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HEMATOPOIESIS
From the Department of Medicine, Division of
Hematology, University of Washington, Seattle.
To determine whether cytokine-induced signals generate unique
responses in multipotential hemopoietic progenitor cells, the signaling
domains of 3 different growth factor receptors (Mpl, granulocyte-colony-stimulating factor [G-CSF] receptor, and Flt-3) were inserted into mouse primary bone marrow cells. To circumvent the
activation of endogenous receptors, each signaling domain was
incorporated into an FK506 binding protein (FKBP) fusion to allow for
its specific activation using synthetic FKBP ligands. Each signaling
domain supported the growth of Ba/F3 cells; however, only Mpl supported
the sustained growth of transduced marrow cells, with a dramatic
expansion of multipotential progenitors and megakaryocytes. These
findings demonstrate that the self-renewal and differentiation of
multipotential progenitor cells can be influenced through distinct, receptor-initiated signaling pathways.
(Blood. 2001;98:328-334) Hemopoietic stem cells (HSCs) are defined by their
ability to self-renew and to contribute to all lineages of mature blood cells.1 Additionally, HSCs appear capable of contributing
to nonhemopoietic tissues including skeletal muscle,2
liver,3 and brain.4,5 Multipotential
hemopoietic progenitor cells (MHPCs), positioned hierarchically
downstream of HSCs, have a more limited capacity for self-renewal and a
more restricted potential for differentiation. Among the earliest
identifiable MHPCs are the common lymphoid6 and
myeloid7 progenitors. HSCs and MHPCs stand poised (or
primed) either to self-renew or to commit themselves to a pathway of
differentiation.8 Multiplex reverse transcription-polymerase chain reaction (RT-PCR) analysis of single isolated CD34+, lineage Capitalizing on the considerable therapeutic potential of HSCs and
MHPCs requires the ability to manipulate their self-renewal and
differentiation. Transcription factors stand at the center of these
processes.10-12 For example, myb-ets-transformed
multipotent chicken progenitor cells can be induced to differentiate
into either megakaryocytes or eosinophils by modulating GATA-1
concentrations,13 whereas differentiation to myeloid cells
can be induced by overexpressing PU.1.14 Self-renewal can
also be controlled, as evidenced by the ability of GATA-2 to inhibit
the self-renewal of multipotent FDCP-mix cells and primary mouse bone
marrow clonogenic progenitor cells.15,16
In contrast to the well-characterized ability of transforming growth
factor- That HSC-MHPC self-renewal and differentiation may be subject to
external modulation is suggested by the expression of receptors for a
wide range of growth factors, including interleukin-3 (IL-3), IL-1 We have previously shown that signaling by the thrombopoietin receptor
Mpl can support the expansion of retrovirally transduced marrow cells
for longer than a month of culture, generating megakaryocytes and cells with the ability to form colonies in semisolid
media.35 Contrasting simple receptor overexpression
experiments, which fail to preclude ligand-activated signaling by
endogenous receptors,36,37 we incorporated the Mpl
signaling domain into an FKBP fusion protein, allowing for its specific
activation using FK1012, a chemical inducer of dimerization
(CID).38,39 Here we test whether Mpl specifically induces
these responses or whether other receptors known to be expressed in
HSCs induce similar responses. Like Mpl, the G-CSF receptor and Flt-3
are both reported to be expressed in HSCs.22-24 Here we
show that Mpl signaling can induce MHPC self-renewal and that this
ability is not provided for by signals emanating from the G-CSF
receptor or Flt-3. Furthermore, we show that though Mpl signaling
allows granulocytes to be generated from committed myeloid progenitors,
signals other than those provided by Mpl are required for granulocytes
to develop from MHPCs.
Retroviral constructs
Retroviral producer lines
Cell proliferation assays Ba/F3 cells were washed twice with phosphate-buffered saline and cultured overnight in IL-3-deficient medium. Then 1 × 104 cells per well were plated in 96-well plates and medium containing either IL-3 or CID to a final volume of 100 µL. Plates were incubated at 37°C in 5% CO2 for 40 hours, and 25 µL of a 5 mg/mL solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added. Cells were incubated for an additional 4 hours at 37°C, and 100 µL lysis buffer (20% sodium dodecyl sulfate-40% dimethylformamide-2% glacial acetic acid, pH 4.7) was added. Plates were incubated at 37°C for 2 hours before assay. The OD570-630 value was determined using an enzyme-linked immunosorbent assay plate reader.Retroviral transduction of primary murine bone marrow cells Female B6D2F1 mice were injected intraperitoneally with 150 mg/kg 5-fluorouracil. Forty-eight hours later, bone marrow cells were harvested and cultured for 48 hours in Dulbecco minimum essential medium containing 16% fetal calf serum (FCS) 5% IL-3-conditioned medium, 100 ng/mL recombinant human IL-6, and 50 ng/mL recombinant murine SCF in a 37°C, 5% CO2 incubator. After 48 hours of prestimulation, cells were transferred onto irradiated (1500 cGy) producer cells and cocultivated using identical growth conditions except for the addition of polybrene (8 µg/mL). Marrow cells were harvested after 48 hours of cocultivation. Transduction efficiency was assessed using G418 at an active concentration determined to be sufficient to kill all nontransduced cells (800 µg/mL) for colonies containing the neo- vector or GFP expression for colonies containing the GFP vector. Cells were plated at a concentration of 1 × 104 cells/mL in cultures containing 30% FCS, 1% bovine serum albumin, 5 × 10 4 M 2-mercaptoethanol,
10% WEHI-3 cell-conditioned medium, and 1% methylcellulose. Cultures
were plated in triplicate and incubated in a highly
humidified 37°C, 5% CO2 incubator. Colonies were scored on day 8.
Semisolid progenitor assay Transduced cells were plated at a concentration of 3 × 104 cells/mL in cultures containing 30% FCS, 1% bovine serum albumin, 5 × 104 M 2-mercaptoethanol, and
1% methylcellulose with or without 100 nM AP20187. Cultures were
plated in duplicate and incubated in a highly humidified 37°C, 5%
CO2 incubator. Colonies were scored on day 8 using an
inverted microscope and standard morphologic criteria.
Suspension cultures After retroviral transduction, marrow cells were cultured in Iscoves modified Dulbecco medium containing 10% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin either in the presence or the absence of FK1012 (100 nM) or AP20187 (100 nM) in a 37°C 5% CO2 incubator. Cells were counted on the days indicated. Cell morphology was analyzed by light microscopy after Wright-Giemsa staining.Clonal assays Marrow cells transduced using the F36Vmpl vector were plated into 96-well plates at densities of 10, 25, 50, and 100 cells per
wellin Iscoves modified Dulbecco medium containing 10% FCS, 50 U/mL
penicillin, and 50 µg/mL streptomycin either in the presence or the
absence of AP20187 (100 nM) in a 37°C, 5% CO2
incubator. GFP-expressing clones were identified using an
inverted fluorescence microscope. A fraction of wells demonstrating
clonal growth was humanely killed for Turks staining, and cell
morphology was evaluated using a 40× inverted light microscope.
Differentiation assays Cells in suspension cultures were washed with phosphate-buffered saline 3 times and cultured in Iscoves modified Dulbecco medium containing 10% FCS, 50 U/mL penicillin, 50 µg/mL streptomycin, and 50 ng/mL SCF plus 3 U/mL erythropoietin for erythroid differentiation or IL-3 (0.1% conditioned media) plus 100 ng/mL GM-CSF for myeloid differentiation. Cells were analyzed by fluorescence-activated cell sorter for expression of the monocytic marker CD11b, the granulocytic marker Gr-1, and the erythroid marker Ter119.Megakaryocyte colony-forming unit assay Megakaryocyte colony-forming unit (CFU-MK) assays were performed using a plasma clot assay. CFU-MK colonies were identified by staining cells with a biotin-labeled anti-CD41 antibody and then by streptavidin-labeled alkaline phosphatase.
Mpl, Flt-3, and G-CSF receptor signaling domains stimulate growth in a factor-dependent cell line Signaling domains from Mpl, Flt-3, and the G-CSF receptor were incorporated into fusion proteins containing CID-binding sites (Figure 1). CID-binding sites consisted of FKBP12, which binds FK1012,38 or of a FKBP12 derivative harboring a single amino acid substitution (F36V) that accommodates binding to a new class of CIDs, including AP20187.42 Membrane targeting was achieved using a 14-amino acid myristylation peptide from c-Src.43 Each construct was incorporated into an MSCV-based retroviral vector and tested for the ability to induce CID-dependent growth in the IL-3-dependent cell line Ba/F3.44 Cell proliferation assays demonstrated that each construct was capable of inducing CID-dependent growth (Figure 2). The reduced cell growth observed at higher CID concentrations is consistent with excessive occupancy of the CID-binding sites, thereby preventing dimerization. CID concentrations that were optimal for growth of Ba/F3 cells were carried forward for studies using transduced primary murine bone marrow cells.
Only Mpl stimulates growth in primary murine bone marrow cells Primary murine bone marrow cells were transduced using the retroviral vectors that had been demonstrated to be functional in Ba/F3 cells. After transduction, marrow cells were cultured in suspension, in the absence of added cytokines, and in the presence or absence of the appropriate CID (Figure 3). Cell growth failed to occur in the absence of CID, whereas cell numbers were consistently higher in the presence of CID. G-CSF receptor signaling through the addition of AP20187 resulted in a delay in cell loss over time, likely reflecting some effect on cell survival. Signaling through Flt-3 produced slight increases in cell numbers over time, indicating a modest ability of this receptor to induce cell growth. In contrast, Mpl consistently induced a sustained growth of transduced marrow cells. Differences between Mpl and the other receptors were not related to the relative efficiency of gene transfer into progenitors (Figure 3, legend). The Mpl effect was highly consistent, allowing for the sustained growth of transduced marrow cells for periods ranging from 98 days to nearly 1 year (Table 1). Cell growth consistently remained strictly CID-dependent (data not shown). These findings indicate that Mpl is able to induce expansion in a subset of primary marrow cells that have an extensive proliferative capacity, whereas the G-CSF receptor and Flt-3 lack this ability.
Receptor-specific differences in cell morphology The types of cells emerging in response to CID varied in accordance with receptor type and time in culture (Figure 4). In all experiments, granulocytes were abundant at early time points but fell to less than 1% by 21 days of culture. For Flt-3 and the G-CSF receptor, the decline in granulocytes was mirrored by a time-dependent rise in the percentage of macrophages. In contrast, macrophages increased only transiently in response to Mpl signaling. Megakaryocytes, present at low levels immediately after transduction, remained at low levels in response to Flt-3 and G-CSF receptor signaling, whereas megakaryocytes rose in response to signaling by Mpl. In particular, Mpl signaling resulted in a dramatic rise in megakaryocytes as assessed not only morphologically but also by staining using an antibody directed against the megakaryocyte-specific antigen, CD41, which was consistently positive in more than 30% of cells (data not shown). These findings demonstrate that lineage output can be modulated through distinct, receptor-initiated signaling pathways.
Mpl induces MHPC self-renewal Marrow cells transduced with the F1mpl or the F36Vmpl vectors were maintained in continuous culture from 3 to 11 months, with cell doubling times ranging from 36 to 72 hours (Table 1). In contrast to the temporal variation in cell types generated during the first 3 weeks, the phenotypes of cells generally stabilized beyond 3 weeks of culture. CD41 were consistently expressed in a significant fraction of cells (30%-80%). Markers of primitive cells (CD34, Sca-1), erythroid cells (Ter119), and myeloid-monocytic cells (Gr-1 and CD11b) were consistently expressed at low levels (less than 5%), whereas c-Kit expression was variable among different experiments (expressed in 5%-90% of cells).In 11 of 13 experiments, pools of transduced marrow cells expanded in
response to Mpl signaling retained the potential for multilineage
differentiation (Table 1 and data not shown). Multipotency was retained
in subclones derived by single-cell plating from pools of
Mpl-transduced marrow cells. Erythroid differentiation was observed in
response to SCF plus erythropoietin, and myeloid differentiation
occurred in response to IL-3 plus GM-CSF (Figure 5). Mpl-expanded cells also retained the
capacity to generate colonies in the spleens of irradiated mice (day 12 CFU-S) (Table 1 and data not shown). These functional attributes
persisted even after cells had been expanded after more than 100 days
of culture and by a factor of more than 1019-fold. In
contrast, Mpl-expanded cells were incapable of long-term repopulation
in lethally irradiated mice and showed no potential for B- or
T-lymphoid differentiation (data not shown). These findings are
consistent with an ability of Mpl to induce the self-renewal of MHPC,
possibly the functional equivalents of the recently described c-kit+ FcRlo CD34+
Sca1
Mpl signaling is permissive for differentiation of committed progenitors To investigate the basis for the temporal variation in cell types emerging in response to Mpl signaling, we performed limiting-dilution assays. Marrow cells transduced using the F36Vmpl vector were plated into 96-well plates at densities of 10, 25, 50, and 100 cells per well, without added cytokines and in the presence or absence of AP20187. To facilitate the detection of clones, the F36Vmpl vector incorporated a GFP reporter that allowed viable cells to be readily identified using an inverted fluorescence microscope. After 7 days of culture, clonogenic cells were detected at frequencies from 1 in 59 to 1 in 89 cells plated. Each clone contained between 8 and 1500 cells, with most clones containing between 50 and 500 cells. Approximately 75% of these clones contained either granulocytes alone or a combination of granulocytes and macrophages (Table 2). The remaining clones contained a mixture of granulocytes and megakaryocytes or were unidentified. The unidentified group might have included nonhemoglobinized erythroid cells that were difficult to classify definitively based on morphologic criteria alone. As seen in our bulk cultures, cell growth failed to occur in limiting-dilution cultures performed in the absence of CID (data not shown).
To further demonstrate the ability of Mpl to support the
differentiation of committed progenitors, marrow cells transduced using
the F36Vmpl vector were cultured in semisolid media containing 100 nM
AP20187 in the absence of added cytokines. Colonies obtained after 8 days included not only colony-forming unit-granulocyte macrophage
(CFU-GM) but also erythroid burst-forming unit (BFU-E) and CFU-MK
(Table 3). These findings confirm that
Mpl can support the terminal differentiation of committed myeloid,
erythroid, and megakaryocytic progenitor cells.
Mpl supports the sustained proliferation of committed megakaryocyte progenitor cells In the limiting-dilution assays, fewer than 1 in 6000 cells were capable of sustaining growth beyond 3 weeks of culture (Table 2). These rare clones contained both morphologically nondescript mononuclear cells and megakaryocytes, whereas granulocytes were consistently absent. These cells were capable of forming megakaryocytic colonies in semisolid media in the presence of thrombopoietin but were incapable of generating myeloid cells in response to IL-3 and GM-CSF or erythroid cells in response to SCF and erythropoietin. They failed to generate colonies when cultured in semisolid media in the presence of IL-3 and were incapable of forming spleen colonies when injected into lethally irradiated mice. The longest lived among these clones was sustained in culture for slightly longer than 100 days. We conclude that these clones represented committed megakaryocyte progenitor cells.
Recent reports demonstrate that the stem cells of normal adults retain enormous plasticity. Hemopoietic stem cells can differentiate into muscle,2 liver,3 and brain,4,5 neural stem cells can differentiate into blood,45 gut, liver, lung, or muscle,46 and muscle stem cells can differentiate into blood.2,47 Furthermore human embryonic stem (ES) cells, capable of contributing to all human tissues, have recently been generated.48 Having firmly established the vast differentiation potential of stem cells, focus now shifts to determining whether, among the many available options, desired fates can be specifically elicited. In practice, this restates the question of whether stem cell fate is intrinsically determined or whether it is subject to extrinsic influence. Our results using chemically activated growth factor receptors suggest that MHPCs can be instructed to adopt specific fates. Mpl supports MHPC self-renewal, yet Flt-3 and the G-CSF receptor both failed to support MHPC self-renewal. The ability of Mpl to support MHPC self-renewal is consistent with its established role in maintaining normal numbers of progenitors and stem cells in vivo.49,50 Our inability to generate MHPCs in clonal assays contrasted with our relatively consistent ability to generate MHPCs from pools of Mpl-transduced marrow cells. In the clonal assays, 50 000 transduced cells were analyzed, whereas pooled cultures routinely allowed between 2 and 5 million transduced cells to be evaluated. We conclude that MHPCs capable of self-renewal in response to Mpl signaling are rare (fewer than 1 in 50 000), thus providing the likely basis for the discrepancy between our results using clones and pools of transduced cells. Mpl preferentially enabled MHPCs to differentiate toward the megakaryocytic lineage. Mpl, the receptor for thrombopoietin, is a primary regulator of platelet production.51 The preferential production of megakaryocytes might have resulted from megakaryocytic progenitors being relatively more responsive to signaling by Mpl than were progenitors committed to other cell lineages. This interpretation is supported by the Mpl-supported sustained growth of megakaryocytic progenitors in our limiting-dilution assays (Table 1). Alternatively, signaling by Mpl might alter the probability with which MHPCs commit to the megakaryocytic pathway. Mpl's specificity in the present study stands in apparent contrast to that in a previous report using knock-in mice. Substitution of the Mpl signaling domain with the corresponding portion of the G-CSF receptor reduced megakaryocyte progenitors by only 30%.32 Although these findings were interpreted as arguing against an instructive role of Mpl in hematopoietic cell fate decisions, we suggest that the absence of an Mpl signal might have been compensated for by alternative signaling pathways that remained available in vivo, such as those activated by IL-6 or IL-11. Alternatively it is possible in our own studies that even though the G-CSF receptor fusion was functional in Ba/F3 cells, it might have lacked properties necessary to generate an "authentic" G-CSF signal in primary MHPCs. Finally, our results suggest that though Mpl is permissive for the terminal differentiation of committed progenitors, signals other than those provided by Mpl are required for MHPCs to generate erythroid or granulocyte progeny. Mpl's ability to support the terminal differentiation of committed progenitor cells is reminiscent of various findings in which ectopically expressed receptors are able to support the terminal differentiation of progenitors in which they are not normally expressed.31-34 These findings suggest that though receptor swapping is permissible in committed progenitor cells, constraints are placed on the interchangeability of receptors within MHPCs. Our results open the possibility that HSCs/MHPCs might be manipulated to perform various functions, depending on the particular receptor signaling domain activated. An especially attractive feature of the CID-based approach derives from the possibility of inducing a population of genetically modified MHPCs to adopt a desired fate in vivo.41 Mpl may induce qualitative or quantitative changes in signaling that are required for MHPC self-renewal and that are not provided for by G-CSF receptor and Flt-3. Alternatively, the G-CSF receptor and Flt-3 may activate pathways that are inhibitory to MHPC self-renewal. Understanding these mechanisms may not be only biologically important, it may have therapeutic implications.
We thank Tim Clackson (Ariad Pharmaceuticals) for AP20187, Christine Halbert and James Yan for technical assistance, and Ken Kaushansky, Belinda Avalos, and Ihor Lemischka for cDNAs.
Submitted January 5, 2001; accepted March 20, 2001.
Supported by National Institutes of Health grants 5R01DK52997, 1R01DK57525, 2P01HL53750, 1P01DK55820, and 2P01DK47754; by an American Society of Hematology Junior Faculty Scholar Award; and by an award from the Fanconi Anemia Research Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: C. Anthony Blau, Dept of Medicine, Division of Hematology, University of Washington, Mailstop 357710, Health Sciences Bldg, Seattle, WA 98195; e-mail: tblau{at}u.washington.edu.
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© 2001 by The American Society of Hematology.
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  H. Abdel-Azim, Y. Zhu, R. Hollis, X. Wang, S. Ge, Q.-L. Hao, G. Smbatyan, D. B. Kohn, M. Rosol, and G. M. Crooks Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl Blood, April 15, 2008; 111(8): 4064 - 4074. [Abstract] [Full Text] [PDF]
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M. A. Weinreich, I. Lintmaer, L. Wang, H. D. Liggitt, M. A. Harkey, and C. A. Blau Growth factor receptors as regulators of hematopoiesis Blood, December 1, 2006; 108(12): 3713 - 3721. [Abstract] [Full Text] [PDF]
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W. Zhao, C. Kitidis, M. D. Fleming, H. F. Lodish, and S. Ghaffari Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway Blood, February 1, 2006; 107(3): 907 - 915. [Abstract] [Full Text] [PDF]
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 C. A. Blau, C. F. Barbas III, A. L. Bomhoff, R. Neades, J. Yan, P. A. Navas, and K. R. Peterson {gamma}-Globin Gene Expression in Chemical Inducer of Dimerization (CID)-dependent Multipotential Cells Established from Human {beta}-Globin Locus Yeast Artificial Chromosome ({beta}-YAC) Transgenic Mice J. Biol. Chem., November 4, 2005; 280(44): 36642 - 36647. [Abstract] [Full Text] [PDF]
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H. L. Bradley, C. Couldrey, and K. D. Bunting Hematopoietic-repopulating defects from STAT5-deficient bone marrow are not fully accounted for by loss of thrombopoietin responsiveness Blood, April 15, 2004; 103(8): 2965 - 2972. [Abstract] [Full Text] [PDF]
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B. Larrivee, D. R. Lane, I. Pollet, P. L. Olive, R. K. Humphries, and A. Karsan Vascular Endothelial Growth Factor Receptor-2 Induces Survival of Hematopoietic Progenitor Cells J. Biol. Chem., June 6, 2003; 278(24): 22006 - 22013. [Abstract] [Full Text] [PDF]
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 C. Brisken, M. Socolovsky, H. F. Lodish, and R. Weinberg The signaling domain of the erythropoietin receptor rescues prolactin receptor-mutant mammary epithelium PNAS, October 29, 2002; 99(22): 14241 - 14245. [Abstract] [Full Text] [PDF]
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C. Challier, L. Cocault, R. Berthier, N. Binart, I. Dusanter-Fourt, G. Uzan, and M. Souyri The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells Blood, August 28, 2002; 100(6): 2063 - 2070. [Abstract] [Full Text] [PDF]
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T. Neff, P. A. Horn, V. E. Valli, A. M. Gown, S. Wardwell, B. L. Wood, C. von Kalle, M. Schmidt, L. J. Peterson, J. C. Morris, et al. Pharmacologically regulated in vivo selection in a large animal Blood, August 28, 2002; 100(6): 2026 - 2031. [Abstract] [Full Text] [PDF]
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K. G. Otto, V. C. Broudy, N. L. Lin, E. Parganas, J. N. Luthi, J. G. Drachman, J. N. Ihle, and C. A. Blau Membrane localization is not required for Mpl function in normal hematopoietic cells Blood, October 1, 2001; 98(7): 2077 - 2083. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
family members to specify cell fate during
embryogenesis,
,
granulocyte-colony-stimulating factor (G-CSF), IL-6, stem cell factor
(SCF),
