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RED CELLS, IRON, AND ERYTHROPOIESIS
Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from
the early mammalian fetus
Samantha J. England,1 Kathleen E. McGrath,1 Jenna M. Frame,1 and James Palis1
1Department
of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY
In the hematopoietic hierarchy, only stem
cells are thought to be capable of longterm self-renewal. Erythroid progenitors
derived from fetal or adult mammalian
hematopoietic tissues are capable of
short-term, or restricted (102- to 105-fold),
ex vivo expansion in the presence of
erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary
erythroid precursors derived from early
mouse embryos are capable of extensive
(106- to 1060-fold) ex vivo proliferation.
These cells morphologically, immunophe-
notypically, and functionally resemble
proerythroblasts, maintaining both cytokine dependence and the potential,
despite prolonged culture, to generate enucleated erythrocytes after 3-4
maturational cell divisions. This capacity
for extensive erythroblast self-renewal is
temporally associated with the emergence of definitive erythropoiesis in the
yolk sac and its transition to the fetal
liver. In contrast, hematopoietic stem cellderived definitive erythropoiesis in the
adult is associated almost exclusively
with restricted ex vivo self-renewal. Primary primitive erythroid precursors,
which lack significant expression of Kit
and glucocorticoid receptors, lack ex vivo
self-renewal capacity. Extensively selfrenewing erythroblasts, despite their near
complete maturity within the hematopoietic hierarchy, may ultimately serve as a
renewable source of red cells for transfusion therapy. (Blood. 2011;117(9):
2708-2717)
Introduction
In the adult, all blood cells are ultimately derived from hematopoietic stem cells (HSCs) that are primarily quiescent yet capable of
extensive self-renewal. The differentiation of HSCs into multipotential and unipotential progenitors is accompanied by a loss both of
proliferative capacity and of self-renewal potential. Immature
erythroid-restricted progenitors, termed erythroid burst-forming
units, have a higher proliferative potential than late-stage erythroid
progenitors, termed erythroid colony-forming units (CFU-E).1
CFU-E subsequently generate a cascade of morphologically identifiable erythroid precursors that undergo 3-4 maturational cell
divisions as they progress from proerythroblast to basophilic,
polychromatophilic, and orthochromatic erythroblast stages.2 Erythroid precursor maturation is characterized by decreased cell size,
hemoglobin accumulation, nuclear condensation, and the cell
surface expression of Ter119.3 Orthochromatic erythroblasts enucleate and soon thereafter enter the blood stream as reticulocytes.
Red blood cell (RBC) production is regulated by several
exogenous factors, including erythropoietin (Epo), cortisol, and
stem cell factor (SCF). Erythropoiesis is critically dependent on
Epo, a glycoprotein hormone that provides a survival signal to
late-stage erythroid progenitors.4,5 Low oxygen levels in tissues
stimulate the production of Epo, resulting in the survival of more
CFU-E and, in turn, an increase in the number of RBCs. The
cellular response to acute hypoxia, termed stress erythropoiesis, is
also regulated, in part, by glucocorticoids, because mice with
diminished glucocorticoid signaling display a delayed recovery
after induction of anemia.6 SCF, a soluble protein that signals
through the Kit receptor, which is expressed by erythroid progeni-
tors and immature precursors, is also necessary for erythroid
differentiation and the early stages of maturation of erythroid
progenitors.7,8
The addition of the synthetic glucocorticoid dexamethasone,
along with SCF and Epo, to cultures of mouse bone marrow or fetal
liver cells induces the outgrowth and proliferation of erythroid
“progenitors” for ⬃ 15 days.6,9-14 The proliferative capacity of
these cells is restricted to ⬃ 102- to 105-fold total expansion.
However, cultures initiated from murine embryonic stem cells
proliferate for longer periods of time.15 Although this difference in
proliferative capacity was ascribed to the embryonic stem cell
origin of the cultures, we asked whether the ex vivo proliferative
capacity of erythroid progenitors derived from the early embryo
may differ from that of their fetal and adult counterparts.
Here, we investigate the ability of erythroid cells cultured from
carefully staged mouse embryos to proliferate ex vivo. Surprisingly, definitive erythroid cells derived from the yolk sac and early
fetal liver are capable not only of restricted (102- to 105-fold) but
also extensive (106- to 1060-fold) proliferation ex vivo, a far greater
proliferative potential than previously recognized. Despite prolonged culture, these immature erythroblasts preserve the potential
to mature into enucleated RBCs, indicating that they are capable of
long-term self-renewal. In contrast, primitive erythroid cells derived from the yolk sac are incapable of either restricted or
extensive self-renewal ex vivo. Our findings raise the possibility
that definitive erythropoiesis is uniquely characterized by the
capacity of immature erythroblasts, lying only 3-4 cell divisions
from terminally differentiated RBCs, to undergo self-renewal cell
Submitted July 29, 2010; accepted November 16, 2010. Prepublished online as
Blood First Edition paper, December 2, 2010; DOI 10.1182/blood-2010-07-299743.
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 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
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© 2011 by The American Society of Hematology
BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
divisions. Extensively self-renewing erythroblasts (ESREs) may
ultimately serve as an in vitro source of RBCs for use in transfusion
therapy.
EXTENSIVE ERYTHROID SELF-RENEWAL
2709
Technologies), and the single-cell suspension was cultured in erythroid
expansion media.
Erythroid cell maturation
Methods
Mice and tissues
All experiments with mice were approved by the University of Rochester’s
Committee on Animal Resources. Outbred ICR mice (Charles River
Laboratories International or Taconic Farms Inc) or C57BL/6J mice
(Charles River Laboratories International or The Jackson Laboratory) were
mated overnight, and vaginal plugs were examined in the morning
(embryonic day 0.3; E0.3). At various gestational ages, mice were killed by
CO2 narcosis, and the embryos were dissected in PB2 (Dulbecco
phosphate-buffered saline [Invitrogen], 0.1% glucose [Invitrogen], and
0.3% bovine serum albumin [Gemini Bioproducts]). Yolk sacs from
E7.5-E9.5 conceptuses were dissociated with 0.008% trypsin (Worthington
Biochemical Corporation) and mechanical trituration. Livers from E11.5E18.5 fetuses, bone marrow, and spleens were mechanically dissociated by
pipetting with a 1000-␮L pipette.
Acute anemia was induced by injecting adult mice intraperitoneally
with 35 mg/kg phenylhydrazine (P26252; Sigma-Aldrich) on days 1 and 2.
Bone marrow and spleen cells were harvested from killed mice on day 4 and
cultured as described in “Erythroid expansion culture.”
Erythroid expansion culture
Cells from each tissue were resuspended ⱕ 2 ⫻ 106 cells/mL in a 24-well
gelatin-coated tissue culture dish (Gibco/BRL) in serum-free “erythroid
expansion media” consisting of either StemPro34 plus nutrient supplement
(Gibco/BRL) or StemSpan SFEM (Stem Cell Technologies) supplemented
with 2 U/mL human recombinant Epo (Amgen), 100 ng/mL SCF (PeproTech), 10⫺6M dexamethasone (D2915; Sigma), 40 ng/mL insulin-like
growth factor-1 (PeproTech), and penicillin/streptomycin (Pen/Strep; Invitrogen). Cholesterol Mix (Sigma) was added to a final concentration of 0.4%
in StemSpan-based erythroid expansion media. The dexamethasone stock
solution was stored in the original glass container and never frozen. After
1 and 3 days of culture, the nonadherent cells were filtered through a 70-␮m
filter (BD Falcon) and transferred to a new gelatin-coated well at a
concentration of ⱕ 2 ⫻ 106 cells/mL. The second transfer was termed day
0 of expansion culture. No adherent cells were present in culture after the
2 well transfers. Total live and dead cell numbers were determined daily by
Trypan Blue (Gibco/BRL) exclusion, and the cell concentration was
brought to 2 ⫻ 106 total cells/mL daily through partial medium changes.
Embryonic stem cell differentiation and culture
PC13 embryonic stem cells (University of Rochester transgenic facility)
were quickly thawed at 37°C and washed with DMEM-ES (Dulbecco
modified Eagle medium [DMEM; Invitrogen], Pen/Strep, sodium bicarbonate [Sigma], 15% fetal bovine serum [Gemini Bioproducts], monothioglycerol [MTG; Sigma], and glutamine [Invitrogen]). Cells were passaged at
least once in DMEM-ES with leukemia inhibitory factor (LIF; Millipore)
on a T25 gelatin-coated flask (BD Falcon). Before differentiation into
embryoid bodies (EBs), cells were grown overnight in IMDM-ES with LIF
(same as DMEM-ES with LIF, except Iscove modified Dulbecco medium
[IMDM; Invitrogen] replaced DMEM). Cells were washed once in PB2 and
resuspended in 4 mL of EB differentiation media (IMDM, 15% fetal bovine
serum [Summit Biotechnology], MTG, ascorbic acid [Sigma], transferrin
[Sigma], Protein-Free Hybridoma Media [PFHM-II; Invitrogen], glutamine, and Pen/Strep) at various cell concentrations (2500-20 000 cells)
into ultra-low adhesion 60 ⫻ 35-mm dishes (Corning). EBs were harvested
daily after 5 days of incubation and dissociated with mechanical trituration
in phosphate-buffered saline (Invitrogen) containing 1mM EDTA (ethylenediaminetetraacetic acid; Sigma) and 0.008% trypsin. Dissociation was
stopped with PB2 containing 10% plasma-derived serum (PDS; Animal
Maturation of erythroid cells proliferating in culture was initiated by
washing the cells with PB2 and resuspending them in “erythroid maturation
media” (1⫻ IMDM, 2 U/mL Epo, 100 ng/mL SCF, 10% Serum Replacement [Invitrogen], 5% PDS, 1⫻ glutamine, 10% PFHM-II, and 12.7 ␮L/
100 mL 1:10 MTG), at 2 ⫻ 106 cells/mL in a gelatin-coated well. Cultures
were maintained daily at 2 ⫻ 106 cells/mL during maturation.
Erythroid cell evaluation
Cells were cytospun (Shandon II; Thermo-Scientific), Wright-Giemsa
stained, premounted, and coverslipped. Images were acquired on a Spot RT
Slider camera (Diagnostic Instruments, Inc) attached to an Eclipse 80i
microscope (Nikon) with the use of Plan Fluor objective lenses (Nikon,
100⫻ magnification, 1.3 NA). Photoshop CS4 Extended software (Adobe)
was used for image processing. Images for supplemental Figure 1 (available
on the Blood Web site; see the Supplemental Materials link at the top of the
online article) were acquired on a Digital Sight-U2 camera (Nikon). The
maturational stages of the erythroblasts were scored with cell and nuclear
size and with nuclear and cytoplasmic staining characteristics. Benzidine
staining was performed as previously described.16 The cell surface phenotype of the expanding erythroblasts was analyzed with the use of
phycoerythrin (PE)–indocyanine 7 CD117 (Kit), PE CD71, and allophycocyanin (APC) Ter119 antibodies (eBioscience) on an LSR-II flow cytometer
(BD Bioscience). Data were analyzed with the use of FlowJo software
(Version 8.8.6; TreeStar).
Analysis of gene expression
RNA was isolated with either Trizol, as previously described,17 or the
RNeasy kit according to the manufacturer’s instructions (QIAGEN). For
the latter approach, cells were disrupted with the use of the kit’s RLT buffer,
and centrifugation through a Qia-shredder column. RNA was then washed
several times on the RNeasy column and eluted with RNase-free water.
cDNA was constructed with the SuperScript III First Strand kit (Invitrogen). Quantitative reverse-transcription polymerase chain reaction (qPCR)
of embryonic (⑀y and ␤H1) and adult (␤1 and ␤2) globins was performed as
previously described with the use of the 18s ribosomal subunit RNA as a
control.17 qPCR was also performed with primer pairs for Epo receptor
(EpoR; 5⬘-CCC AAG TTT GAG AGC AAA GC-3⬘, 5⬘-TGC AGG CTA
CAT GAC TTT CG-3⬘), Epo (5⬘-CCA CCC TGC TGC TTT TAC TC-3⬘,
5⬘-CTC AGT CTG GGA CCT TCT GC-3⬘), SCF (5⬘-CCG TGA CCT TGT
GTG GAT GAT TC-3⬘, 5⬘-TGG GTT TTC AGC ACT CAG ACG-3⬘), and
cMyb (5⬘-AAG ACC CTG AGA AGG AAA AGC G-3⬘, 5⬘-GTG TTG GTA
ATG CCT GCT GTC C-3⬘), all with an annealing temperature of 57°C, and
Kit (5⬘-CTC ACA TAG CAG GGA GCA CA-3⬘, 5⬘-ACA ACT CAC CCA
CAC GCA TA-3⬘) and glucocorticoid receptor (Nr3c1; 5⬘-AGG CCG CTC
AGT GTT TTC TA-3⬘, 5⬘-TAC AGC TTC CAC ACG TCA GC-3⬘), both
with an annealing temperature of 60°C.
Carboxyfluorescein diacetate, succinimidyl ester
Carboxyfluorescein diacetate, succinimidyl ester (CFSE) staining was
performed according to the manufacturer’s directions (Invitrogen). After
staining with CFSE, cells were cultured in erythroid expansion media as
described in “Erythroid expansion culture.” A portion of the culture was
removed daily and stained with antibodies for Kit, CD71, propidium iodide
(Molecular Probes) for live/dead discrimination, and Vybrant DyeCycle
VioletStain (Invitrogen) or Draq5 (Biostatus Limited) to visualize DNA
content. Stained cells were analyzed with an LSR-II flow cytometer, and the
data were analyzed with FlowJo software.
Isolation of primary proerythroblasts
Primitive, embryonic-definitive, and adult-definitive proerythroblasts were
purified from E9.5 yolk sac, E11.5 fetal liver, and adult bone marrow,
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2710
BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
ENGLAND et al
respectively, with the use of fluorescent-activated cell sorting. Briefly, live
(DAPI⫺ [4⬘-6⬘-diamidine-2-phenylindole]; Invitrogen) and Ter119low(APC)
primitive proerythroblasts were isolated from the E9.5 yolk sac; large (high
forward scatter; FSC), live (DAPI⫺), and Kithigh(PE)/Ter119low(APC)
embryonic-definitive proerythroblasts were isolated from E11.5 fetal liver;
and large (high FSC), live (DAPI⫺), and Kithigh(PE)/Ter119low(APC)
adult-definitive proerythroblasts were isolated from bone marrow.
added, and the samples were centrifuged for 5 minutes at 8000g to collect
supernatant for cortisol analysis.
Results
Yolk sac–derived erythroid cells exhibit extensive ex vivo
proliferative potential
Cortisol measurement
Cortisol levels in E11.5 and E12.5 embryonic tissues as well as adult
peripheral blood were determined with the use of an enzyme-linked
immunosorbent assay (ELISA), performed according to the manufacturer’s
protocol (Parameter Cortisol ELISA kit; R&D Systems). Wells were read at
450 nm with the use of an AD340 plate reader (Beckman Coulter).
Amniotic fluid was collected from E11.5 and E12.5 embryos with the use of
a 30-gauge syringe. Glass micropipettes were used to gently aspirate blood
directly from the heart of each embryo. The livers were placed in 25 ␮L of
dilution buffer (ELISA kit) and triturated. More dilution buffer (75 ␮L) was
We previously established that primitive and definitive erythroid
progenitors emerge in 2 overlapping waves from the murine yolk
sac between E7.5 and E10.5 of gestation.18 To examine the
potential of these erythroid lineages to generate ex vivo–
proliferating erythroblasts, cells from spatially and temporally
defined murine tissues were cultured in the presence of either
StemPro34 or StemSpan supplemented with Epo, SCF, insulin-like
growth factor-1, and dexamethasone. Consistent with published
results,19 cell cultures derived from adult bone marrow showed a
Figure 1. Cell cultures initiated from embryonic
tissues are capable of yielding extensively proliferating erythroid cells. (A) Cells were grown in erythroid
expansion media. Erythroid cells derived from adult bone
marrow proliferated ⬃ 103-fold, whereas those from the
3 independent E9.5 yolk sac cultures each proliferated
⬎ 1012-fold. (B) Cells were grown as in panel A but were
maintained at indicated cell concentrations. Cells exhibit
a slowed growth rate and increased death at concentrations of ⱖ 4 ⫻ 106 cells/mL or higher, but no changes in
kinetics were observed at concentrations ⬍ 2 ⫻ 106cells/
mL. (C) Most of the extensively proliferating cells resemble proerythroblasts (ProE) and basophilic erythroblasts (BasoE). The cultures also contain a small number
of polychromatophilic (PolyE) and orthochromatic
(OrthoE) erythroblasts, as well as reticulocytes
(mean ⫾ SEM; N ⫽ 16). (D) Ter119 and Kit levels of ex
vivo extensively proliferating erythroblasts, adult bone
marrow (BM) cells, and E12.5 fetal liver (FL) cells. Most
extensively proliferating erythroblasts are Kithigh/Ter119low.
Kithigh/Ter119low cell populations (red circles), with similar
forward scatter (FSC) characteristics are found in the
adult marrow and E12.5 fetal liver. One of 3 representative experiments is shown. (E) Extensively proliferating
erythroid cells express small amounts of adult (␤1 and
␤2), but no embryonic (⑀y and ␤H1), ␤ globin transcripts
(mean ⫾ SEM; N ⫽ 3). In contrast, circulating blood
cells from E12.5 of gestation, composed predominantly
of primitive erythroblasts, express both embryonic and
adult ␤-globin gene transcripts.
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
EXTENSIVE ERYTHROID SELF-RENEWAL
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Figure 2. Most of the cells in the expansion culture
divide once daily and maintain their large size and
Kithigh phenotype. (A) Staining characteristics of ex
vivo extensively expanding erythroid cultures indicate
that most of the cells divide once daily. There is also a
minor population of cells that divide 2 or 3 times in
24 hours. Results from 1 of 3 experiments are shown.
(B) The major cell population that divides only once
daily is composed of large (FSChigh), nucleated
(DNAhigh), Kithigh cells that retain these characteristics
from day to day (blue lines), shown for 24 and 48 hours
of culture after CFSE staining. In contrast, the minor,
rapidly dividing population is composed of smaller
(FSClow), Kitlow cells, many of which are enucleated
(DNAlow/⫺; red lines). These results suggest that the
minor population consists of terminally maturing erythroid cells.
restricted (102- to 105-fold) capacity to proliferate ex vivo (Figure
1A). Similarly, cultures derived from E9.5 yolk sac initially
underwent a “restricted” phase of exponential growth that resulted
in a gradual increase in terminally mature RBCs. Unlike adult
marrow–derived cultures, however, the restricted phase of these
E9.5 yolk sac–derived cultures was followed by the continued
proliferation of erythroblasts for weeks to months (Figure 1A). We
routinely obtained cell cultures that underwent extensive (1010- to
1030-fold) expansion. The longest culture resulted in 1064-fold
cellular expansion over 203 days, before the culture was electively
terminated (data not shown). This extensive ex vivo proliferative
potential has not previously been described for cells harvested from
wild-type murine embryos.
To determine whether cell concentration altered growth rate or
cell survival, cultures were maintained at 2 ⫻ 102, 2 ⫻ 104,
2 ⫻ 105, 2 ⫻ 106, 4 ⫻ 106, and 2 ⫻ 107 cells/mL. Cells exhibited
decreased growth rate when maintained at 4 ⫻ 106 cells/mL, and a
cessation of growth combined with massive cell death occurred at
the highest concentration (Figure 1B). In contrast, growth rate was
not affected by concentrations as low as 200 cells/mL (Figure 1B).
No differences in the cellular kinetics of restricted or extensive
cellular proliferation were found between StemPro34-based and
StemSpan-based erythroid expansion media.
Expansion cultures are composed of immature erythroid
precursors
To begin to investigate the cellular identity of the proliferating
cells, we examined their morphology after Wright-Giemsa staining.
As shown in Figure 1C, the cells constituting the expansion
cultures morphologically resemble proerythroblasts and basophilic
erythroblasts with a small percentage of more mature polychromatophilic and orthochromatic erythroblasts. Benzidine staining, to
identify hemoglobin-containing cells, showed that 13.5% ⫾ 5.6%
(mean ⫾ SEM; N ⫽ 21) of the cells were benzidine positive,
consistent with the morphological observations that the proliferating cultures consisted primarily of immature erythroblasts along
with a small percentages of maturing erythroblasts.
We recently determined that proerythroblasts in the murine
bone marrow are large cells that coexpress Kit, CD71, and low
levels of the erythroid-specific marker Ter119.20 Flow cytometric
analysis of the extensively proliferating erythroid cells also showed
a high FSC and the cell surface expression of Kit, CD71, and low
levels of Ter119, a phenotype similar to primary proerythroblasts in
the bone marrow and fetal liver (Figure 1D circled cells).
We also assayed the pattern of ␤-globin genes expressed by the cells
present in extensively proliferating cultures. As shown in Figure 1E,
only the adult (␤1 and ␤2) globins were expressed. The globin
expression is derived primarily from the small subpopulation of
maturing cells (data not shown). Despite the yolk sac origin of these
cultures, we could not detect the embryonic (⑀y and ␤H1) globins that
are expressed by primitive erythroid cells.21 These results are consistent
with a definitive erythroid identity of the cells in culture.
To assess the contribution of individual cells to the expanding
culture’s overall growth rate, we performed CFSE staining. Evaluation of the culture over several days showed a major cell
population undergoing a uniform decrease in CFSE fluorescence
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ENGLAND et al
BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
Figure 3. ESREs are capable of terminal erythroid
maturation. (A) Proliferating ESREs transferred from
erythroid expansion media to erythroid maturation
media mature from proerythroblasts to orthochromatic
erythroblasts and enucleated erythrocytes over 3 days.
The lower panel is a composite of cells from a single
photograph of a cytospin preparation. (B) The kinetics
of ESRE maturation. Over the course of 3 days ESREs
transition from immature erythroblasts into ⬎ 90%
enucleated erythrocytes (mean ⫾ SEM; N ⫽ 16 independent maturation cultures). (C) Proliferating ESREs
have a high FSC and are KithighTer119low cells (upper
left). After maturation, the cells decrease in size (FSC;
upper right compared with lower right), down-regulate
Kit, and up-regulate Ter119 (upper left compared with
lower left). One of 3 representative experiments is
shown. (D) ESREs grown in StemPro34-based erythroid expansion media, when transferred to erythroid
maturation media, yield an 8-fold increase in cell
number over the course of 3 days (mean ⫾ SEM;
N ⫽ 36). ESREs grown in StemSpan-based erythroid
expansion media, when transferred to erythroid maturation media, yield a 19-fold increase in cell number
over the course of 3 days (mean ⫾ SEM; N ⫽ 13).
intensity each day, as well as a minor cell population undergoing an
increased loss of CFSE fluorescence intensity each day (Figure
2A). The major cell population is composed of large (FSChigh),
Kithigh, CD71high, DNAhigh cells (Figure 2B). In contrast, the minor
cell population is composed of small (FSClow), Kitlow cells, of
which 48.2% ⫾ 6.5% are enucleated (compared with the major
population which is 1.9% ⫾ 0.3% enucleated; n ⫽ 8; Figure 2B).
These data indicate that immature erythroblasts, which constitute
the majority of the cells in these cultures, are dividing daily and are
responsible for the exponential cell growth that occurs in the
cultures. Furthermore, these data suggest that the minor cell
population consists of maturing erythroid cells that divide more
rapidly than once a day as they mature into RBCs.
Extensively proliferating erythroblasts mature into enucleated
erythrocytes
In vivo, proerythroblast maturation into enucleated RBCs is
characterized by the progressive accumulation of hemoglobin.2 To
test the ability of extensively proliferating erythroblasts to mature,
we cultured them in erythroid maturation media, which contains
IMDM, Epo, SCF, serum replacement, PDS, glutamine, PFHM-II,
and MTG. After 3 days, ⬎ 97% of cells from all cultures (N ⫽ 49)
were benzidine-positive and consisted primarily of enucleated
erythrocytes and a small number of late-stage (orthochromatic)
erythroblasts (Figure 3A-B). Consistent with benzidine positivity,
qPCR analysis showed an 11.4-fold and 6.3-fold up-regulation of
the ␤1- and ␤2-globin genes, respectively, after 3 days of maturation. However, no ⑀y- or ␤H1-globin gene expression was detected
(data not shown), consistent with the definitive erythroid identity of
these cells.17
Maturation of extensively proliferating erythroblasts is characterized not only by structural changes and globin gene upregulation but also by changes in cell surface phenotype. Analysis
by flow cytometry showed marked down-regulation of Kit and
up-regulation of Ter119 associated with a decrease in FSC (Figure
3C), changes that characterize the maturation of erythroid precursors in vivo, in both the fetal liver and the postnatal bone
marrow.3,20
Proerythroblasts normally undergo 3-4 maturational cell divisions in vivo to generate enucleated erythrocytes.2 We examined
the kinetics of proliferating erythroblasts cultured for 3 days in
erythroid maturation media. Cells that had originally proliferated in StemPro34-based erythroid expansion media exhibited
an 8-fold increase in cell number when placed in erythroid
maturation media (Figure 3D). This corresponds on average to
3 maturational cell divisions. Cells that had originally proliferated in StemSpan-based erythroid expansion media exhibited a
19-fold increase in cell number during maturation, corresponding to ⬃ 4 maturational cell divisions (Figure 3D). The extra
division for StemSpan-expanded cells occurs in the first 24 hours
of maturation. These results, taken together, provide functional
evidence that the cultures consist of extensively proliferating
immature erythroblasts that are, on average, 3-4 divisions from
enucleated erythrocytes.
Ex vivo extensively proliferating erythroblasts self-renew
We next asked whether the proliferating erythroblasts maintain the
potential to terminally mature throughout the extensive life of the
expanding culture. As shown in Figure 4A, we removed cells from
a proliferating culture at days 19, 26, 50, 57, and 72 and examined
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
EXTENSIVE ERYTHROID SELF-RENEWAL
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Developmental origin of ESRE potential
Figure 4. ESREs maintain their ability to mature despite extensive proliferation ex vivo. (A) The capability of ESREs to mature was tested sequentially
(open time points) during continued ESRE culture. (B) ESREs from time
points listed in panel A were placed into erythroid maturation media, and the
kinetics of erythroid cell maturation was analyzed by morphological evaluation
of cytospun cells. ESREs maintain the capability to completely mature with the
same kinetics after 3 days in erythroid maturation media despite prolonged
culture ex vivo.
their ability to mature ex vivo. At all time points tested, ⱖ 80% of
the cells matured into enucleated erythrocytes over the course of
3 days (Figure 4B). These results indicate that maturation potential
is preserved despite daily cell divisions over months of ex vivo
proliferation. We therefore named these cells extensively selfrenewing erythroblasts (ESREs).
Our initial studies indicated that erythroblasts capable of extensive
ex vivo self-renewal could be generated from the E9.5 yolk sac and
that these ESREs were “definitive” in nature on the basis of their
globin expression pattern (Figure 1E). To better define the developmental origin of ESRE potential and to determine whether primitive erythroid precursors can also self-renew when cultured ex
vivo, we examined temporally and spatially defined embryonic,
fetal, and adult hematopoietic tissues from outbred mice for ESRE
potential.
No ex vivo erythroblast proliferation, restricted or extensive in
nature, was generated from the E7.5 yolk sac, the time and place
during embryogenesis when primitive, but not definitive, erythroid
progenitors are present18 (Figure 6). The earliest tissue that
generated ex vivo self-renewing erythroblasts was the E8.5 yolk
sac, the time point when and the place where definitive erythroid
potential is first detected in the murine conceptus.18 A large
proportion of cultures established from E8.5 yolk sac, E9.5 yolk
sac, and E12.5 fetal liver generated not only restricted but also
extensive erythroid self-renewal (38 of 70; Figure 6). ESRE
potential from these tissues and developmental time points was
more frequently generated with the use of StemSpan-based erythroid expansion media (15 of 16; 94%) than with StemPro34based erythroid expansion media (23 of 54; 43%). Our results
support the hypothesis that ESRE potential is associated with the
transient wave of definitive hematopoiesis that emerges from the
yolk sac, before HSC emergence, and transitions to the fetal liver.
In contrast to fetal tissues, culture of adult bone marrow or adult
spleen cells, which are derived from HSCs, resulted in restricted,
but almost never in extensive, erythroid self-renewal potential (1 of
26; Figure 6). Tissues from fetal and adult C57BL/6J mice
generated cultures of erythroid cells with extensive and restricted
self-renewal capacity similar to those from outbred mice (supplemental Table 1).
Embryonic and fetal development takes place in a hypoxic
environment, raising the possibility that hypoxia is a determinant of the proclivity for fetal but not adult tissues to give rise to
extensive ex vivo erythroblast self-renewal. We therefore asked
whether the hypoxic stress after the rapid induction of anemia
might lead to the emergence of ESRE potential either in the bone
ESRE proliferation and survival are cytokine dependent
To determine whether Epo, SCF, and dexamethasone are each
required for the continued proliferation of ESRE cultures, we
removed each factor individually during the restricted and extensive expansion phases of ex vivo culture. Cells deprived of Epo, or
of SCF, stopped proliferating within 2 days of cytokine removal,
and all cells in the cultures stained Trypan-positive within 5 days
(Figure 5). Removal of dexamethasone resulted in a transient
increase in cell numbers as the cells completed rapid maturational
cell divisions. During restricted self-renewal, the cultures deprived
of dexamethasone ultimately favored the slow growth of mast cells
after the erythroid cells had matured or died. Our results, taken
together, indicate that ESREs remain dependent on Epo, SCF, and
dexamethasone for both restricted and extensive ex vivo
self-renewal.
Figure 5. Epo, SCF, and dexamethasone are each required both for restricted
and for extensive erythroid self-renewal. When Epo, SCF, or dexamethasone is
individually removed from the culture during restricted self-renewal or during
extensive self-renewal, proliferation is halted because of cell death, decreased
proliferation, or terminal maturation. Results of 1 of 3 independent experiments is
shown. *Culture was ultimately composed of mast cells.
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ENGLAND et al
erythroblasts, we examined the expression of cMyb in primaryprimitive, fetal-definitive, and adult-definitive proerythroblasts,
as well as in ESREs. Consistent with published data,23 we were
unable to detect cMyb transcripts in E9.5 primitive erythroblasts
(Figure 7B). In contrast, cMyb transcripts are expressed in fetal
liver and bone marrow proerythroblasts. Furthermore, extremely high levels of cMyb transcripts were detected in ESREs
(Figure 7B).
Epo, SCF, and cortisol are found in the fetal liver
Figure 6. Generation of erythroid cell cultures with restricted and extensive
self-renewal from embryonic, fetal, and adult hematopoietic tissues derived
from ICR mice. No restricted or extensively proliferating erythroid cultures could be
established from E7.5 yolk sac (YS) cells. Cultures of erythroid cells with restricted
self-renewal potential were generated from later YS, fetal liver (FL), spleen, and bone
marrow (BM). The highest frequency of erythroid cultures exhibiting extensive
self-renewal potential was derived from E8.5-E14.5 embryos, which is associated
with the emergence of a transient wave of definitive erythroid potential in the yolk sac
and its transition to the early fetal liver. Cultures derived from “stressed” BM and
spleen from mice made anemic with phenylhydrazine were only capable of restricted
self-renewal.
marrow or in the spleen, the site of stress erythropoiesis in
the adult mouse. However, the culture of bone marrow and
spleen cells from phenylhydrazine-treated anemic mice yielded
erythroblasts with restricted, but not extensive, self-renewal
capacity (Figure 6). Taken together, our results from both
outbred and C57BL/6J mice indicate that ex vivo erythroid
self-renewal potential is associated with definitive hematopoiesis, and extensive ex vivo erythroid self-renewal is primarily
associated with early definitive hematopoiesis that emerges in
the yolk sac and transitions to the fetal liver.
Expression of the receptors for Epo, SCF, and cortisol in
primary proerythroblasts
Epo, SCF, and glucocorticoids are each necessary to obtain both
restricted and extensive ex vivo self-renewal of definitive erythroblasts. To investigate why primitive erythroid cells fail to selfrenew in response to these factors, we examined the mRNA
expression levels of their receptors, EpoR, Kit, and glucocorticoid
receptor (Nr3c1), in primary primitive and definitive proerythroblasts. Primitive proerythroblasts were isolated from E9.5 yolk sac,
fetal-definitive proerythroblasts were isolated from E11.5 livers,
and adult-definitive proerythroblasts were isolated from bone
marrow. EpoR, Kit, and Nr3c1 transcripts were expressed at similar
levels in fetal- and adult-definitive proerythroblasts (Figure 7A).
Although primitive proerythroblasts also expressed similar levels
of EpoR compared with adult-definitive proerythroblasts, they
contained 94-fold and 169-fold lower levels of Kit and glucocorticoid receptor, respectively (Figure 7A). These results suggest that
primitive erythroblasts fail to self-renew because they lack the
ability to respond both to SCF and to glucocorticoids.
cMyb has been shown to be a target of glucocorticoid
receptor signaling.22 Given the extremely low levels of glucocorticoid receptor gene expression in primitive, but not definitive,
During murine ontogeny, the erythron undergoes a massive
expansion in the fetal liver between E12.5 and E16.5, leading to
a 275-fold increase in circulating definitive erythrocytes over
the course of these 4 days.21,24 We asked whether Epo, SCF, and
cortisol, the exogenous factors driving erythroid precursor
self-renewal ex vivo, are present in vivo. Both Epo and SCF
transcript levels were markedly higher in the fetal liver than in
the kidney and bone marrow, their adult sites of synthesis,
respectively (Figure 7C). Cortisol was found in amniotic fluid,
fetal liver, and peripheral blood at both E11.5 and E12.5,
although at lower concentrations than that found in adult serum
(Figure 7D). These results indicate that all 3 factors are present
in the fetal liver and raise the possibility that erythroblast
self-renewal might occur in vivo.
Discussion
The ex vivo culture of primary erythroid cells from the fetal liver,
cord blood, and bone marrow of mammalian species in the
presence of Epo, SCF, and dexamethasone results in significant, but
ultimately restricted, proliferation of definitive erythroblasts.6,9-15,19,25-28 Here, we report that the yolk sac and early fetal
liver of murine embryos give rise to erythroblasts that can
proliferate for months ex vivo in serum-free conditions. The
majority of the cells divide daily while maintaining their phenotype
as large, Kithigh cells. These ESREs retain their ability to terminally
mature into enucleated RBCs, even after 106- to 1060-fold expansion, all the while remaining entirely dependent on the combinatorial action of Epo, SCF, and glucocorticoid signaling for their
survival and proliferation. ESREs can be grown at very low cell
concentrations, indicating that their proliferation is not dependent
on a more complex set of exogenous factors.
Erythropoiesis is characterized by the progressive maturation of
lineage-committed progenitors, capable of colony formation in
semisolid media, to morphologically recognizable precursors. To
better define the cellular identity of ESREs along this continuum,
we analyzed their morphology, gene expression, cell-surface
phenotype, and kinetics of cellular maturation. ESREs are large
cells with uncondensed chromatin and basophilic cytoplasm,
similar to the morphology of proerythroblasts. Like proerythroblasts, ESREs express moderate-to-high levels of Kit and CD71
and low levels of Ter119 on their cell surface.20 Furthermore, like
proerythroblasts, ESRE maturation is associated with an approximate 8- to 16-fold increase in cell number.2 Taken together, these
findings indicate that proliferating ESREs are erythroid cells at the
immature precursor stage of maturation, just 3-4 cell divisions from
an enucleated RBC. It is surprising that immature erythroblasts,
cells so close to terminal maturity within the hematopoietic
hierarchy, are capable both of restricted and of extensive selfrenewal when cultured in vitro.
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
EXTENSIVE ERYTHROID SELF-RENEWAL
2715
Figure 7. Expression of Epo, SCF, and cortisol and their receptors. (A) Primary definitive proerythroblasts (ProEs) express receptors for Epo (EpoR), glucocorticoids (Nr3c1), and
SCF (Kit). In contrast, primary primitive ProEs, derived from E9.5 YS, express similar levels of EpoR but nearly undetectable levels of Nr3c1 and Kit (mean ⫾ SEM; N ⫽ 3; *P ⬍ .001,
t test). (B) ESREs expressed high levels of cMyb transcripts compared with fetal liver and bone marrow definitive proerythroblasts. cMyb transcripts were not detected in primitive
proerythroblasts. (C) Epo and SCF transcripts are expressed at higher levels in the mid-gestation fetal liver than in the adult bone marrow (BM) or kidney (mean ⫾ SEM; N ⫽ 3).
(D) Cortisol levels were detected by ELISA in fluid from embryonic and adult mouse tissues (mean ⫾ SEM; N ⫽ 3). BD indicates below detection.
Our studies indicate that ex vivo erythroid self-renewal is
strictly associated with definitive erythroid cells, because ESREs
exclusively express adult globins and are similar in size to primary
immature definitive erythroblasts. Erythroid ontogeny is characterized by the overlapping development of 3 distinct lineages.29 The
first lineage, primitive erythropoiesis, which transiently emerges
from the yolk sac of the mouse conceptus at E7.25, generates a
semisynchronous wave of erythroblasts that mature in the circulation and uniquely express embryonic globins.21,30 The second
lineage is a transient definitive erythroid lineage that emerges from
the yolk sac at E8.5-E9.5 as a wave of definitive erythro-myeloid
progenitors that are thought to colonize the fetal liver by E10.5.18,31,32
The third erythroid lineage is a continuous, definitive erythroid
lineage generated by HSCs that emerge from arterial beds at E10.5,
colonize the liver around E12.5, and ultimately colonize the bone
marrow in the perinatal period.33-35 Given this backdrop of
erythroid ontogeny, we analyzed the emergence of erythroidrestricted and extensive self-renewal potential in murine embryos.
The potential for restricted ex vivo erythroblast self-renewal first
emerges with that of definitive erythroid progenitors in the E8.5
yolk sac, is found in the liver throughout fetal life, and persists
postnatally in the bone marrow and spleen, sites of adult murine
erythropoiesis. In contrast, the presence of extensive ex vivo
erythroid self-renewal potential is correlated temporally and spatially with the transient definitive erythroid lineage that emerges
from the yolk sac and colonizes the early fetal liver.
In contrast to definitive erythroid precursors, primitive
erythroid precursors do not self-renew ex vivo in the presence of
Epo, SCF, and dexamethasone. Given that these factors are each
required in concert for ex vivo self-renewal, it is probable that
the lack of primitive erythroid self-renewal potential is because
of the extremely low levels of Kit and glucocorticoid receptor
expression in primitive erythroblasts. We also found that cMyb
is preferentially expressed in primary definitive versus primitive
proerythroblasts, consistent with its known role in definitive, but
not primitive, erythropoiesis.23,36 cMyb is a target of glucocorticoid receptor signaling and can maintain ex vivo erythroblast
proliferation in the absence of glucocorticoid signaling.22,37
Constitutive activation of cMyb blocks the maturation of murine
erythroleukemia cells.38 Interestingly, we found high levels of
cMyb transcripts in ESREs, suggesting that cMyb may play a
role in the self-renewal of definitive erythroblasts. Taken
together, these data indicate that there are marked differences in
cytokine signaling between primitive and definitive erythropoiesis and that these differences correlate with the ability of
definitive, but not primitive, erythroid precursor cells to undergo
self-renewal cell divisions. We postulate that induction of
self-renewal divisions may serve as a mechanism to regulate
definitive erythropoiesis. The rapid increase in immature erythroid precursor cell numbers would ultimately result, after
3-4 rapid maturational cell divisions, in the acute expansion of
RBC output.
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
ENGLAND et al
The frequency with which ESRE cultures are established from
embryonic and fetal tissues is influenced by the make-up of the
culture media. Erythroid cells in culture were more likely to
extensively self-renew if they were grown in StemSpan-based than
in StemPro34-based erythroid expansion media. Although there
were no differences in the growth kinetics during self-renewal,
ESREs cultured in StemSpan-based erythroid expansion media
yielded twice as many RBCs after their transfer into erythroid
maturation media. For these reasons, we favored the use of
StemSpan. However, the proprietary nature of these serum-free
media prevented us from exploring the causes for their differential
effects.
The emergence of blood in cultures of differentiating embryonic
stem cells appears to recapitulate yolk sac hematopoiesis, because
both are characterized by overlapping waves of primitive and
definitive erythroid progenitors.18,39 Carotta et al15 reported the
prolonged outgrowth of erythroid “progenitors” with a definitive
erythroid phenotype from days 6-9 EBs. We have generated
cultures of ESREs from murine embryonic stem cells that are
indistinguishable from ESREs derived from primary embryonic
tissues (supplemental Figure 1). These results raise the possibility
that ESREs derived from human embryonic stem cells and induced
pluripotent stem cells may ultimately serve as a renewable source
of RBCs for cell replacement therapy.
Acknowledgments
We thank Anne Koniski, Paul D. Kingsley, and Michael Bulger for
help with cell culture and for helpful discussions throughout these
studies.
This work was supported by grants from New York Stem Cell
Science (NYSTEM) and from NIH/NIDDK (DK09361).
Authorship
Contribution: S.J.E. designed and performed experiments, analyzed data, and wrote the paper; J.M.F. performed experiments;
K.E.M. designed experiments and analyzed data; J.P. designed
experiments, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: James Palis, University of Rochester Medical
Center, Department of Pediatrics, Center for Pediatric Biomedical
Research, 601 Elmwood Ave, Rochester, NY 14642; e-mail:
[email protected].
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2011 117: 2708-2717
doi:10.1182/blood-2010-07-299743 originally published
online December 2, 2010
Immature erythroblasts with extensive ex vivo self-renewal capacity
emerge from the early mammalian fetus
Samantha J. England, Kathleen E. McGrath, Jenna M. Frame and James Palis
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