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Expression of Growth Factor Receptors in Unilineage Differentiation
Culture of Purified Hematopoietic Progenitors
By U. Testa, C. Fossati, P. Samoggia, R. Masciulli, G. Mariani, H.J. Hassan, N.M. Sposi, R. Guerriero, V. Rosato,
M. Gabbianelli, E. Pelosi, M. Valtieri, and C. Peschle
We have evaluated the expression of growth factor receptors (GFRs) on early hematopoietic progenitor cells IHPCS)
purified from human adult peripheral blood and induced in
liquid suspensionculture t o unilineagedifferentiationlmaturation through the erythroid (E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol lineage. The receptors for
basic fibroblast GF (bFGF1, erythropoietin (Epo), thrombopoietin (Tpo), and macrophagecolony-stimulating factor (MCSF) have been only assayed at mRNA level; the majority
of GFRs have been evaluated by both mRNA and protein
analyses: the expression patterns were consistent at both
levels. In quiescent HPCs the receptors for early-acting [fk3
ligand (FL), c-kit ligand (KL), bFGF, interleukin-6 (IL-611 and
multilineage [IL-3, granulocyte-macrophageCSF (GM-CSFII
HGFs are expressed at significant levels but with different
patterns, eg, kit and ftf3 are detected on a majority and
minority of HPCs, respectively, whereas IL-3Rs and GMCSFRs are present on almost all HPCs. In the four dfferentiation pathways, expression of early-acting receptors shows
a progressive decrease, more rapidly for bFGFR-1 and fct3
than for c-kit; furthermore, c-kit is more slowly downmodulated in the E and Mk than the G and Mo lineages. As a
partial exception, IL-6Rs are still detected through the early
or late stages of maturation in the Mk and M o lineages,
respectively. IL-3R expression is progressively and rapidly
downmodulated in both E and Mk pathways, whereas it
moderately decreases in the Mo lineage and is sustained in
the G series. The expression of GM-CSFR is gradually downmodulated in all differentiation pathways, ie, the receptor
density markedly decreases but late erythroblasts are still
partially GM-CSFR’ and terminal G, Mk and Mo cells are
essentially GM-CSFR+. Expression of receptors for late-acting cytokines is lineage-specific.Thus, EpoR, G-CSFR. TpoR,
and M-CSFR exhibit a gradual induction followed by a sustained expression in the E, G, MK, and M o lineages, respectively. In the other differentiation pathways the expression
of these receptors is either absent or initially low and thereafter suppressed. These observations are compatible with
the following multi-step model. (1) The early-acting GFRs
are expressed on quiescent HPCs with different patterns,
whereas the multilineage GFRs are present on 290% to 95%
HPCs. (2) Multilineage GFs, potentiated by early-acting
HGFs, trigger HPCs into cycling. HPC proliferation/differentiation is followed by declining expression of the early-acting
GFRs and in part of multilineage GFRs (see above). (3)Multilineage GFs trigger the expression of the unilineage GFRs
(see Testa U, et ai: 6/ood81:1442, 1993). Interaction of each
unilineageGF with its receptor leads t o sustained expression
of the receptor (possibly via transcription factors activating
the receptor promoter) and thus mediates differentiation/
maturation through the pertinent lineage.
0 7996 by The American Society of Hematology.
H
derived growth factor receptor (PDGFR); (2) Rs belonging
to the tyrosine kinase superfamily,” namely the receptors
for CSF-I (M-CSF) and steel factor, encoded by the c-fms
and c-kit proto-oncogenes, respectively, and the $t3/FLK2
receptor; and (3) Rs belonging to the peculiar highly conserved ‘‘cytokines receptor ~uperfamily”’~~’~
which includes
IL-2RP chain, IL-4R, ILdR, IL-3Ra chain, GM-CSFRa
chain, G-CSFR, and EpoR.
Elucidation of HGFR control mechanisms is obviously
crucial to unveil the cellular and molecular basis of hematopoiesis, particularly at the level of HSCsMPCs. Our laboratory has shown that purified PB HPCs express high-affinity
HGFRs, with prevalent IL-3R, a lower level of IL-6Rs and
GM-CSFRs and barely detectable levels of EpoRs.14 Subse-
EMATOPOIESIS IS sustained by a pool of hematopoietic stem cells (HSCs) that can extensively self-renew
and differentiate into progenitor cells (HPCs).’ HPCs are
committed to a specific lineage(s) and are functionally defined as colony- or burst-forming units (CFUs, BFUs), ie,
early and late HPCs of the erythroid series (BFU-E and
CFU-E, respectively), the megakaryocytic lineage (BFUMK, CFU-MK), the granulocyte-monocytic series (CFUGM, CFU-G, CFU-M), as well as multipotent CFUs for all
four lineages (CFU-GEMM). HPCs in turn differentiate into
morphologically recognizable precursors that mature to terminal elements circulating in peripheral blood (PB).
The survival, growth, and differentiation of hematopoietic
cells is at least in part regulated by a network of hematopoietic growth factors (HGFs) termed colony-stimulating factor
(CSF) or interleukin (IL).’ Erythropoietin (Epo), G-CSF,
thrombopoietin (Tpo), and M-CSF essentially stimulate late
unilineage HPCs (CFU-E, CFU-G, CFU-MK, and CFU-M,
respectively) and the derived differentiated precursors.
Multilineage HGFs, including IL-3 and GM-CSF, affect
CFU-GEMM and early unilineage or bilineage HPCs (BFUE, BFU-MK, and CFU-GM).’ Other cytokines Vft3 and ckif ligands (FL, KL), basic fibroblast GF (bFGF), leukemia
inhibitory factor (LIF), and IL-61 act on the primitive stages
of hematop~iesis.~-~
The availability of recombinant HGFs favored analysis of
their cell-surface receptors (Rs). HGFRs have been recently
cloned and classified into three different categories: (1) Rs
belonging to the Ig superfamily,” eg, IL-1R and platelet-
Bfood, Vol88, No 9 (November 1). 1996 pp 3391-3406
From the Department of Hematology-Oncology Istituto Superiore
di Sanitci; Blood Transfusion Center, University “La Sapienza, ”
Rome, Italy; and Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA.
Submitted March 13, 1996; accepted July 4, 1996.
Address reprint requests to C. Peschle, MD, Thomas Jefferson
University, Thomas Jefferson Cancer Institute, Bluemle Life Sciences
Bldg, Room #528-233, S 10th St, Philadelphia, PA 19107-5541.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section I734 solely to
indicate this fact.
0 19% by The American Society of Hematology.
OOO6-497I#6/8809-~0$3.OO/O
3391
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3392
TESTA ET AL
quent studies by double labeling of total human'' or Rhesus
monkey bone marrow (BM) ~ e l l s ' ~with
~ ' ' anti-CD34 and
either anti-IL-3R or GM-CSFR monoclonal antibodies
(MoAbs) confirmed that CD34+ cell subsets express IL-3Rs
andor GM-CSFRs. Furthermore, studies on fractionated BM
CD34' cells showed that small cells possess a higher IL6R, IL-3R, and GM-CSFR density than large ones.''
These studies did not provide insight into the expression
pattern of HGFRs during HPC differentiatiordmaturation
along the diverse hematopoietic lineages. We have recently
developed methodology that enables stringent purification
and abundant recovery of PB HPCS~.'~-''
and their selective
unilineage differentiation along the erythroid (E),19-24
granulocytic (G),23.24
megakaryocytic (Mk),*' or monocytic ( M o ) ~
lineage. The availability of these experimental tools allowed
us to explore the expression of HGFRs at different stages
of HPC differentiatiordmaturation restricted along the E, G,
Mk, or Mo pathway.
MATERIALS AND METHODS
HGFs and Culture Medium
Recombinant human IL-3 (rhIL-3; 2 X IO6 Ulmg), rhGM-CSF,
and rhIL-6 (2 X 10* Ulmg) were supplied by the Genetics Institute
(Cambridge, MA); rhEpo (1.2 x lo5 U/mg) and bovine basic fibroblast GF (rbFGF; 2 x lo7 U/mg) by Amgen (Thousand Oaks, CA);
and rhFL (1.9 X lo6 U/mg) and rhKL (1 X 1O5 Ulmg) by Immunex
(Seattle, WA). rhG-CSF (1 X 10' Ulmg) and rhM-CSF (6 x lo7U/
mg) were purchased from R&D Systems (Minneapolis, MN), and
rhTpo was generously provided by Genentech (San Francisco, CA).
Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) was prepared weekly before each purification experiment.
Adult PB HPC Purc9cation
Adult PB was obtained from male donors after informed consent
as described.' HPCs were purified as reported7.I9and modified.*@"
Briefly, (IA) PB samples were separated over a Ficoll-Hypaque density gradient (Pharmacia, Piscataway, NJ), and (IB) PB mononuclear
cells (PBMCs) resuspended in IMDM containing 20% heat-inactivated fetal calf serum (FCS; GIBCO) for three cycles of plastic adherence. Thereafter, (11) cells were separated by centrifugation on a discontinuous Percoll gradient (Biochrom KG, Berlin, Germany). (111)
Step 111 purification was potentiated (Step IIIP) as
HPC Clonogenetic Assay
In FCS' cultures purified HPCs were seeded ( I X 10' cells/mL/
dish, two or three plates per point) and cultured in 0.9% methylcellulose, 40% FCS in IMDM supplemented with a-thioglycerol
mol/L) (Sigma, St Louis, MO), ferric ammonium citrate (10 mg/
mL), pure human transferrin (0.7 mg/mL), and different HGFs as
detailed below at 37°C in a 5% COz/5% OZ/90%NZ humidified
atmosphere. In FCS- cultures,26FCS was substituted by bovine serum albumin (BSA), pure human transferrin, human low-density
lipoproteins, insulin, sodium pyruvate, L-glutamine (2 X lo-' mol/
L), rare inorganic elementsz7 supplemented with iron sulfate (4 X
IO-* mom), and nucleosides. Both FCS+ and FCS- cultures were
supplemented with FL (100 ng/mL), KL (100 ng), IL-3 (100 U),
GM-CSF (10 ng), Epo (3 U), M-CSF (250 U), and G-CSF (500 U).
CFU-GEMM, BFU-E, and CFU-GM colonies were scored on day
14-15 and 16-18 in FCS" and FCS- cultures, respectively.
Unilineage HPC Liquid Suspension Culture
Step IIIP HPCs were seeded ( 5 X IO4 cells/mL) and grown in
liquid suspension culture supplemented with the following HGFs:
(1) in FCS- erythropoietic culture, very low doses of 1L-3 (0.01 U/
mL) and GM-CSF (0.001 ng) and a saturating level of Epo ( 3 U)"'-'';
(2) in FCS- granulopoietic culture, low amounts of IL-3 ( I U) and
GM-CSF (0.1 ng) and saturating amounts of G-CSF (500 U)23,'4;
(3) in FCS- megakaryocytopoietic culture, saturating doses of Tpo
(100 ng)*'; and (4) in FCS+ monocytopoietic culture, saturating
amounts of both FL (100 ng) and M-CSF (500 U).' Cells were
periodically counted and analyzed for morphology, membrane phenotype, and HGFR expression at both mRNA and protein levels.
Unilineuge HPC Clonogenetic Culture
HPCs growing in unilineage liquid suspension cultures were assayed in unilineage clonogenetic cultures. These were performed by
adding the HGF stimuli used for unilineage liquid suspension cultures (see above) in FCS- (erythropoietic, granulopoietic, megakaryocytopoietic cultures) or FCS' (monocytopoietic culture) medium
(see above). Mk colonies were scored on day 10- I2 and other colonies on day 14-16, The scoring threshold was 100 cells/colony for
erythroid, granulocytic, and monocytic clones and 3 celldcolony for
megakaryocytic ones.
Control Cells
Normal human adult fibroblasts, PB granulocytes, and monocytes
were obtained by standard procedures. Fetal liver erythroblasts were
obtained as described." Human TF-1, K562, U937, KG I , HL-60,
and HEL leukemic cell lines were grown under standard conditions.
Immunojluorescence HPC Analysis
MoAbs. Anti-CD34 (8G12 clone), -HLA-DR, -CD38, -CD61,
-VLA-4, -CD3 I , -CD58, -CD44, -CD4 (Becton Dickinson, Mountain
View, CA), +kit, -gp170 (Immunotech, Marseille, France), and
-Thy- I (PharMingen, San Diego, CA) MoAbs directly conjugated
with fluorochrome [fluorescein isothiocyanate (FITC) or phycoerythrin (PE)] were used to characterize the membrane phenotype of
step IIIP HPC.
Labeling procedure. Cells were washed twice in phosphate-buffered saline (PBS) and then incubated for 60 minutes at 4°C in the
presence of an appropriate amount of MoAb. After three washes
with cold PBS, cells were resuspended in PBS containing 2.5%
formaldehyde and then analyzed by FACS (FACScan; Becton Dickinson) for fluorescence intensity with a program suitable for doubleimmunofluorescence analysis. At least 4,000 cells were analyzed for
each determination. The gating for analysis of the living cell population was determined by propidium iodide staining.*x
Fluorescence Analysis of HGFR Expression
Two types of reagents were used: ( I ) anti-HGFR MoAbs either
simply purified or fluorochrome-conjugated; and (2) recombinant
HGFs either biotin or fluorochrome conjugated. All reagents are
listed in Table 1.
Anti-IL-3, -c-kit, -jt3 MoAbs. PE-labeled anti IL-3R a-chain
MoAb, clone 9G5"' was purchased from PharMingen; PE-labeled
anti-c-kit was obtained from Immunotech: PE-labeled anti-jf3,
clone SF1.3403" was kindly supplied by Dr 0. Rosnet (Marseille,
France). These antibodies were incubated as indicated above for
fluorochrome-conjugated antibodies.
Control experiments for exogenous ligand versus MoAb competition were performed by incubating the step IIIP cells with a fixed
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GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
3393
Table 1. Reagents Used for HGFR Expression Analysis on Step lllP HPCs Induced to Unilineage Differentiation
Fluorescence
Receptor
Early-acting HGFs
Multilineage HGFs
MoAb
Ligand
bFGFR
-
-
kit
f1r3
+ [PE-labeled MoAb (Immunotech)]
+ [PE-labeled M o A ~ ~ ~ I
+ [Biotin-conjugatedKL (R&D)I
IL-6R
-
IL-3R
+ [PE-labeledM o A ~ ~ ~ I
GM-CSFR
Unilineage HGFs
RT-PCR (Ref No.)
+[MoA~~~I
-
+ [Biotin-conjugatedIL-6 (R&D)I
+ [PE-labeledGM-CSF (R&D)I
-
EpoR
+ [PE-labeled G-CSF (R&D)I
G-CSFR
M-CSFRlfms
TpoRlmpl
amount of anti-IL-3R or anti-jf3 MoAb in absence or presence of
increasing concentrations of IL-3 (0.01, I, or 100 U/mL) or FL (1,
10, or 100 ng/mL), respectively. No competition was observed for
IL-3 at the dosage added in E and G cultures (data not shown).
To assay the binding of anti-jt3 MoAb in Mo culture, cells were
first (1) washed twice in neutral PBS, treated with 1 mL of cold PBS
(titrated to pH 3.0 with HCI) for 1 minute at 4°C to remove the large
majority of surface-bound FL, washed once with neutral PBS; and then
(2) incubated with PE-labeled anti-jt3 MoAb: this procedure allows to
remove greater than 95% surface-bound FL. Control experiments for
exogenous ligand versus MoAb competition showed that IT,at the
concentration used in the Mo culture medium, causes a 50% reduction
of anti-jr3 MoAb binding to the cells (data not shown).
Anti-GM-CSFR MoAb. Purified unconjugated anti-GM-CSFR
a-chain MoAb, clone 17-A3' was purchased from PharMingen. Cells
were first incubated for 60 minutes at 4°C in the presence of 2.5
mg/mL of this antibody, washed with cold PBS, and then incubated
for 30 minutes at 4°C with biotin-conjugated sheep anti-mouse IgGs
(Cappel, West Chester, PA). After washing with cold PBS, cells
were incubated with Quantum Red-labeled streptavidin (Sigma) and
then analyzed by fluorescence-activated cell sorting (FACS).
Control experiments for exogenous ligand versus MoAb competition were performed by incubating the step IIIP cells in the presence
of a fixed amount of anti-GM-CSFR MoAb combined or not with
increasing concentrations of GM-CSF (0.001, 0.1, or 100 ng/mL).
No competition was observed at the cytokine dosage supplemented
in E and G cultures (data not shown).
Ligands. Biotin-conjugated c-kit and IL-6 were obtained from
R&D Systems. Cells were incubated for 60 minutes at 4°C with this
reagent, incubated for an additional 30 minutes at 4°C with PElabeled streptavidin (R&D Systems), and then analyzed by FACS.
PE-labeled GM-CSF and G-CSF were obtained from R&D Systems. The cells were incubated for 60 minutes at 4°C with this
reagent, washed, and then analyzed by FACS.
G-CSF was added at saturating level in G culture. To assay the
binding of PE-labeled G-CSF in G culture, cells were first (1) washed
twice in neutral PBS, treated with 1 mL of cold PBS (titrated to pH
3.0 with HCl) for 1 minute at 4°C to remove the large majority of
surface-bound G-CSF, washed once with neutral PBS; and then (2)
incubated with PE-labeled G-CSF this procedure allows removal
of greater than 95% surface-bound G-CSF (see Results). As for GMCSF, we previously showed that the GM-CSF concentrations used
'~
in E and G cultures cause less than 5% receptor o c c ~ p a n c y(see
also Results).
The specificity of biotinylated and PE-conjugated ligands (KL,
IL-6, GM-CSF, G-CSF) was assessed by using TFI erythroleukemic
cells incubated with ligand alone or combined with an appropriate
amount of blocking antiligand MoAb: the difference in fluorescence
intensity between these two conditions reflects the level of specific
binding and the number of receptors (Fig 1).
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)Analysis of HGFR mRNA
Total RNA, extracted by the guanidium isothiocyanate method3*
in the presence of 12 mg of Escherichia coli rRNA as a carrier, was
normalized by dot hybridization with a human rRNA probe." The
normalized RNA was reverse transcribed according to the manufacturer's instructions (GIBCO-BRL, Gaithersburg, MD). The RT-PCR
was normalized for P2-mi~roglobulin'~
(amplification within the linear range was achieved by 20 PCR cycles: denaturation at 95°C for
30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C
for 45 seconds). To evaluate the expression of HGFR genes, an
aliquot of RT-RNA (corresponding to -2 ng RNA) was amplified
within the linear range by 30 PCR cycles. Each sample was electrophoresed in 2% agarose, transferred to nylon membrane, and hybridized with a specific probe. Positive controls (detailed in Results) were
normalized for P2-microglobulin and included in all PCR assays. For
negative controls, an aliquot of RNA (-2 ng) from each sample
was amplified to exclude the presence of contaminant DNA; a mock
reaction was also included. PCR cycles: 95"C/30 seconds, 55"C/30
seconds, 72"C/45 seconds (except when otherwise indicated).
Primers and Probes
bFGFR-I (see ref 33). Primer 5': AAGACCTGGACCGCATCGT (2342-2360); Primer 3': TTACAGCTGACGGTGGAGT
(2581-2599); Probe: AGCTCTACGTGCTCCTCAGGGGAGGA
TTCCG (2440-2470).
c-kit (see ref 34). Primer 5': CG'ITGACTATACAGTTCAGCGAC (1 180-1201); Primer 3' CTAGGAATGTGTGTAAGTGCCTCC (843-864); Probe: GATCAGCAAATGTCACAACAACCTT GGA (1032-1059).
IL-6R (see ref 35). Primer 5': TCTCACTGCCATGCCAGCT
(1478-1496); Primer 3': CTGTTGAAAACGACCAGGC (20352053); Probe: TCAGCCTGCTCCAGCTGTTCAGCTGGTT GAGGTITCAA (1565-1603).
IL-3R a-chain (see refs 36 and 37). Primer 5': AACGACAAGCTGGTGGTCT (105 1-1069); Primer 3' AGGTCCAGCAGCCAGTCT (1 195-1212); Probe: CTGGTCACTGAAGTACAGGTCGTGCAGA AAACTTGAGAC (1089-1 140).
GM-CSFR a-chain (see ref 38). Primer 5': CTACCGCGAAGAGGTCTT ( I 3 13-1331); Primer 3': CCAAGTAGCTGGGATGAT
(1638-1655); Robe: GCCAGGCGCGGTGGCTCACGCCTGTAA
TCCCAGCA (1479-15 14). Annealing at 53°C.
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TESTA ET AL
3394
labeled
Ligand
Negative
Control
"1 1
I
J
'oak
l
0100W
102
104
L
0100
102
1
1"
zk,
0
100
102
,
I
100
102
,
~
104
"I
"1
'"h
0100
102
l
0100W
102
104
L
Fluorescence
)
104
[k,
,
104
c-kit
-
( G S )
.
[G-CSFR)
1w
Fig 1. Flow cytometric analysis of HGFR expression in TF-1 erythroleukemic cells. (Left panels) Cells stained with phycoerythrin-conjugated streptavidin (SA-RPE) (negative control). (Right panels) Cells
treated with labeled ligands (PE-labeled GM-CSF and G-CSF, biotinlabeled IL-6 and c-kit) combined with SA-RPE. White area, specific
binding; black area, nonspecific binding in the presence of an excess
of anticytokine blocking MoAb. The low level of PE-GM-CSF binding
is probably due to the saturating GM-CSF level (10 ng/mL) in the
culture medium.
EpoR (see ref 39). Primer 5': TCATGGACCACCTCGGGGCGT (2-19); Primer 3' TAGCGGATGTGAGACGTCATG (519539); Probe: TCTGGTGTTCGCTGCCTACAGCCGACA CGTCGAGC (314-348). Annealing at 54°C.
G-CSFR (see ref 40). Primer 5 ' : TCCCAGCATGTCTATGCC
( I7 12-1729); Primer 3': AGCCATGAGGTGGATGTG ( 19581975); Probe: CACTACACCATCTTCTGGACCAACGTCC AG
(1850-1879). Annealing at 50°C.
M-CSFRgms (see ref 41). Primer 5': AGAGCATCTTCGACTGTG (3946-3964); Primer 3': GAACTTGGCTGTTCACCAG (405 1-4068); Probe: ACGTCTGGTCCTATGGCATCTTGCTCTGG (3984-4012). Annealing at 50°C.
TpoR/mp/ (see ref 42). Primer 5': AGCTGATTGCCACAGAAACC (557-576); Primer 3': ACTTGGGGAGGTCTGCTTTG
(665-684); Probe: CCAGTCTCCATGTGCTCAGCCCACAAT
GCC (621-650). Annealing at 56°C.
RESULTS
HPC Purijication and Membrane Phenotype
Early HPCs were purified from PB by a methodology
previously de~cribed'.'~and recently
The
step IIIP cell population obtained by the modified purification procedure is characterized by 90% CD34' cells and
80% HPC frequency, coupled with 45% HPC re~overy.~"'~
In the purified HPC populations used here, the HPC clonogenetic assay was optimized by adding to the formerly used
HGFs (KL, IL-3, GM-CSF, Epo) saturating dosages of GCSF, M-CSF, and FL: in a series of 12 consecutive experiments the HPC frequency and recovery increased to 90.3 t
2.1 (mean t SEM) and 69.0 t 5.3, respectively (Fig 2),
compared with 80.3 t 3.2 and 61.9 t 5.1, respectively,
obtained in the same experiments with the formerly used
HGFs (not shown). The colony type frequency was: BFUE, 33.5% ? 1.8%, CFU-GM (including CFU-G, -M, -GM),
44.9% t 2.8%, and CFU-GEMM, 11.5% t 0.9%. With
respect to evaluation of BFU-MK colonies and different
types of GM clones, additional clonogenic experiments involving treatment with the above HGFs combined with Tpo
(100 ng/mL) showed: BFU-MK, 1.5% t 0.4% (scored on
day 10-12, see Materials and Methods), CFU-G, 19.8% t
0.6%, CFU-M, 7.7% t 1.7%, CFU-GM, 8.8% t 1.6%.
Step IIIP cells are intensively CD34' (Fig 3) over the
negative background and 5% to 15% of these are positive
for Thy-I and gp170 membrane antigens, which are expressed on putative HSCs and primitive HPCS.~'." The large
majority of step IIIP HPCs are CD38' and HLA-DR', but
5% to 10% are CD34'/CD38'"" and CD34'kILA-DR""".
Only a minority (5% to 10%) of CD34' step IIIP cells are
positive for a few lineage-specific differentiation antigens,
such as CD4. Finally, step IIIP cells are largely positive
for adhesion molecules, including CD31, CD58, CD44, and
VLA-4.
Control Experiments f o r HGFR Assay
Control experiments, particularly with respect to competition at receptor level between endogenous HGF and labeled
ligand or MoAb, have been detailed in Materials and Methods.
Expression of HGFRs in Quiescent HPCs
In a first set of experiments we evaluated HGFR expression in quiescent step IIIP HPCs by immunofluorescence
(Fig 4) and RT-PCR (Fig 5A and B).
Immunofluorescence studies with PE-labeled anti-Jt3 and
anti-c-kif MoAbs showed that 35% and 80% of cells were
jt3' and c-kit', respectively. Futhermore, 40% to 45% of
cells were labeled by biotin-labeled IL-6.
Studies with anti-IL-3R a-chain or anti-GM-CSFR achain MoAbs showed that the large majority (ie, 290% to
95%) of cells were IL-3R' and GM-CSFR'. These observations were confirmed by studies with PE-labeled GM-CSF
(see below): however, the percentage of GM-CSFR' cells
was slightly lower (>70%) than that obtained using antiGM-CSFR MoAb (see below).
Finally, studies with PE-labeled G-CSF showed a reactivity with only a minority of cells.
RT-PCR analysis. mRNAs encoding HGFRs for earlyacting (bFGF, KL, and IL-6) and multilineage (IL-3 and
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GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
3395
10'1
lo,
Fig 2. Total cell number, percentage of CD34'
cells, HPC frequency, and recovery in the step lllP
purification procedure (mean k SE values from 12
separate experiments). (Left) Total cell number was
evaluated at different steps of purification, including
the Ficoll cut (Step I), Percoll gradient (Step II), and
at the end of the sequential passages on magnetic
beads (Step IIIP). (Right) The percentage of CD34*
cells in step lllP was evaluated by flow cytometry.
Step lllP HPC (BFU-E + CFU-GM + CFU-GEMM) frequency was evaluated by addition of IL-3, GM-CSF,
Iml
I*
1001
Y
i :
s
d
1.
stop I
step II
Epo, KL, FL, M-CSF, and G-CSF (see Results). HPC
recovery, percentage of HPCs after step IMP, as compared with their number in PBMCs (Step I).
GM-CSF) cytokines werc expressed at significant levels in
quiescent HPCs (Fig 5A). In contrast, mRNAs encoding
HGFRs for late, unilineage cytokines (Epo, G-CSF, M-CSF,
and Tpo) were barely (Fig SB, top panels) or not (Fig SB,
bottom panels; for TpoR also top panels) expressed in quiescent HPCs in different experiments (top and bottom panels
show two separate representative HPC purification and culture experiments).
HGFRs Expression During HPC E, G, Mk, and Mo
Differentiation/Matiiratioii
The unilineage differentiation culture systems. Experiments were performed to evaluate the expression kinetics of
HGFRs during unilineage E, G, Mk, and Mo differentiation/
maturation of step HIP HPCs in liquid suspension culture.
The main features of these culture systems have been extensively reported4."." and are further detailed in Table 2.
Day 0 step IIIP HPCs, assayed in E, G, Mk, or Mo clonogenetic cultures (ie, in unilineage semisolid cultures treated
with the same HGF stimuli of corresponding unilineage liquid cultures), generated only the expected E, G, Mk, or
Mo colonies, respectively. The frequency of these colonies
varied for the different lineages, ie, 28%, 16%. 7%, and 4%
for the E, G, Mk, and Mo lineages, respectively (Table 3).
During the first week of E, G,Mk, or Mo liquid culture
the HPC number, assayed in secondary unilineage semisolid
cultures, showed a gradual increase, ie, an increasing number
of selectively E, G,Mk, or Mo colonies, respectively: this
indicates unilineage HPC proliferation, ie, growth of E-, G-,
Mk-, or Mo-committed HPCs in the corresponding culture4.19.21.25 (and Table 2 and results not shown). Cell proliferation was associated with progressive HPC differentiation,
as shown by a gradual decrease of the size of the unilineage
colonies generated in secondary semisolid culture (Table 3)
and the percentage of CD34' cell^.^.'^.^^ In the Mo lineage
the decrease of colony size from day 0 to day 7-9 was less
rapid than for the other lineages (Table 3): in the second
step lllP
CDM*C.ll
Frequency
fmgennw
FI.C(WIKY
L
E2ER
I
v
step lllP
culture week the survival of a miniscule CFU-M population
(eg. -3% of total cells on day 12) is seemingly related to
the capacity of FL to stimulate the proliferation of primitive
HPCs and CFU-GM.4
Unilineage cultures were supplemented with an early-acting HGF (FL in Mo culture) or multilineage HGFs (IL-3,
GM-CSF in E and G culture, in part Tpo in Mk culture).g6
Addition of these HGFs may allow the proliferation of HPCs
not restricted to unilineage differentiation. To investigate this
aspect, HPCs were sequentially collected from unilineage
cultures and assayed in semisolid medium supplemented
with saturating levels of KL, FL, multilineage (IL-3, GMCSF), and unilineage (Epo, G-CSF, Tpo, M-CSF) HGFs:
these clonogenetic culture conditions allow to assay the differentiation potential of the HPCs growing in liquid unilineage culture. The results (not shown here, see Discussion)
indicate that other categories of HPCs survive in unilineage
cultures, ie, HPCs with CFU-GEMM/CFU-GM potential in
E culture, with CFU-GEMM/BFU-E potential in G and Mo
culture, with CFU-GEMM/BFU-E/CFU-GMpotential in Mk
culture: it is crucial that the frequency of these HPCs rapidly
declines in the first culture week, ie, these HPCs are either
gradually channeled into the unilineage differentiation pathway or undergo apoptosis.
During the second week of culture the cell clonogenetic
capacity is lost (ie, the size of generated clusters is below
the colony scoring threshold, except for a few monocytic
colonies in Mo culture, see above): HPCs enter into the
maturation compartment and progressively express the morphologic, antigenic, and functional pattern of maturing E,
G, Mk, and Mo precursors in the respective cultures.
The cell number amplification from day 0 through day
14-16 sharply differs in the four unilineage culture systems:
IO'-fold for the E series, 102-fold for the G lineage, 30and 12-fold for the Mo and Mk pathways, respectively.
These differences seemingly reflect the frequency and particularly the proliferative potential of the different types of
HPCs in step IIIP cells.
-
-
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
3396
1000
+
+
600
U
v)
Y
200
0 200 ' 600
FSC -b
100
100
1 02
CD34
+
104
'
1000
100
104
...
-
10-
100
102
CD34 -b
102
CD34
-
102
'
FL1
100
102
104
HLA-DR -+
'p
100
- 100
0
'
+
102 '
CD34 -b
.
100
102
+
.
1
104
loo!-- I ,
, ,
1 00
1 02
CD34 -+
164
loo!,
100
I, .,
,
102
CD34 -&
I
104
I
104
1
. .
I
104
TESTA ET AL
102
CD44
+
104
Expression of Early-Acting HGFRs
RT-PCR analysis (Fig 5A) indicated that the mRNA expression of early-acting HGFRs (bFGFR-I, c-kit, and IL-6R)
was generally characterized by a progressive decline in both
E and G differentiation pathways. bFGFR-I mRNA declined
more rapidly during E than G differentiation, whereas the
decrease of c-kit mRNA is slower in the E than G pathway.
In previous studies' j t 3 mRNA showed an abrupt decrease
in the E pathway and a slower decrease in the G series. IL6R mRNA also declined to undetectable or low level in E
and G maturation, respectively.
Immunofluorescence studies with anti-jt3 or anti-c-kit
MoAbs confirmed the pattern observed at mRNA level (see
Figs 6A and B, and 7A). Thejt3' cell frequency sharply
loo! - I ,
, ,
100
102
CD34 -b
I
104
Fig 3. Flow cytometric analysis of step lllP cells with a panel
of MoAbs (a representative experiment is shown). Cells were
labeled with either a PE- or FITCconjugated anti-CD34 or appropriately labeled anti-Thy-1,
-gp170, -CD38, -HLA-DR, -CD4,
-CD31, -CD58, -CD44. or - V I A 4
MoAbs. On the top line the first
two panels from left to right
show cell scatter (iodide propidium staining) and negative control.
decreased during E and G differentiation, slightly more
rapidly in the former than the latter lineage (Figs 6A and
7A). The number of c-kit+ cells slowly decreased in the E
and G series to low levels at day 12 or 9, respectively
(representative results in Fig 6B): thus, the decline was less
rapid in the E than the G lineage (Fig 7A). Similar results
were obtained using biotin-conjugated c-kit (data not
shown).
Further immunofluorescence experiments were performed on Mk and Mo cultures (Fig 7A): t h e j t 3 and c-kit
expression pattern in these lineages was similar to that
observed for the E and G series. In Mo culture, however,
we cannot exclude downmodulation o f j t 3 by the saturating
FL level present in the medium (see below studies on G-
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GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
FLI
FSC-
t
2
104
104
102
* 102
3397
-b
t
+
0
100
100
100
100
102
104
CD34 -b
Kl
104
104
w 102
w 102
t
2f
9
=?
102
104
CD34 -b
4
;:
$..
,.
100
100
102
104
CD34 -b
x
w 102
l w y , .":-;'
100
102
104
CD34 +
;::rl
v
v5
a.
0
100
I00
level by RT-PCR and in all lineages at protein level by
immunofluorescence.
RT-PCR analysis. ( I ) As shown in Fig 5A, IL-3R
mRNA was briskly downmodulated in the E series to undetectable level at day 9, while it was sustained or slightly
decreased in the G pathway; (2) GM-CSFR mRNA was
characterized by a gradual decrease to low or undetectable
level in advanced maturation in the E pathway, compared
with a moderate decrease in the maturing G lineage.
Fluorescence studies with anti-GM-CSFR and -1L3R
MoAbs and PE-labeled GM-CSF provided results in line
with those observed at mRNA level.
IL-3R assay. At day 0-2, HPCs differentiating along either the E and G lineages were almost all IL-3R'. Starting
from day 5 IL-3R' cells rapidly decreased in E culture,
whereas expression in the G lineage was essentially unmodified, as evaluated in terms of IL3R' cell frequency (60%
to 70% IL-3R' cells at day 12) and fluorescence intensity.
IL3R expression progressively and sharply declined during
Mk differentiation, almost as rapidly as in the E lineage.
The decrease of IL3R expression was slow in the Mo series
and a significant proportion (-50%) of mature monocytes
displayed IL-3Rs.
GM-CSFR assay. Similar results were obtained using
anti-GM-CSF MoAb (Fig 6F and results not shown) and
PE-labeled GM-CSF (Fig 7B). However, the MoAb assay
was more sensitive: this is seemingly caused by amplification
of the MoAb fluorescence signal (see Materials and Methods) rather than by receptor occupancy by exogenous GMCSF in E and G culture (the supplemented cytokine concentration causes only 4%
receptor oc~upancy,'~
see above).
During the first 5 culture days virtually all cells differentiating along the E and G lineages were GM-CSFR', particularly by MoAb assay. In the second week, E precursors
exhibited a gradually reduced reactivity with anti-GMCSFR MoAb, ie, a moderate decrease of percent positive
cells combined with a pronounced decline of fluorescence
intensity. G precursors remained prevailingly (by ligand
assay, Fig 7B) or completely (by MoAb assay, Fig 6F) GMCSFR', but showed a sharp decrease of fluorescence level.
Interestingly, the expression patterns of GM-CSFR evaluated
by RT-PCR (Fig 5A) and MoAb (Fig 6F) assays are very
similar.
In the Mk and Mo pathways GM-CSFR expression is
characterized by a sustained majority of GM-CSFR' cells,
coupled with a gradual decrease of fluorescence intensity,
particularly in the Mk pathway (Fig 7B; also results not
shown performed by MoAb assay).
102
104
CD34 +
100
100
102
104
CD34 -b
Fig 4. Flow cytometric analysis of HGFR expression on quiescent
step lllP cells la representative experiment is shown). Cells were
labeled with FITC-conjugated anti-CD34 and appropriately labeled
anti-flt3 MoAb, anti-c-kit MoAb, biotin-labeled IL-6, anti-IL-3R
MoAb, anti-GM-CSFR MoAb, or PE-labeled G-CSF. (Top panels) Cell
scatter (iodide propidium staining) and negative control.
CSFR in G culture): further studies are in progress to evaluate this aspect.
Experiments with biotin-conjugated IL-6 showed that IL6R expression ( I ) rapidly decreased to undetectable levels
in the E series, (2) slowly decreased in the G series, (3)
was sustained until day 7 and then decreased during Mk
differentiation, and (4)increased through day 9 and remained
detectable until terminal maturation in Mo lineage (Fig 7A,
see also Fig 6C).
Expression of Multilineage HGFRs
The expression pattern of HGFRs specific for IL-3 and
GM-CSF was investigated in the E and G lineages at mRNA
Expression of Late-Acting Unilineage HGFRs
The expression of EpoR, G-CSFR, TpoR, and M-CSFR
was investigated in the four differentiation pathways by RTPCR analysis (Fig 5B).
These HGFRs, only scarcely expressed in quiescent HPCs,
were gradually induced and then sustainedly expressed in
the pertinent lineage. Conversely, they are barely or not
expressed in the other lineages: (1) EpoR mRNA was transiently expressed at low level in the G and Mk lineages from
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TESTA ET AL
3398
A
( Day
0 2 5 7 9
12
14
0 2 5 7 9 1 2 1 4
Q
Posmm CONTROL
0
bFGFR- 1
Fibroblasts
c-Kit
K562
**
11-6R
u937
m
KGI
m
HI.-60
0
11-3R
' 0 -
-*-
*I*
GM-CSFR
l32m
0 2 5 7 9
EpoR
G-CSFR
4
12 14
0
*
m
-
I
0 2 5 7 9 1214
El
POSITIVE CONTROL
Erythrobl.
1)1
Granuloc.
(I
M-CSFR/hTlS
Monoc.
TpoR/mpl
HE1
PosmvE CONTROL
Erythrobl.
Granuloc.
4
P2m
POSINVE CONTROL
EpoR
G-CSFR
Granuloc.
4
*
.*.'.,
Erythrobl.
0
Granutoc.
0
HEL
0
M-CSFR/fms
TpoR/mpl
hm
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GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
3399
Fig 5. (A) bFGFR-I, a k i t IL-6R. ILSR, and GM-CSFR mRNA levels during in vitro erythroid (E)or granulocytic (GIdifferentiation of step lllP
HPCs, as evaluated In representative experiments by RT-FCR. @2-Microgfobulin(Plmt controls are presented. Positive controls are also prasented. Similar results were obtained in two other experiments. IB) EpoR, G-CSFR, M-CSFR, and TpoR mRNA levels during arythroid (E),
granulocytic 101. megakaryocytic IMk), and monocytic (Mo) differentiation of step lllP HPCs, as evaluated by RT-PCR in two repreaentative
HPC purification cultura axperiments (top and bottom panels).Ptm controls are presented. Positive controls are also presented. Similar results
were obtained in two other experiments.
day 2 to 5 and day 9 to 14 respectively, while it was not
detected in the Mo series; ( 2 ) G-CSFR mRNA was transiently expressed at low level in day 2-5 E culture, and barely
detected in the Mk and Mo series until initial maturation; ( 3 )
M-CSFR was transiently expressed at low level in day 2 E
culture, barely detected in G lineage until maturation and
absent in the Mk series; and (4) TpoR mRNA was barely
or not detected in E, G and Mo cultures.
These findings were confirmed by fluorescence studies
quantifying the binding of PE-labeled G-CSF to HPCs differentiating along the E, G, Mk, and Mo lineages (Figs 6G and
7B). The G-CSF binding, barely present on quiescent HPCs,
was mildly induced in E, Mk,and Mo differentiation at day
2-5, then progressively decreased. In the G lineage the early
receptor induction was followed by temporary downmodulation and then a rebound up to the initial induction level.
Two series of control experiments indicated that the saturating G-CSF level added in the G culture partially downmodulates membrane G-CSFRs. Thus, (1) step IIIP cells
were grown from day 0 to 2 in a modified G culture system,
treated with standard IL-3 and GM-CSF concentrations (1
U/mL and 0.1 ng/mL, respectively) combined or not with
graded concentrations of G-CSF up to the level in G culture
( 5 , 5 0 , or 500 U/mL); thereafter, G-CSF bound to cell mem-
brane was removed (see Materials and Methods) and GCSF binding was measured with PE-labeled G-CSF. Cells
cultivated in the presence of 500 U/mL G-CSF displayed a
lower G-CSF binding capacity than those grown without GCSF or with 5-50 U/mL G-CSF (Table 3). (2) Unilineage
HPC differentiation along the G pathway was induced by
treatment with standard IL-3/GM-CSF levels without G-CSF
addition (in this modified unilineage culture, G-cell growth
is slower than in standard G culture, results not shown):
interestingly, granulopoietic cells displayed higher percentages of G-CSFR' cells in G-CSF- modified G culture than
G-CSF' standard G culture (Fig 7B). The G-CSFR expression patterns evaluated at protein level in G-CSF- culture
(Fig 7B) and at mRNA level in G-CSF' culture (Fig 5B)
are strictly consistent.
DISCUSSION
The analysis of key mechanisms controlling human hematopoiesis has been hindered by lack of (1) methodology for
stringent purification and abundant recovery of HPCs and
( 2 ) culture systems for unilineage differentiation/maturation
of the purified HPCs. Thus, little is known on HGFR expression in hematopoiesis, particularly at discrete stages of HPC
differentiatiodmaturation along the different lineages. We
Table 2. Step lllP HPCs Unilineage Culture Systems: Analysis of Cell DifferentiationlMaturation(mean from at least three experiments)
Day 0 Assay of
HPCs"
Colony Type and No./100 Cells
Unilineage Culture System
Erythroid
(El
BFU-E
CFU-MK
CFU-G
CFU-M
28
0
0
0
First Unilineage Culture Week: Assay of
Differentiating HPCs'
Second Unilineage Culture Week Analysis
of Maturing Precursors
(a) HPC No. ( X l @ ) (b)Colony Size (cells
no./colony)
Lineage-Specific
Maturation
Marker (%
positive cells)
dO
(a)
+
14
+
d7
199
-
-,
d9
d8
0
-
d14-16
Glycophorin A
Morphology (%
values)
614-16
Polychromatic
+
orthochromatic
erythroblasts
Megakaryocytic (Mk)
Granulocytic-neutrophilic (GI
Monocytic (Mo)
0
0
0
7
0
0
0
16
0
0
0
4
(b)
1,300
(a)
(b)
3.5
24
(a)
+
400
+
22.8
3
8
+
24
(b)
700
+
250
(a)
2
+
11
(b)
2,000
-t
800
+
HPC assay in clonogenic semisolid culture supplemented with unilineage HGF stimulus.
t Not evaluated; ie, below colony scoring threshold.
+
-+
-t
+
-+
-,
+
N.E.t
19
+
98
0
N.E.t
CD42b
24 +
95
0
CDllb
N.E.t
37
<400
23
+
Late MK cells
+
98
Metamyelocytes
granulocytes
98
CD14
16
98
98
Monocytes/
macrophages
94
97
+
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3400
Table 3. Binding of PE-Conjugated G-CSF to Step IMP Cells Grown
for Two Days in Absence or Presence of Graded
Concentrations of Exogenous G-CSF
TESTA ET AL
differentiation: it is suggested that these HPCs are either
gradually channeled into the unilineage pathway or undergo
apoptosis (see Results). Thus, both selective and inductive
phenomena may underlie HPC differentiation in unilineage
G-CSF Binding
cultures. Accordingly, studies on the differentiation of primiG-CSF Concentration
Fluorescence Intensity
tive HPCs’ and multilineage GF-dependent cell linesS5sugIUlmL)
% Positive Cells
(arbitrary units)
gest that late-acting HGFs modulate HPC differentiation
None
14
43
allowing the survival of selected categories of HPCs. whose
5
71
42
differentiation capacities are intrinsically determined. How50
55
37
ever, the HPC differentiation program, although partially
500
39
31
preprogrammed, is in part induced by HGF(s) through mechanisms other than anti-apoptotic ones.56Furthermore, we observed that the percentage of apoptotic cells is (1) low (ie,
< 10%) in E and G cultures, where the initially responsive
have recently developed novel culture techniques to selecHPC
subpopulation is relatively large, and (2) more elevated
tively channel HPC proliferation along the different sefies.4,19-25 On this basis, we have analyzed the expression
(ie, 530%) in Mk and Mo cultures, where the initially responsive HPC subpopulation is relatively small (results not
of an extensive series of HGFRs on 90% purified HPCs
shown).
undergoing strictly unilineage E, G, Mk, or Mo differentiaIn the second culture week, we monitored a gradual wave
tionlmaturation.
of unilineage maturation of morphologically recognizable
The HGFR assays provided consistent results. The mRNA
precursors along the E, G, Mk, or Mo pathway through
levels of c-kit, IL-6R, IL-3R, and GM-CSFR, evaluated by
terminal maturation.
semiquantitative RT-PCR, were compatible with correExpression of early-acting HGFRs (bFGFR- 1, jt3, c-kif)
sponding protein results obtained by MoAb andor labeled
consistently
decreases during hematopoietic differentiation.
ligand assay(s): this suggests that posttranscriptional mechaIn
line
with
these observations, bFGF, FL, and K L act on
nisms do not play a major role in the expression of these
primitive
stages
of hematop~iesis?.~:
FL amplifies the numHGFRs.
ber of putative stem cells (long-term culture initiating cells
In protein assays, receptor occupancy by its ligand supple[LTC-ICs]): whereas all of these cytokines potentiate the
mented in culture was carefully considered: a series of conaction of other HGFs on primitive HPCs [blast CFU (CFUtrol studies insured that this potential bias did not obscure
B), high proliferative potential colony-forming cells (HPPresults and interpretation thereof (see Results).
CFC)J and more differentiated HPCS.?-~
c-kit expression deAs previously mentioned, we reported that quiescent
creases less rapidly in the E and Mk than in the G and
HPCs purified from PB express IL-3Rs at elevated level and
Mo lineages: these results are consistent with the prevailing
IL-6Rs/GM-CSFRs at moderate level, whereas EpoRs were
erythroid differentiation of c-kit’ HPCs in cultures supplebarely dete~tab1e.l~
We confirm these results and further
mented with Epo, G-CSF, KL, and multilineage HGFs.”
show that receptors for early-acting HGFs (bFGF, flt3, and
IL-6R expression similarly declines in the E and C linIU)are clearly expressed on the purified HPCs, whereas the
eages, but is maintained until the early or late stages of
receptors for other unilineage HGFs (G-CSF, M-CSF, and
maturation in the Mk and Mo lineages, respectively. The
Tpo) are barely or not expressed. This pattem is in line with
persistent expression in the two latter series is consistent
BM studies showing that: (1) flt3R is selectively expressed
with the two biologic activities of IL-6, which stimulates in
on CD34’ cells4’; (2) c-kit is present on early/late HPCS,~~.‘’ vitro” and i n vivosx megakaryocytopoiesis and potentiates
whereas ILdR is present on different HPC subsets including
the proliferation of monocytes in clonogenetic5’ and uniline34+/33- and 34+/33+cells5’; (3) M-CSFR,” G-CSFR,” and
age Mo culture (our unpublished observations).
TpoR” are scarcely expressed on CD34’ cells. However,
The expression kinetics of multilineage HGFRs differs in
these studies were carried out on BM CD34+ cells displaying
the four lineages. The IL-3R is rapidly downmodulated in
a low or moderate HPC clonogenetic efficiency (comprised
E and Mk differentiation, while the expression slowly debetween 2% and 35%). c-kit was also detected on putative
clines in the Mo pathway and is sustained in the G lineage,
HSCS.’~
thus leading to significant IL-3R expression on terminal Mo
In the unilineage cultures HPC differentiation and precurand particularly G cells. The GM-CSFR, although downsor maturation gradually progress in the first and second
modulated in all series with respect to receptor density, is
week, r e s p e c t i ~ e l y (see
~ ~ ~also
~ - ~Table
~
3).
still detected by MoAb assay on virtually all terminal G,
HPC differentiation in the first week deserves discussion.
Mo, and Mk cells. These expression patterns are in line with
The E, G, Mk, and Mo culture systems are respectively fed
the biologic activity of IL-3 and GM-CSF: (1) both stimulate
by BFU-E, CFU-GM, CFU-Mk, and CFU-M. The proliferatthe proliferation of early HPCs and their differentiation along
the G, Mo, E, and Mk pathways“; (2) their combined action
ing HPCs gradually differentiate along the unilineage pathchannels purified HPCs into G differentiationlmaturation
way, as indicated by the progressive decrease of ( I ) their
coupled with a 5 5 % to 10% Mo cell component’y~2’~22;
(3)
proliferative potential and (2) CD34 expression. Early-acting
both sustain the functional activity of neutrophils and monoand multilineage HGFs added to these cultures may initially
cytes.‘”.’‘
allow the survival of HPCs not restricted to the unilineage
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
In the unilineage culture systems the assay of late-acting
HGFRs at protein level bears two limitations, caused by the
addition of the unilineage HGF at saturating level: thus, (1)
the HGF unilineage HGFR interaction may interfere with
the receptor assay by ligand or MoAb (see G-CSFR results);
more important, (2) the HGF/HGFR interaction causes receptor internalization6’ and may therefore lead to an underestimation of HGFR expression in the unilineage differentiation pathway (as indicated by experiments on G-CSFR
expression in G culture supplemented or not with graded GCSF levels; see Results).
The expression pattern of unilineage HGFRs (EpoR, GCSFR, TpoR, and M-CSFR) has been extensively analyzed
by RT-PCR. This assay allows only a semiquantitative evaluation of HGFR mRNA levels: however, the observed expression patterns are markedly differentiated and permit a comparative stage- and lineage-specific analysis. It is also
noteworthy that the expression of E ~ o Rand~ M-CSFRffl
~
genes is largely regulated via transcriptional mechanisms.
Our observations indicate that the unilineage HGFRs are
gradually induced and then sustainedly expressed in the pertinent lineage (ie, expression of EpoR, G-CSFR, M-CSFR,
and TpoR is sustained in the E, G, Mo, and Mk series,
respectively). In some cases, they are initially and transiently
expressed at low level in other lineages (eg, EpoR in G
lineage and G-CSFR in E lineage): hypothetically, this may
reflect the initial differentiation of a minority of multipotent/
bipotent HPCs (see above). The low EpoR expression on
maturing Mk cells is compatible with studies in the mouse
indicating presence of EpoRs on rodent megakaryocyte^.^^
Previous studies have provided limited insight into these
phenomena: thus, analysis of total murine BM cells by 1251[G-CSF] autoradiography6’ and human erythroid colonies
by ‘2sI-[Ep]s.67indicated that the G-CSFR and EpoR are
expressed on G and E lineage cells, respectively.
Altogether, these novel observations allow a comprehensive analysis of HGFR expression in discrete hematopoietic
cell populations at sequential stages of differentiatiodmaturation along the four main lineages.
The findings suggest a two-step model for HGFRs expression during hematopoietic differentiation, which lends further support to the HGFR cascade transactivation hypothesis
proposed by our group.14 The first step involves interaction
of multilineage HGFs with their receptors on HPCs: this
leads to progenitor proliferatioddifferentiation, which is followed by expression of unilineage HGFRs. The second one
entails interaction of a unilineage HGF with its receptor,
leading to sustained receptor expression and unilineage differentiatiodmaturation. This model is also in line with the
GM-CSF induction of E ~ o R . ~ ’
It is generally assumed that each late-acting HGFs downmodulates its own receptor at membrane leve1.6’ However,
our results indicate that each unilineage HGFR is sustainedly
expressed in the pertinent series. By implication, it is suggested that the unilineage HGF/HGFR interaction triggers
molecular mechanisms sustaining the receptor expression,
3401
which override the receptor internalization otherwise induced by HGFmGFR interaction. Accordingly, optimal MCSFR expression in a c - h s + FDC-P1 cell line requires the
continuous presence of exogenous M-CSF.~*It is also noteworthy that a combined IL-3/GM-CSF stimulus induces G
lineage differentiatiodmaturation coupled with G-CSFR exp r e s ~ i o n ’ ~ ~(and
’ ~ ~present
~’
results): it is apparent, therefore,
that unilineage HGF/HGFR interaction is sufficient but not
necessary to induce unilineage differentiation and sustained
HGFR expression.
The expression of unilineage HGFRs may be controlled
by lineage-specific transcription factors (TF) activating the
receptor promoter. In this regard, (1) the erythroid TF
GATA-1, sustainedly expressed following interaction of Epo
with its receptor, maintains EpoR tran~cription.~~.~’
(2) A
functional linkage between lineage-specific TFs and unilineage HGFRs is supported by the similarity of their expression
pattern in unilineage HPC differentiation, eg, both erythroidspecific TFs (GATA-1, NF-E2, TAL-1) and EpoR are sustainedly expressed in the erythroid lineage, but only transiently induced at lower level in the early differentiation
stages of the G ~ . e r i e s ~ (and
* ~ ’ this
~ ~ ~report).
~
( 3 ) In the monocytic lineage optimal PU.1 expression is required for MCSFR expression.@Hypothetically, the expression of other
late-acting HGFRs may be similarly mediated via lineagespecific TF(s).
Altogether, our observations are compatible with the following two-step molecular hypothesis. (1) Multilineage
HGFs trigger HPCs into proliferatioddifferentiation: this
causes induction of lineage-specific TFs, which in turn induce unilineage HGFR(s); (2) in the unilineage HPC differentiatiodmaturation pathway expression of the pertinent,
lineage-specific TF(s) and HGFR is sustained by self-maintaining molecular loop(s) at the level of TF and HGFR promoters.
Hematopoietic commitment has been interpreted in terms
of stocha~tic,~~-~’
inductive? or hybrid74models. The inductive model proposes that binding of HGF(s) to their receptor(s) induces differentiation, in that it determines lineage
choice in multipotent HPCs by activating the transcription
of a lineage-specific gene program. The stochastic model
proposes that multipotent HPCs do not require exposure to
external inducing stimuli to undergo lineage restriction:
HGFs simply permit the proliferation of intrinsically committed cells and the subsequent expression of the mature
cell phenotype. Our results are in part compatible with both
models. Hypothetically, (1) the differential expression of
receptors for early-acting HGFs on quiescent HPCs may
reflect a stochastic process in early commitment. (2) Consistent expression of multi-lineage HGFRs on quiescent HPCs
allows the induction of progenitor cycling by IL-3/GM-CSF.
(3) Expression of unilineage HGFR(s) on the proliferating
HPCs may be stochastic. (4) The sustained expression of
each unilineage HGFR through the corresponding differentiatiodmaturation pathway represents an inductive process,
mediated by the pertinent unilineage HGF. The unilineage
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
A
B
I
I
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El
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le
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1w
Day 0
El
Day 2
Day 2
KI
Day 5
Day 5
..
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jJ
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CD34
...:
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CD34
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-
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-
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I
Day 0
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Day 0
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J
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Day 2
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t
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Day 7
ka
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Day 9
:...
-.a'..
Day 9
I.
Day 12
CD34
4
CD34
4
CD34
Fig 6.
-
CD34
-
Day 12
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS
E
3403
Day 0
J
El
Day 2
Day 5
t
Day 2
PI
Day 5
Day 7
w
c!
f,
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Day 7
d
d
Day 9
Day 9
Day 12
CD34
-D
CD34
4
CD34
-
Day 12
Day 0
Day l 2
Day 14
CD34
-
CD34
-
Fig 6. Representative experiments of flow cytometric analysis for fit3 (A), c-kit (6).IL-6R (C), IL-3R (D and E), GM-CSFR
IF), and G-CSFR (G) during selective erythroid (E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol differentiation of step lllP HPCs. c-kit and GM-CSFR were evaluated by
specific MoAbs. flt3, c-kit, GM-CSFR, and G-CSFR expression
is shown in E and G lineages, IL-6R expression in Mk and M o
lineages, and IL-3R in all four lineages. See also mean ? SEM
expression values in Fig 7A and B.
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3404
TESTA ET AL
(Gi
IMOi
IM,j
L
100
8
20
0
3
6
9
1
2
0
3
6
9
12
m
B
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. m,
[GM-CSFR
]
1W
+ ml
0
3
6
9
wvr
1
2
w
0
3
6
9
bys
1
2
0
3
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9
DWS
HGF prevailing in each microenvironmental niche may
channel HPCs expressing its receptor into the COKespondh2
lineage; unipotent HPCs and differentiated precursors growing in the absence of a sufficient level of the pertinent unilineage HGF seemingly undergo apoptosis.'
1
2
0
3
6
9
tmyt
1
2
Fig 7. Percentage of f/t3+, c&it+, IL-6R+ (AI, IL-3R+, GMCSFR+, and G-CSFR+ (B) cells
during step lllP HPC erythroid
(E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol
differentiation. The receptor
density of IL-3R and GM-CSFR is
also presented, as evaluated in
terms of mean fluorescence intensity (arbiiary units). In the
case of G-CSFR, representative
data in G-CSF- culture are also
shown (see Results). fh3, c-kit,
and IL-3R were evaluated by
M o w ; lL-6R. OM-CSFR, and GCSFR by labeled ligand. (further
details in Materials and Methods). Mean 2 SEM v d u r from
three separate experiments. See
also representative experiments
in Fig 6A-G.
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1996 88: 3391-3406
Expression of growth factor receptors in unilineage differentiation
culture of purified hematopoietic progenitors
U Testa, C Fossati, P Samoggia, R Masciulli, G Mariani, HJ Hassan, NM Sposi, R Guerriero, V
Rosato, M Gabbianelli, E Pelosi, M Valtieri and C Peschle
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