine PGK promoter and the downstream control of the human -globin IVS2 and polyadenylation signal (22 23 ). In the Ad5-TRIIDN-IRES vector, a bicistronic transcript linking the TRIIDN and GFP genes by the IRES is under the control of the murine PGK promoter and the rabbit -globin IVS2 and polyadenylation signal. In the Ad5-TRIIDN-block vector, expression cassettes for TRIIDN and for the GFP gene are controlled individually and separately under the murine PGK promoter and the rabbit -globin IVS2 and polyadenylation signal are constructed in adenoviral vectors. These adenoviral vectors were generated as described in Materials and Methods.

The K562 and HeLa cells were cultured and transduced with adenoviral vectors as previously described (23). The GFP gene expression was assessed 40 h posttransduction by flow cytometric analysis.

Signaling assays

Bovine aortic endothelial cells (BAEC) at passage 16 cultured in 10% CO₂ containing atmosphere were grown to 70% confluence in low-glucose DMEM containing 10% calf serum. Cells were transduced with adenoviral vectors at a multiplicity of infection (MOI) of 200 as described for the transduction of K562 cells. Sixteen hours posttransduction, cells were starved for 6 h and then stimulated with either TGF-3 at 2.5 ng/ml, BMP-6 at 50 ng/ml, or activin-A at 50 ng/ml for 1 h. TGF-3 is known to signal through the same TGF- receptors as TGF-1. The phosphorylation status of Smad2 and the BMP-Smads (Smad 1/5/8) in these ligand-stimulated cells was analyzed by Western blot analysis as described previously (26). Briefly, cells were rinsed with PBS and harvested in a lysis buffer containing 125 mM of NaCl, 10 mM of Tris-HCl (pH 7.5), 1 mM of EDTA, 1 mM of PMSF, 1.5% aprotinin, and 1% Triton X-100. Cell lysate was disrupted with sonication (Sonifier cell disruptor, B-30; Branson Sonic Power, Danbury, CT) and thereafter boiled for 5 min. The cellular proteins were separated on 8% SDS-PAGE gel and transferred to Hybond-C nitrocellulose membranes (Amersham, Little Chalfont, Buckinghamshire, U.K.). After blocking the nonspecific binding of proteins to the membrane, primary Abs against phosphorylated Smad2 and BMP-Smads (26) were used at a 1,000-fold dilution in TBST (10 mM of Tris (pH 7.4), 150 mM of NaCl, 0.1% Tween 20), a Smad 2-specific Ab (27) was used at a 750-fold dilution in TBST, and a mAb against actin (Chemicon International, Temecula, CA) was used at a 10,000-fold dilution. The secondary HRP-conjugated goat anti-rabbit IgG Ab (Amersham) was used at a 10,000-fold dilution in TBST. Detection was by ECL.

For analyzing the Ad5-TRIIDN-IRES-mediated blockage of TGF- signaling in CD34⁺ cells, CD34⁺ cells were cultured in serum-free medium and transduced with the adenoviral vectors as described below except with the support of KL, FL, and MGDF. CD34⁺/GFP⁺ cells were sorted. After an overnight culture in the same medium, these cells were stimulated with human TGF-1 at 10 ng/ml for 1 h and harvested in the same lysis buffer as for BAECs for examining the phosphorylation status of the Smad2 protein.

Adenoviral vector transduction of umbilical cord blood CD34⁺ cells and cell sorting conditions

Umbilical cord blood samples were obtained from normal deliveries according to institutional guidelines. CD34⁺ cells were isolated from pooled cord blood samples by using a CD34⁺ cell progenitor isolation kit as described previously (22). When not used immediately, CD34⁺ cells were frozen in 10% DMSO and 90% FCS in liquid N₂. Freshly isolated or thawed frozen CD34⁺ cells were cultured in X-VIVO 15 medium (BioWhittaker, Walkersville, MD) containing 1% BSA (StemCell Technologies, Vancouver, British Columbia, Canada), 10^-4 M of 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% L-glutamine (referred to as serum-free medium) with the support of MGDF at 100 ng/ml in a 6-well plate at a cell density of 6.6 x 10⁵ cells/well/2 ml medium. Six to 7 h later, these CD34⁺ cells were transduced with adenoviral vectors at a MOI of 500. The adenoviral vectors were preincubated with the polyamidoamine dendrimer reagent SuperFect (Qiagen, Hilden, Germany) at a concentration of 15 µg/ml as described previously (22, 23). Forty hours later, cells were harvested and stained with an allophycocyanin-conjugated anti-CD34 mAb and PE-conjugated mAb mixture consisting of Abs to CD3, CD14, CD15, CD19, CD20, CD38, CD56, and glycophorin A or the isotype-matched control Abs at saturating concentrations. The stained cells were resuspended in PBS containing 7-aminoactinomycin D at 1.0 µg/ml. The 7-aminoactinomycin D negatively stained cells were analyzed and sorted for CD34⁺CD38^-Lin^-GFP⁺ cells as shown in Fig. 2.

View larger version (46K): [in this window] [in a new window]   FIGURE 2. Adenoviral vectors mediate high levels of GFP expression in cord blood CD34+CD38-Lin- cells. Pooled cord blood CD34+ cells from multiple donors were cultured and transduced with the indicated adenoviral vectors at a MOI of 500 as described in Materials and Methods. GFP expression and cell surface marker expression were assessed at 40 h posttransduction with flow cytometry. The flow cytometric analysis was set up with the control cells cultured in serum-free medium with the support of KL, FL, and MGDF. The adenoviral vector-transduced cells were cultured in serum-free medium with the support of MGDF alone. R3 represents the cell population with the CD34+CD38+Lin+ immunophenotype and R2 represents the cell population with the CD34+CD38-Lin- immunophenotype. Percentages of GFP-expressing cells in each cell population and their mean channel intensity were shown in each histogram. Data represent three independent experiments.

Sorted CD34⁺CD38^-Lin^- cells were seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a density of 1 cell/well or lower. In most of the experiments, a control group was set up in which cells were seeded in 20 µl of serum-free medium with the support of a cytokine mixture consisting of 25 ng/ml KL, 25 ng/ml G-CSF, 10 ng/ml IL-3, 25 ng/ml FL, 25 ng/ml MGDF, and 25 ng/ml IL-6. Assuming that the vast majority of the CD34⁺CD38^-Lin^- clones can be recruited into proliferation by this cytokine combination, the data from this group represent the maximal recruitment efficiency. Cell growth was scored after 10–12 days culture with wells containing three or more cells. For analyzing the effect of overexpressing TRIIDN on the viability of CD34⁺CD38^-Lin^- cells, cells were first seeded in 10 µl of serum-free medium in the absence of cytokine. After 24–72 h of cytokine deprivation, 10 µl of serum-free medium containing the same cytokines as in the control group, but with double concentration, were added to each well to stimulate the proliferation of the survived clones. Surviving cells were scored after 10–12 days of further culture. For analyzing the effect of overexpressing of TRIIDN on the proliferation of primitive progenitor cells, sorted CD34⁺CD38^-Lin^- cells were seeded in 20 µl of serum-free medium containing MGDF (100 ng/ml) or KL, FL, and MGDF (all at 100 ng/ml). The cell proliferation was scored at days 10–12 of the culture. For the experiment shown in Fig. 6, the TGF-1 was added at 10 ng/ml and cell proliferation was scored at day 6 of the culture.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Transient overexpression of TRIIDN blocks the effect of exogenous TGF-1 on CD34+CD38-Lin- cells. Umbilical cord blood CD34+ cells were cultured with the support of MGDF and transduced with the indicated adenoviral vector as described in Materials and Methods. Transgene-expressing CD34+CD38-Lin- cells were sorted and seeded in Terasaki plates at 1.0 cell/well in 20 µl of serum-free medium with the support of KL, FL, and MGDF in the absence or presence of human TGF-1 as indicated. Data shown are the numbers of total wells containing three or more cells and of the wells containing >50 cells () at day 6 of culture. Each group was initiated with 180 wells. The p values were calculated using the 2 test. Data represent means ± SEM of two independent experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Because primitive hemopoietic progenitor cells are a heterogeneous cell population and only a small fraction of the cells are stem cells, adenoviral vector-mediated gene delivery should ideally be targeted to the cells with a primitive immunophenotype. Furthermore, adenoviral vector-mediated gene delivery into these cells needs to allow the discrimination between cells expressing the transgene and those not expressing the transgene. We wanted to design adenoviral vectors for simultaneous expression of a functional gene and a marker gene in primitive hemopoietic progenitor cells. As depicted in Fig. 1, we have generated two different adenoviral vectors encoding the TRIIDN and GFP genes. In Ad5-TRIIDN-IRES, the expression of TRIIDN and GFP is linked in the same transcript by an IRES. The expression of this transcript is under the control of the PGK promoter and the rabbit -globin IVS2 and polyadenylation signal. In Ad5-TRIIDN-block, the expression of TRIIDN and GFP is controlled separately and individually by the PGK promoter and the rabbit -globin IVS2 and polyadenylation signal. Umbilical cord blood CD34⁺ cells cultured in serum-free medium with the support of MGDF were transduced with adenoviral vectors at a MOI of 500 using SuperFect (22). Forty hours posttransduction, cells were analyzed for GFP expression and the expression of CD34, CD38, and lineage markers. The expression levels of the GFP gene were compared between cells with a primitive immunophenotype and cells with a more mature immunophenotype. As shown in Fig. 2, with all three adenoviral vectors tested, we consistently observed that higher percentages of cells with CD34⁺CD38^-Lin^- immunophenotypes were expressing GFP (up to 75%) compared with those in cells with CD34⁺CD38⁺Lin⁺ immunophenotypes (<15%). The levels of GFP expression in CD34⁺CD38^-Lin^- cells were two to four times higher compared with those in CD34⁺CD38⁺Lin⁺ cells. We were particularly interested in comparing the GFP expression in CD34⁺CD38^-Lin^- cells mediated by these vectors. The GFP expression level in CD34⁺CD38^-Lin^- cells transduced by Ad5-TRIIDN-IRES was 60% compared with that in cells transduced by the Ad5-GFP control vector. The Ad5-TRIIDN-block mediated slightly higher levels of GFP expression in the CD34⁺CD38^-Lin^- cells compared with those in the Ad5-GFP control vector-transduced CD34⁺CD38^-Lin^- cells. However, the Ad5-TRIIDN-block-mediated GFP expression levels in the CD34⁺CD38^-Lin^- cells were 2-fold higher compared with those in CD34⁺CD38^-Lin^- cells transduced by Ad5-TRIIDN-IRES. We further addressed whether the different levels of GFP expression in CD34⁺CD38^-Lin^- cells are cell type-dependent or vector construct-dependent. HeLa and K562 cells were transduced with these three adenoviral vectors. As shown in Fig. 3, the same pattern in the relative levels of GFP expression between these vectors was observed both in HeLa and K562 cells. These data show that the difference in design of the adenoviral vector construct determines the relative levels of GFP expression.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Adenoviral vector-mediated GFP expression in K562 and HeLa cells. The K562 and HeLa cells were cultured and transduced with the indicated adenoviral vectors as described in Materials and Methods. The K562 cells were transduced at a MOI of 200 and GFP expression was assessed 40 h posttransduction. The HeLa cells were transduced at a MOI of 100 and GFP expression was assessed 24 h posttransduction. Percentages of GFP-expressing cells and their mean channel intensity were shown in each histogram. The data represent three independent experiments.

Inhibition of TGF- signaling by adenoviral vector-mediated expression of TRIIDN

To assess whether the adenoviral vector-mediated overexpression of TRIIDN is capable of blocking TGF- signaling, signaling studies were first performed in BAECs. BAECs were chosen as these cells can be transduced with high efficiency with adenoviruses and these cells respond to TGF-, BMP, and activin (M.-J. Goumans, unpublished results). As shown in Fig. 4, the phosphorylation of Smad2 can be observed in the Ad5-GFP-transduced BAECs upon TGF- stimulation. In the BAECs transduced by the Ad5-TRIIDN-IRES, the TGF--mediated Smad2 phosphorylation was strongly inhibited. However, in the BAECs transduced by the Ad5-TRIIDN-block, the TGF--mediated phosphorylation of Smad2 was only slightly diminished compared with that in the cells transduced by the control Ad5-GFP vector. We also addressed whether adenoviral vector-mediated overexpression of TRIIDN could affect the signaling pathways of the other TGF- superfamily members. As also shown in Fig. 4A, activin-A-mediated Smad2 phosphorylation was only slightly reduced by TRIIDN and BMP-6-induced BMP-Smad phosphorylation was not affected by the overexpression of the TRIIDN (Fig. 4B). These results are consistent with previous reports in which TRIIDN constructs were found to inactivate TGF- signaling, but not signaling by other members of the TGF- superfamily (28, 29). Our data show that the Ad5-TRIIDN-IRES-mediated overexpression of TRIIDN is sufficient for disrupting TGF- signaling in a specific manner, whereas the Ad5-TRIIDN-block-mediated overexpression of TRIIDN is not. Therefore, all the subsequent experiments were performed with the Ad5-TRIIDN-IRES.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Ad5-TRIIDN-IRES-mediated overexpression of TRIIDN specifically blocks TGF- signaling in primary endothelial cells. BAECs were transduced with the indicated adenoviral vectors and stimulated with TGF-3, activin-A (A), or BMP-6 (B) as described in Materials and Methods. Cell lysates were separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane. The phosphorylation status of Smad2 and BMP Smads were analyzed as described in Materials and Methods. The detection of Smad2 and actin proteins demonstrates the equal loading of proteins between each sample. Lysates from COS-7 cells transfected with pcDNA3 plasmids encoding TRI and Smad2 or ALK-1 and Smad1 (44 ) were used as positive controls for phosphorylated Smad2 and phosphorylated BMP-Smads, respectively. Data shown represent two independent experiments with similar results.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Ad5-TRIIDN-IRES-mediated overexpression of TRIIDN specifically blocks Smad2-mediated TGF- signaling in CD34+ cells. A, The phosphorylation status of Smad2 was examined in the nontransduced unsorted parallel CD34+ cultures with or without TGF-1 stimulation as described for the GFP-expressing CD34+ cells from B. Lysate from COS-7 cells transfected with plasmids encoding constitutively active TRI and Smad2 was used as a positive control for Smad2 phosphorylation. B, Umbilical cord blood CD34+ cells were cultured in the serum-free medium with the support of KL, FL, and MGDF and transduced with the indicated adenoviral vectors as described in Materials and Methods. CD34+/GFP+ cells were sorted and further cultured under the same conditions for 16 h and stimulated with TGF-1 at 10 ng/ml for 1 h. The phosphorylation status of Smad2 in these cells was analyzed as described in Fig. 4. Equal loading of Smad2 protein and total protein levels from each sample was verified by Abs against Smad2 or actin. Data shown represent two independent experiments with similar results.

Overexpression of TRIIDN transiently blocks the inhibitory effects of exogenous TGF- in CD34⁺CD38^-Lin^- cells

TGF- potently inhibits the growth and survival of primitive hemopoietic progenitor cells (7, 9). First, we studied whether overexpression of TRIIDN can reduce or abolish the effects of exogenously added TGF- on CD34⁺CD38^-Lin^- cells. Sorted transgene-expressing CD34⁺CD38^-Lin^- cells were plated in Terasaki plates at 1 cell/well in serum-free medium with a cytokine combination consisting of KL, FL, and MGDF in the presence or absence of TGF-. Cell proliferation was evaluated at day 6 of culture. As shown in Fig. 6, the combination of early acting cytokines KL, FL, and MGDF supported the growth of about two-thirds of CD34⁺CD38^-Lin^- cells and their growth was markedly inhibited by the exogenously added TGF-. Overexpression of TRIIDN significantly rescued more CD34⁺CD38^-Lin^- clones from the inhibitory effect of exogeneous TGF-. However, the proliferation potential of the rescued clones was compromised, because no large clones were found from the TRIIDN-expressing CD34⁺CD38^-Lin^- cells in the presence of TGF-. Furthermore, when the same plates were evaluated at days 10–12 of culture, most of the cells were of small size with nontransparent cytoplasm (data not shown), possibly due to the proapoptotic effect of the exogenously added TGF-. These data show that adenoviral vector-mediated overexpression of TRIIDN can transiently block the signaling effects of exogenously added TGF-.

Overexpression of TRIIDN enhances the survival of CD34⁺CD38^-Lin^- cells in serum-free conditions

Primitive hemopoietic progenitor cells have been demonstrated to produce TGF- (10, 30, 31) and TGF- may act in an autocrine manner in CD34⁺CD38^-Lin^- cells. Therefore, we studied the effect of overexpressing TRIIDN on the survival of the CD34⁺CD38^-Lin^- cells in the absence of exogenously added TGF-. Transgene-expressing CD34⁺CD38^-Lin^- cells were plated in Terasaki plates at a cell density of 1 cell/well in serum-free medium in the absence of cytokines. After 24–72 h of cytokine deprivation, a cytokine mixture consisting of KL, G-CSF, IL-3, FL, MGDF, and IL-6 was added to rescue the surviving clones. As shown in Fig. 7, 60% of the CD34⁺CD38^-Lin^- cells survived 24 h of cytokine deprivation and 20–30% of the CD34⁺CD38^-Lin^- cells survived 48 h of cytokine deprivation. Overexpression of TRIIDN significantly enhanced the survival of CD34⁺CD38^-Lin^- cells to 90% at 24 h and 40% at 48 h of cytokine deprivation. This result directly demonstrates that at the clonal level, the autocrine TGF- signaling acts as a negative regulator for ex vivo survival of CD34⁺CD38^-Lin^- cells and blockage of this autocrine TGF- signal can enhance the survival of primitive progenitor cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Transient overexpression of TRIIDN enhances the survival of CD34+CD38-Lin- cells in the absence of cytokines. Umbilical cord blood CD34+ cells were cultured with the support of MGDF and transduced with the indicated adenoviral vector as described in Materials and Methods. Transgene expressing CD34+/CD38-/Lin- cells were sorted and seeded in a Terasaki plate at 1.0 cell/well in 10 µl of serum-free medium in the absence of cytokines. Serum-free medium (10 µl) containing KL, G-CSF, IL-3, FL, MGDF, and IL-6 at concentrations described in Materials and Methods was added at the indicated times of cytokine deprivation to stimulate the proliferation of the surviving clones. The growth of these clones was evaluated after 10–12 days of further culture as described in Fig. 6. The numbers of the total clones and the clones with >50 cells () are shown. Each group was initiated with 180 wells. Values of p were calculated by comparing the percentages of survived clones at each cytokine deprivation time point between Ad-GFP- and Ad-TRIIDN-transduced CD34+CD38-Lin- cells using the 2 test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data represent means ± SEM of two independent experiments.

Overexpression of TRIIDN enhances the proliferation of CD34⁺CD38^-Lin^- cells

We further investigated whether transient overexpression of TRIIDN affects the proliferation of CD34⁺CD38^-Lin^- cells. Transgene-expressing CD34⁺CD38^-Lin^- cells were plated in Terasaki plates at 1 cell/well in serum-free medium in the presence of MGDF alone or a cytokine combination of KL, FL, and MGDF. Cell proliferation was evaluated between 10 and 12 days of culture. The maximal-responding clones were assessed by plating the cells with the support of a combination of cytokines consisting of KL, G-CSF, IL-3, FL, MGDF, and IL-6. As shown in Fig. 8, in the presence of MGDF alone, between 55 and 70% of the control CD34⁺CD38^-Lin^- clones were stimulated to proliferate. The overexpression of TRIIDN significantly increased the recruitment of proliferating CD34⁺CD38^-Lin^- cells to 81–100%. In contrast, KL, FL, and MGDF recruited >80% of the control CD34⁺CD38^-Lin^- clones into proliferation; the significantly increased recruitment of proliferation of CD34⁺CD38^-Lin^- cells by overexpression of TRIIDN was evident only in one of three experiments performed. It has been reported that MGDF alone is capable of promoting the survival of primitive progenitor cells (32), whereas the synergistic effects of KL, FL, and MGDF can induce the proliferation of these cells (7). Our data show that autocrine TGF- inhibits the survival and proliferation of CD34⁺CD38^-Lin^- cells. However, these effects are most likely to become evident when the exogenous proliferating signals are maintained at lower levels. When CD34⁺CD38^-Lin^- cells are exposed to strong proliferation signals, the autocrine TGF- plays a marginal effect on the proliferation of CD34⁺CD38^-Lin^- cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Transient overexpression of TRIIDN significantly enhanced the recruitment of CD34+CD38-Lin- cells into proliferation in the presence of MGDF alone. Umbilical cord blood CD34+ cells were cultured in serum-free medium with the support of MGDF and transduced with the indicated adenoviral vectors as described in Material and Methods. Transgene-expressing CD34+/CD38-/Lin- cells were sorted and seeded in Terasaki plates at 1.0 cell/well in 20 µl of serum-free medium. Cells were either supported by a cytokine mixture consisting of KL, G-CSF, IL-3, FL, MGDF, and IL-6 for assessing the maximal responding clones, by a combination consisting of KL (K), FL (F), and MDGF (M), or by MGDF alone for assessing the recruitment under proliferation conditions or under survival conditions. Cell proliferation was scored at days 10–12 of culture for wells containing three or more cells. Data shown are means ± SEM of the percentages of recruitment into proliferation in the presence of KL, FL, and MDGF or MGDF alone compared with the maximal recruitment. Values of p were calculated using the 2 test. Data represent two experiments initiated with 180 wells and one experiment initiated with 120 wells.

Overexpression of TRIIDN accelerates the growth kinetics of CD34⁺CD38^-Lin^- cells

Because our data show that transient blockage of autocrine TGF- provides a proliferative advantage to CD34⁺CD38^-Lin^- cells when cells are supported by MGDF, we further investigated whether the cell proliferation kinetics were affected. Transgene-expressing CD34⁺CD38^-Lin^- cells were plated in Terasaki plates at a statistical cell density of 0.5–0.8 cell/well in serum-free medium with the support of MGDF. Each well was monitored for initial cell division(s) 24 h postplating. Two or more cells per well were considered as cell proliferation. As shown in Fig. 9, the CD34⁺CD38^-Lin^- cells transduced with Ad5-TRIIDN-IRES proliferated significantly faster than the CD34⁺CD38^-Lin^- cells transduced with the Ad5-GFP control vector, as this group contained significantly more wells with two and more cells and fewer wells with only one cell. These data suggest that blockage of autocrine TGF- signaling exerted a pronounced proliferative effect on CD34⁺CD38^-Lin^- cells at the early stage of the culture.

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Transient overexpression of TRIIDN accelerated the kinetics of CD34+CD38-Lin- cell proliferation. Umbilical cord blood CD34+ cells were cultured in serum-free medium with the support of MGDF and transduced with the indicated adenoviral vectors as described in Material and Methods. Transgene-expressing CD34+/CD38-/Lin- cells were sorted and seeded in Terasaki plates at 0.5–0.8 cell/well in serum-free medium with the support of MGDF. Cell numbers per well were counted at 24 h of the culture. Data shown are the percentages of the wells with one cell () and wells with two and more cells (). Data are means ± SEM derived from four experiments. The p value was calculated using the 2 test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Adenoviral vector-mediated gene expression in primitive hemopoietic progenitor cells is not well established and the current adenoviral vector transduction protocols do not transduce 100% of the primitive progenitor cells; therefore, it is essential to design adenoviral vectors capable of expressing the functional gene and a marker gene in the same cell. Our previous studies using the GFP gene have demonstrated that the GFP transgene under the control of the PGK promoter and the -globin gene IVS2 and polyadenylation signal can be expressed at high levels in the putative stem cells capable of repopulating nonobese diabetic SCID mice (22, 23). By using these regulatory elements, we have generated Ad5-TRIIDN-IRES and Ad5-TRIIDN-block vectors for expressing TRIIDN and GFP in the same target cells. Consistent with our previous findings, we found that significantly higher percentages of the cells with the CD34⁺CD38^-Lin^- immunophenotype were expressing the GFP gene after adenoviral vector transduction compared with those in the more mature CD34⁺CD38⁺Lin⁺ cells. The Ad5-TRIIDN-block vector allowed higher levels of GFP gene expression than that of the Ad5-TRIIDN-IRES vector, however, the TRIIDN expression driven by the internal PGK promoter in the Ad5-TRIIDN-block is not sufficient to block the TGF- signaling. The difference in the levels of GFP gene expression and TRIIDN gene expression is dependent on the viral vector constructs as demonstrated in CD34⁺CD38^-Lin^-, K562, and HeLa cells. The expression of two genes controlled by the two identical promoters such as in Ad5-TRIIDN-block is usually 3- to 5-fold lower for both genes compared with a single cassette vector (24). However, the PGK promoter contains an orientation and position-independent enhancer (37), making it likely that in the Ad5-TRIIDN-block, the second PGK promoter enhances the first PGK promoter, such that the GFP gene expression is markedly higher compared with the Ad-GFP control vector, whereas the expression of the TRIIDN gene is not sufficient to block TGF- signaling.

The role of TGF- signaling in the hemopoietic system is mainly based on the studies where TGF- ligand, neutralizing Abs, or antisense oligonucleotides against the production of TGF- have been added to the proliferating progenitor cells in vitro. Exogenous TGF-1 inhibits the proliferation of the primitive hemopoietic progenitor cells (9, 38) and may also induce apoptosis in primitive hemopoietic progenitor cells (12, 39). Blocking the TGF- synthesized in the culture system by TGF-1-specific antisense oligonucleotides or neutralizing Abs recruited a primitive cell population that usually remains quiescent into proliferation and multilineage colonies were formed (14, 15). Most of these studies were performed in serum containing medium in the presence of multiple cytokines. Because TGF- can be produced by primitive hemopoietic progenitor cells, adding exogenous TGF- may hyperactivate the TGF- signaling pathway. In the studies using antisense oligonucleotides or neutralizing Abs, it has been unclear to which extent TGF- signaling is blocked. In our studies, the disruption of TGF- signaling in the primitive progenitor cells is achieved by overexpression of TRIIDN in these cells, and we show that the Smad2-mediated signaling is blocked. Furthermore, overexpression of TRIIDN on CD34⁺CD38^-Lin^- cells reverted the negative effects on the survival of the exogenous TGF- in these cells. To directly demonstrate the effects of autocrine TGF- signaling on primitive hemopoietic progenitor cells, the CD34⁺CD38^-Lin^- cells were plated in single cell cultures in serum-free medium. Autocrine TGF- signaling has a negative effect on the survival of CD34⁺CD38^-Lin^- cells. Inhibition of autocrine TGF- signaling did provide survival advantages to CD34⁺CD38^-Lin^- cells, possibly due to up-regulation of antiapoptotic genes (31). In contrast to studies using neutralizing Abs or antisense oligonucleotides (10, 14, 15, 16, 17), our data show that when primitive hemopoietic progenitor cells are exposed to the synergistic proliferation signals of early acting cytokines, inhibiting the autocrine TGF- signaling does not provide significant proliferating advantages. This discrepancy may be due to the fact that different culture conditions induce the proliferation of divergent cell populations. It is also possible that the primitive hemopoietic progenitor cells from different sources may behave differently in response to TGF- signaling and signaling of the other cytokines (40). The third possibility is that overexpression of TRIIDN blocks the signaling of all TGF- isoforms, whereas the Ab or antisense oligonucleotide based approach may only block the signaling of one TGF- isoform.

Our findings have several implications. TGF- signaling is tightly controlled. Both the primitive hemopoietic progenitor cells and the stromal cells in the bone marrow are demonstrated to produce TGF-, such that the TGF- signaling in the primitive hemopoietic progenitor cells can be triggered by both autocrine and paracrine TGF- (10, 11, 30, 31). Persistent loss of TGF- signaling in the HSCs and the primitive hemopoietic progenitor cells is associated with transplantable autoimmune manifestations and may possibly be involved in the development of leukemias (41, 42, 43). Transient disruption of TGF- signaling is needed to bypass these complications. Our results show that adenoviral vector-mediated overexpression of TRIIDN can transiently render hemopoietic progenitor cells with a HSC immunophenotype unresponsive to TGF- signaling in a cell autonomous manner. We believe that this model is highly useful to investigate the role of TGF- in HSCs. Furthermore, this study provides a background for transient gene expression based manipulation of putative HSCs. Transient modulation of signaling pathways can be used to manipulate the self-renewal and differentiation of HSCs.

	Acknowledgments

 
We gratefully acknowledge Dr. Saemundur Gudmundsson and the staff of the Department of Obstetrics and Gynecology, Malmö, for collecting umbilical cord blood samples. We are grateful to Drs. Ken Iwata, Kuber Sampath, and Yuzuru Eto for recombinant ligands. We thank Anna Fossum and Zhi Ma for excellent cell sorting. We are grateful to M.-J. Goumans for insightful discussions. We thank Niels-Bjarne Woods for proofreading of the manuscript. We are particularly indebted to A. Haimovitz-Friedman for BAECs.

	Footnotes

 
¹ These studies were generously supported by grants from Lund University medical faculty, the Swedish Cancer Society, the Swedish Medical Research Council, and the Swedish Gene Therapy Program (to S.K.); Kungliga Fysiografiska Society, Crafoordska Foundation, Georg Danielsson Foundation, and funds of Lunds Sjukvårdsdistrict (to X.F.); and Netherlands Heart Foundation (99-046) and Netherlands Organization for Scientific Research (MW902-16-295) (to P.t.D).

² Address correspondence and reprint requests to Dr. Stefan Karlsson, Department of Molecular Medicine and Gene Therapy, Lund University, BMC A12, 221 84 Lund, Sweden. E-mail address: Stefan.Karlsson{at}molmed.lu.se

³ Abbreviations used in this paper: HSC, hemopoietic stem cell; TRI, TGF- receptor type I; TRII, TGF- receptor type II; TRIIDN, a dominant negatively acting mutant of TRII; MGDF, megakaryocyte growth and development factor; KL, c-kit ligand; FL, Flt3 ligand; BMP, bone morphogenetic protein; Ad5, adenovirus 5; GFP, green fluorescence protein; IVS2, intervening sequence 2; PGK, phosphoglycerate kinase; IRES, internal ribosomal entry site; BAEC, bovine aortic endothelial cell; MOI, multiplicity of infection.

Received for publication June 22, 2001. Accepted for publication November 12, 2001.

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