RAPID COMMUNICATION
Cellular and Molecular Characterization of the Role of the FLK-2/FLT-3
Receptor Tyrosine Kinase in Hematopoietic Stem Cells
By Francis C. Zeigler, Brian D. Bennett, Craig T. Jordan, Susan D. Spencer, Susanne Baumhueter,
Kathleen J. Carroll, Jeffrey Hooley, Kenneth Bauer, and William Matthews
The flk-2/flt-3 receptor tyrosine kinase was cloned from a
hematopoieticstem cell population and is considered
to play
a potential role in the developmental fate of the stem cell.
Using antibodies derived against the extracellular domain
of the receptor, we show that stem cells from both murine
fetal liver and bone
marrow can expressflk-2/flt-3. However,
in both these tissues, there are stem cell populations that
do not express the receptor. Cellcycle analysis showsthat
stem cells that do not express the receptor have a greater
percentage of the population in Go when compared with
the flk-2/flt-3-positive population. Development of agonist
antibodies to the receptor shows aprolierative role for the
receptor in stem cell populations. Stimulation with an agonist antibody gives rise
to an expansion ofboth myeloid and
lymphoid cells and this effect is enhanced by the addition
of kit ligand. These studies serve to further illustrate the
importance ofthe flk-2/flt-3 receptor in theregulation of the
hematopoietic stem cell.
0 1994 by The American Societyof Hematology.
T
long-term bone marrow cultures, further suggesting that flk2/flt-3 may transduce growth signals in hematopoietic stem
cells.'" In this study, we have produced a series of antibodies
to the flk-2/flt-3 receptor that have allowed us to define the
cellular characteristics of hematopoietic populations expressing flk-2/flt-3. Furthermore, we have shown a proliferative
role of flk-2/flt-3 agonist antibodies on the hematopoietic
stem cell. This proliferative capacity is restricted to stem
cell populations and gives rise to an expanded population of
more mature hematopoietic cell types.
HE IMPORTANCE OF signal transduction through receptor protein tyrosine kinases (rptks) in cellular proliferation and morphogenesis hasbeen extensively demonstrated.'.*A role for rptks in hematopoiesis, both at the level
of the undifferentiated stem cell and in committed cell lineages, has been clearly illustrated by signalling through the
c-kit receptor tyrosine kinase and its cognate ligand, kit ligand
A search to uncover other tyrosine kinase receptors that may play a role in hematopoietic stem cell development led to the discovery of a novel receptor tyrosine kinase
designated fetal liver kinase 2 (flk-2): This receptor was
also independently cloned from the placenta by Rosnet et
a15 and termed flt-3. The flk-2/flt-3 receptor tyrosine kinase
shares structural homologywith the subclass I11 receptor
tyrosine kinases such as pdgfr, c-kit, and c-frn~.~.'
Northern blot and polymerase chain reaction (PCR) analysis of flk-2/flt-3 message showed expression largely confined
to primitive hematopoietic cell populations, including stem
cell populations capable of long-term reconstitution of lethally irradiated mice.4 These findings led to the hypothesis
that flk-2/flt-3 isinvolved in the early steps of hematopoietic
cell differentiation and that it may be involved in stem cell
self-renewaL4
Several studies have now shown the capacity of the flk2/flt-3 receptor to signal in a variety of intracellular pathways.6,' Recently, the flk-2/flt-3 ligand has been identified
and demonstrated to increase thymidine incorporation in
early hematopoietic precursors and enhance the formation
of hematopoietic colonies in methylcellulose
Additionally, antisense oligonucleotides directed against the human homologue of flk-2/flt-3 inhibited colony formation in
From the Departments of Molecular Biology, Cell Genetics, and
Immunology, Genentech, Inc, South San Francisco, CA.
Submitted June 3, 1994; accepted July 22, 1994.
Address reprint requests to William Matthews, PhD, Genentech,
Inc, 460 Point San Bruno Blvd, South San Francisco, CA 94080.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8408-0047$3.00/0
2422
MATERIALS ANDMETHODS
Isolation of hematopoietic stem cell populations. Hematopoietic
stem cell populations were isolated from AA4+ cells derived from
midgestation fetal liver as previously described." The AA4+ cells
were fractionated into Sca' and Sca- subpopulations using the Ly6
A/E phycoerythrin conjugate (Pharmingen, San Diego, CA). The
AA4'CD34+ or CD34- populations were derived using an affinity
purified rabbit antimouse CD34.I'
Antimurine flk-2/flt-3 antibodies were raised in rabbits or Syrian
hamsters using a flk-2Mt-3-IgG chimeric protein as immunogen.
Hamster antimurine flk-2 hybridomas IC2-514 and IC2-310 were
derived from Syrian hamster splenocytes fused with murine myeloma P3X63 Ag8U.I . l 3 For fluorescence-activated cell sorter
(FACS) analysis, the IC2-514 monoclonal was purified by protein
A affinity and conjugated to biotin, phycoerythrin, or fluorescein
isothiocyanate (FITC). flk-2/flt-3 specificity of the antibodies was
assessed by flow cytometry analysis of BAF-3 cells transfected with
the full-length receptor. The nontransfected parental line was used
as control (data not shown). The 579A rabbit polyclonal sera and
IC2-5 14 monoclonal antibody were also shown to immunoprecipitate the flk-2/flt-3 receptor from 35S-methioninelabelled transfected
cell lines (data not shown). The antimouse c-kit biotin conjugate
was purchased from Pharmingen and all secondary and Lin cocktail
antibodies were purchased from Caltag (South San Francisco, CA).
Bone marrow hematopoietic stem cells were obtained from 8- to
12-week-old C.57BV6 Ly 5.1 (A20.1) or 5.2 (AL1-4A2) mice. The
mononuclear cell fraction was isolated by density gradient centrifugation (Accudenz; Accurate Biochemicals, Westbury, NY) and
stained with the Lin cocktail antibodies as previously described."
Lin stained cells were removed via magnetic bead depletion (Dynal,
Inc, Great Neck, NJ).I4The Lid" population was then stained using
the appropriate antibodies. Viable cells were selected by propidium
iodide ( 1 pg/mL) exclusion and separated on an Elite flow cytometer
(Coulter Electronics, Hialeah, FL).
Competitiverepopulation. Stem cell populations were isolated
Blood, Vol 84, No 8
(October 15), 1994: pp 2422-2430
ROLE OFFLK-2/FLT-3
IN HEMATOPOIETIC STEM CELLS
from young adult C57 BY6 Ly 5.1 mice. Young adult male C57BU
6Ly 5.2 mice were obtained from the National Cancer Institute
(NCI; Bethesda, MD) and used as recipients. A minimum of five
animals was used per experimental group. Whole body irradiation
(1,100 cGy, 190 cGy/min) was administered as a single dose from
a I3’cs source. One million or 5 X I d bone marrow cells from
the 5.2 mice were used as competitor. Titration experiments were
performed with an AA4+Sca+ stem cell population to ensure we
were in the linear range of the competitive repopulation assay. In
general, the repopulation capacity of I X 10“ cells of the potential
stem cell populations being compared were measured relative to the
competitor population. Cell doses were equivalent to the representation of each population in the fetal liver or marrow. Cells were
administered via tail vein injection and peripheral blood samples
(50 to 100 pL) were obtained via the rem-orbital sinus 4 weeks,
12 weeks, and 6 months postreconstitution. The percentage of Ly
5.1 (A20.1) donor cells was determined by staining with biotinconjugated Ly 5.2 (AL1-4A2) monoclonal (A20.1.7). Briefly, peripheral blood was collected in 10 U/mL heparin, I mmolR. EDTA
in phosphate-buffered saline (PBS) and immediately placed on ice.
Erythrocytes were removed by the addition of 5 v01 2% dextran
T500 in PBS followed by incubation at room temperature for 20
minutes. The red blood celldepleted supernatant was then centrifuged for 5 minutes at approximately 200g. The pellet was resuspended in 450 pL dH20on ice followed immediately by the addition
of 50 pL 1.5 m o a NaCl and 500 pL ofPBS/IO% fetal bovine
serum (PBSFBS) and centrifuged as before. The resultant pellet
was resuspended in PBSFBS before staining. This procedure removed nearly 100% of the erythrocytes (the remaining red blood
cells were excluded by size gating) while leaving the leukocytes
95% viable by propidium iodide exclusion. FACS staining of Ly
5.2 peripheral blood mononuclear cells prepared by density gradient
centrifugation for Ly 5.2 antigen was nearly identical to cells prepared by lysing. Controls for the specificity of Ly 5.2 staining included naive Ly5.1 cells (negative control), naive Ly 5.2 cells
(positive control), and cells from animals repopulated with Ly 5.2
competitor cells only. To confirm repopulation by the donor Ly 5.1
cells in all lineages, peripheral blood cells and the bone marrow
mononuclear fraction were stained in conjunction with Ly 5.2 for
the following antigens: B220 (B-cell lineages), CD4/8 (T-cell lineages), and Gr-]/Mac-l (myeloid lineages).
The relative repopulating ability of each putative stem cell population was compared by calculating repopulation units (RU) as described by Harrison et aI.l5 Each RUrepresents the same repopulating ability as 1 X ld fresh marrow cells. Harrison et all’ have
confirmed that, for long-term repopulating stem cells, the stem cell
content of fresh marrow is 1 stem cell per ld total marrow cells.
Therefore, the number of RU from each donor population is defined
by the following equation”: % donor repopulation = 100(RU/RU
+ C); where C is the number of fresh competitor marrow cells/l@
and RU = %(C)/(100 - %).
Cloning of murineflk-2/flt-3 receptor. The murine flk-2/flt-3 receptor was cloned by reverse transcriptase-PCR (RT-PCR) from
RNA isolated from midgestation mouse fetal livers. Six sets of overlapping primers were designed to the murine flt-3 sequence? These
primers were designed to amplify three segments of the gene, nucleotides 1-1307, 1308-1992, and 1993-3096. The PCR products were
then subcloned into pRK5.A. The sequence of the full-length flk-2/
flt-3 gene was identical to the published murine flt-3 gene. A flk-2/
flt-3-IgG extracellular domain-IgG fusion gene was constructed and
fusion protein produced as previously described.I6
Assays for agonistic antibodies. The B M - 3 cell line was electroporated with the full-length murine flk-2/flt-3 receptor as previously described.” Tyrosine phosphorylation experiments were performed as previously described.’* Briefly, B M - 3 cells were
2423
incubated with hybridoma supernatant or polyclonal antisera diluted
1:20 for 30 minutes, lysed in IP lysis buffer (1% N
P
4
,1 mmoVL
EDTA, 200 mmoyL NaCI, 50 mmoVL TrisCI, pH 8.0, 2 mmol/L
phenylmethylsulfonyl fluoride [PMSFI, 2.5 mmoyL Na3V04), and
immunoprecipitated with 579A antimurine flk-2/flt-3 polyclonal
sera. Immunoprecipitated lysates were separated on a 4% to 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gradient gel and analyzed by antiphosphotyrosine Western
blot. For the thymidine incorporation assay, B M - 3 cells were
starved of interleukin-3 (IL-3) for 24 hours and then stimulated with
antibody overnight, followed by an 8-hour pulse of 1 pCi [’H]
thymidine. Thymidine incorporation was then determined using a
cell harvester. After the discovery of IC2-310 agonist activity, the
hybridoma supernatant was purified by protein-A affinity chromatography. The purified antibody retained activity at concentrations between 10 and 40 pg/mL. We routinely used 40 pg/mL in all subsequent in vitro assays.
Colony assays. Standard myeloid methylcellulose colony assays
were performed using complete methylcellulose medium (M3430
Stem Cell Technologies, Inc, Vancouver, British Columbia, Canada)
with the addition of 50 ng/mL KL (R & D Systems, Minneapolis,
MN). Colonies were counted after 10 days in culture; only colonies
of greater than 50 cells were scored. Lymphoid colonies were produced using base methylcellulose (Stem Cell Technologies) with 50
ng/mL KL and 50 ng/mL murine L 7 (R&D system^).'^ Cytospin
analyses of the resultant colonies were performed as previously described?’
Stromal cell production. Fetal liver stromal cell lines were isolated by infecting primary cultures of fetal stroma with the recombinant retrovirus Sv40tsA58 as previously described?’ One of the
resultant stromal cell lines, designated 7-4, was used in these experiments.
Suspension culture assays. Hematopoietic stem cell populations
were seeded at 10‘‘ cells/mL in Dulbecco’s modified Eagle’s medium
(DMEM)/F12 media supplemented with IGF-1 at 10 ng/mL (Genentech, Inc, South San Francisco, CA), KL at 50 ng/mL(R & D
Systems), PDGF-BB at 2 ng/mL,and bFGF at 25 ng/mL (Boehringer
Mannheim, Indianapolis, IN).
Stromal cell/stem cell coculture assays. Hematopoietic stem cell
populations were seeded at 10‘‘ cells/mL on the fetal liver stromal
line 7-4 in DMEM/F12 media supplemented with 10% fetal calf
serum (FCS). Cocultures were incubated at 37°C for 7 days and the
contents of each well were then removed for analysis. The results
obtained from each in vitro assay system were confirmed in a minimum of three independent experiments. Growth factors were used
at the following concentrations: KL at 50 ng/mL; IL-3 at 1 ng/
mL (Genzyme, Cambridge, MA); granulocyte-macrophage colonystimulating factor (GM-CSF) at 0.2 ng/mL (R & D Systems); PDGF
B/B at 2 ng/mL, and bFGF at 2.5 n g / d (Boehringer Mannheim).
Cultured hematopoietic cells were phenotyped by staining using
direct antibody conjugates to the following antigens: CD4, CD8,
MAC-I, GR-1, and B220 (Caltag Inc, South San Francisco, CA).
All antibodies were titered against peripheral blood white blood
cells and controls for specificity included irrelevant isotype-matched
direct conjugates at similar dilutions. Staining profiles were also
obtained in the presence of an FcR blocking antibody (Pharmingen).
Cell cycle analysis. Two-step acridine orange staining was performed as detailed previously?223Briefly, cells that had been sorted
after dual-parameter immunofluorescence staining were centrifuged
and resuspended in RPM1 1640 cell culture medium with 10% FBS
at a finalconcentration of l@/mL.To 0.3 mL of this cell suspension,
a solution consisting of 0.45 mL of 0.1% Triton X-100 in 0.15 N
NaCl and 0.08 N HCI was added and the mixture was incubated for
45 seconds on ice. To this mixture, 1.8 mL of a solution consisting
of 12 pm acridine orange (Polysciences, Inc, Warrington, PA) in
2424
ZEIGLER ET AL
0.2 mom NaZHP04,0.1 mom citricacid,
molL Na-EDTA,
and 0.15 mom NaCl was added and the sample was immediately
analyzed by flow cytometry. Green fluorescence(DNA content) was
collected through a 560-nm dichroic long-pass filter coupled with a
525 2 15 nm bandpass while red fluorescence(RNA) was simultaneously collected through a 630-nm long-pass filter.
The G, subpopulation was defined on the basis of the red fluorescence (RNA content) of peripheral blood mononuclear cells (PBMC)
stained in parallel to the previously sorted samples. A cursor was
placed at the position corresponding to the red fluorescence intensity
of 97% of PBMC, with cellshavinghigher RNA contentsabove
this position classified as cycling (ie, G , , S, and G2M) populations
and those at or below the cursor classified as G,,.2' Enumeration of
the proportions of cycling cells was performed by conventional cell
cycle analysis using the algorithm of Dean and Jettz4 available in
the Multicycle software (Phoenix Flow Systems, San Diego, CA).
RESULTS
Characterization ofJIk-2@t-3expression in hematopoietic
cell populations. Investigation of flk-2/flt-3mRNA expression showed that flk-2/flt-3 was preferentially expressed in
primitive hematopoietic cell populations. However, it was
unknown whether the flk-2/flt-3 receptor was expressed on
hematopoietic stem cells capable of long-term engraftment
of lethally irradiated hosts. The production of hamster monoclonal antibodies raised against the murine flk-2/flt-3 receptor allowed this question to be addressed. Additionally, to
characterize further murine hematopoietic stem cell populations, an affinity-purifiedrabbit antimurine CD34 polyclonal
antibody was raised as previously described."
The stem cell expression of CD34 and flk-2 was characterized using previously defined stem cell populations. In the
fetal liver, the AA4.1 antibody (AA4) delineates a population
of approximately 1% of the cells in which all the totipotent
stem cell activity resides." The AA4+ population can be
further enriched using antibodies to the Sca-l antigen (Ly6A/
E). TheAA4+Sca+ population contains allthe totipotent
stem cell activity: Experiments using the CD34 polyclonal
antibody demonstrated that60% of the AA4' cells are
CD34+ and that all AA4+CD34+cells are positive for c-kit
expression. Furthermore, all AA4'Sca' cells are also CD34+
(data not shown). Repopulation studies showed that all the
long-term reconstituting stem cell activity resides in the
AA4+CD34+kit+population and that the AA4+CD34-kit+
population does not repopulate (Table 1). Similarly, all the
colony-forming potential in methylcellulose assays was
found in the AA4+CD34+ki+fraction (30 colonies per 1 X
IO3 AA4+CD34+kit+cells plated v 0 colonies per 5 X lo3
AA4+CD34-kit+ cells plated).
We used the AA4+Sca+ and the AA4+CD34+populations
to investigate stem cell expression of the flk-2/flt-3 receptor
(Fig 1). FACS analysis of these populations showed that all
of the stem cell populations could be subdivided into flk-2'
or flk-2- fractions (Fig I). Repopulation studies demonstrated that the flk-2hIt-3 receptor can be expressed on stem
cells but that both flk-2/flt-3-positive and flk-21flt-3-negative cell fractions give rise to long-term reconstitution (Table
1). In experiments using bone marrow as the source for stem
cells, the mononuclear fraction was segregated into a Linl"
population by immunomagnetic bead separation using the
Lin cocktail of antibodies to mature hematopoietic cell
types.I4 The resulting Lid" population contained both Lin'"
and Lin- cells by FACS analysis; however, for simplicity,
it will be referred to as Lid". The Lid" mononuclear cells
were fractionated into a Lin'OSca' stem cell fraction (Fig
1). In accordance with the fetal liver populations, this bone
marrow stem cell population also gave rise to both flk-2/flt3-positive or flk-2Mt-3-negative subpopulations. Both of
these subpopulations gave rise to long-term repopulation
(Table l). The Lin'"CD34' bone marrow population also
gave rise to long-term repopulation (data not shown).
To compare the relative repopulating abilities of each of
the different donor populations from the same experiment,
RU were calculated as described in Materials and Methods
(Table l). These experiments showed the high stem cell
content of the AA4+CD34+kit+and AA4+Sca' subpopulations. Interestingly, in both the AA4+CD34+ fraction from
fetal liver and the Lin'"Sca+ fraction from bonemarrow,
the flk-2- cell populations had a statistically significant increase in RU when compared with the relevant flk-2+ population (AA4+CD34+flk-2. > AA4+CD34'flk-2+ P 2 .05;
Lin'"Sca+flk-2~2 Lin'OSca'flk-2+ P 2 .001; Table 1).
Cell cycle analysisofJIk-2/@tt-3-expressingstem cell populations. Cell cycle analysis of hematopoietic stem cell
populations has previously indicated that stem cell populations are heterogeneous in relation to cell cycle s t a t ~ s . ~ ~ ~ * ~
Furthermore, enhanced repopulation has been attributed to
those stem cells in the G&, phase compared with the actively proliferating S/G2Nsubset.26If flk-2Mt-3 expression
on stem cells represents a potentially more committed stem
cell population, exhibiting decreased repopulation capacity,
this may be reflected in the cell cycle status. Additionally,
it has been suggested that flk-2/flt-3 mRNA expression is
correlated with cycling stem cell^.^'^^^ Therefore, we determined the cell cycle status of the fetal liver stem cell population AA4+Sca+ with respect to flk-Uflt-3 expression (Fig 2).
We used a two-step acridine orange staining technique to
differentiate G, from G, phase cells." When coupled with
conventional cell cycle analysis of the DNA content histogram, this method allows for the simultaneous analysis of
the G,,, G , , S, and G2M cell cycle phase compartments of
any cell population. Using this technique, we showed that a
major difference between these two subpopulations was the
greatly increased percentage of cells residing in Go in the
AA4+Sca+flk-2- population (Fig 2). Additionally, the percentage of cells in S/GzN phase of the cell cycle argues
that these two fetal liver stem cell populations contain many
actively cycling cells. Similarly, the bone marrow stem cell
population, Linl"Sca+,was also found to contain a greater
percentage of Go cells in the flk-2- population (Lin'"Sca+flk2-G0 [39%] v Linl"Sca+flk-2+G0[26%]).
Cell cycle analysis of the AA4+kit' stem cell populations
demonstrated a much lower percentage of cells in Go when
compared with the AA4'Sca+flk-2- population. However,
little difference was found between the AA4'kit+flk-2+ (GO
[12%], G, [39%], S [41%], GJM [8%]) and the AA4'kit'flk2- (G,, [7%], G, [47%], S [52%], G2/h4,[5%1) populations.
Development of agonist antibodies to theJIk-2@-3 receptor. We approached functional studies of the biologic role
OF
2425
ROLECELLS
FLK-2ffLT-3STEM
IN HEMATOPOIETIC
Table 1. Competitive Rupopulation Assays of Purified Cell Populations
12 wk
4 wk
RU
% 5.1
Cell Population
72 2 3
6823
41 t 12
58 2 18
75 2 4
2 2 2
50 2 8
57 2 12
83
89
24
25
4
84
40
53
~
7
30
0
10
13
RU
~~~~~
90
92
66 2 10 68
77 +- 8
25
70
23 2 6
26
2 10 67
13
11
96 5.1
RU
% 5.1
~
AA4'Sca+flk-2'
AA4'Sca'flk-Z(-)
AA4+CD34+flk-2+
AA4'CD34+flk-2(-)
AA4TD34'kit'
AA4+kitfCD34(-)
LinioSca+flk-2+
LinioSca+flk-2(-)
24 wk
24
41
10
17
39
3
7
11
22
22
2 9
84 2 4
23
423
24 2 5
2 12
45
57
11
26
23
0
3
20
Fetal liver and bone marrow stem cell populations were isolated as described. All AA4+ populations were from the fetal liver and all Lin'"
populations were from the bone marrow. Long-term repopulation by fetal liver- or bone marrow-derived populations was determined using
competitive repopulationanalysis of genetically marked fetal liver or bone marrow in C57BU6 mice allelic at the Ly 5 locus, designated Ly 5.1
(A20.1) and Ly 5.2 (AL1-4A2). Five animals were used per stem cell populationas described in Materials and Methods. Ly 5.1 expression in the
Ly 5.2 mice was determined 4, 12, and 24 weeks postengraftment. Only the 24-week time point isshown. RU were calculated as described in
Materials and Methods. Contribution of thestem cell populations to all lineages was determined by staining both peripheral blood leukocytes
and bone marrowcells for the following
antigens: 8220. CD-4/8, and Gr-1MAC-1. These experiments showed that alllineages were reconstituted
by donor5.1 cells (data not shown). Additionally, no preferential reconstitution of any lineage by the different donor
populations was observed.
C
BM
F
T
CD-34
c-kit
flk-2
Fig l. Fractionationof fetal liver and bone marrow stam cell populations. Fractionation of AA4 cells from day 14 gestation fetal liver. AA4+
cells were enriched by immunapanning. AA4+ cells were subsequently stained using antibod- against Sea-1, CD34, flk-2, and okit. AA4*
calls were stained for (A) flk-2 and Sca-l; IB) c-kit and CD34; IC)flk-2 and CD34; and (D) flk-2 and *kit. Fractionation of tin" bone marrow
progenitor cells with antibodies against Sca, flk-2, c-kit, andCD34. Lin* bone mamow progenitor cells wereisolated by indirect magnetic bead
panning. The tin cocktail comprised RA3-6B2,YTS 191.1, YTS 169.4,8C5, ?ER-119. M1/70.15, and CG16. The Lin" bone marrow cells were
and flk-2 (shown as a dot-plot because of the very small population of LinbSca+flk-2- cells in the
stained for (E)flk-2 and CD34 and (F) %-l
marrow). These experiments were repeated a minimum of four times and gave similar staining profiles on each occasion.
2426
ZEIGLER ET AL
,ml
101
102
10-1
Sca-FITC
27.6% GO
5.1% G1
51.7% S
15.6% @M
I'
0
l o o 101201
Sca-FITC
103
I
I
200
400
600
800
RED FLUORESCENCE (RNA CONTEND
loo0
Fig2. Cell cycleanalysisofhematopoieticstemcellpopulations.Dual-parameterfluorescencehistograms
of AAI'Sca+flk-2+-enriched
(upper left) and AA4+Sca+flk-2+-enriched (upper right)
stem cell populationsafter cell sorting. The lower panels illustrate corresponding red
fluorescence histograms obtained after acridine orange for AA4+Sca+flk-2- (lower
left) and for AA4+Sca'flk-2+ populations(lower right). The
used to discriminate
(lower fluorescenceintensity)fromcycling cells (see Materialsand
cursorillustratedthefluorescenceintensity
Methods). The percentages of cells in each phase of the cell cycle is providedin the inset.
of the flk-2/flt-3 receptor using antibodies raised to the extracellular domain of the receptor. A polyclonal antibody
(579A) was raised in rabbits by producing a flk-2/flt-3-IgG
extracellular domain fusion protein and using this as antigen.
The 579A polyclonal antisera was subsequently shown to
be capable of activating tyrosine phosphorylation of the flk2/flt-3 receptor (Fig 3). Therefore, the flk-2-IgG fusion protein was used to raise monoclonal antibodies in Syrian hamsters. The hybridomas resulting from the subsequent fusions
were screened for the ability to activate the flk-2/flt-3 receptor.
Agonistic activity of these resultant hybridomas was determined using two assay systems. A phosphotyrosine assay
using the full-length murine flk-2/flt-3 receptor expressed
in the IL-3-dependent cell line BAF-3" and a thymidine
incorporation assay using L-3-dependent BAF-3 cells expressing the full-length receptor.
In the phosphotyrosine assay, the transfectednontransfected BAF-3 lines were treated with various antibodies
followed by immunoprecipitation of the flk-2/flt-3 receptor
using the 579A polyclonal antibody. Immunoblotting of the
immunoprecipitated material using antiphosphotyrosine antibodies demonstrated the phosphorylation of the flk-uflt-3
receptor in response to both IC2-310 or 579A (Fig 3). The
phosphorylated flk-2Mt-3 receptor migrated at an apparent
molecular weight of approximately 160 kD. Similar molecular weight values for the full-length receptor have been obtained in other st~dies.6,'~
In the thymidine incorporation
assay, cells were starved of E - 3 for 24 hours andthen
incubated with antibody. Both 579A and IC2-310 gave a
significant stimulation of thymidine uptake in the transfected
BAF-3 cells (Fig 4). The specificity of these responses was
demonstrated by the lack of response to irrelevant antibodies
and to hamster IgG.
Hematopoietic assays of the jlk-2r agonist monoclonal
antibody IC2-310. To assist in the evaluation of the biologic function of the IC2-310 agonist antibody, we developed
a Dexter culture assay system using immortalized stromal
cell lines from the fetal liver (see Materials and Methods).
Stem cell populations were plated onto the fetal liver stromal
line 7-4. The stem cell content was determined by competitive repopulation analysis prior to and after 7 days of coculture. After 7 days of coculture the resulting cell populations
could only sustain short-term repopulation of the irradiated
host as evidenced by contribution of donor cocultivated cells
at 4 weeks postengraftment (data not shown). However, after
this early time point no further contribution from the cocultivated cells was observed.
OF
2427
ROLECELLS
FLK-2FLT-3
STEM
IN HEMATOPOIETIC
Fig 3. Phosphotyrosine assay
usingflk-2lflt-3 receptor transfected into IL-3-dependent cells.
Transfected BAF-3 cells (BAF-3T)
and nontransfected BAF-3 cells
(BAF-31were starved of 11-3 and
treated as indicated below.Cells
were lysed, immunoprecipitated
with 579A polyclonal sera, and
run out onSDS-PAGE 4%t o 12%
gradient gels. Gels werethen
transferred t o nitrocellulose and
Western blotted
using
the
antiphosphotyrosineantibody4610.
(AI No treatment of BAF-3T; (B)
579A polyclonal sera on BAF-3T;
(C) IC2-310monoclonal antibody
on BAF-3T; ID) lC2-334 antihuman flk-2 on antibody BAF-3T;
(E) notreatment of BAF-3; (F)
579A polyclonalon BAF-3; (G)
IC2-310 monoclonalon BAF-3;
(H) lC2-334 antihuman
flk-2
monoclonal antibody on BAF-3.
A B C D E F G H
97kD
-
Cocultivation upon the 7-4 stromal cell line gave rise to
a dramatic expansion in cell number (Table 2). Lineage analysis of the resultant cell populations was performed using
flow cytometric analysis and Wright-Giemsa staining of cytospin material. These analyses showed the presence of immature progenitor, myeloid, and lymphoid cells (Table 3).
Stem cells plated in the presence of the IC2-310 agonist
antibody gave rise to a greater proliferative event than seen
on 7-4 alone. However, IC2-3 I O did not induce proliferation
1-
Baf3-T
Fig 4.
Baf3
Thymidineincorporation assay usingflk-2/flt-3 receptor
into
IL+dependent cell
line
3 2 ~ cells
.
were darved ~f
11-3 overnight and then stimulated for24 hours and thymidine incorporation was determined. Both transfected BAF-3 cells (BAF-3T) and
the parent BAF-3 cell line were stimulatedwith (A) media alone, (B)
rabbit preimmune sera, (C) 579A polyclonal antibody, and (D) 1C2310 agonist antibody. Assays wereperformed in duplicateand repeated in three independent experiments.
of the non-flk-2/flt-3-expressing stem cell populations or
the non-stem cell populations AA4+Sca-. The lack of effect
on the AA4'Sca- population is noteworthy because we can
detect low levels of flk-2/flt-3-positive cells in this population (data not shown). Furthermore, IC2-310 had no effect
on the non-flk-2/flt-3-expressing stem cell populations
(Table 2). FACS analysis of these cells again demonstrated
the presence of several potential lineages (Table 3). As with
cells grown on 7-4 alone, the IC2-310 stimulated cells were
only capable of repopulating in the short term (data not
shown). The proliferative event enhanced by the IC2-310
antibody was greatly increased in combination with KL (Table 2). Cytospin analysis of the cocultivated cells showed a
significant decrease in the percentage of blast cells with a
concomitant increase in the percentage of cells from the
myeloid lineages, including myeloblasts, myelocytes, promyelocytes, or metamyelocytes (Table 3). Collectively, these
data show the overall proliferation resulting from stimulation
of the flk-2/flt-3 receptor. Furthermore, they illustrate that,
in the context of cocultivation on the 7-4 stromal cell line,
this proliferative event was accompanied by differentiation
to more mature hematopoietic phenotypes.
To develop stroma-free suspension cultures, we investigated the ability of various growth factor combinations to
support the growth of hematopoietic cells in the absence of
stroma. We found that a combination of IGF-I, bFGF, KL,
and PDGF-BB was a very potent stimulator of multilineage
expansion of stem cells cocultivated on 7-4, with expansion
exceeding 200-fold over 7 days. Suspension cultures performed in the presence of these factors, without stroma, gave
rise to a 41- 2 IO-fold expansion in cell number. The addition
of IC2-3 10 to this growth factor cocktail increased expansion
to 85- 2 12-fold. These resultant cells Once again represented
multiple lineages as assessed byFACS analysis (data not
shown). However, Stem
plated in suspension with IC2310 alone did not give rise to viable cultures.
2428
ZEIGLER
AL
Table 2. Cell Proliferation Assays on Fetal Liver Stroma
~
Control
Cell Population
IC2-310
33 t 14
32 :t 3
8 t 0.71
6 t 0.71
12 2 2.6
25 2 5
12 2 3
52 2 7
AA4+kitiflk-2'
AA4'kittflk-2( )
AA4+Sca '
AA4 'Sea( - )
AA4'CD34'kit'
AA4+CD34+flk-2'
AA4+CD34+flk-2(~
)
Lin''CD34'flk-2'
KL
52 ir 2
28 i 1
31 t 2.1
13
6 t 0.84
22 t 2.3
52 If- 11
14 5 5
173 t 38
210 i 18
71 i 12
120 -t 14
12 t 0.35
95 i 6.1
KL
+ IC2-310
276 t 12
84 t 5
180 i 13
5 0.35
129 t 7
Stem cell populations were isolated as described. Ten thousand cells from the relevant stem cell population were plated onto 7-4 stromal
line and incubated at
37°C. After 7 days, hematopoietic cells were removed from the stroma and cell counts were performed.
Data are presented
as fold expansion of the initial10,000 cells. Assays were performed in duplicate wells and repeated in three independent experiments. Control
wells were media containing hamster-lgG at 40 pg/mL.
Effect of IC2-310 monoclonal antibody on methylcellulose
colony formation. Hematopoietic colony assays were performedtodetermine theeffects of theIC2-310 antibody
on the colony-forming potential of primitive hematopoietic
populations. The methylcellulose assays were performed in
the presenceof WEHI-conditioned media supplemented
with
KL to test the myeloid potential ofthe inputcell"' or, alternatively, in thepresence of IL-7 and KL to test the B-lymphoid
potential."
Stem cell populations plated directly into methylcellulose
with the addition of IC2-310aloneshowed no induction
Table 3. Lineage Analysis From Cell Proliferation Assays
Cytospin Analysis
of colony growth. However, IC2-310 did promote a small
increase in methylcellulose colony formation when added in
combination with WEHI-conditioned media compared with
the use of WEHI-conditioned media alone (135 -t 3 v 104
-+ 7 per lo3 cells plated). The most dramatic increases in
colony formation using IC2-310 were observed when stem
cells were first cocultivated on the stromal cell-line 7-4.
Hematopoietic stem cell populations plated onto 7-4 cells
and then removed after 7 days were capable of yielding both
myeloid colony-formingcells(CFC)andlymphoidCFC.
When thecocultivation on 7-4 was
performed in the presence
of IC2-310 agonist antibody there was an approximate 10fold increaseinmyeloid
CFC and aninefold increase in
lymphoid CFC (Fig 5). Cytospin data showed the myeloid
colonies to be of mixed lineage but principally they represented the granulocyte/macrophage subset (datanot shown).
%
AA4 Sea~
alone
30
Media
IC2-310
52
IC2-310/KL
IC2-31O/GM-CSF
IC2-310/KL/GM-CSF
% Blasts
% lg MNC
% Myeloid
Lymphoid
28
10
15
12
3
47
13
35
31
23
47
52
70
12
8
3
2
4
myeloid
mycloid+lC2-310
FACS Analysis
AA4 'Sea+
Control
310
% MACl+
65
76
Yo
% Gr-l'
64
46 63
% 8220'
CD4iCD8'
11
22
36
U.
U
200
Cytospin differentials from the AA4'SCAI
stem cell population after
cocultivation with stromal cell line 7-4 for 7 days.Cells were coculti100
vated in the presence of the growth factors indicated.
"Blasts" are
cells of immature phenotype characterized by intermediate to high
nuclear to cytoplasmic ratio with basophilic nuclei and pale cytoof intermediate
plasm. Large mononuclear cells (Lg MNC) are cells
0
size and low nuclear to cytoplasmic ratio. The majority of these cells
show pale to light blue cytoplasm and coarsely reticulated chromatin.
Some of these cells are smaller, more basophilic, and more clearly
Fig 5. Hematopoietic cells were seeded into methylcellulose after
plasmacytoid."Myeloid"
are cellsofmaturemyeloidlineages.
being cocultivated on the7-4 fetal liver stromal line for 7 days in the
"Lymphoid" are cells displaying lymphocyte or plasma cell characterpresence or absence of IC2-310 agonist antibody. Themethylcellulose
istics. For FACS analysis, cells from all control wells or all IC2-310cultures were established under conditions favoring either myeloid
treated wells were pooled and stained using antibodies against MAC- or lymphoid colonies as described in Materials and Methods. Colo1 Gr-l, B220, and CD4/8. These data are presented from one represennies were then scored for CFC after 10 days in culture (see inset).
a minimum of
tativeexperiment.Theexperimentswererepeated
Assays were performed in triplicate and repeated in three independent experiments.
three times.
2429
ROLE OF FLK-2FLT-3 IN HEMATOPOIETIC STEM CELLS
Analysis of the colonies produced in the presence of IL-7
and KL demonstrated a B220+IgM- phenotype. Once again,
the proliferative effect of IC2-310 was restricted to the stem
cell population. No effect was seen on the AA4'Sca- cell
population that does not contain stem cells (Fig 5). These
results support the observations that the IC2-310 antibody
is only capable of stimulating proliferation of hematopoietic
cell populations containing early progenitors.
DISCUSSION
Hematopoiesis is dependent on the capacity of the hematopoietic stem cell to self-renew and commit to a pathway of
differentiation. In recent years, progress has been made in
identifying some of the molecules that are involved in these
events3' The flk-2Mt-3 receptor was cloned from an enriched
stem cell population and it was speculated that it may play
a role in determining the early developmental fate of the
stem cell: However, previous studies of this receptor have
all determined expression at the level of the &A.
The
development of monoclonal antibodies that recognize the
native receptor allowed us to examine the expression of this
molecule on stem cell populations.
Reconstitution experiments of lethally irradiated mice
show that the flk-2/flt-3 receptor tyrosine kinase is expressed
on stem cell populations, but, quite clearly, not all stem cells
express flk-2/flt-3. This findingwas confirmed in all the
different fetal liver and bone marrow stem cell populations
isolated. Cell cycle analysis of the stem cell populations
from fetal liver and bone marrow demonstrated that the flk2- stem cell fractions had significantly fewer cycling cells
than the corresponding flk-2+ fraction. Furthermore, in both
the AA4+CD34' and the Lin'"Sca+ populations, the flk-2+
subpopulations had significantly less repopulating capacity
than their flk-2- counterparts. These data suggest the hypothesis that the flk-2/flt-3 receptor is expressed by a subset of
hematopoietic stem cells destined to differentiate to more
committed progenitor cells. This hypothesis gains support
from studies that have demonstrated decreased radioprotective capacity in cycling stem cellsz6and from the expression
of flk-2/flt-3 mRNAin stem cell fractions believed to be
actively
The mostwidely used marker for the study of human
hematopoietic cells is cell surface expression of CD34. Various functional assays have shown that the CD34' subpopulation from human marrow contains virtually all primitive hematopoietic ~ e l l s . ~The
' . ~ ~monospecific polyclonal antibody
to murine CD34 clearly demonstrated that, in accordance
with the human homologue, the stem cell activity in murine
hematopoiesis is confined to the CD34' fraction. Therefore,
the phenotype of the murine hematopoietic stem cell from
the fetal liver is AA4+Lin1"Sca+CD34+kit+flk-2'.From the
bone marrow, the phenotype of the stem cell appears to be
Lin'"Sca'kit+CD34+flk-2'.
From the current experiments, it is clear that activation of
the flk-2/flt-3 receptor promotes the proliferation and differentiation of hematopoietic stem cells when they are cocultivatedwith stroma. This proliferation is most clearly evidenced by the increases in both cell number and CFC
obtained on activation of stem cells with the IC2-3 10 agonist
antibody. Conversely, the agonist antibody has little effect
on non-stem cell populations. In these experiments, activation of flk-2/flt-3 expressing stem cells does not lead to
an increase in the number of cells capable of long-term
reconstitution. However, it is possible that the assays used
on the stromal cell line 7-4 initiate a differentiation program
that could prevent self-renewal of the most primitive stem
cell. We are currently addressing the effects of the agonist
antibody in vivo, where the ability of the stem cell to self
renew should not be compromised.
Recently, the cognate ligand of the flk-2/flt-3 receptor has
been ~ l o n e d .Based
~ . ~ on thymidine incorporation studies and
methylcellulose assays, the flt-3 ligand induces proliferation
of fetal liver and bone marrow primitive hematopoietic population~.'.~Aswith IC2-310, the proliferative effects are
greatly enhanced in cooperation with KL. It is interesting to
note that we see greater proliferative effects of KL on the
AA4+kit+flk-2+stem cell population than on its flk-2- counterpart. These results may indicate that the expression of flk2/flt-3 and c-kit on stem cell populations represent potentially
different stages of stem cell development. However, with
the current availability of reagents to activate these receptors,
the opportunity exists to dissect the early proliferative events
of the stem cell. Such information should prove valuable for
the biology of hematopoiesis as well as for clinical transplantation therapy.
ACKNOWLEDGMENT
We thank Dr David Goeddel and Dr Tim Austin for critical comments on the manuscript. We also gratefully acknowledge Christopher Donahue and James Chin for expert flow cytometry. The patient
and expert assistance of Sunita Sohrabji in preparing the manuscript
is greatly appreciated. We also thank Linda Werner and Gabrielle
Hatami for assistance with the cytospin analyses.
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