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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2559-2568
HEMATOPOIESIS
Identification and characterization of a bipotent (erythroid and
megakaryocytic) cell precursor from the spleen of
phenylhydrazine-treated mice
Alessandro Maria Vannucchi,
Francesco Paoletti,
Silvia Linari,
Cristina Cellai,
Roberto Caporale,
Pierluigi Rossi Ferrini,
Massimo Sanchez,
Giovanni Migliaccio, and
Anna Rita Migliaccio
From the Division of Hematology, University of Florence and Azienda
Ospedaliera Careggi, Florence, Italy; and the Laboratory of Cell
Biology and Laboratory of Clinical Biochemistry, Istituto Superiore di
Sanità, Rome, Italy.
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Abstract |
We have identified a cell population expressing erythroid (TER-119)
and megakaryocyte (4A5) markers in the bone marrow of normal mice. This
population is present at high frequency in the marrows and in the
spleens involved in the erythroid expansion that occurs in mice
recovering from phenylhydrazine (PHZ)-induced hemolytic anemia.
TER-119+/4A5+ cells were isolated from the
spleen of PHZ-treated animals and were found to be blast-like
benzidine-negative cells that generate erythroid and megakaryocytic
cells within 24-48 hours of culture in the presence of erythropoietin
(EPO) or thrombopoietin (TPO). TER-119+/4A5+ cells represent a late
bipotent erythroid and megakaryocytic cell precursors that may exert an
important role in the recovery from PHZ-induced anemia.
(Blood. 2000;95:2559-2568)
© 2000 by The American Society of Hematology.
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Introduction |
All the circulating elements of the blood derive from
rare marrow cells through a complex process that involves extensive proliferation, lineage commitment, and cell differentiation and maturation.1,2 This process is modulated/regulated by
cellular interactions with a wide range of cytokines.3,4
One working model proposed for the mechanism of hematopoietic
commitment suggests that the cells program their differentiation toward
a particular lineage by progressive restriction of their
differentiation potential.5 This model predicts the
generation of bipotent cell precursors as one of the steps leading to
erythroid and megakaryocytic differentiation.6
The existence of this common precursor was suggested by the
observations that the majority of the human7-10 and
murine11,12 erythroleukemic and megakaryoblastic cell
lines, as well as the blasts freshly isolated from human erythroblastic
(M6) and megakaryoblastic (M7) leukemia,13,14 coexpress
erythroid and megakaryocytic markers. These data could be explained
either as lineage infidelity of leukemic cell
differentiation15 or according to the hypothesis that
leukemic cells retain certain properties of the normal cells from which
they derive.5 Thus, in the case of these cell lines and
primary leukemic blasts, they may have retained the properties of their
common precursor.16
A linkage in the control of erythropoiesis and megakaryocytopoiesis was
also suggested by the biological activity of erythropoietin (EPO) and
thrombopoietin (TPO), the growth factors primarily responsible for
differentiation toward these lineages.17,18 EPO and TPO were first identified because their levels specifically increase in the
serum of animals recovering from anemia19 or
thrombocytopenia,20 respectively. However, animals whose
EPO17 (or TPO18) serum levels are exogenously
increased develop not only increased numbers of circulating red cells
(or platelets), but also higher numbers of megakaryocytic (or
erythroid) progenitor cells in the marrow. Transgenic mice
overexpressing the human TPO receptor (Mpl) exhibit not only
chronic thrombocytosis but also enhanced erythroid recovery following
5-fluorouracyl treatment.21 Furthermore, the phenotype of
mice that are genetically unable to express either one of these growth
factors,22,23 as well as of their corresponding
receptors,22,24,25 is characterized by reduced levels of
both erythroid and megakaryocytic progenitor cells. Alternatively,
because the erythropoietin receptor (EpoR) and Mpl are
coexpressed on erythroid and megakaryocytic progenitors26
and erythroid progenitors from mice lacking EpoR differentiate
in vitro in the presence of recombinant TPO,27 it is also
possible that EPO and TPO are, at least partially, redundant in their
control of the early stages of erythroid/megakaryocytic differentiation.
The hypothesis of a common cell precursor was invoked to explain the
observation that not only the promoter regions of EpoR and
Mpl, but also those of all the other erythroid- and
megakaryocytic-specific genes investigated, contain functional binding
domains for a common panel of transcription factors.28
Furthermore, mice in which the expression of either
Nef2,29,30 Gata1,31,32 or
Fog6 (a recently described multitype
zinc finger protein that modulates the biological activity
of GATA)33 is impaired by gene disruption express a
similar phenotype characterized by deficiency in both erythroid and
megakaryocytic cell differentiation. Moreover, forced expression of
Gata1 in avian myelomonocytic cells34 or in the murine myeloid cell line M135 induces both erythroid and
megakaryocytic differentiation. Evidence that megakaryocytic gene
promoters are active into erythroid cells is provided by the
observation that transgenic mice expressing a suicide gene under the
control of the glycoprotein IIb (GpIIb)-specific promoter
region are both thrombocytopenic and anemic.36 The fact
that these mice contain reduced levels of multipotential progenitor
cells supports the concept that activation of lineage-specific
promoters precedes the establishment of the full differentiation
program.37,38
Despite this accumulating data, common erythroid and megakaryocytic
cell precursors have not been identified. Little evidence has been
provided for a cell precursor whose frequency specifically increases in
vivo by either increasing the serum levels of EPO39 or
TPO40 or by forcing murine stem cells to express
Mpl constitutively.41 Moreover, no cell precursor
has been found to specifically decrease in tissues from
Epo/EpoR-22 or
Tpo/Mpl-42,43 deficient mice. Human44 and murine45 bipotent progenitor cells
have been recently described but have not yet been isolated.
The specific aim of this study was to identify and describe the
properties of an in vivo candidate cell for the bipotent precursor.
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Materials and methods |
Mice
C57BL mice (2-4 months old) were purchased as needed
from Charles River (Calco, Italy). In experimental mice,
phenylhydrazine (PHZ; 60 mg/kg body weight; Sigma Chem, St. Louis, MO)
was injected intraperitoneally for 2 consecutive days.46,47
On each of the 3 days after the second PHZ injection, 5 mice were
killed by cervical dislocation, and the bones and spleens removed under
sterile conditions for further analysis. All the procedures were
approved by the institutional animal care committee.
Phenotypic analysis of the cells
Cell morphology was analyzed according to standard criteria on
cytocentrifuged (Shandon, Astmoor, UK) slides stained with May-Grunwald-Giemsa. Hemoglobin-containing cells were identified by
benzidine staining.48 Megakaryocytes were identified by
copper-thiocoline acethylcolinesterase E (ACHE) staining.49
Cell viability was assessed either by trypan-blue (in optical
microscopy) or by propidium iodide (5 µg/mL, Sigma) (in flow
cytometry) exclusion. The cells were immunophenotyped with the
following antibodies: phycoerythrin conjugated (PE)-TER-119 [Ly-76, a
rat immunoglobulin G2b (IgG2b) monoclonal
antibody recognizing an antigen expressed on erythroid cells from
erythroblasts to erythrocyte 50; fluorescein conjugated (FITC)-CD4, FITC-CD8, FITC-B220 (all from PharMingen, San Diego, CA),
and FITC-4A5 [a rat monoclonal IgG2a antibody specific for murine megakaryocytes and platelets (gift from Dr S
Burstein)51 that recognizes glycoprotein V (Burstein S,
personal communication, April 1999)]. Cells, suspended in
Ca++-, Mg++-free phosphate buffered saline
(PBS) supplemented with 1% (v/v) bovine serum albumin, 2 mmol/L EDTA
(that minimizes the adhesion of platelets, or of their membranes, to
the cell surface), and 0.1% NaN3, were labeled with each
antibody (~1 µg/106 cells) for 30 minutes on ice. The
cell fluorescence was analyzed either with the FACScan flow cytometer
(Becton Dickinson, San Jose, CA) or with a Coulter Epics Elite ESP
(Coulter, Miami, FL) cell sorter. Cells incubated with appropriately
labeled isotype controls (PharMingen) were used to gate the
nonspecific fluorescence signal. Before the analysis, mature red cells
were depleted by hypotonic lysis (0.38% ammonium chloride for 15 minutes on ice).
Cell purification
Monocellular spleen suspensions were prepared by cutting the spleens
into small fragments in 5 mL of Ca++- and
Mg++-free PBS containing 10% (v/v) fetal bovine serum
(FBS, Boehringer Mannheim, Mannheim, Germany) and by passing the cell
suspension through progressively smaller needles. Marrow cells were
flushed from the femurs with a syringe containing 2 mL PBS-10% FBS.
Marrow and spleen light density mononuclear cells were isolated by
centrifugation over Ficoll-Paque ( = 1.077 g/mL; Pharmacia
Biotech, Uppsala, Sweden) at 800g for 20 minutes at room
temperature. Light density spleen cells were either analyzed as such or
further enriched for erythroid/megakaryocytic precursors by
immunomagnetic selection or cell sorting. For the former, adherent
cells were first removed by 2 cycles of adherence to plastic at
37°C for 1 hour. The nonadherent fraction was then depleted of B
and T lymphocytes by binding to Dynabeads (Dynal AS, Oslo, Norway)
magnetic microspheres coated with anti-B220 (Pan-B) and anti-Thy 1.2 (Pan-T) antibodies as described by the manufacturer. The not-bound
cells were incubated with FITC-conjugated 4A5 (~1
µg/106 cells) for 30 minutes on ice, washed twice with
PBS containing 0.5% (w/v) bovine serum albumin and 2 mmol/L EDTA, and
finally suspended in 80 µL of buffer and 20 µL of MACS microbeads
(Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) conjugated with
goat antirat IgG F(ab)2. The cells were washed twice and
loaded on a MS+/RS+ column placed in a MACS
separator. 4A5 were recovered in the effluent fraction,
whereas the 4A5+ cells were flushed out of the column. Both
fractions were then incubated with PE-TER-119 for fluorimetric
analysis. For cell sorter separation, light density cells were
incubated with FITC-4A5 and 4A5+ cells sorted with a
Coulter tuned at 488 nm. The sorted cells were then incubated with both
PE-TER-119 and FITC-4A5 and double-positive cells sorted again. The
sorted fractions were then reanalyzed for purity with the cytometer. In
some experiments, purified cells were labeled again with FITC-4A5 and
PE-TER-119 and examined under a fluorescent microscope (Axiookop Zeiss,
GmbH, Jena, Germany). Fluorescence emission was captured and analyzed
with the CytoVision program (Applied Imaging, Santa Clara, CA) after
nucleous counterstaining with DAPI
(4',6'-diamidino-2-phenylindole; Vysis, Downers
Grove, IL).
Liquid culture of spleen cells from PHZ-treated animals
Light density and purified cells
(TER-119+/4A5± cells) were obtained from
the spleens of PHZ-treated mice at days 1-2 following treatment. These
cells were resuspended in IMDM supplemented with 10% Nutridoma
(2-5 × 105 cells/mL) and incubated for up to 48 hours either without growth factors (GF) or in the presence of EPO (5 U/mL) or TPO (100 ng/mL) at 37°C in 5% CO2.
Semisolid culture of normal progenitor cells
Light density marrow cells (0.25-1.0 × 105
cells/plate) isolated from normal mice were seeded into FBS-free
semisolid culture plates (Stem Cell Technologies, Vancouver, BC).
Colony growth was stimulated with the following combinations of
recombinant growth factors: (i) rat stem cell factor (100 ng/mL), mouse interleukin 3 (IL-3; 10 ng/mL) (both from Sigma) and
either human EPO (2 U/mL; Boehringer Mannheim, Mannheim, Germany) for
burst-forming unit erythroid (BFU-E) growth52
or (ii) mouse granulocyte-colony stimulating factor (G-CSF) and
granulocyte-macrophage colony stimulating factor (GM-CSF) (50 ng/mL
each; both from Sigma) for colony-forming unit-granulocyte
macrophage (CFU-GM) growth52 or (iii)
human TPO (50 ng/mL; PrepoTech, London, UK) for colony-forming
unit-megakaryocyte (CFU-Mk) growth.53 The
growth of CFU-E-derived colonies was stimulated with EPO alone (2 U/mL).54 The cultures were incubated at 37°C in a
humidified incubator containing 5% CO2 in air and scored
either 3 days (for CFU-E-derived colonies) or 7 days (for CFU-GM-,
BFU-E-, and CFU-Mk-derived colonies) following initiation of culture.
Morphologically recognizable colonies that were clearly separated from
the others (ie, the average distance from adjacent colonies was at
least twice their diameter) were individually collected under sterile
conditions, washed once in PBS, and suspended in 0.5 mL of Trizol
(GIBCO BRL, Paisley, UK). In some experiments, single BFU-E- and
CFU-GM-derived colonies were harvested at day 5 of culture and
replated in secondary serum-deprived cultures stimulated with either
EPO (5 U/mL) or TPO (100 ng/mL) alone (1 colony × 200
µL/well). After 4 days of incubation at 37°C in 5% CO2, the wells were scored for the presence of
CFU-E-derived colonies and megakaryocytes.
The frequency of BFU-E and CFU-E in the spleen of anemic mice and in
the purified cell fractions was determined in triplicate cultures
(105 or 104 cells/plate, respectively) under
the conditions described above. To improve accuracy, the frequency of
CFU-Mk was determined in triplicate semisolid agar cultures (0.3%
Bacto-agar, 2.5 × 105 light density and
1.0 × 104 purified cells/plate) containing
L-glutamine (2 mmol/L), L-serine (8 mg/L),
sodium pyruvate (1 mmol/L), L-asparagine (16 mg/L), FBS
(15% v/v); murine IL-3 (10 ng/mL), murine interleukin 6 (IL-6, 100 ng/mL; Sigma), and human TPO (50 ng/mL) in McCoy's 5A medium (GIBCO
BRL). After 7 days of incubation, the agar was dehydrated and
megakaryocytic colonies identified in situ by ACHE
staining.53
RNA isolation and semiquantitative RT-PCR analysis
Total RNA was prepared using a commercial guanidine
thiocyanate/phenol method (Trizol, GIBCO BRL) as described by the
manufacturer. Glycogen (20 µg; Boehringer Mannheim) was added to each
sample as a carrier. Total RNA (1 µg) was reverse transcribed at
42°C for 30 minutes in 20 µL of 10 mmol/L Tris-HCl, pH 8.3, containing 5 mmol/L MgCl2, 1 U RNAse inhibitor, 2.5 U
Moloney Murine Leukemia Virus reverse-transcriptase, and 2.5 µmol/L
random hexamers (all from Perkin-Elmer, New Jersey). The expression of
 - and  -globin, EPO receptor (EpoR),
acetylcholine esterase (AchE), GpIIb, TPO receptor
(Mpl), and myeloperoxidase (Mpo) was analyzed by
amplifying reverse-transcribed complementary DNA (cDNA; 2.5 µL) in
the presence of the specific sense and antisense primers (100 nmol/L
each) described elsewhere.55,56 The reaction was performed
in 100 µL of 10 mmol/L Tris-HCl, pH 8.3, containing MgCl2
(2 mmol/L), dNTP (200 µmol/L each), 0.1 µCi of
[32P]dCTP (specific activity 3000 Ci/mmol; Amersham
Italia, Cologno Monzese, Italy) and 2 U AmpliTaq DNA polymerase.
Primers specific for -actin (50 nmol/L each) were added to
each amplification after the first 10 cycles as a control for the
amount of cDNA used in the reaction.55,56 PCR conditions
were as follows: 60 seconds at 95°C, 60 seconds at 60°C, and 60 seconds at 72°C, using a GeneAmp 2400 Perkin-Elmer thermocycler.
All the RT-PCR presented were done in the linear range of amplification
defined by preliminary experiments to be, for most of the genes
analyzed, between 20-30 cycles with the exception of Mpl (30-38 cycles) and - and -globin (18-24 cycles).
Positive (RNA from adult marrow) and negative (mock cDNA) controls were
included in each experiment. Aliquots (20 µL) were removed from the
PCR mixture after amplification, and the amplified bands separated by
electrophoresis on 4% polyacrylamide gel. Gels were dried using a
Bio-Rad apparatus (Hercules, CA) and exposed to Hyperfilm-MP (Amersham
Italia) for 2 hours at  70°C. All the procedures were according
to standard protocols.57
Statistical analysis
Statistical analysis was performed by analysis of variance
(ANOVA test) using Origin 3.5 software for Windows (Microcal Software, Northampton, MA).
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Results |
Expression of erythroid and megakaryocytic markers in the marrow and
spleens of normal and PHZ-treated mice
PHZ-treated mice develop a profound anemia from which they recover
by augmenting splenic erythropoiesis (Tables
1 and 2). Spleens from PHZ-treated animals are 1.8- to 3.7-fold larger than normal spleens. On day 1, they contained high numbers of BFU-E (140 ± 50 colonies/104 cells), relatively low numbers
of CFU-E (21 ± 11 colonies/104 cells), and many
(83% ± 5%) benzidine-negative blasts (Table 1). On day 3, they
contained fewer BFU-E (15 ± 5 colonies/104 cells),
more CFU-E (273 ± 39 colonies/104 cells), and even
greater numbers (69% ± 15%) of benzidine-positive erythroblasts
distributed along all maturation stages (Table 1). The numbers of
megakaryocytic progenitors (0.8 ± 0.05 CFU-Mk/104
cells, Table 1) and ACHE-positive cells (below detection limits) in the
spleen remained low throughout the phase of recovery from anemia.
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Table 1.
Number of progenitor cells and benzidine-positive cells
in the light density fraction of the spleens from normal mice and from
mice 1 and 3 days after PHZ treatment*
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Table 2.
Frequency (in percent) of TER-119+ and/or
4A5+ cells in the marrow and spleens of normal mice and
of mice recovering from the anemia induced by phenylhydrazine (PHZ;
day 1-3)
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Flow cytometry analysis of the expression of erythroid (TER-119) and
megakaryocytic (4A5) markers on cells from the bone marrow and the
spleen of normal and PHZ-treated mice is shown in Figure 1A and 1B. The frequency of
TER-119+ and 4A5+ cells increased both in the
marrow and in the spleen during the recovery from PHZ-induced anemia
(Figure 1A, 1B, and Table 2). In addition, a third cell population
coexpressing TER-119 and 4A5 was identified in the bone marrow of
normal mice (Figure 1A). TER-119+/4A5+ cells
represented 1.3% ± 0.6% of normal bone marrow cells and their
frequency increased up to 3.8% (P < .01) in the marrow of PHZ-treated animals (Figure 1A and Table 2). A significant proportion (1.3%-8.3%) of TER-119+/4A5+ cells were also
detected in the light density spleen cells from PHZ-treated mice. The
analysis of the erythroid and megakaryocytic markers expressed by
marrow and spleen cells from PHZ-treated animals was verified by
examining the binding of both TER-119 and 2D5, an antibody that
recognizes GPIIB (Burstein S, personal communication,
April 1999). Dual positive
TER-119+/2D5+ cells were identified
in normal marrow and in the marrow and spleens from PHZ-treated mice.
Their frequency was found to be identical to the frequency of
TER-119+/4A5+ cells (Table 2 and result
not shown).
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| Fig 1.
Expression of erythroid and megakaryocytic markers in
hematopoietic tissues from normal and phenylhydrazine (PHZ)-treated
mice.
(A) Flow cytometric analysis of the expression of TER-119 (Y axes) and
4A5 (X axes) by bone marrow (top panels) and spleen (bottom panels)
light density cells from normal and PHZ-treated (1, 2, and 3 days after
the second injection) mice. The cells were also incubated with
isotype-matched irrelevant antibodies as negative controls and the
results presented on the left panels. R1, R2, and R3 indicate the gates
used to define cells expressing TER-119 and 4A5 alone or coexpressing
the 2 antigens. (The corresponding cell frequencies as determined in
multiple experiments are presented in Table 2.) Propidium
iodide-positive cells were less than 1% and were excluded by
appropriate gating. (B) Hystogram analysis of the staining with
FITC-IgG2 (right) or with FITC-4A5 (left) of normal (black
line) and PHZ-treated (day 1, gray line) light density spleen cells
gated in the TER-119-positive (R1+R3 of Figure 1A) area. (C)
Semiquantitative RT-PCR analysis of the expression of erythroid
(-globin and EpoR), megakaryocytic (GpIIb,
AchE, and Mpl) and myeloid (Mpo) genes in
spleen cells obtained from either normal or PHZ-treated (days 1 and 3)
mice. Actin was amplified to control for the amount of cDNA
used in each reaction. -globin and actin were
amplified for 18, 21, and 24 cycles; Mpl for 25, 30, and 35 cycles; and all the other genes for 27, 30, and 33 cycles (increasing
numbers of cycles are indicated by a triangle on the top of the
panels). The results are representative of those obtained in 3 separate
experiments.
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Semiquantitative RT-PCR analysis of expression of erythroid- and
megakaryocytic-specific genes from the spleens of normal and
PHZ-treated mice is presented in Figure 1C. Of the genes analyzed, only
-globin was readily amplified from normal spleen cells. AchE was amplified from those cells only after 33 cycles. In
contrast, as predicted by the flow cytometry data, not only erythroid
(-globin and EpoR) but also megakaryocytic
(GpIIb, AchE, and Mpl) genes were amplified
with high efficiency from day 1 PHZ-spleens. With progression of the
anemia (day 3 following PHZ-treatment), erythroid-specific genes
(-globin and EpoR) were amplified even more
efficiently (maximal amplification noted after 18 and 30 cycles,
respectively), whereas amplification of megakaryocytic genes was either
barely (GpIIb) or not (AchE and Mpl)
detectable. Mpo was never amplified, even at high numbers of
cycles, from the spleens of either normal or PHZ-treated mice.
Isolation and characterization of
TER-119+/4A5+ cells from the spleens of
PHZ-treated mice
TER-119+/4A5+ cells were purified from the
spleen of day 1-2 PHZ-treated animals because of the high
TER-119+/4A5+ cell content of this tissue
[~1-1.75 × 106
TER-119+/4A5+ cells/spleen = 5% of
40-70 × 106 light density cells/2 PHZ-treated
spleens (Table 3)].
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Table 3.
Purification of
TER-119+/4A5+ double-positive cells from
the spleen of phenylhydrazine (PHZ)-treated mice*
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The first purification method was discovered serendipitously. We had
found that day 1 PHZ-treated cells cultured for 18 hours in the absence
of GF are enriched for cells that do not express -globin by
RT-PCR unless incubated for 2 hours with EPO.55 When
analyzed for surface antigen expression, the GF-starved cells were
found to contain few TER-119+ cells (7%) and to be
enriched (~5 times, from 5.8% to 30%) for TER-119+/4A5+ cells (Table 3). The majority
(60%) of them were TER-119/4A5 cells
expressing either B220 (B cells) or CD4/CD8 (T cells) (Table 3).
GF-starved cells contained some BFU-E (5 ± 2
BFU-E/104 cells) and no benzidine-positive (Table 1) or
ACHE-positive (data not shown) cells.
The physical isolation of TER-119+/4A5+ cells
is described in Figure 2 and Table 3. For
immunomagnetic selection, day 1-2 PHZ-treated light density spleen
cells were first depleted of monocytes by 2 cycles of adherence to
plastic, then depleted of lymphocytes by panning on panT/panB-coated
microspheres (B/T cells), and finally
positively selected for 4A5+ cells by the 4A5-coated
immunomagnetic beads. The cell fraction that did not bind to the beads
(4A5) was isolated as a negative control. Alternatively,
TER-119+/4A5+ cells were isolated from
light-density spleen cells by 2 consecutive sortings, the first 1 on
cells labeled with 4A5 only. The 4A5+ cells were then
labeled with both 4A5 and TER-119 and sorted again.
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| Fig 2.
Isolation of TER-119+/4A5+
cells from the spleens of phenylhydrazine (PHZ)-treated animals.
(A) Immunomagnetic isolation. Flow cytometric analysis of the
expression of TER-119 (Y axes) and 4A5 (X axes) in nonadherent, B- and
T-depleted (B/T),
TER-119+/4A5, and
TER-119+/4A5+ cell fractions purified from the
spleens of PHZ-treated mice. The gates, which identify cells expressing
TER-119 and 4A5 alone or coexpressing the 2 antigens, were set to
include only propidium iodide-negative cells. Gating and negative
controls (not shown) are as in Figure 1A. Similar results were obtained
in 7 additional purifications. The corresponding cell frequencies are
summarized in Table 3. (B) Cell sorter isolation. Flow cytometric
analysis of the expression of TER-119 (Y axes) and 4A5 (X axes) in
light density spleen cells, in 4A5+ cells isolated during
the first sorting, and in the double
TER-119+/4A5+ cells isolated with the second
sorting. The gates, which identify cells expressing TER-119 and 4A5
alone or coexpressing the 2 antigens, were set to include only
propidium iodide-negative cells. Gating and negative controls (not
shown) are as in Figure 1A. Similar results were obtained in 3 additional purifications. The corresponding cell frequencies are
summarized in Table 3. (C) Analysis of immunomagnetically purified
TER119+/4A5+ cells using fluorescence
microscopy and image analysis. Nuclei were counterstained (blue) with
DAPI. FITC-4A5 fluorescence (green) and PE-TER-119 fluorescence (red)
were individually analyzed and captured (original magnification
×100). (D) Semiquantitative RT-PCR analysis of the expression of
erythroid (- and -globin and EpoR) and
megakaryocytic (AchE, Mpl, and GpIIb) genes in
B/T cells or in
TER-119+/4A5 and
TER-119+/4A5+ cells (purified by double
sorting) from the spleens of PHZ-treated mice. Actin
complementary DNA (cDNA) was amplified as well as control of the total
cDNA used in each reaction. -globin, GpIIb, and
actin were amplified for 20, 25, and 30 cycles, whereas all the
other genes were amplified for 25, 30, and 35 cycles (increasing
numbers of cycles are indicated by a triangle on top of the panels).
Similar results were obtained in 3 separate experiments.
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With immunomagnetic selection, a total of
[~2 × 106 cells (~66% of which
TER-119+/4A5+ on reanalysis) were obtainedper
75 × 106 original light density spleen cells
(Figure 2A and Table 3). Because the theoretical
TER-119+/4A5+ cell content of the starting
population was 4.3 × 106 cells, ~32% of all the
TER-119+/4A5+ cells were recovered with this
procedure. The major contaminants were found to be single
TER-119+ cells (10%-15%) and B and T lymphocytes
(11%-15%; Table3). A lower number of cells
(~2 × 105) were recovered at the end of the
purification by sorting. However, in this case, the purified
cells contained a higher proportion of
TER-119+/4A5+ (~82% on reanalysis) cells,
and the majority of contaminants were represented by neutrophils (the
lymphocytes were excluded by size gating).
All the purified TER-119+/4A5+ cell fractions
(irrespective of the method used) were enriched for benzidine- and
ACHE-negative cells (Table 1 and 4) with the morphology of
blasts (Figure 3B) that coexpressed
fluorescein and phycoerythrin membrane-associated fluorescence by
microscopic analysis (Figure 2C). Progenitor cells were not detected
among the TER-119+/4A5+ cells (Table 1). In
contrast, the TER-119+ cell fractions, isolated as control,
contained benzidine-positive erythroblasts and some CFU-E (3 ± 2
CFU-E/104 cells) (Table 1).
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| Fig 3.
Differentiation of
TER-119+/4A5+ cells isolated from the
spleens of phenylhydrazine (PHZ)-treated mice in serum-deprived
cultures stimulated with either erythropoietin (EPO; 5 U/mL) or
thrombopoietin (TPO; 100 ng/mL).
(A) Flow cytometric analysis of the expression of TER-119 and 4A5 in
cells isolated by immunomagnetic selection as such (left panel) and
after 24-48 hours of culture in the presence of either EPO (center
panels) or TPO (right panels). Negative controls (not shown), analysis
conditions, and cell gating are the same as in Figure 1A. The
frequencies of the cells expressing TER-119 and/or 4A5 observed in
individual experiments are shown in Table 4. (B) May-Grunwald Giemsa
staining of Ter119+/4A5+ cells purified from
the spleens of PHZ-treated mice as such (left panel) or cultured for
24-48 hours in the presence of either EPO (center panels) or TPO (right
panels). The same cell preparations presented in Figure 2A
(magnification ×400). (C) Semiquantitative RT-PCR analysis of the
expression of erythroid (-globin) and megakaryocytic
(AchE and GpIIb) genes in
TER-119+/4A5+ cells purified from the spleens
of PHZ-treated mice at baseline (left) or cultured for 24 hours in the
presence of EPO (center) or TPO (right). Actin complementary DNA (cDNA)
was amplified to control for the total amount of cDNA used in each
reaction. The same cell fractions are shown in the top right panels of
Figure 3A. -globin and actin were amplified for 18, 21, and 24 cycles and AchE and GpIIb for 27, 30, and 33 cycles (increasing numbers of cycles are indicated by a triangle on
top of the panels). Similar results were obtained in 3 additional experiments.
|
|
The semiquantitative RT-PCR analysis for the expression of erythroid
and megakaryocytic genes in the purified cell fractions is presented in
Figures 2D and 3C. Both erythroid and megakaryocytic genes were readily
amplified from the fraction depleted of B and T lymphocytes
(B/T cells) (Figure 2D), whereas, as
expected on the basis of their morphology, only the erythroid genes
were amplified from fractions enriched for TER-119+ cells
(Figure 2D). All of the genes analyzed were amplified also from
TER-119+/4A5+ cells. However in this case,
EpoR and Mpl, as well as -globin and
GpIIb, were amplified with high efficiency, but amplification of -globin and AchE was detectable only after the
highest number of PCR cycles (Figure 2C).
To verify if the purified TER-119+/4A5+ cells
had the potential to differentiate into erythroid and megakaryocytic
cells, they were cultured for up to 48 hours in the presence of EPO or
TPO under serum-deprived culture conditions (Table
4 and 5).
TER-119+/4A5+ cells survived poorly in the
absence of GF, and very few (20%-30%) of the original
dual-labeled cells remained detectable after 48 hours of culture under
those conditions. In cultures stimulated with either EPO or TPO, the
total number of cells remained constant for 24 hours (Table
5), and ~50% of the original cell number was still detectable after
48 hours (Table 4 and 5). In contrast, the frequency of
TER119+/4A5+ cells declined
progressively and only 7%-10% of the cells expressed both antigens by
48 hours (Figure 3A and Table 5). The decline in the frequency of
double-positive cells was paralleled by an increase in the frequency of
cells positive for 1 antigen. In cultures stimulated with EPO, there
was a significant increase in the number of single TER119+
cells (60% by 24 hours), whereas, in cultures stimulated with TPO, the
frequency of both TER-119+ (60% by 48 hours) and
4A5+ (16% by 24 hours) cells was significantly
increased (Table 5). These surface phenotypic changes were accompanied
by morphological changes: cells cultured with EPO became
benzidine+ and acquired a predominantly erythroid
morphology (Figure 3B). Orthochromatic erythroblasts,
conceivably in the enucleation phase, were clearly recognized at
48 hours. Following culture with TPO, both erythroblasts
and megakaryocytes were recognized (Figure 3B).
Semiquantitative RT-PCR analysis of the genes expressed by the cultured
TER-119+/4A5+ cells is presented in Figure 3C.
The expression of all the genes analyzed increased when
TER-119+/4A5+ cells were cultured for 48 hours:
-globin was amplified after 18 cycles from cells cultured
either with EPO or TPO, whereas substantial amplification of
GpIIb and AchE was obtained only from cells cultured
with TPO (Figure 3C). This last result reflects the high frequency of
single 4A5+ (Table 4) ACHE+ (Table
5) cells detected in those cultures.
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Table 5.
Total cell number and frequency of
TER-119+ and/or 4A5+ cells in cultures of
purified double-positive TER-119+/4A5+
cells from the spleen of day 1-2 phenylhydrazine-treated
mice*
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|
As a negative control, purified single TER-119+ cells were
also cultured under the same conditions. Very low numbers (5%-10%) of
TER-119+ cells survived 24-48 hours even in the presence of
EPO or TPO and did not provide amounts of cDNA sufficient for gene
expression analysis (not shown).
Expression of erythroid and megakaryocytic-specific genes in single
colonies derived from normal progenitor cells
To evaluate the time span that normal hematopoietic progenitors
remain bipotent, the expression of lineage-associated genes in single
colonies derived from marrow progenitor cells was assessed by RT-PCR
(Figure 4). cDNAs were obtained from single
colonies derived from either early (CFU-GM, BFU-E, and CFU-Mk) or late (CFU-E) progenitor cells induced to proliferate and differentiate in
semisolid cultures by appropriate combinations of growth
factors.49-51 The presence in the cDNA libraries of
differentiation-associated genes (erythroid: -globin and
EpoR; megakaryocytic: GpIIb, AchE, and
Mpl; and granulocytic: Mpo) was then evaluated by PCR.
To control for adequate amounts of cDNA, actin fragments were
concurrently amplified from each library. Actin-specific cDNA
fragments were amplified from 137 of a total of 140 single colonies
processed (97% efficiency). Only actin-positive libraries were
included in the analysis presented in Figure 4.
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| Fig 4.
RT-PCR analysis of the expression of erythroid
(-globin and EpoR), megakaryocytic (GpIIb,
AchE, and Mpl), and myeloid (Mpo) genes in
single colonies derived from early erythroid, megakaryocytic, and
myeloid (BFU-E, CFU-Mk, and CFU-GM, respectively) or late erythroid
(CFU-E) progenitor cells.
RNA was prepared from individual colonies (lanes 1-4), reverse
transcribed, and PCR-amplified for either 24 (-globin) or 35 (all the other genes) cycles. Actin-specific fragments were
amplified from all the PCR reactions presented (not shown). Lane C
shows the data obtained using complementary DNA (cDNA) from normal bone
marrow (positive control). The results obtained with 4 representative
colonies are shown in each panel. The ratio on the right of each panel
specifies the actual number of single colonies that were positive for
that particular gene as a function of the total number of colonies
analyzed. The cloning efficiency was 32 ± 10 BFU-E-, 6 ± 2
CFU-Mk-, 10 ± 4 CFU-E-, and 55 ± 20 CFU-GM-derived
colonies per 105 normal bone marrow cells, and the number
of colonies analyzed corresponds to the colonies detected in 1.5, 4, 3, and 0.5 dishes, respectively. BFU-E = burst-forming unit erythroid;
CFU-E = colony-forming unit-erythroid; CFU-GM = colony-forming
unit-granulocyte macrophage; CFU-Mk = colony-forming
unit-megakaryocyte.
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|
Megakaryocytic-specific cDNAs (GpIIb, AchE, and
Mpl) were amplified from all 25 CFU-Mk-derived colonies and
from a high proportion (86%-95%) of the single BFU-E-derived
colonies analyzed (Figure 4). Conversely, erythroid-specific cDNAs
(-globin and EpoR) were amplified not only from all
the BFU-E-derived colonies but also from 96%-100% of the
CFU-Mk-derived colonies analyzed (Figure 4). It is possible that the
amplification of erythroid (or megakaryocytic) genes from libraries
prepared from single colonies was due to contamination from the
originally plated marrow cells. To exclude this possibility, cDNAs were
also prepared from 10 separate methylcellulose samples (10-20 µL
each; ie, the same volume necessary to harvest a single colony),
randomly removed from regions of the dish not containing colonies.
Because actin was never amplified from those samples (not
shown), we believe that cell contamination does not contribute in a
detectable way to cDNA libraries prepared from the single colonies. It
is also possible that the BFU-E- and CFU-Mk-derived colonies analyzed
were CFU-Mix-derived colonies whose myeloid component had not been
recognized by morphological evaluation. Although CFU-Mix-derived
colonies were rare (2-4 colonies/105 cells), cDNA libraries
were prepared from 17 of them and specific genes were amplified from
these libraries as well. -globin, EpoR, GpIIb, and Mpo were amplified from all 17 CFU-Mix-derived colonies analyzed, and AchE and Mpl
were amplified from 16 of them (94%). The fact that Mpo was
never amplified from the colonies identified as erythroid or
megakaryocytic (Figure 4) supports the morphological observation that
these colonies did not contain detectable myeloid cells.
The presence of erythroid and megakaryocytic genes was also analyzed in
cDNA libraries prepared from 27 single CFU-GM-derived colonies and
from 12 single CFU-E-derived colonies (Figure 4). Mpo was
readily amplified from all 27 CFU-GM- derived colonies analyzed,
whereas erythroid- and megakaryocytic-specific cDNAs were
never amplified from these cDNAs (Figure 4). In contrast, erythroid
(-globin- and EpoR-) but not megakaryocytic
(GpIIb, AchE, or Mpl) genes were readily
amplified from libraries prepared from single CFU-E-derived colonies.
Because CFU-E-derived colonies contain at most 50 cells, it is
possible that the failure to detect megakaryocytic genes in those
libraries was due to technical limitations. To exclude this
possibility, cDNA libraries prepared from 7 separate pools of 5-20 individual CFU-E-derived colonies were analyzed as well. Although
-globin was readily amplified from each of the pools, a very
faint GpIIb band was amplified from only 2 of them and
AchE and Mpl were never amplified (data not shown).
To evaluate whether the cells within a BFU-E-derived colony
had the potential to generate megakaryocytic cells, single
BFU-E-derived colonies were harvested at day 5 and transferred into
secondary cultures stimulated with either EPO or TPO (Table
6). Twenty-two single colonies of the total
24 harvested grew in secondary cultures, giving rise mostly to
CFU-E-derived colonies (5 ± 3), with few (3 ± 2) single
megakaryocytes, in the presence of EPO, and to many (on average
22 ± 9) single megakaryocytes when stimulated with TPO. Single GM
colonies, similarly cultured into secondary cultures as control,
proliferated very poorly (17%) under these conditions, giving rise to
few clusters of macrophages and only in the presence of TPO.
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|
Table 6.
Differentiation potential of the cells within
single BFU-E-derived colonies transplanted at day 5 in secondary
cultures stimulated with either EPO or TPO
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|
| |
Discussion |
The failure to establish in vivo murine models of pure
erythropoiesis by genetic strategies has suggested a linkage between erythroid and megakaryocytic differentiation involving the presence of
bipotent cell precursors.6,22-25,29-34 In this regard, a
myeloid progenitor cell, capable of giving rise to cells of all the
myeloid lineages, has been recently isolated and characterized (Traver D, et al, unpublished data, 1999). This myeloid progenitor
generates in vitro 2 additional progenitor cell populations, 1 exclusively committed to the myelomonocytic lineage and the other 1 committed to the erythroid/megakaryocytic lineage.58 A
similar bipotent E/Mk progenitor cell has been recently described in
culture of human44 and murine45 bone marrow
cells. This progenitor cell is apparently different from BFU-E and
CFU-Mk that give rise to pure colonies containing only erythroid and
megakaryocytic cells, respectively. These data suggested that, in the
hemopoietic cell hierarchy, the last bipotent E/Mk cell is upstream to
the BFU-E level.
Our working hypothesis was that a bipotent E/Mk cell should not
only be present in hematopoietic tissues of normal mice, but its
frequency should be increased following recovery from PHZ-induced anemia. To prove this hypothesis, we have investigated the expression of erythroid and megakaryocytic markers in the bone marrow and spleens
of normal and PHZ-treated mice. These organs of PHZ-treated animals contained increased levels of both erythroid
(TER-119+) and megakaryocytic (4A5+) cells
(Figure 1A). Furthermore, at day 1, PHZ-treated spleens expressed high
levels of both erythroid (-globin and EpoR) and megakaryocytic (GpIIb, AchE, and Mpl) genes
(Figure 1B). Therefore, despite the fact that PHZ-treated animals have
been used as animal models for pure erythropoiesis,48
megakaryocytopoiesis is also increased (at least transiently) in the
tissues from these mice.
A population of TER-119+/4A5+ cells was noticed
in the bone marrow of normal mice and its frequency increased
from 1.3% to 3.8%-4.7% after PHZ-treatment (Figure 1A
and Table 2). High numbers (1.3%-8.3% of the light density cells) of
TER-119+/4A5+ cells were also detected in
PHZ-treated spleens (Figure 1A, 1B, and Table 2). The presence of EDTA
in the buffers used to manipulate the cells makes it unlikely that the
antibodies were reacting against platelets (or their
membranes) adherent to the surface of erythroblasts.
Furthermore, fluorescence was expressed uniformly on all the cell
surfaces under microscopic examination (Figure 2C). Therefore, TER-119
and 4A5/2D5 staining identifies cells expressing both
erythroid and megakaryocytic markers whose frequency increases in
hematopoietic tissues from mice recovering from PHZ-induced anemia. These results indicate the
TER-119+/4A5+ cells as a potential candidate
for the E/Mk precursor.
TER-119+/4A5+ cells were purified by several
means from the spleens of PHZ-treated mice. GF starvation enriched
TER-119+/4A5+ cells by ~5-fold, whereas both
immunomagnetic selection and cell sorting provided fractions
enriched by ~11-fold. In the case of immunomagnetic selection,
~35% of the original TER-119+/4A5+ cells
were recovered at the end of the purification (Table 3). The
TER-119+/4A5+ cell fractions contained
benzidine-negative (Table 1) and ACHE-negative (Table 4) blasts (Figure
3B) expressing highly detectable levels of EpoR, Mpl,
-globin, and GpIIb, and low levels of
-globin and AchE by RT-PCR (Figure 2D and 3C). The
fact that TER-119+/4A5+ cells were recognized
by 2D5 (not shown) indicates that GpIIb expression is at the
single-cell level. It was also interesting that
TER-119+/4A5+ cells expressed almost as much
-globin but much less -globin than single
TER-119+ cells (Figure 2D). Because activation of
-globin precedes that of -globin in the erythroid
differentiation pathway,59 the difference in the levels of
- and -globin expression is consistent with the
hypothesis that TER-119+/4A5+ cells are early
precursor cells.
Although the TER-119+/4A5+ cells survived in
the absence of GF for 18 hours, ~80% of them died in the
absence of EPO or TPO for 24-48 hours (Table 5) even in the
presence of G-CSF or stem cell factor (data not shown).
After 24-48 hours of culture in the presence of EPO,
TER-119+/4A5+ cells gave rise to a cell
population composed mainly (60%-75%) of single
TER-119+ cells. After the same length of time, but in
the presence of TPO, both TER-119+ (62%) and
4A5+ (11%-16%) cells were observed. It is unlikely that
these differentiated cells developed from progenitor cells
because of the low numbers of BFU-E, CFU-Mk, and CFU-E
detected in the purified fractions. These results indicate
that TER-119+/4A5+ blasts
differentiate into erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of EPO or TPO.
The observation that late differentiated cells, such as the
TER-119+/4A5+ cells described in this
manuscript, are still bipotent E/Mk precursors is apparently in
contradiction with the report that, by careful morphological
evaluation, normal murine BFU-E- and CFU-Mk-derived colonies do not
contain cells for the other lineage. To clarify this point,
we analyzed the expression of erythroid- and megakaryocytic-specific genes in single colonies derived in vitro from normal erythroid, megakaryocytic, and myeloid progenitor cells. Almost all (~90%) of the colonies deriving from BFU-E and CFU-Mk, but none of the colonies derived from CFU-GM and CFU-E, contained significant numbers
of cells expressing erythroid and megakaryocytic genes (Figure
4). To confirm that the expression of megakaryocytic genes by a
single BFU-E-derived colony was an index of the megakaryocytic potential of its cells and not of lineage-infidelity within the erythroid cells for megakaryocytic gene expression, single
BFU-E-derived colonies were harvested at day 5 and transferred into
secondary cultures stimulated only with TPO. A significant proportion
(92%) of the single erythroid colonies gave rise to
ACHE+ cells with a clear megakaryocyte morphology when
cultured in the presence of TPO. This result indicates that,
although megakaryocytes are not observed within BFU-E-derived
colonies, almost all of them contain megakaryocyte precursors. Because
we had analyzed a fair amount of all the colonies present in a culture
dish (see legend to Figure 4), we conclude that normal BFU-E, and
probably also CFU-Mk, are still bipotent for E/Mk differentiation.
Therefore, as suggested by Axelrad et al,60 the normal E/Mk
precursor is downstream to the BFU-E level.
The precise relationship of TER-119+/4A5+ cells
with the progenitor cell compartments remains to be established. In
fact, although progenitor cells were never detected among these cells,
the normal bone marrow contains progenitor cells intermediate between
BFU-E and CFU-E, the day 3 BFU-E61 that do not grow in the
absence of serum (Migliaccio G, unpublished observation). It is also
possible that TER-119+/4A5+ cells are generated
by an alternative differentiation pathway during which cells remain
bipotent after the CFU-E level to play an important physiological role
in the recovery from PHZ-induced anemia. However, irrespective of their
precise relationship with the progenitor cell compartments,
TER-119+/4A5+ cells may provide a valuable tool
to define the gene activation hierarchy during erythro-megakaryocytic
differentiation in vivo.
| |
Acknowledgments |
The authors wish to thank Drs Sam Burstein and Thalia Papayannopoulou
for suggestions and helpful discussions.
| |
Footnotes |
Submitted May 18, 1999; accepted December 20, 1999.
Supported by grants from Associazione Italiana per le Leucemie,
Florence ("30 ore per la vita"), MURST 60%; and Grant CEE ERB-B104-CT96-0646 of the IV Framework Program of the
European Community, Brussels, Belgium; by funds from
Associazione Donatori Midollo Osseo, Florence, Italy; by a donation
from Famiglia Yuja; and by institutional funds from Istituto Superiore
di Sanità, Rome, Italy. C.C. was the recipient of a
fellowship from FIRC (Milano, Italy).
Reprints: Alessandro Maria Vannucchi, Bone Marrow
Transplantation Unit, Division of Hematology, Azienda Ospedaliera Careggi, 50134, Florence, Italy; e-mail: a.vannucchi{at}dfc.unifi.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction