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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1947-1958
Flt3high and Flt3low CD34+
Progenitor Cells Isolated From Human Bone Marrow Are Functionally
Distinct
By
Katharina S. Götze,
Manuel Ramírez,
Kelly Tabor,
Donald Small,
William Matthews, and
Curt I. Civin
From the Division of Pediatric Oncology, Departments of Oncology and
Pediatrics, Johns Hopkins University School of Medicine, Baltimore MD;
and Genentech Inc, South San Francisco, CA.
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ABSTRACT |
We generated monoclonal antibodies against the human Flt3 receptor
and used them to study the characteristics of normal human bone marrow
cells resolved based on Flt3 expression. Human CD34+ or
CD34+lin marrow cells were sorted into two
populations: cells expressing high levels of Flt3 receptor
(Flt3high) and cells with little or no expression of Flt3
receptor (Flt3low). Flt3 receptor was detected on a subset
of CD34+CD38 marrow cells, as well as on
CD34+CD19+ B lymphoid progenitors and
CD34+CD14+CD64+ monocytic
precursors. Flt3 receptor was also present on more mature
CD34CD14+ monocytes. In colony-forming
assays, Flt3high cells gave rise mainly to colony-forming
unit-granulocyte-macrophage (CFU-GM) colonies, whereas
Flt3low cells produced mostly burst-forming unit-erythroid
colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the
G0 phase of the cell cycle, whereas Flt3high
cells were predominantly in G1. Cell numbers in the
suspension cultures initiated with Flt3high cells were
maintained in the presence of Flt3 ligand (FL) alone, and increased in
response to FL plus kit ligand (KL). In contrast, cell numbers in the
suspension cultures started with Flt3low cells did not
increase in the presence of FL, or FL plus KL. Upregulation of Flt3
receptor on Flt3low cells was not detected during
suspension culture. CD14+ monocytes were the major cell
type generated from
CD34+linFlt3high cells in
liquid suspension culture, whereas cells generated from CD34+linFlt3low cells were
mainly CD71+GlycA+ erythroid cells. These
results show clear functional differences between
CD34+Flt3high and
CD34+Flt3low cells and may have implications
concerning the in vitro expansion of human hematopoietic progenitor
cells.
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INTRODUCTION |
PLURIPOTENT HEMATOPOIETIC stem cells are
characterized by the ability to self-renew as well as differentiate
into various lineage-committed progenitor cells, which in turn produce
large numbers of mature blood cells.1 The majority of
hematopoietic stem and progenitor cells in the bone marrow are in cell
cycle dormancy.1-3 These cells are thought to enter active
cell cycle in response to certain cytokines, and subsequent stages of
differentiation are associated with upregulation of growth factor
receptors on the cell surface.3,4 To date, several
hematopoietic growth factor receptors involved in the proliferation and
differentiation of early progenitor cells have been identified.
Knowledge of the cellular expression of these receptors may make it
possible to delineate the earliest signalling events in hematopoiesis
and further characterize the pluripotent stem cell.
One such receptor that plays a role in regulating early hematopoiesis
is the tyrosine kinase Flt3 (Flk2/STK-1), cloned by three groups
including our own.5-8 Flt3 receptor has also been designated CD135.9 It is structurally related to c-kit
(kit, CD117), the receptor for kit ligand (KL). By themselves, Flt3 ligand (FL) or KL show only weak proliferative
effects.10-12 However, they synergize with each other and
various other factors (eg, interleukin-3 [IL-3], IL-6, and
granulocyte-macrophage colony-stimulating factor [GM-CSF]) to
stimulate proliferation and colony formation of early progenitor
cells.10-17 The Flt3 and c-kit receptors share structural
features and have similar effects, but clear differences exist between
the two receptors and their respective ligands. Both FL and KL induce
the proliferation of myeloid progenitors.14-19 However, KL
but not FL stimulates mast cells20 and erythroid precursors,21,22 and FL but not KL promotes the expansion
of early B lymphoid progenitors23-25 and dendritic
cells.26 It has been found that primitive murine
hematopoietic progenitors that are quiescent express low levels of kit,
whereas actively cycling progenitor cells are
kithigh.27,28 Expression of kit is
downregulated as progenitor cells mature, resulting in stage-related
expression of murine kit receptor.27 Similarly, the
majority of murine stem cells residing in the G0 stage of
the cell cycle show little or no expression of Flt3
receptor,29 whereas actively cycling murine stem cells have
increased Flt3 mRNA and protein expression.29,30
Recently, it has been reported that human CD34+
stem/progenitor cells possessing long-term engrafting ability express
detectable but low levels of kit receptor.18 Whether Flt3
receptor is expressed on human hematopoietic stem cells is still a
matter of controversy. Early studies showed that antisense
oligonucleotides directed against the gene for human Flt3
receptor suppressed colony formation by CD34+ progenitor
cells in semisolid culture and strongly inhibited the generation of
granulo-monocytic colonies from long-term bone marrow
cultures.6 More recently, it has been shown that FL alone
increases the number of primitive long-term culture-initiating cells in
cultures initiated from CD34+CD38
cells,19 fetal liver cells,31 or peripheral
blood stem/progenitor cells.32 Taken together, these
results indicate that signalling through Flt3 receptor is important in
very early human hematopoiesis, possibly involving the earliest stem
cells.
In the present study, we used monoclonal antibodies (MoAbs) against the
human Flt3 receptor to examine its expression on human CD34+ bone marrow cells. CD34+ progenitor cells
were fractionated into two subpopulations: cells expressing high levels
of Flt3 receptor (Flt3high) and cells with little or no
expression of Flt3 receptor (Flt3low). The results show
that CD34+Flt3low cells clearly differ from
CD34+Flt3high cells with respect to
differentiation potential, cell cycle status, and responsiveness to FL.
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MATERIALS AND METHODS |
Cells.
Human bone marrow cells were isolated by density-gradient
centrifugation (Ficoll-Hypaque; Pharmacia, Piscataway, NJ). Human marrow was obtained from the posterior iliac crests of consenting healthy adults under an Institutional Review Board approved protocol. CD34+ cells were isolated using immunomagnetic microspheres
as previously described.33 The purity of the isolated
CD34+ cell preparations ranged from 88% to 95%.
CD34+ cells were further depleted of more mature cells
expressing CD3, CD14, CD15, CD19, and CD71 lineage antigens by using
Dynabeads (Dynal, Lake Success, NY) following the manufacturer's
instructions. The human hematopoietic cell lines ML-1 and TF-1 and the
murine cell line 32D were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) as were TF-1 cells and 32D cells transfected with the gene for human Flt3 receptor (TF-1/S.9; 32D-Flk2). Media for growth of TF-1 and TF-1/S.9 cells also contained 10 ng/mL
recombinant human IL-3; media for growth of 32D and 32D-Flk2 cells
contained 10 ng/mL recombinant murine IL-3. Parental human 293 cells and 293 cells transfected with human Flt3 receptor (293-Flk2) were maintained in Dulbecco's modified Eagle's medium containing 10%
FBS. G418 (0.5 mg/mL) was added to selective media of transfected cell
lines.
MoAbs against Flt3 receptor.
For production of MoAbs 2G3.9.14 (IgG2b) and 4A6.6.3
(IgG2a), BALB/c mice were immunized in each rear footpad
with either 106 transfected 293-Flk2 cells, or
106 transfected 32D-Flk2 cells on days 0, 7, 14, 27, and 76 or days 0, 3, 7, 10, and 101, respectively. To produce MoAb 1E7.3.2
(IgG1), BALB/c mice were immunized in each rear footpad
with 2.5 µg of soluble human Flt3/Flk2-IgG suspended in MPL/TDM
adjuvant on days 0, 3, 7, 14, 17, 21, and 122. Four days after the last
immunization, lymph nodes were harvested and fused with P3/X63-Ag8U1
myeloma cells34 with 35% polyethyleneglycol as
described.35 Hybridoma cell lines secreting antibody
specific for human Flt3/Flk2 receptor were assayed either by
enzyme-linked immunosorbent assay using CD4-IgG versus Flt3/Flk2-IgG or
by flow cytometry using transfected 293-Flk2 or 32D-Flk2 cells versus
control cells. Selected hybridomas were cloned twice by limiting
dilution then further characterized for their binding capabilities.
Ascites was produced in BALB/c mice, and MoAbs were purified on a
protein G affinity column. Protein concentration was determined by
absorbance at 280 nm using an extinction coefficient of 1.4.
Other antibodies.
Phycoerythrin (PE)-conjugated purified mouse antihuman CD34, CD3 and
secondary rat antimouse IgG1, and IgG2a plus
IgG2b, as well as Peridinin Chlorophyll Protein
(PerCP)-conjugated mouse antihuman CD34 and matching IgG1 isotype
control, were purchased from Becton Dickinson (San Jose, CA).
Fluorescein isothiocyanate (FITC)-conjugated purified secondary goat
antimouse Ig (polyclonal) and CD71 were also obtained from Becton
Dickinson. PE-conjugated mouse antihuman CD19, CD45, and CD117 were
bought from Pharmingen (San Diego, CA), as were CyChrome-conjugated
CD38, CD19, CD64, and IgG isotype controls. PE-conjugated purified
mouse antihuman CD14 and CD15 were purchased from Dako (Carpinteria,
CA). PE-conjugated and FITC-conjugated isotype control antibodies were
obtained from Sigma (St Louis, MO). A pool of polyclonal murine
antibodies (containing IgG1, IgG2a,
IgG2b, and IgG3 subclasses) used as a negative
control was also purchased from Sigma.
Immunophenotyping.
Two- or three-color flow cytometry was performed on a FACSort flow
cytometer (Becton Dickinson) using the CellQuest (Becton Dickinson)
software for data acquisition and analysis. Instrument alignment and
compensation were accomplished using Calibrite beads (Becton Dickinson)
following the manufacturer's instructions. Directly conjugated MoAbs
were used to stain hematopoietic cells for all antigens except Flt3
receptor on which an indirect staining procedure was used as described
previously.36 Briefly, cells were resuspended in
immunofluorescence assay (IFA) medium (0.01 mol/L HEPES, 0.15 mol/L
NaCl, 0.1% NaN3, 4% bovine serum, pH 7.4) containing 2%
human AB+ serum. Indirect staining was performed by adding
unconjugated anti-Flt3 receptor antibodies to CD34+ cells
for 30 minutes at 4°C. Cells were then washed twice in IFA medium
and resuspended in 100 µL of either a PE-conjugated rat or
FITC-conjugated goat antimouse IgG secondary antibody diluted 1:50 in
IFA medium containing 2% human AB+ serum and either 2%
rat or goat serum, respectively. After a 30-minute incubation at
4°C, cells were washed twice, and 15 µg of mouse IgG1
(Sigma) was added to each tube to block unsaturated valencies of
antimouse IgG.37 After 15 minutes, directly conjugated antibodies were added to the cells for 30 minutes at 4°C, followed by 2 washes with 2 mL of ice-cold IFA medium and fixation with 1%
formaldehyde. Determinations of background staining using matching isotype control MoAbs at the appropriate concentrations were performed in parallel in every experiment. For control of indirect staining, a
pool of polyclonal mouse antibodies was used as the primary antibody.
The same secondary antibody (PE or FITC) used for Flt3 staining was
used.
Immunostained cells were analyzed by flow cytometry. For cell sorting,
104 events were acquired from the sample tube and isotype
control tube to set gates. Fluorescence-activated cell sorting (FACS) gates were defined to include the 30% brightest Flt3-expressing cells
(defined as Flt3high) versus the 30% lowest
Flt3-expressing cells (defined as Flt3low) within the
CD34+ cell population (see Fig 5).
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| Fig 5.
Clonogenic capacity of
CD34+Flt3high versus
CD34+Flt3low marrow cell subpopulations.
Cells were FACS sorted as shown in Fig 4 and plated at a density of 500 cells per culture dish in methylcellulose. Colonies were scored after
14 days of culture. (A) Colony formation from
CD34+Flt3high versus Flt3low
cells. Data represent mean ± SD of triplicate cultures from three individual experiments. (B) Colony formation of
CD34+linFlt3high versus
CD34+linFlt3low cells. Data
represent mean ± SD of triplicate cultures from one experiment. *
P < .001; ** P < .004.
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Clonal colony assays.
Freshly isolated, FACS sorted subsets of CD34+ cells
cultured in vitro for 3 to 9 days were plated in 1 mL methylcellulose containing 30% FBS, 1% bovine serum albumin (BSA),
104 mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine
and human cytokines (Stem Cell Technologies Inc, Vancouver, Canada) in
35 mm2 tissue culture dishes in triplicate and incubated at
37°C, 5% CO2. Human cytokines were present at the
following concentrations: KL 50 ng/mL, GM-CSF 20 ng/mL, IL-3 20 ng/mL,
IL-6 20 ng/mL, granulocyte colony-stimulating factor (G-CSF) 20 ng/mL,
and erythropoietin (Epo) 3 U/mL. After 14 days of culture, plates were
scored for the presence of erythroid (BFU-E), granulo-monocytic
(CFU-GM), and multilineage (CFU-Mix) colonies by standard morphological criteria using an inverted microscope. To determine the replating efficiency of populations derived from each FACS sorted cell fraction, day-14 colonies were replated into secondary and tertiary clonal cultures. For this, methylcellulose plates were flushed several times
with RPMI medium until all cells were removed. Cells were then pooled,
washed twice in RPMI to remove residual methylcellulose, counted, and
replated in triplicate as described previously. Two types of colonies
were scored in these 2o and 3o cultures:
"CFU-C," which were defined as colonies containing  50
cells/colony, and "blast clusters," consisting of 10 to 50 cells/colony.31
Liquid suspension cultures.
For suspension cultures, CD34+ cells sorted on the basis of
Flt3 receptor expression were cultured in 24-well tissue culture plates
containing RPMI supplemented with 10% FBS and various growth factor
combinations for 3 to 9 days. Human growth factors were used at the
following concentrations: FL 100 ng/mL (generously provided by Dr
Stewart Lyman, Immunex Corp, Seattle, WA), KL 50 ng/mL, IL-3 20 ng/mL,
IL-6 20 ng/mL, GM-CSF 20 ng/mL, and Epo 3 U/mL (Amgen, Thousand Oaks,
CA). At various timepoints, cells were removed from the wells by
vigorous pipetting. After washing, cells were counted by trypan blue
dye exclusion, stained, and analyzed for expression of leukocyte
differentiation antigens by flow cytometry as described previously.
Cells were also plated in methylcellulose (see above). In some
experiments, cytocentrifuge slides were prepared and cells were stained
with Wright-Giemsa for morphological analysis.
Cell cycle analysis.
CD34+ cell samples were stained and analyzed for
simultaneous expression of the proliferation-associated nuclear antigen
Ki67, DNA content, and a cell surface antigen (ie, Flt3 receptor) using a modification of the flow cytometric assay described by Jordan et
al.38 Briefly, cells were washed after isolation and
resuspended in IFA medium. All subsequent steps were carried out at
4°C. Cells were first stained indirectly with MoAbs against Flt3
receptor (or isotype control) as described previously using a
PE-conjugated secondary antibody. After the final wash, cells were
fixed in 1% formaldehyde (ultra-pure, EM grade; Polysciences,
Warrington, PA) in phosphate-buffered saline (PBS) and incubated on ice
for 30 minutes. An equal volume of 0.2% Triton X-100 in PBS was added, and samples were left at 4°C overnight for permeabilization. The following day, the fixed and permeabilized samples were washed and
resuspended in IFA buffer and stained with anti-Ki67-FITC (Dako Corp,
Carpinteria, CA). Finally, cells were washed and resuspended in PBS
containing 1% FBS and 0.5 µg/mL 7-actinomycin D (7-AAD) then
incubated at 4°C overnight before analysis. Peripheral blood lymphocytes (resting cells) as well as lymphocytes stimulated into
cycle with phytohemagglutinin were used as negative and positive controls for Ki67 staining, respectively.39 An
isotype-matched, irrelevant control mouse hybridoma supernatant (MOPC
21; Sigma) was used as an additional control.
Replicate samples were stained for Flt3 receptor versus propidium
iodide (PI). For this, cell samples were first stained for Flt3
receptor using the same MoAb cocktail and an FITC-conjugated secondary
antibody as described previously, washed twice and resuspended in
400-µL ice-cold PBS. Cells were fixed by the addition of 1.2 mL
ice-cold 95% ethanol and kept on ice for 20 minutes. Cells were then
spun down and resuspended in 1 mL cold PBS containing 0.05 mg/mL PI.
DNAse-free RNAse was added to a concentration of 50 µg/mL, and cells
were incubated for 30 minutes at room temperature.
Samples were stored at 4°C until analysis by flow cytometry. DNA
histograms were analyzed using ModFit LT software (Verity Software
House Inc, Sunnyvale, CA) to determine the distribution of
cells in G0/G1, S, and G2/M phases
of the cell cycle.
Statistical analysis.
Statistical analysis was performed using the Stata software program
(Stata Press, College Station, TX). Results are expressed as mean ± standard deviation (SD) or standard error (SE). Results were considered
significant if the P value was  .05. We used the two-sample
Wilcoxon rank-sum test (Mann-Whitney two-sample statistic) to compare
clonogenic output of the Flt3high versus
Flt3low cell populations.
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RESULTS |
Specificity of MoAbs against Flt3 receptor.
Screened candidate murine monoclonal antihuman Flt3 receptor
antibodies, produced and initially identified as described previously, were retested for their ability to specifically bind to Flt3 receptor on the surface of hematopoietic cell lines previously characterized for
Flt3 mRNA expression. The human erythroleukemia TF-1 cell line had been
previously transfected with the full length human Flt3 receptor.
Transfected TF-1 cells (TF-1/S.9) expressed Flt3 receptor by Western
blot (data not shown). Because parental TF-1 cells normally express
kit, but not Flt3 receptor, staining for kit was used as a positive
control for both the transfected and parental TF-1 cells. All 11 monoclonal antihuman Flt3 MoAb clones tested selectively stained the
Flt3-transfected TF-1/S.9 cells but not parental TF-1 cells, as
determined by flow cytometry. In contrast, antihuman kit MoAbs stained
both parental and TF-1/S.9 cells (data not shown). The same anti-Flt3
MoAbs also stained (previously) transfected Flt3-transfected 32D cells,
but not parental 32D (murine) cells. This confirmed that the anti-Flt3
MoAbs were specific for Flt3 receptor. Staining intensity was
relatively weak, likely because of low expression of Flt3 receptor on
the transfected cells. Therefore, the anti-Flt3 MoAb preparations were
titered using the human myeloid leukemia ML-1 cell line, which has been
shown to express Flt3 receptor at high levels.6,40 Staining
with optimal concentrations of single MoAbs was bright on ML-1 cells
but weak on human CD34+ cells. Therefore, antibodies were
tested in combinations, in an attempt to maximize Flt3 receptor
labeling intensity. Three of the antibodies tested (clones 4A6.6.3
[IgG2a], 1E7.3.2 [IgG1], and 2G3.9.14
[IgG2b]) produced additive staining when used in combination, and the cocktail of these three MoAbs produced the highest
intensity of staining of any combination, both on ML-1 and
CD34+ cells (Fig 1). Therefore,
this MoAb cocktail was used to label Flt3 receptor in all subsequent
experiments.
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| Fig 1.
Fluorescence intensity of human ML-1 cells (left panel)
and purified human CD34+ marrow cells (right panel)
labeled with MoAb against Flt3 receptor and stained with
FITC-conjugated secondary antibody. (1) Unstained cells (dotted line),
(2) cells stained with mouse isotype control antibody (thin solid
line), (3) cells stained with a single MoAb against Flt3 receptor (MoAb
4A6.6.3; dashed line, shown only for ML-1 cells), (4) cells stained
with a combination of three anti-Flt3 receptor MoAbs (MoAbs 4A6.6.3,
1E7.3.2, and 2G3.9.14; thick solid line). The mean fluorescence
intensity (MFI) of each histogram is provided below the panels. The
results shown are representative of two experiments.
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Flt3 receptor expression on normal human marrow mononuclear cells.
Flt3 receptor was present at very low levels on 32% of human marrow
CD34+CD38 cells and at relatively high
levels on 52% of CD34+CD38+ cells
(Fig 2C: comparison of the left versus
right lower quadrants). Among the CD34+ cells, the highest
levels of Flt3 receptor were found on cells coexpressing high amounts
of CD38. Flt3 receptor was also expressed at moderate to high levels on
75% of the mononuclear cell subsets expressing little or no CD34 but
high levels of CD38 (Fig 2D: comparison of the left versus right upper
quadrants). Dual light scatter analysis suggested that these
CD34CD38++Flt3high cells
were monocytes. Confirming this, marrow mononuclear cells strongly
positive for the CD14 monocyte marker coexpressed Flt3 (Fig 3A). Flt3 receptor was not present
either on mature CD15+ granulocytes, or on mature
CD34CD19+ B lymphocytes (data not
shown). Finally, Flt3 receptor was detected on a small percentage of
mature CD34CD14 cells, which were
not further characterized (Figs 2D and 3A).
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| Fig 2.
Analysis of Flt3 receptor expression on human marrow
mononuclear cells using three-color flow cytometry. Cells were stained for CD34, CD38, and Flt3 receptor. A scatter gate was first drawn to
exclude dead cells (R1, not shown). (A) Expression of CD34 and CD38 on
all live marrow mononuclear cells. Two cell populations were defined by
gates: (R2) CD34+ and (R3) CD34. (B)
Expression of CD34 and Flt3 receptor on total marrow mononuclear cells.
Using the gates set in (A), expression of Flt3 receptor versus CD38 was
examined on gated CD34+ cells (C) or CD34
cells (D). Percentages of cells are provided in each plot by quadrant.
Quadrants were drawn based on isotype control profiles (E). The results
shown are representative of three experiments.
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| Fig 3.
FACS analysis of Flt3 labeling of unfractionated marrow
mononuclear cells and purified CD34+ cells. (A)
Expression of Flt3 receptor versus CD14 on unfractionated marrow
mononuclear cells. Cells were first gated to exclude dead cells (not
shown). Quadrants were drawn as shown in (B). In the results of
immunostaining of purified CD34+ cells, expression of
Flt3 receptor was correlated with (C) CD38, or (D) CD117 (kit). For (C)
and (D), a CD34+ cell gate had been set first (not
shown). (E) A blast gate was set (not shown), then a gate was set on
the brightest Flt3+ cells (R1). Expression of CD14 and
CD64 on CD34+ cells gated on R1 (F). The results shown
are representative of two (A) or three (B to F) experiments.
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Flt3 receptor expression on purified CD34+ and
CD34+lin marrow cells.
As was observed above in analyses of unfractionated mononuclear cells,
analysis of purified stem/progenitor cells showed that Flt3 receptor
was clearly present, albeit at low levels, on a subset of
CD34+CD38+ cells (Fig 3C). Fifty-three percent
of cells in the CD34+CD38 cell fraction
expressed Flt3 receptor (ie, in Fig 3C, comparison of the lower
quadrants showed that a total of 1.7% of the CD34+ cells
had the phenotype
CD34+CD38Flt3+, whereas
1.5% were
CD34+CD38Flt3). Of
the CD34+CD38+ cells, 46% expressed Flt3
receptor. Those cells exhibiting the highest level of Flt3 expression
also stained brightest for CD38. CD34+ cells that were low
or negative for kit were also low or negative for Flt3 receptor,
whereas the brightest kit+ cells were Flt3++.
However, those CD34+ cells staining most intensely for Flt3
receptor had an intermediate level of kit fluorescence (Fig 3D). This
population exhibited the light scattering properties of monocytic
cells. Coexpression of CD14 and CD64 confirmed that these
CD34+Flt3+ cells were monocytic precursors (Fig
3E and F). CD34+Flt3+kit+ cells
expressed low levels of CD71, whereas very high levels of CD71 were
observed on a fraction of
CD34+Flt3kit+ cells,
suggesting that they were committed erythroid precursors (data not
shown). Flt3 receptor was also detected on a subset of
CD34+CD19+ B lymphoid precursors (data not
shown).
We further studied the expression of Flt3 receptor on CD34+
bone marrow cells further depleted of the relatively mature cells expressing lineage antigens (ie, CD3, CD14, CD15, CD19, and CD71), hereafter referred to as CD34+lin cells.
The lineage-depleted cells were highly purified (97%
CD34+lin), and enriched in
CD34+CD38 cells (5.2% in the
CD34+lin versus 2.9% in the
CD34+ cell population, data not shown). There were no cells
expressing CD14, CD15, CD19, or CD3 detectable in the
CD34+lin cell fraction (data not shown).
CD71 was expressed in low amounts on some
CD34+lin cells. Lineage depletion did
not change the percentage of cells with the phenotype
CD34+CD38Flt3+
significantly, but the percentage of cells that were
CD34+CD38Flt3
increased from 1.5% to 3.5% (ie, 64% of the cells in the
CD34+CD38lin cell
population were Flt3 compared with 47% of
CD34+CD38 cells, data not shown). In the
CD34+CD38+ cell population, the expression of
Flt3 receptor did not change significantly.
FACS sorting of Flt3high versus Flt3low
CD34 and
CD34+lin bone marrow cells.
CD34+ or CD34+lin bone
marrow cells were sorted by flow cytometry into two populations based
on expression of Flt3 receptor. After gating on CD34+ cells
with low side light scatter and low to moderate forward light scatter
properties ("blast gate"), sorting gates were set to obtain the
30% brightest Flt3+ cells (operationally defined as
Flt3high) and the 30% of cells negative or dimmest for
Flt3 receptor (Flt3low) within the total CD34+
population (Fig 4). The cell population
expressing intermediate amounts of Flt3 was not obtained. For each
population obtained by FACS sorting, a small number of cells was
reanalyzed by flow cytometry (Fig 4C). Dual light scatter analysis of
the reanalyzed cell populations showed that the Flt3low
cells were consistently smaller than the Flt3high cells.
This difference in cell size was confirmed by Wright-Giemsa staining of
cytospins, which in addition showed that
CD34+Flt3high cells also had a broader rim of
cytoplasm than CD34+Flt3low cells (data not
shown).
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| Fig 4.
FACS sorting of Flt3low and
Flt3high CD34+ cells. Cells were stained
using antibodies against CD34, CD45, and Flt3 receptor. (A) Gates were
first set on light scattering (R1) and fluorescence (R2) to acquire
only CD34+ cells with low side light scatter and low to
moderate forward light scatter properties ("blast gate") with the
isotype control staining for these cells shown in the rightmost panel.
(B) With gates R1 and R2 activated, sorting gates were set for
acquistion of CD34+ cells negative or dimmest for Flt3
receptor (Flt3low = R3) and CD34+ cells
brightest for Flt3 receptor (Flt3high = R4). (C)
Reanalysis of CD34+ cells obtained after FACS sorting for
Flt3 receptor expression using the gates shown in (B). No gates were
activated for this reanalysis. Cells obtained by gating on R3
(Flt3low) are shown in the left plot, and cells obtained by
gating on R4 (Flt3high) are shown on the right. The cell
staining profiles in each FACS sorting experiment performed were
similar to those shown here. Gates R3 and R4 were consistently drawn to
each contain 30% of cells within the total population defined by gates
R1 and R2.
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Clonogenic capacity of FACS sorted Flt3high versus
Flt3low populations.
FACS sorted CD34+Flt3high versus
CD34+Flt3low cells were plated in hematopoietic
colony-forming assays. CD34+Flt3high cells gave
rise primarily to granulo-monocytic colonies (38 ± 13 CFU-GM, 16 ± 4 BFU-E per 500 sorted cells, P < .001), whereas CD34+Flt3low cells formed mostly erythroid
colonies (12 ± 7 CFU-GM, 56 ± 12 BFU-E per 500 sorted cells,
P < .001; Fig 5). There was no
significant difference in the numbers of more primitive multilineage
colonies from CD34+Flt3high versus
CD34+Flt3low cells (4 ± 1 and 3 ± 2 CFU-Mix per 500 sorted cells, respectively, P > .1).
In a separate experiment (Fig 5B), the FACS sorted
CD34+linFlt3high cell
population produced more granulo-monocytic colonies than erythroid
colonies (23 ± 13 CFU-GM, 8 ± 6 BFU-E per 500 sorted cells,
P < .004). In the
CD34+linFlt3low population,
few colonies were seen (0 CFU-GM, 2 ± 2 BFU-E per 500 sorted cells,
P < .004). There was no significant difference in the number
of CFU-Mix in the CD34+lin+Flt3high
versus Flt3low populations (4 ± 4 CFU-Mix in the
Flt3low fraction, 4 ± 2 CFU-Mix in Flt3high
cell fraction per 500 cells, P > .1).
In three experiments performed starting with
CD34+lin cells, colonies formed from
CD34+linFlt3low versus
CD34+linFlt3high cells after
14 days in methylcellulose were replated into secondary and tertiary
cultures. The Flt3high and Flt3low cell
populations formed approximately the same number of blast clusters in
2o culture (6 ± 7 blast clusters from
Flt3low, 11 ± 6 from Flt3high cells,
P = .07). In 3o culture, the number of blast
clusters generated was very low (1 ± 1 blast clusters from
Flt3low, 3 ± 1 from Flt3high cells,
P = .07). However, all the plates contained many single blast
cells, which were too numerous to be enumerated. There was no
significant difference in the number of CFU-C formed in 2o
culture (17 ± 10 CFU-C from Flt3low, 23 ± 10 from
Flt3high cells, P = .1) or 3o culture
(2 ± 2 colonies from Flt3low, 3 ± 1 colonies from Flt3high, P = .7).
Cell cycle analysis of FACS sorted cell populations.
We used surface, intracellular, and DNA (SID) analysis to
examine the cell cycle status of purified human CD34+ and
CD34+lin bone marrow cells with respect
to Flt3 receptor expression. This assay, as described by Jordan et
al,38 allows for discrimination between G0 and
G1 stages of the cell cycle. Samples were stained for
Ki67 antigen, which is not expressed in quiescent cells
but is upregulated when cells enter cell cycle. Simultaneous staining with 7-amino actinomycin D (7-AAD; Molecular Probes, Eugene,
OR) was used to measure cellular DNA content. Using these
two parameters in combination with cell surface staining for Flt3
receptor, all phases of the cell cycle were delineated for each cell
population of interest.
As shown for a representative experiment in
Fig 6, SID analysis showed that
CD34+Flt3low cells had a greater percentage of
cells in G0 (41%) compared with Flt3high cells
(1% in G0). The percent cells in G1 was larger
in the Flt3high population than in the Flt3low
cell fraction (75% versus 37%, respectively). There were no
appreciable differences in the percentage of cells in
S/G2/M phases in these three subpopulations (total
CD34+ versus Flt3high versus
Flt3low). However, the proportion of the CD34+
cells that were Flt3high and in G2/S/M phases
was twice as high as those that were Flt3low and in
G2/S/M (10% versus 5%).
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| Fig 6.
Cell cycle status of
CD34+Flt3high versus
CD34+Flt3low cells. (A) Based on 7-AAD
labeling, a gate was set to exclude dead cells (R1). (B) Next, a
"blast gate" was set (R2). (C) With both R1 and R2 activated,
gates were drawn to discriminate Flt3low (R3) from
Flt3high (R4) cells. Histograms of Flt3 receptor (bold
line) and IgG isotype control (dashed line) staining are shown. The
plots in the lower panels show the Ki67 versus 7-AAD staining profiles
for (D) total CD34+ cells, (E)
CD34+Flt3low, and (F)
CD34+Flt3high subpopulations. Cells in the
lower left quadrant of each plot are defined as being in
G0, cells in the upper left quadrant as G1, and
cells in the upper right quadrant as G2, S, or M phases of
the cell cycle. For the three surface marker-defined subpopulations, the percentage of cells in each phase of the cell cycle is provided in
the table. The numbers in parentheses represent the percentages of
cells with the given immunophenotype and cell cycle stage within the
total CD34+ cell population. This experiment was
performed three times with CD34+ cells and twice with
CD34+lin cells. Staining profiles were
similar each time.
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Replicate samples were stained for Flt3 versus PI. Analysis of PI
staining was confirmatory (data not shown).
In vitro expansion of FACS sorted cells in response to FL.
Based on the cell cycle analyses, it was of interest to determine
whether Flt3high versus Flt3low cells
proliferate in response to FL, and whether Flt3low
cells upregulate expression of Flt3 receptor. FACS sorted
CD34+linFlt3high versus
CD34+linFlt3low cells
were incubated in RPMI medium containing 10% FBS and various growth factor combinations. Figure 7 shows
the cell counts in these cultures over 9 days in three independent
experiments.
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| Fig 7.
Effect of various cytokines on the survival and
proliferation of FACS sorted
CD34+linFlt3high versus
CD34+linFlt3low cells in
liquid suspension culture. Ten thousand Flt3high or
Flt3low cells were cultured in RPMI containing 10% FBS and
the designated cytokines, as described in Materials and Methods. Cells
were counted by trypan blue exclusion. (A) and (B) time course of
proliferation. Numbers shown are mean values of three independent
experiments. The combination of FL plus five growth factors is shown
separately in (B), because it would be off-scale in (A). (C) Fold
expansion of sorted cells after 9 days in suspension culture in the
same three experiments. The bars show cell expansion as the mean number of live cells per input cell. (A) ( ) Flt3 low; ( ) Flt3 low + FL; ( ) Flt3 low + FL + KL; ( ) Flt3 low FL + KL + IL-3;
() Flt3 high; ( ) Flt3 high FL; ( ) Flt3 high FL + KL; ( )
Flt3 high FL + KL + IL-3. (B) () Flt3 low; () Flt3
high. (C) ( ) FL + KL, IL-3 + GM-CSF + Epo; () FL + KL + IL-3; () FL + KL; ( ) FL; () medium only. Error bars indicate
the standard errors.
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Cultures of CD34+linFlt3high
cells had approximately constant cell numbers for 6 days in the
presence of FL alone, but cell numbers declined by day 9. In the
presence of FL and KL, cell numbers were maintained for 6 days then
increased twofold from day 6 to day 9 of culture. The addition of IL-3
enhanced the effects of FL and KL, with moderate cell expansion at 6 days of culture and a 2.5-fold increase in cell number at day 9. In
contrast, the numbers of cells in the cultures of
CD34+linFlt3low cells fell
rapidly in the presence of FL alone, FL plus KL, or FL plus KL plus
IL-3 (Fig 7A and C).
Extensive proliferation was observed when either
CD34+linFlt3high and
Flt3low cells were cultured in the combination of FL plus
five other growth factors (GFs; KL, IL-3, IL-6, GM-CSF, and Epo).
Although the cultures of
CD34+linFlt3low cells had a
slightly longer initial lag phase, cultures of both subpopulations had
expanded 100-fold after 9 days of culture (Fig 7B and C).
Cell surface antigen expression of cells generated in suspension
culture.
We also examined the cell surface antigen expression of cells produced
in the above suspension cultures (Table 1).
CD34+linFlt3high cells
cultured in FL plus five GFs developed predominantly into CD14+ monocytes, although a few erythroid cells were also
detected. The expression of Flt3 receptor gradually decreased over time in cultures of Flt3high cells, but a population of cells
expressing Flt3 receptor at moderate levels was still present after 9 days in culture. A similar pattern of downregulation was observed for
kit.
When CD34+linFlt3high cells
were cultured in FL alone, or in FL plus KL, most cells still expressed
Flt3 and kit receptors after 9 days. In fact, a subset of these cells
remained strongly positive for Flt3 and kit receptor. CD34+
cells were not detected after 9 days with any of the GF combinations used.
The numbers of cells in the cultures initiated with
CD34+linFlt3low cells
decreased or did not increase to a significant extent when cultured
with FL alone or FL plus KL, making it impossible to assess the
phenotype of these small numbers of cells.
CD34+linFlt3low cells
cultured in FL plus five GFs produced mainly erythroid cells, which
were kit+, CD71++, and Glycophorin
A++. CD14 was not detected on any cells generated from
CD34+linFlt3low cells.
Interestingly, upregulation of Flt3 receptor was not observed on cells
in cultures started with Flt3low cells by using any
combination of cytokines at any of the timepoints examined. The
expression of Flt3 receptor on cultured
CD34+linFlt3low cells
remained low to negative, whereas the expression of kit receptor
decreased from high levels at day 3 to moderate or negative levels at
day 9.
Clonogenic capacity of
CD34+linFlt3high
versus Flt3low marrow cells after suspension culture.
Cells generated in suspension cultures in the experiments above were
assayed in colony-forming assays (Table 2).
The total numbers of blast cluster-forming cells (see Methods for
definition31) increased from day 6 to day 9 of suspension
culture in the combination of FL plus five GFs for both the initially
cultured Flt3high (sixfold) and Flt3low
(twofold) cells. The total numbers of CFU-C (see Methods
for definition31) formed from the cultured cells derived
from both the
CD34+linFlt3high and
Flt3low cell populations increased dramatically (27-fold
for Flt3high and 37-fold for Flt3low cells)
from day 0 to day 6. At day 9, the total numbers of CFU-C were still
13-fold greater than initial levels for the Flt3high and
threefold for the Flt3low cultures.
In contrast, when Flt3high cells were cultured in FL alone
for 9 days, the total numbers of CFU-C did not change significantly from day 0 to day 9. The number of CFU-C produced by initially Flt3high cells cultured in FL plus KL was lower than with
FL alone, but the formation of blast clusters from initially
Flt3high cells was increased 14-fold by the additional
presence of KL in the suspension culture.
The production of CFU-C and clusters from initially Flt3low
cells cultured in FL alone or FL plus KL declined significantly from day 0 to day 9. This paralleled the decline in the total cell numbers
in these cultures. Both the numbers of CFU-C and clusters from
initially Flt3low cells were increased in the presence of
FL plus KL in contrast to the cultures containing FL alone (10-fold and
4-fold, respectively).
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DISCUSSION |
In this study, we assessed the phenotype and biology of normal human
bone marrow CD34+ cells expressing high versus low levels
of Flt3 receptor. Previously published results have suggested that Flt3
receptor is present on all human CD34+ cells.16
However, biotinylated FL, not antibody, was used to label cells in that
study, and staining intensity was low. The production of MoAbs against
the human Flt3 receptor enabled us to label Flt3+ versus
Flt3 cells and to FACS sort CD34+
progenitor cells into two subpopulations: cells expressing high levels
of Flt3 receptor (Flt3high) and cells with little or no
expression of Flt3 receptor (Flt3low). Cells expressing
intermediate levels of Flt3 were not studied, because our goal was to
compare cell subsets that differed distinctly in Flt3 expression levels
rather than to comprehensively study the entire spectrum of marrow cell
types.
Hematopoietic colony-forming cell assays showed a clear difference
between Flt3high and Flt3low populations.
CD34+Flt3high cells contained mainly
granulo-monocytic colonies, whereas
CD34+Flt3low cells contained erythroid colonies
almost exclusively. This observation is in line with previous
studies showing that FL has no effect on human
erythropoiesis.11,12,16 Flt3 was detected on monocytic precursors (CD34+CD64+CD14+) and B
lymphoid precursors (CD34+CD19+), as well as on
more mature monocytic cells
(CD34CD38+CD14+). This
agrees with reports that FL induces proliferation and differentiation
of monocytic precursors.32 Furthermore, cells generated
from CD34+linFlt3high cells
in liquid culture were predominantly CD14+ monocytes,
whereas cells generated from
CD34+linFlt3low cells were
erythroid (CD71+, Glycophorin A+). The
detection of Flt3 receptor on mature
CD34CD14+ cells is in apparent contrast
to earlier results from our laboratory, in which Flt3 mRNA was not
detected in bone marrow cells that had been twice (doubly) depleted of
CD34+ cells.6 It is possible that, as
progenitor cells mature, Flt3 protein is still expressed on the cell
surface, although Flt3 mRNA has already declined to undetectable
levels. Alternatively, the discrepancy may be caused by differences in
sensitivity of the methods used, or monocytic cells may have been
nonspecifically depleted in the prior study.
It has been postulated that Flt3 receptor is a marker for hematopoietic
stem cells.5-8 Although we could detect Flt3 receptor on a
subset of CD34+CD38 cells, a population
enriched for primitive stem/progenitor cells, the staining intensity
was relatively weak. Levels of Flt3 receptor increased concomitantly
with increasing levels of CD38. In addition, depletion of the more
mature component of the CD34+ cell population (ie, those
CD34+ cells expressing "lineage antigens") resulted
in an enrichment of
CD34+CD38Flt3 cells.
Analysis of FACS sorted cell populations also showed that Flt3low cells were consistently smaller than
Flt3high cells, with only a narrow halo of cytoplasm. Taken
together, these results suggest that the earliest progenitor cells may
lack or have only low levels of Flt3 receptor. To further investigate this hypothesis, we performed cell cycle analyses.
Transplantable murine stem cells expressing Flt3 receptor have
previously been shown to be actively cycling, whereas murine stem cells
negative for Flt3 receptor were predominantly in
G0.29 The same study also showed that the
repopulating capacity of murine Flt3+ stem cells was
significantly less than that of murine Flt3
cells.29 Based on these observations, it was postulated
that expression of Flt3 receptor was stage related on murine
hematopoietic stem cells with a pattern similar to that of
kit.27 Our analysis of the cell cycle status of human
CD34+ (and CD34+lin) marrow
cells showed that CD34+Flt3low progenitor cells
are primarily in cell cycle dormancy, whereas the majority of
CD34+Flt3high cells are actively cycling. We do
not think it is likely that our procedure of labeling cells with
anti-Flt3 MoAbs and FACS sorting activated the cells to begin cell
cycling, because Flt3high cells did not proliferate
detectably in the absence of FL (see below and Fig 7). However, we have
not determined whether one or more of the antibodies used in our study
can activate Flt3 receptor (ie, act as an agonist imitating the effect
of FL) by other tests such as ability to induce proliferation of
transfected cell lines.
FACS sorted CD34+Flt3high cells gave rise
primarily to granulo-monocytic colonies, whereas
CD34+Flt3low cells formed mostly erythroid
colonies. Removal of the relatively mature cells expressing lineage
antigens from the CD34+ cell population before FACS
separation of Flt3high versus Flt3low cells
resulted in depletion of a large proportion of the colony-forming cells, somewhat obscuring this difference (Fig 4). It is not entirely clear from our results whether CD34+ cells expressing high
levels of Flt3 receptor are already definitely committed to
differentiate towards the granulo-monocytic lineage rather than the
erythroid lineage, or if the presence of Flt3 receptor is simply
permissive for differentiation.
When CD34+linFlt3high cells
were incubated in short-term liquid suspension cultures, the cell
numbers in these cultures remained stable over several days in FL alone
and increased moderately in response to FL plus KL. This is in
accordance with the recent observation that FL induces proliferation of
human CD34+CD38 progenitor
cells,41 and can accelerate cell cycling of hematopoietic progenitors.42 Taken together, these results suggest that
Flt3 receptor is expressed on those progenitor cells poised to enter cell cycle and proliferate.43 The numbers of cells in the
cultures initiated with
CD34+linFlt3low cells did
not increase in response to FL alone or FL plus KL. Interestingly, we
did not detect upregulation of Flt3 receptor on Flt3low
cells. This would seem to argue against a stage-related expression of
Flt3 receptor on human hematopoietic progenitor cells. It is possible
that the conditions needed to induce expression of Flt3 receptor on
Flt3low cells (eg, additional perhaps unknown cytokines,
adhesion factors, or bone marrow microenvironmental factors) were not
present in our in vitro system. In a recently published work, primitive
murine lin progenitor cells also failed to
proliferate in response to FL as the sole growth factor.44
The clonogenic output of the FACS sorted
CD34+Flt3high cells after liquid suspension
culture was higher than that of the
CD34+Flt3low cells, no matter what was the
combination of cytokines used in the cultures. The lower clonogenic
output of the Flt3low cells cultured in the presence of FL
alone or FL plus additional GFs may be a reflection of the
unresponsiveness of Flt3low cells to FL. In addition, the
phenotypic analysis of cells generated in suspension culture showed
that a fraction of Flt3high cells remained strongly
positive for Flt3, as well as for kit, when cultured in FL plus KL,
suggesting that at least some of these cells did not differentiate
further. This is in line with previous work showing severely impaired
hematopoiesis in mice deficient for both Flt3 and kit
receptor43 and m |
Abstract
Introduction
Abstract