|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 430-436
GENE THERAPY
Expansion of genetically modified primary human hemopoietic cells
using chemical inducers of dimerization
Robert E. Richard,
Brent Wood,
Hui Zeng,
Liqing Jin,
Thalia Papayannopoulou, and
C.
Anthony Blau
From the Divisions of Hematology and Medical Genetics, the
Department of Medicine, and the Department of Laboratory Medicine,
University of Washington School of Medicine, Seattle, WA.
 |
Abstract |
The inability to deliver a therapeutic gene to a sufficient
percentage of hematopoietic stem cells is the major obstacle to using
gene therapy to treat blood disorders. Providing genetically corrected
stem cells with a reversible growth advantage could solve this problem.
To this end we have employed small synthetic molecules that can
reversibly dimerize and activate fusion proteins which contain a growth
factor receptor signaling domain. We have shown that the thrombopoietin
receptor (mpl) signaling domain can be used in this system to expand
transduced multipotential progenitor cells from mouse bone marrow. In
the present study we tested a similar retroviral vector in human
CD34-selected cord blood cells. Following transduction, cells cultured
in the presence of the dimerizing molecule AP1903 expanded
13.8- to 186-fold relative to cells cultured in the absence of AP1903.
The cell type that emerged in suspension culture was erythroid.
Contrary to our results in the murine system, cell expansion was
transient. Activation of mpl caused the disappearance of BFU-E followed
by a transient increase in CFU-E. In contrast, mpl activation had no
discernable effect on transduced myeloid progenitor cells.
AP1903-mediated expansion was restricted to transduced cells, as
demonstrated by immunohistochemical staining. These findings indicate
that synthetic dimerizing molecules can be used to expand primary human hematopoietic cells.
(Blood. 2000;95:430-436)
© 2000 by The American Society of Hematology.
| |
Introduction |
Inefficient gene delivery into the human hematopoietic
stem cell presents what is arguably the single most formidable obstacle to stem cell gene therapy. One approach to achieving a therapeutically relevant frequency of genetically modified stem cells is to employ selection. Present methods to achieve selection rely on the transfer of
a gene that confers resistance to a subsequently administered cytotoxic
drug.1 In this setting, selection produces a preferential elimination of unmodified cells.
An alternative approach for achieving selection uses a gene that
confers a reversible growth advantage in the presence of a synthetic
drug. In this setting, unmodified cells fail to respond to the drug,
leading to the preferential expansion of the genetically corrected
population. Conditional cell growth can be accomplished through the use
of fusion proteins composed of a growth factor receptor signaling
domain fused to a binding site for a synthetic drug called
a chemical inducer of dimerization (CID).2,3 In
order for the effects of selection to persist, it is expected that
expansion must occur among genetically modified stem cells. Therefore,
in order for this approach to produce a permanent increase in
genetically modified cells, signaling molecules that are capable of
inducing expansion among stem cells must be identified. A molecule that
is a candidate for having this capacity is the thrombopoietin receptor (mpl).
In the context of a viral oncogene, the mpl signaling domain produces a
myeloproliferative disorder in mice.4 In addition to the
important role of mpl in megakaryocyte growth and differentiation, it
has also been demonstrated that mpl plays a role in the maintenance of
multipotential progenitors5,6 and stem
cells.7,8,9 In previously published studies,10
we demonstrated that transfer of a gene encoding a fusion protein
which contains the signaling domain of mpl allows for a marked
and sustained expansion of multipotential murine progenitor cells in
the presence of CID. In the studies presented we evaluated whether
CID-mediated activation of the mpl-signaling domain could stimulate
expansion of genetically modified primary human CD34-selected cord
blood cells.
| |
Materials and methods |
Our materials included cytokines human stem cell factor (hSCF),
human interleukin-3 (hIL-3), and hIL-6 (Peprotech, Rocky Hill, NJ) and
recombinant human erythropoietin (Ortho Biotech, Raritan, NJ).
Throughout this report, stated G418 concentrations indicate active drug concentrations.
Retroviral construct
The MFM (MSCVF36Vmpl) retroviral vector is identical to
the MSCV-based vector described in our previous report,10
with the exception that a point mutation was introduced into the FK506 binding protein (FKBP) domain to produce a substitution at amino acid
position 36 from phenylalanine to valine using primer-directed mutagenesis. The mpl signaling domain contained in this vector is
derived from murine mpl.
Retroviral producer lines
Retroviral producer lines were generated as previously
described.10 In brief, the MFM vector was transfected into
the ecotropic packaging cell line PE501. After 48 hours,
supernatant was collected and used to transduce the Gibbon ape leukemic
virus (GALV)-packaging cell line, PG13.11 We tested
G418-resistant clones for genetic stability by Southern analysis
testing of KpnI-digested genomic DNA. A clone was obtained with
a titer of 5 × 105 colony-forming
units/mL. Retroviral supernatant was collected from subconfluent
monolayers of MFM/PG13 producer cells after incubation for 48 hours at
33°C.
Isolation of CD34-selected cord blood cells
CD34-selected cells were isolated from normal human umbilical cord
blood scheduled for disposal after delivery. Mononuclear cells from
total cord blood were separated from red blood cells using density
gradient centrifugation (with a density of 1.077) on the cell
separation medium (Lymphoprep; Mediatech, Herndon, VA). Adherent cells
were removed by incubating the total cell suspension on tissue culture
plates for 1 hour at 37°C. Immunomagnetic selection of the CD34+
cells was accomplished using the MACS system (Miltenyi Biotec, Auburn,
CA), and the resulting purity of the CD34-selected cord blood cells is
shown in Table 1.
Transduction of CD34-selected cord blood cells
CD34-selected cells were placed in 6-well Costar plates
that had been coated with CH296 fibronectin fragment
(Retronectin; Takara Biomedicals, Otsu, Japan) according to the
manufacturer's instructions. Cells were cultured for 24 hours at
32°C with 7.5% CO2 in Iscove's modified Dulbecco's
medium (IMDM) supplemented with fetal bovine serum (FBS), 10%;
recombinant human interleukin 6 (rhIL-6), 100 ng/mL; rhIL-3, 50 ng/mL;
recombinant human stem cell factor (rhSCF), 50 ng/mL; and protamine
sulfate, 4 µg/mL. Retroviral supernatant was added at a multiplicity
of infection of approximately 4:1. After 24 hours the supernatant was
removed, and fresh viral supernatant was added. After an additional
24-hour incubation, nonadherent cells were removed, counted, and
divided among the experimental conditions. Transduction efficiency was determined by performing methylcellulose assays both in the presence and absence of G418 (1.2 mg/mL). In 1 experiment
(Table 1), transduction conditions were modified by the addition of a
24-hour preincubation step in which the cells were cultured in DMEM
with 16% FBS in the presence of IL-3, IL-6, Flt3L, and SCF (50 ng/mL each).
An additional method for transduction, based on the report of Dao and
Nolta,12 was tested as indicated in Table 1. CD34-selected cord blood cells were preincubated for 12 hours (ExVivo 15;
Biowhittaker, Walkersville, MD) with hIL-6, hIL-3, hSCF, and
Flt3L (50 ng/mL each). Retronectin-coated plates were
preloaded with retrovirus by incubating supernatant on the plates 3 times for 15 minutes each incubation. Cells were then placed in wells
containing an equivalent volume of retroviral supernatant, which was
collected in DMEM with 10% FBS. Anti-transforming growth factor  (anti-TGF) antibody (R&D Systems, Minneapolis, MN) was added at a
concentration of 5 µg/mL and incubated for 12 hours at 37°C with
5% CO2.
Suspension culture
After retroviral transduction, CD34-selected cord blood
cells were washed and cultured (either in the absence or presence of
AP1903 [100 nmol/L]) in IMDM containing FBS, 10%; penicillin, 50 units/mL; and streptomycin, 50 µg/mL. Human serum (10%) was also
included in the cultures listed in experiments 1 and 2 (Table 1).
Clonogenic assays in semisolid media
Clonogenic assays were performed in triplicate in the presence of
methylcellulose, 1.2%; FBS, 30%; bovine serum albumin
(BSA), 1%; 2-mercaptoethanol (SIGMA, St. Louis, MO),
5 × 10-4 mol/L; hIL-3, 5 ng/mL; hSCF, 50 ng/mL; and
human erythropoietin (h-epo), 5 units/mL (for measurements of erythroid
burst-forming units [BFU-Es]). Cells were cultured in a humidified
37°C incubator with 5% CO2. Colonies were counted on
day 14.
Erythroid colony-forming unit (CFU-E) and megakaryocyte (CFU-Mk) assays
were performed using a plasma clot assay as previously described.13 CD34-selected cells were plated in 10% human
plasma, 10% BSA, and 10% bovine citrated plasma in
addition to penicillin, 50 units/mL; streptomycin, 50 µg/mL;
CaCl2, 2 mmol/L; and thrombin, 0.25 units/mL. Growth
factors included erythropoietin (epo), 2 units/mL; IL-3, 5 ng/mL; IL-6,
3.5 ng/mL; SCF, 50 ng/mL; and thrombopoietin conditioned media. CFU-E
colonies were analyzed at days 5 to 7, while CFU-Mk colonies were
analyzed after 13 days of culture. Individual plasma clots were removed
and flattened on a gelatin-treated slide. For CFU-E assays, clots were
fixed with glutaraldehyde and stained with hematoxylin and benzidine.
CFU-Mk colonies were identified by staining with a
biotin-labeled anti-CD41 antibody. This was followed by staining with a
streptavidin-labeled alkaline phosphatase (Vector Laboratories,
Burlingame, CA) in accordance with the recommendations of the manufacturer.
Flow cytometry
Cells were centrifuged and resuspended in phosphate-buffered saline
(PBS)/BSA/Azide, then incubated with directly conjugated primary
antibodies phycoerythrin-labeled (PE-labeled) antiglycophorin A and
PE-labeled anti-CD33 (Becton Dickinson, Franklin-Lakes, NJ) at 4°C
for 30 minutes. The cells were then centrifuged, washed once with
PBS/BSA/Azide, resuspended in PBS/BSA/Azide, and analyzed with a flow
cytometer (Coulter XL-MCL; Coulter Electronics, Miami, FL). We
collected 10 000 gated cellular events during analysis for each
specimen. Cellular events were identified on the basis of CD45
expression (PE-Cy5 anti-CD45; Immunotech, Westbrook, ME), and
forward/side scatter gating was used to exclude cellular debris and
aggregates. Gates were set by comparing each specimen
with isotype-matched negative control antibodies.
Immunohistochemical staining
Immunocytochemistry was performed on cytospin preparations
of CD34-selected cord blood cells using the monoclonal antibody HA .11 (BAbCO, Richmond, CA). This antibody recognizes the influenza hemaglutinin epitope tag (Figure 1A). After
fixation in 95% ethanol, cytospins were incubated overnight at 4°C
with HA .11 in PBS. This was followed by secondary staining with a
biotinylated goat antimouse antibody (Signet Laboratories, Dedham, MA)
at room temperature for 20 minutes. Slides were then incubated with
peroxidase-labeled streptavidin (Signet Laboratories) for 20 minutes.
The peroxidase reaction was performed by mixing 0.5 mg/mL
3-amino-9-ethylcarbazole (Signet Laboratories) in 0.15 mol/L Tris-HCl
with 0.1% hydrogen peroxide. Cells were counterstained with methylene
blue (Biochemical Sciences, Swedesboro, NJ). The proportion of positive
cells was evaluated under high-power light microscopy. At least 500 cells were scored from each sample.
View larger version (16K):
[in this window]
[in a new window]
| Fig 1.
The MFM vector can mediate a dramatic expansion of mouse
bone marrow cells in the presence of AP1903.
(A) MFM is an MSCV-based retroviral vector. The gene encoding the
F36Vmpl fusion protein is transcribed from the long terminal repeat
(LTR). The neo gene is transcribed from the phosphoglyceate kinase
(PGK) promoter. In Figure 1A, M indicates myristylation domain; HA,
epitope tag from influenza hemagglutinin; F36V, the CID-binding domain;
and mpl, the intracellular signaling domain of murine mpl. (B) Mouse
bone marrow was transduced with the MFM vector and then incubated in
the absence ( ) or presence ( ) of AP1903 (100 nmol/L). The
transduced mouse bone marrow cells cultured in the absence of AP1903
died over a 2-week period, while the cells cultured in the presence of
AP1903 exhibited a dramatic expansion, similar to our previously
published results using FK1012.10
|
|
| |
Results |
Experiments were performed to test whether CID-mediated activation
of the mpl-signaling domain can function to expand primary human CD34+ cells.
CID-mediated expansion of transduced CD34+ cord blood
cells
The MFM vector used for our studies (Figure 1) encodes a fusion
protein that contains a myristylation domain which targets the molecule
to the cell membrane, a CID-binding domain, the cytoplasmic portion of
murine mpl, and an epitope tag. The CID-binding domain has been
modified to accommodate binding to a new class of synthetic CIDs.14 In contrast to dimerizing agents, such as
FK1012, which can bind endogenous FKBPs, a new synthetic
dimerizer, AP1903, has been developed to reduce association with
naturally occurring FKBPs. The AP1903 dimerizer specifically binds to a
mutated FKBP termed F36V, which contains a phenylalanine to valine
substitution. This combination of ligand-FKBP has been shown to allow
for CID-mediated activation of the Fas signaling
pathway.14 Retroviral transfer of the MFM vector into Ba/F3
cells allows for AP1903-mediated cell growth as reported previously for
FK1012 (data not shown). In addition, retroviral transfer of the MFM
vector into primary mouse bone marrow cells results in dramatic cell
growth that is dependent on AP1903 (Figure 1B). This is similar to our
previous findings using FK1012.10 The MFM vector was used
to generate a PG13-based producer cell line11 for
transduction of human CD34-selected cord blood cells.
Transductions of CD34-selected cord blood cells were carried
out as described in "Materials and Methods." The presence of a
neo gene in the MFM vector allowed gene transfer rates to be determined
by performing colony assays both in the presence and absence of G418.
Rates of gene transfer into progenitors ranged between 10% and 66%. A
summary of our experiments is shown in Table 1. Following transduction,
cells were placed in IMDM with 10% FBS, either in the presence or
absence of AP1903, at a concentration previously determined to be
optimal for the expansion of transduced mouse bone marrow cells (100 nmol/L). Mock-transduced cells that were incubated either in the
presence or absence of AP1903 provided an additional control. The
effects of AP1903 were evaluated in the absence of growth factors other
than those present in serum.
As shown in Figure 2, some degree
of cell expansion was observed in each of the conditions tested. Both
MFM-transduced cells cultured in the absence of AP1903 and
mock-transduced cells displayed a 3-fold increase in cell number, which
peaked at days 5-10 of culture and fell thereafter. This relatively
modest level of cell growth in the controls may be attributable to the
persistent effects of growth factors that were present at the time of
the transduction procedure. However, MFM-transduced cells cultured in
the presence of AP1903 exhibited a maximal 186-fold increase in cell
number by day 15. In contrast to results obtained using primary mouse bone marrow cells,10 sustained growth of transduced human
hematopoietic cells was not observed. The addition of Flt3 ligand (50 ng/mL) to AP1903 failed to result in improved cell growth (data not
shown). The ability of AP1903 to induce a transient expansion of
MFM-transduced cord blood cells was highly reproducible. In each of 7 independent experiments (Figure 3A),
MFM-transduced cells cultured in the presence of AP1903 were expanded
between 13.8-fold and 186-fold relative to cells cultured in the
absence of AP1903. In contrast, AP1903 had no effect on the growth of
mock-transduced cells (Figure 3B).
View larger version (16K):
[in this window]
[in a new window]
| Fig 2.
Expansion of transduced cord blood cells in the presence
of AP1903.
CD34-selected cord blood cells were transduced using the MFM vector
(Table 1, Experiment 3). Following transduction, cells were cultured
either in the presence ( ) or absence () of AP1903. Controls
included mock-transduced cells cultured in the presence ( ) or
absence ( ) of AP1903.
|
|
View larger version (10K):
[in this window]
[in a new window]
| Fig 3.
MFM-transduced CD34+ cord blood cells consistently
expand in the presence of AP1903.
The maximal level of cell expansion of MFM-transduced (A) and
mock-transduced (B) cord blood cells was plotted for each of the
experiments listed in Table 1. Each line represents one experiment. The
addition of AP1903 resulted in a 13.8-fold to 186-fold expansion
compared with cells cultured in the absence of AP1903.
|
|
Cells expanded in the presence of CID belong primarily to the
erythroid lineage
The lineage of cells expanding in response to AP1903 was evaluated
by Wright-Giemsa staining and flow cytometry at various time points
during the culture. In cultures that contained AP1903, the predominant
cell type was erythroid, as confirmed by benzidine staining (data not
shown) and flow cytometry using an antibody directed against
glycophorin A (Figure 4). Glycophorin A
positive cells were evident by day 7 of the culture, and by day 21, 89% of cells cultured in AP1903 were glycophorin A positive, while fewer than 1% of cells displayed the myeloid/monocytic marker CD33. An
absence of myeloid cell expansion in response to AP1903 was
confirmed by determining absolute neutrophil counts during the course
of the culture (data not shown). The absence of myeloid cell expansion
occurred despite having achieved transduction rates into
granulocyte/macrophage colonies (CFU-GM) of up to 66% (Table 1). In
the absence of AP1903, cultures contained a large percentage of dying
cells; most surviving cells displayed CD33.
View larger version (38K):
[in this window]
[in a new window]
| Fig 4.
Cells expanded in the presence of AP1903 are
predominately erythroid.
CD34-selected cord blood cells were transduced with the MFM vector and
incubated in the presence or absence of AP1903. On the days indicated,
aliquots of cells were removed from the suspension culture and tested
by flow cytometry for binding of antibodies directed against
glycophorin A and CD33. The cells incubated without the drug died
following day 7 and were therefore not available for analysis.
|
|
We next investigated whether CID-mediated activation of the
mpl-signaling domain could stimulate an expansion of clonogenic progenitor cells. Cells were removed from the culture at various time
points and analyzed for progenitor content as previously described. A
time-dependent decline in BFU-E, CFU-GM, and CFU-Mk numbers was
observed, both in the presence and absence of AP1903 (Figure
5, panels A, C, and D respectively). In
contrast, a transient expansion of CFU-E in response to AP1903 was
observed. This time frame correlated with the disappearance of BFU-E
and presaged the emergence of glycophorin A positive cells (Figure 5,
panel B). These observations are consistent with CID-mediated
differentiation of transduced BFU-E, which results in an orderly
progression to CFU-E and then terminal differentiation of erythroid
cells.
View larger version (21K):
[in this window]
[in a new window]
| Fig 5.
AP1903 produces a transient expansion of CFU-E.
Total BFU-E (A), CFU-E (B), CFU-GM (C), and CFU-Mk (D) values were
determined for cord blood transduced with the MFM vector, expanded in
the absence and presence of AP1903. Cell expansion occurred in the
presence of AP1903, similar to previous experiments (data not shown).
BFU-e, CFU-GM, and CFU-Mk values decreased regardless of whether CID
was present. CFU-E increased on day 7, then diminished, and was
dependent on the presence of AP1903. = + AP1903;
=  AP1903.
|
|
Cell expansion in response to AP1903 is restricted to the
transduced population
To evaluate the possibility that the erythroid cell expansion was
the indirect result of growth factor secretion in response to the CID,
we directly examined the expanded cells for the presence of the F36Vmpl
fusion protein. Immunohistochemical analysis was performed using an
antibody directed against the HA epitope tag present on
the fusion protein (Figure 1). Up to 86% of cells expanded in the
presence of AP1903 expressed the fusion protein (Figure 6A, B, and C; Table
2). In contrast, only 9%-14% of cells
cultured in the absence of CID expressed the fusion protein. This
frequency was similar to the rate of gene transfer into progenitors as
assessed by colony assays. The persistence of low-frequency HA positive cells in the absence of CID suggests that transduced cells were neither
preferentially expanded nor eliminated from culture in the absence of
selection. The restriction of AP1903-mediated expansion to the
transduced cell population suggests that AP1903 exerted a direct
differentiative effect on genetically modified erythroid progenitor
cells.
View larger version (108K):
[in this window]
[in a new window]
| Fig 6.
CD34+ cord blood cells transduced with the MFM vector
and expanded with AP1903 express the F36Vmpl transgene.
Immunohistochemical staining was performed to identify cells that
contain the HA epitope as part of the F36Vmpl transgene. (See
"Materials and Methods" for a complete description of the
method.) Cord blood cells were transduced with the MFM vector and then
incubated in the presence (A) or absence (B) of AP1903 for 14 days
prior to staining for the HA epitope. Panel C shows mock transduced
cells incubated for 14 days and then stained for the presence of the HA
epitope.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
CD34-selected cord blood cells transduced with the MFM
vector and expanded with AP1903 express the F36Vmpl transgene
|
|
AP1903 can support BFU-E and CFU-Mk development in semisolid culture
and can synergize with other cytokines
To directly analyze progenitor response to AP1903 in the context of
other cytokines, cord blood cells were cultured in semisolid media with
AP1903 alone or in combination with SCF and IL-3. To provide
positive controls, progenitor assays were performed in the presence of
erythropoietin, SCF, and IL-3 (Figure 7,
panel C). Colony assays performed in the presence of AP1903 gave rise to BFU-E but failed to generate CFU-GM beyond those observed in the
absence of CID (Table 3). The addition of
SCF and IL-3 to the culture conditions resulted in an increase in the
total number of BFU-E as well as in an increase in the size of the
BFU-E colonies (Table 3 and Figure 7, panels A and B), suggesting
synergy between the fusion protein and SCF and/or IL-3. The average
size of colonies cultured in the presence of AP1903 were 2400 cells/colony; AP1903/SCF/IL-3, 11 400 cells/colony; and epo/SCF/IL-3,
27 600 cells/colony. Plasma clot assays were performed in the presence
and absence of AP1903 to determine if CFU-Mk development could be
supported in the absence of added cytokines. Figure 7 (panel D) shows a
representative megakaryocyte colony that developed in the presence of
AP1903. Activation of the mpl signaling domain through CID-mediated
dimerization was able to support colony growth from transduced BFU-E
and CFU-Mk but was unable to support colony growth from transduced
CFU-GM.
View larger version (122K):
[in this window]
[in a new window]
| Fig 7.
Erythroid and megakaryocytic progenitors transduced with
the MFM vector develop into colonies in the presence of AP1903 alone.
Immediately after transduction with the MFM vector, CD34+ cord blood
cells were plated in colony assays with different combinations of drug
and cytokines. Displayed in panels A-C are representative BFU-E
colonies with the following conditions: (A) BFU-E assays with AP1903
alone; (B) BFU-E assays with AP1903, SCF, and IL-3; (C) BFU-E assays
with epo, SCF, and IL-3; and (D) a representative megakaryocytic colony
from plasma clot assays plated in AP1903 alone. The cells were stained
for CD41 expression, as detailed previously.
|
|
| |
Discussion |
The inability to deliver genes to more than a small
fraction of human stem cells poses a major obstacle to gene therapy for a wide range of hematological disorders. Using selection in vivo has
important theoretical advantages. A clinically effective method for
selection may allow stem cells to be harvested from a patient, transduced, and reinfused with little or no conditioning. The transduced cell population could then be expanded through in
vivo administration of the corresponding selective drug
regimen. Two requirements must be met. First, achieving a sustained
expansion of the selected cell population requires that selection be
imposed among stem cells. Sorrentino and colleagues1,15
have recently demonstrated that stem cell selection can be accomplished
through transfer of the dihydrofolate reductase gene followed by
administration of drugs that preferentially eliminate unmodified
progenitors and stem cells. Second, clinical applicability requires
that the drugs used for selection be well-tolerated.
An alternative to preferentially eliminating nontransduced
cells is to preferentially expand the transduced cell
population. Toward this goal we have adopted a system that allows
intracellular signaling to be reversibly controlled using the CID,
FK1012.2,16 In previous studies we have used genes that
encode fusion proteins containing the epo receptor,3
c-kit,17 or c-mpl10 signaling domains to
generate FK1012-dependent cell lines. Furthermore, FK1012-mediated
activation of the mpl signaling domain produces a dramatic expansion of
transduced primary murine bone marrow cells. Identical results have
been obtained using the alternative CID, AP1903, and its cognate
binding site, the F36V-modified FKBP domain (Figure 1).
Two highly reproducible features of this system merit comment. First,
expansion of murine bone marrow cells in the presence of CID is
sustained. Murine bone marrow cells can be expanded for longer than 300 days in serum-containing cultures with FK1012 but without the addition
of cytokines. Second, cells expanded in response to FK1012 include
multipotential progenitor cells and differentiated megakaryocytes. The
results that we have obtained using human cells differ significantly
from our results in mice. CID-mediated activation of the mpl signaling
domain in human CD34-selected cord blood cells produced only a
temporary expansion, which was exhausted after 28 days of culture.
Transduced human BFU-E, CFU-GM, and CFU-Mk all failed to amplify in
response to the CID, while CFU-E rose transiently due to apparent
differentiation of transduced BFU-E.
What factors might account for the different responses to mpl
activation between mouse and man? There are several possibilities. First, mpl-activated signaling pathways in the human cellular environment may be identical to those in the mouse but incapable of
eliciting expansion among human progenitor cells. In particular, human
stem cell expansion may require environmental signals in addition to
those provided by mpl. In this context it is interesting to note that
bcr-abl, an oncogene with a demonstrated capacity for stimulating human
stem cell expansion, can produce preferential cell expansion only in
the in vivo setting.18,19 This suggests that the selective
advantage conferred by this oncogene requires supplemental signals from
the environment. Second, there may be a difference in the frequency of
the multipotential, expandable progenitors in human cord blood when
compared with 5 fluorouracil-treated mouse bone marrow. Third, the
lack of human progenitor cell expansion may be attributable to
qualitative or quantitative differences in mpl-activated signaling
pathways between the murine and human hematopoietic systems. This
possibility implies that human progenitor cell expansion might be
achievable if the signaling pathways that are activated in the mouse
system could be precisely replicated. We are presently testing a
retroviral construct that contains the cytoplasmic portion of human
mpl. A fourth possibility is that our transduction conditions failed to
deliver the vector into the human equivalent of the transduced
pluripotent mouse cell that was capable of expanding for over 300 days.
Preliminary experiments in the NOD-SCID (non-obese
diabetic severe combined immunodeficiency) mouse model indicate that
3% to 5% of SCID repopulating cells have been transduced (data not
shown). Nevertheless, these findings do not exclude the possibility
that transduction of an even more primitive cell type may be required
in order to achieve a sustained proliferative effect. The possibility
remains that although the MFM vector may be the proper `seed' to
stimulate primitive cell growth, we may have not delivered it into the
necessary cellular `soil.'20
Our findings demonstrate that mpl signaling in primary human
hematopoietic cells is sufficient for full erythroid and megakaryocytic differentiation. The ability of mpl to permit differentiation of
erythroid progenitor cells is in agreement with 2 previously published
reports.21,22 Thrombopoietin administration was permissive for erythroid differentiation of fetal liver cells derived from the
erythropoietin receptor null mouse,22 while ectopic
expression of c-mpl in transduced mouse bone marrow cells allowed for
thrombopoietin-dependent differentiation of transduced
BFU-E.21 In supporting full erythroid maturation,
CID-mediated mpl signaling acts in a manner similar to the combination
of epo and SCF.23 In our studies, CD34+ cells were exposed
to SCF during the transduction procedure, possibly rendering transduced
BFU-E competent to respond to mpl activation. In contrast, we did not
observe differentiation of transduced CFU-GM when mpl was activated, as
has been noted in a previous report.21 These findings
indicate that signals emanating from activated cytokine receptors are
not strictly interchangeable between progenitors of all lineages.
These studies demonstrate that CIDs can specifically deliver a
mitogenic signal to genetically modified primary human hematopoietic cells. Further studies are required to define and manipulate signals that are capable of inducing human stem cell expansion.
| |
Acknowledgments |
The authors would like to thank Denise Farrar and Donna Ceniza for
expert technical assistance, David W. Emery for technical advice, Mo
Dao and Jan Nolta with help developing transduction conditions, and
Michael Gilman and Tim Clackson (Ariad Pharmaceuticals) for providing
AP1903. We would also like to thank Ortho Biotech of Raritan, NJ, for
the generous gift of recombinant human erythropoietin.
| |
Footnotes |
Submitted May 17, 1999; accepted August 31, 1999.
Supported by grants from the National Institutes of Health (NIH 1R01
DK5299701, 1R01 DK57525, 5P01 HL53750, 5P30 DK47754), a Junior Faculty
Award from the American Society of Hematology, and a grant from the
Fanconi Anemic Research Fund.
Reprints: C. Anthony Blau, Mailstop 357710, Health Sciences
Building, University of Washington, Seattle, WA 98195; e-mail:
tblau{at}u.washington.edu.
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.
| |
References |
1.
Allay JA, Persons DA, Galipeau J, et al.
In vivo selection of retrovirally transduced hematopoietic stem cells.
Nat Med.
1998;4:1136-1143[Medline]
[Order article via Infotrieve].
2.
Spencer DM, Wandless TJ, Schreiber SL, Crabtree GR.
Controlling signal transduction with synthetic ligands.
Science.
1993;262:1019-1024[Abstract/Free Full Text].
3.
Blau CA, Peterson KR, Drachman JG, Spencer DM.
A proliferation switch for genetically modified cells.
Proc Natl Acad Sci U S A.
1997;94:3076-3081[Abstract/Free Full Text].
4.
Souyri M, Vigon I, Penciolelli JF, Heard JM, Tambourin P, Wendling F.
A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors.
Cell.
1990;63:1137-1147[Medline]
[Order article via Infotrieve].
5.
Kaushansky K, Broudy VC, Grossmann A, et al.
Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.
J Clin Invest.
1995;96:1683-1687.
6.
Alexander WS, Roberts AW, Nicola NA, Li R, Metcalf D.
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl.
Blood.
1996;87:2162-2170[Abstract/Free Full Text].
7.
Sitnicka E, Lin N, Priestley GV, et al.
The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells.
Blood.
1996;87:4998-5005[Abstract/Free Full Text].
8.
Kimura S, Roberts AW, Metcalf D, Alexander WS.
Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin.
Proc Natl Acad Sci U S A.
1998;95:1195-1200[Abstract/Free Full Text].
9.
Kaushansky K.
Thrombopoietin and the hematopoietic stem cell.
Blood
1998;92:1-3[Free Full Text].
10.
Jin L, Siritanaratkul N, Emery DW, et al.
Targeted expansion of genetically modified bone marrow cells.
Proc Natl Acad Sci U S A.
1998;95:8093-8097[Abstract/Free Full Text].
11.
Kiem HP, Heyward S, Winkler A, et al.
Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.
Blood.
1997;90:4638-4645[Abstract/Free Full Text].
12.
Dao MA, Taylor N, Nolta JA.
Reduction in levels of the cyclin-dependent kinase inhibitor p27(kip-1) coupled with transforming growth factor beta neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells.
Proc Natl Acad Sci U S A.
1998;95:13,006-13,011[Abstract/Free Full Text].
13.
Papayannopoulou T, Brice M, Blau CA.
Kit ligand in synergy with interleukin-3 amplifies the erythropoietin-independent, globin-synthesizing progeny of normal human burst-forming units-erythroid in suspension cultures: physiologic implications.
Blood.
1993;81:299-310[Abstract/Free Full Text].
14.
Clackson T, Yang W, Rozamus LW, et al.
Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.
Proc Natl Acad Sci U S A.
1998;95:10437-10442[Abstract/Free Full Text].
15.
Spencer HT, Sleep SE, Rehg JE, Blakley RL, Sorrentino BP.
A gene transfer strategy for making bone marrow cells resistant to trimetrexate.
Blood.
1996;87:2579-2587[Abstract/Free Full Text].
16.
Spencer DM, Belshaw PJ, Chen L, et al.
Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization.
Cur Biol.
1996;6:839-847[Medline]
[Order article via Infotrieve].
17.
Jin L, Asano H, Blau CA.
Stimulating cell proliferation through the pharmacologic activation of c-kit.
Blood.
1998;91:890-897[Abstract/Free Full Text].
18.
Young JC, Witte ON.
Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.
Mol Cell Biol.
1988;8:4079-4087[Abstract/Free Full Text].
19.
McLaughlin J, Chianese E, Witte ON.
In vitro transformation of immature hematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphia chromosome.
Proc Natl Acad Sci U S A.
1987;84:6558-6562[Abstract/Free Full Text].
20.
Westervelt P, Ley TJ.
Seed versus soil: the importance of the target cell for transgenic models of human leukemias.
Blood.
1999;93:2143-2148[Free Full Text].
21.
Goncalves F, Lacout C, Villeval JL, Wendling F, Vainchenker W, Dumenil D.
Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.
Blood.
1997;89:3544-3553[Abstract/Free Full Text].
22.
Kieran MW, Perkins AC, Orkin SH, Zon LI.
Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.
Proc Natl Acad Sci U S A.
1996;93:9126-9131[Abstract/Free Full Text].
23.
Wu H, Klingmuller U, Acurio A, Hsiao JG, Lodish HF.
Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.
Proc Natl Acad Sci U S A.
1997;94:1806-1810[Abstract/Free Full Text].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
Y. Nagasawa, B. L. Wood, L. Wang, I. Lintmaer, W. Guo, T. Papayannopoulou, M. A. Harkey, C. Nourigat, and C. A. Blau
Anatomical Compartments Modify the Response of Human Hematopoietic Cells to a Mitogenic Signal
Stem Cells,
April 1, 2006;
24(4):
908 - 917.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Staerk, C. Lacout, T. Sato, S. O. Smith, W. Vainchenker, and S. N. Constantinescu
An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor
Blood,
March 1, 2006;
107(5):
1864 - 1871.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. E. Richard, M. Weinreich, K.-H. Chang, J. Ieremia, M. M. Stevenson, and C. A. Blau
Modulating erythrocyte chimerism in a mouse model of pyruvate kinase deficiency
Blood,
June 15, 2004;
103(12):
4432 - 4439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction