Erythroid cells in vitro: from developmental biology to blood

transfusion products
Anna Rita Migliaccioa,b, Carolyn Whitsetta,c and Giovanni Migliacciod
a
Tisch Cancer Institute, Mount Sinai School of
Medicine, bMyeloproliferative Research Consortium
(MPD-RC), cNew York Blood Center, New York, New
York, USA and dDepartment of Cell Biology and
Neurosciences, Istituto Superiore Sanità, Rome, Italy
Correspondence to Professor Anna Rita Migliaccio,
PhD, Tisch Cancer Institute, Mount Sinai School of
Medicine, One Gustave L Levy Place, Box 1079,
New York, NY 10029, USA
Tel: +1 212 2416974; fax: +1 212 8765276;
e-mail: annarita.migliaccio@mssm.edu
Current Opinion in Hematology 2009,
16:259–268
Purpose of review
Red blood cells (RBCs) transfusion plays a critical role in numerous therapies.
Disruption of blood collection by political unrest, natural disasters and emerging
infections and implementation of restrictions on the use of erythropoiesis-stimulating
agents in cancer may impact blood availability in the near future. These considerations
highlight the importance of developing alternative blood products.
Recent findings
Knowledge about the processes that control RBC production has been applied to the
establishment of culture conditions allowing ex-vivo generation of RBCs in numbers
close to those (2.5
1012 cells/ml) present in a transfusion, from cord blood, donated
blood units or embryonic stem cells. In addition, experimental studies demonstrate that
such cells protect mice from lethal bleeding. Therefore, erythroid cells generated ex vivo
may be suitable for transfusion provided they can be produced safely in adequate
numbers. However, much remains to be done to translate a theoretical production of
approximately 2.5
1012 RBCs in the laboratory into a ‘clinical grade production
process’.
Summary
This review summarizes the state-of-the-art in establishing ex-vivo culture conditions for
erythroid cells and discusses the most compelling issues to be addressed to translate
this progress into a clinical grade transfusion product.

Keywords
alternative blood transfusion products, cord blood, erythroid cells, erythropoietin,
ex-vivo expansion

Curr Opin Hematol 16:259–268

ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
1065-6251

ditions are common [3]. In emergencies, national plans

Introduction
The safety and adequacy of the blood supply is a national
[1–4] and international (http://www.who.int/bloodsafety/
universalbts/en/) [5] priority. In developed countries, the
blood supply appears to be adequate. For example, in
2007, blood collection and utilization data from the USA
indicate that the number of screened allogeneic blood
donations that passed all tests exceeded by 7.8% of the
total number of units transfused [1]. WHO reports, how-
ever, indicate that in developing countries national
supplies are rarely sufficient to meet existing needs
(http://www.who.int/bloodsafety/universalbts/en/) [5].
Recent changes in health policies that restrict the use
of erythropoiesis-stimulating agents in patients with can-
cer [6,7] and reports of increased morbidity and mortality
associated with transfusion of older blood units (if con-
firmed) [8] may decrease the blood supply in developed
countries in the future (Table 1) [9–13]. Sporadic blood
shortages related to holidays and extreme weather con-
1065-6251 ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
call for sharing of blood resources across geographic areas,
accessing frozen blood inventories and sponsoring emer-
gency blood drives. However, the infrastructure to main-
tain blood collection and distribution systems may be
disrupted during a disaster.
Even when blood supplies are adequate, finding blood for
certain patients is difficult. Chronically transfused
patients may become immunized to inherited poly-
morphic cell surface determinants (minor blood group
antigens) [12] (Table 1). Clinicians often choose to
transfuse patients at high risk for alloimmunization with
fully matched blood. Regional, national and international
programs have been developed to identify blood for
alloimmunized patients needing rare blood types [9].
National rare donor registries collaborate through the
WHO International Rare Donor Panel to provide rare
blood wherever it is needed [9]. Despite unprecedented
efforts to meet the need of these special patients, there is
DOI:10.1097/MOH.0b013e32832bcaa2

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 260 Hematopoiesis

Table 1 Factors that negatively affect the blood supply

Donor population Increased travel-related deferrals
Epidemics/emerging infections
Identification of new transfusion transmitted infections
Ethnically imbalanced donor/patient populations
Societal and cultural factors
Patient population Size
Limitation on the use of EPO
Increases in the minimal hematocrit for transfusion
Increases in the numbers of stem cell and solid-organ transplants
Lack of best transfusion practice guidelines
Innovative surgical treatments
Special needs
Neonates (fresher units preferred)
Increase in alloimmunized patientsa
Increasing demand for rare blood types and CMV-negative blood
Blood quality Product modifications that decrease shelf life (irradiation and washing)
Possible toxicity of older units may decrease inventory (current shelf life of blood 42 days)
Natural disasters Disrupt blood collection and manufacturing infrastructure
Interrupt communications between blood banks and local hospitals
Increase in patient population
Decrease in donor population
CMV, cytomegalovirus; EPO, erythropoietin. Data from [9–13].
a
In the USA, examples of programs dedicated to the identification of blood for alloimmunized patients include the Blue-tag Program in Philadelphia,
Precise Match in New York City (both regional programs) and the American Rare Donor Program (a national program). International efforts are
described in [9].

a shortage of certain rare blood types, hence the need to
identify alternative RBC transfusion products.
Frozen blood is the alternative transfusion product
usually provided for alloimmunized patients when liquid
units are not available [9]. Additional alternative trans-
fusion products not yet approved for clinical use in the
USA or Europe are represented by hemoglobin (Hb)
solutions and universal RBC transfusion products
(Table 2 [14,15]). RBCs generated in vitro from human
hematopoietic stem/progenitor cells (HSCs) derived
from cord [16] and adult [17] blood or human embryonic
stem cells (hESCs) [18] may represent an important
resource for providing blood to patients with rare blood
types and supplementing the general blood supply
during emergencies. We will review the recent progress
made in establishing ex-vivo culture conditions for ery-
throid cells and discuss the most compelling issues to be
addressed to translate this progress into a clinical grade
transfusion product.
Expansion of human erythroid cells in culture
Erythroid differentiation begins at the level of the plur-
ipotent HSC [19]. HSCs are instructed by specific growth
factors to generate a hierarchy of progressively lineage-
restricted erythroid progenitor cells (EPCs), a process
termed commitment. EPCs, morphologically indistin-
guishable from HSCs, are defined by specific antigen
and mRNA expression profiles and give rise, after several
divisions, to the first morphologically recognizable ery-
throid cell, the proerythroblast. Proerythroblasts are still

Table 2 Red blood cell products and transfusion alternatives

Product/alternative Advantages Disadvantages

Frozen blood [9]
Hb solutions [14] (recombinant/stabilized)
Universal red cell transfusion products [15]
(enzymatic cleavage/chemical masking of
ABO and Rh antigens)
EPO [6,7]
EPO, erythropoietin; Hb, hemoglobin; Rh, rhesus.
Satisfactory storage life (>10 years)
Approved in South Africa for
limited clinical use
Generates group O cells
Licensed
Not immediately available
Cell loss during deglycerolization
Expiration 24 h after thawing (14 days closed system)
Available only near blood centers and large hospitals
that have special facilities
Not approved for human use in Europe or the USA
(approved in Europe for veterinary use only)
Short intravascular survival
Possible toxicity
Currently not approved for clinical use
Not applicable to all blood group antigens
Clinical indications limited
Expensive
Adverse reaction profile not defined for many
clinical conditions

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capable of proliferating and generating additional mature
erythroid precursors (basophilic, polychromatic and ortho-
chromatic erythroblasts), which eventually lose prolifera-
tive potential and undergo enucleation [20]. The number
of erythroblasts produced by this continuous passage
through distinct cellular compartments is determined both
by the number of EPCs recruited and, more importantly,
by the number of divisions allowed to occur within each
cell compartment preceding the polychromatic erythro-
blasts stage.
Polycythemia is the first manifestation of Cushing’s dis-
ease [21], a syndrome associated with chronic stimulation
of the glucocorticoid receptor, whereas anemia is fre-
quently associated with estrogen therapy in breast cancer
[22]. These observations suggested that, in addition to
erythropoietin (EPO), erythropoiesis may also be
regulated by nuclear receptors, such as glucocorticoid
receptor and the estrogen receptor (ESR), and prompted
early studies that identified the ability of dexamethasone
(DXM, a glucocorticoid receptor agonist) [23] and estra-
diol [24] to synergize with EPO in inducing generation of
erythroid colonies in cultures of mononuclear cells
(MNCs) from adult bone marrow or blood. Recently,
the role for these hormones in the regulation of hema-
topoiesis has been confirmed by the observation that
mice lacking glucocorticoid receptor recover poorly from
chemically induced anemia [25], a finding which led to
the discovery that addition of DXM to a combination of
growth factors used to stimulate murine EPCs results in
the generation of larger numbers of erythroblasts in
culture [26]. Now that recombinant growth factors are
available, it is clear that DXM and estradiol do not affect
the number of EPCs recruited in the maturation process
but rather the cellular output from each EPC (Fig. 1).
Figure 1 Estradiol and dexamethasone do not increase the number of erythroid progenitor cells that form colonies in vitro but the
cellular output from each erythroid progenitor cell
Number and morphology of erythroid colonies obtained in culture of adult blood MNCs stimulated with optimal concentrations of SCF, IL-3 and EPO in
the presence of DXM, estradiol or both, as indicated. Magnification 10
. DXM, dexamethasone; EPO, erythropoietin; IL, interleukin; MNCs,
mononuclear cells; SCF, stem cell factor.
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Erythroid cells in vitro Migliaccio et al. 261
The two hormones act by delaying the transition of
proerythroblasts to the next maturation stage, favoring
a higher number of proliferative events. Addition of
DXM and estradiol, alone or in combination, increases
the numbers of erythroblasts that can be obtained in
human cultures [16,17,27–32]. Cultures that increase
erythroblasts are defined as phase 1, expansion or human
erythroid massive amplification (HEMA) cultures (Fig. 2
[17,27–33,34,35]). Calculation of the number of eryth-
roblasts that can be obtained from MNCs or CD34þ cells
from an average adult or cord blood unit under such
conditions indicates that at least cord blood cells can
generate numbers of erythroblasts similar to those pre-
sent in a transfusion (1.7
1011 vs. 2.5
1012) (Table 3
[36,37]).
As the purpose of expansion cultures is to optimize
erythroblasts proliferation, a different set of conditions
was designed to induce erythroblasts maturation (Fig. 2)
[16,17,27–29,30–32]. Ex-vivo expanded erythroblasts are
transferred into maturation cultures, also referred to as
phase 2, stimulated with EPO and insulin-like growth
factor (IGF)1. Under these conditions, erythroblasts pro-
gress to the orthochromatic stage (Fig. 3 [38,39]). Enu-
cleation remains inefficient (5–10%) and requires the
addition of MS-5 cells [29,33] that probably exert in vitro
the functions performed by macrophages in vivo [40] (to
provide a surface to facilitate rolling over during enuclea-
tion [41], to facilitate iron uptake, or both [42]) (Fig. 2).
Functionality studies proved that murine RBCs obtained
in vitro from ESCs under these conditions protect mice
from lethal bleeding [43].
Given that EPCs represent a minority of CD34þ cells,
culture conditions have been designed to induce CD34þ
 262 Hematopoiesis

Figure 2 Anatomy of a hypothetical ex-vivo red blood cell production process

Anatomy of an ex-vivo red blood cell production process

Preculture Phase 1 Phase 2

(commitment) (expansion) (maturation)

IL-3Rα IL-3Rβ GM-CSFRα IL-3Rβ GM-CSFRα EpoR EpoR

IL-3Rα IL-3Rα
EpoR EpoR
HSC EPC Red blood
EPC ProEBs ProEBs cells
gp130
gp130 Mp1 gp130 Mp1
Flt3 Kit Mp1 Kit Kit Mp1 Mp1

MS-5

Culture components Culture components Culture components

Starting cells: Cord blood (or adult blood)
CD34+ cells
Growth factors: FL; SCF; TPO; IL-6; GF-1
Starting cells: Cord blood or adult blood MNC; Starting cells: Erythroblasts obtained in
cord blood or adult blood CD34+ cells; phase 1
precultured of EPCs. Growth factors: EPO; IGF-1
Growth factors: SCF; IL-3/GM/CSF; EPO; IGF-1
Hormones: DXM; estradiol

Biomarkers Biomarkers Biomarkers

Potency of the donor: Genetic
polymorphisms, age, sex and ethnicity.
Potency of the EPC: Phenotypic profiling
(CD235a, CD36, CD71, others); gene
expression profiling (GATA1/PU-1/or
GATA1/GATA2 mRNA ratio), others;
Functional assays (colony formation).
Quality control: Phenotypic (CD235a, CD36,
CD71), or expression (GATA1 level) profiling;
functionality assay (apoptosis).
Potency as transfusion product: Xenogeneic
animal transfusion models.
Quality control: Phenotypic (CD235a, CD36,
CD71), or expression (GATA1 level) profiling.
Potency as transfusion product:
Xenogeneic animal transfusion models.

Under development Transfusion product Transfusion product

Starting cells: hESC; hiPC.
Epigenetic modifiers: HDAC inhibitors.
methylases inhibitors
Advantages: Present in cord blood transfusions,
easy to store and thaw; in-vivo maturation may
deplete iron; longer efficacy; reduce the frequency
of transfusion in chronically transfused patients.
Disadvantages: Time required for efficacy not
known; possible altered antigenicity; altered Hb
expression.
Advantages: Large clinical experience.
Disadvantages: Glycerol dependent storage;
the process of enucleation requires feeder
layers of murine origin; feeder layers of human
origin are under development.

On the basis of the results obtained by our group [17] and others [27–29,30–32], a process to produce ex-vivo RBCs will be composed of three
sequential cultures: preculture (from HSCs to EPCs), expansion (from EPCs to erythroblasts) and maturation (from erythroblasts to enucleated RBCs).
cultures. Growth factor receptors expressed by the populations seeded in each culture and by their progeny are indicated. These expression patterns
guided the choice of growth factors used to stimulate each phase of the production process. The components (starting populations, growth factors and
additives) of each phase of the production are specified in blue boxes. Biomarkers suggested for quality control of cells generated in each culture are
indicated in green boxes. The pink boxes indicate advantages and disadvantages of using erythroblasts and enucleated RBCs as transfusion products
and the promising areas of research in the development of preculture systems. The very first process to generate ex-vivo RBC transfused products may
include only expansion cultures. It is envisioned that the first applications of ex-vivo generated RBC transfusion will be drug delivery [35] or possibly
transfusion of alloimmunized sickle cell patients, patients transplanted with cord blood stem cells, or both. These applications must be preceded by
proof of concept experiments in animal models. DXM, dexamethasone; EPO, erythropoietin; EPCs, erythroid progenitor cells; FBS, fetal bovine serum;
Hb, hemoglobin; HDAC, histone deacetylase; hESC, human embryonic stem cell; HSCs, hematopoietic stem cells; MNCs, mononuclear cells; RBC(s),
red blood cell(s); SCF, stem cell factor.

cells to generate EPCs that would, in turn, be used in
expansion cultures. The purpose of these precultures/
commitment cultures is to increase the number of EPCs,
and therefore of erythroblasts, that can be generated
ex vivo from a single blood donation. Preculture con-
ditions have been developed for cord blood CD34þ cells
[32] but, in principle, the same conditions could be used
to generate EPCs from adult blood CD34þ cells (Fig. 2).
The transition from HSCs to EPCs is associated with
extensive chromatin remodeling that ensures activation
of the expression of erythroid-specific genes [44].
These epigenetic modifications involve acetylation/de-
acetylation of specific histones that relaxes/condenses the
chromatin, favoring and inhibiting, respectively, gene
expression. Once the chromatin configuration is estab-
lished, methylation of specific DNA regions ensures
that the chromatin modeling is inherited by the cel-
lular progeny. These plastic processes are regulated by
families of enzymes called methylases, histone acetyl-
transferases (HATs) and histone deacetylases (HDACs)
[44]. The plasticity of this process has been recently
exploited to reprogram mouse and human somatic cells
by inducing ectopic expression of the four genes Oct4,
Sox2, Klf4 and cmyc (induced pluripotency cells or iPCs)
[45]. Exposure of murine fibroblasts to HDAC inhibi-
tors increased 100-fold the otherwise low efficiency rate

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 Erythroid cells in vitro Migliaccio et al. 263

Table 3 Calculation of the theoretical number of erythroid cells that would be generated by mononuclear cells and CD34R cells from
an average cord blood and adult blood unit cultured under expansion conditions
Adult blood Cord blood

Volume/unit (ml) 450–500 60–120

WBCs/unit (cells/ml) 4.5
109 9
108
HEMA culture seeded with MNCs
MNCs concentration (%) 10 10
Yield of the purification (%) 90–100 90–100
Total number of MNCs in culture (cells/ml) 4.3
108 8.6
107
Fold increase in culture 30 2000
Total number of erythroblasts obtained after 10–14 days of culture (cells/ml) 1.2
1010 1.7
1011
HEMA culture seeded with CD34þ cells

Total number of CD34þ cells per unit (cells/ml) 106( ) 1.8
106
CD34þ/MNC (%) 0.15 0.8
Yield of the purification (%) 50–100 50–100
Total number of CD34þ cells cultured (cells/ml) 4.8
105 6.6
105
Fold increase in culture 103 105
Total number of erythroblasts obtained after 10–14 days of culture (cells/ml) 4.8
108 6.6
1010
A unit of blood contains 2.5
1012 RBCs. The average frequencies of MNC and CD34þ cells in adult blood and cord blood are from [36,37]. The
frequencies of these cells in different units may vary by 1–2 logs. In spite of the 2-log increase obtained in cultures started with CD34þ cells, higher
numbers of erythroblasts are generated in cultures of MNCs. This is due to both loss of CD34þ cells during purification and to the fact that some EPCs
are CD34. EPCs, erythroid progenitor cells; HEMA, human erythroid massive amplification; MNC(s), mononuclear cell(s); RBCs, red blood cells;
WBCs, white blood cells.

of this reprogramming [46], suggesting that addition of
HDAC inhibitors to commitment cultures might allow the
generation of higher numbers of EPCs as well (Fig. 2).
A clinical grade production process for
ex-vivo generation of red blood cell
transfusion products
In the USA, ex-vivo generated RBC transfusion products
fall into the category of biological medicinal products.
The identity of these products must be precisely docu-
mented by defining cell composition, nature of the pro-
duction process and potency in xenogeneic animal
models. In addition, internal controls must be devised
to monitor the quality of cells at each stage of production
and the safety of the product. We will discuss further how
the knowledge of the biology of erythroid cells can guide
the definition of an ex-vivo generated RBC transfusion
product.
Cellular composition of the product
A conservative approach would be to define the cellular
composition of ex-vivo expanded RBC products as ‘enu-
cleated RBCs’. The advantage of this definition is that
these cells are currently used for transfusion therapy.
However, the high costs, long production times and
presence of xenogeneic material (Fig. 2 and Table 4)
decrease the likelihood that this product will be produced
under clinical grade conditions in the near future.
In principle, ex-vivo generated erythroblasts could also
serve as the transfusion product. These cells are present
in the circulation during fetal development, in neonates
with pyruvate kinase deficiency and in adults, under
conditions of stress [20]. Erythroblasts are also present
in large numbers (4
107 cells/ml) in cord blood, which
is occasionally used for transfusions in developing
countries [47]. In 1918, Ende and Ende [48] reported
that the majority of the RBCs circulating in a patient
transfused with 40 ml of cord blood were donor derived
for up to 2 months following the transfusion. This obser-
vation and the consideration that erythroblasts may
undergo 4–64 further divisions suggest that ex-vivo
generated erythroblasts may be more effective as trans-
fusion products than regular transfusions (Table 4). Sup-
port for this hypothesis is provided by the fact that ex-
vivo generated erythroblasts, as those present in cord
blood, express the adhesion receptors [chemokine (CXC
motif) receptor 4, a4 integrin and P-selectin ligand],
necessary to establish proper cell interactions for matu-
ration once injected in vivo [49].
The definition of the cellular composition of the product
must also take into account the fact that the biological
properties of erythroblasts change during ontogenesis
[20]. Therefore, erythroblasts generated from cord blood,
adult blood, precultured cord blood or adult blood and
hESCs will likely express different biological properties.
These differences include size, levels of activity of the
glycolytic enzymes (higher in cord blood cells) and of
carbonic anhydrase (higher in adult blood cells), expres-
sion of different isozymes (phosphoglycerate kinase,
acetylcholinesterase, etc.), HbF or HbA and antigenic
profiles (human leukocyte antigen class II antigens may
be expressed by cord blood but not adult blood-derived
erythroblasts, cord blood or adult blood erythroblasts may
express the i or I antigen).
Nature of the production process
The nature of a production process is usually discussed in
terms of vessel types, incubation conditions, cell manip-
ulation/collection/storage, and so on. All these issues are

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 264 Hematopoiesis

Figure 3 Biological properties of ex-vivo generated human erythroblasts

(a) Antigenic profiling of erythroblasts obtained after 10 days in expansion culture and of the progeny generated by these erythroblasts upon exposure
for 4 days to EPO in maturation cultures. Expansion cultures were seeded with adult blood MNCs and stimulated with SCF, IL-3, EPO, DXM and
estradiol. These cultures generated almost pure (>90%) erythroblasts, as demonstrated by CD36/CD235a staining. CD235a recognizes glycophorin
A. CD36 represents an early marker for erythroid commitment and recognizes the thrombospondin receptor, an antigen acquired by those
hematopoietic cells that had became EPCs in response to EPO [38]. Double CD36/CD235a staining divides erythroblasts into four populations:
EPCs (CD36high/CD235a), proerythroblasts (CD36high/CD235alow), basophilic–polychromatophilic (CD36high/CD235ahigh, basoerythroblast) and
orthochromatic (CD36/CD235ahigh, orthoerythroblast) erythroblasts [39]. Reticulocytes are identified as small CD36/CD235ahigh cells that stain
negative for DNA. The gatings identifying the four populations are indicated by rectangles. Erythroblasts obtained after 10 days of expansion cultures
are mainly composed of proerythroblasts and basoerythroblass. Removal of basoerythroblasts from the culture by density selection allows generation
of at least 20-fold more erythroblasts because these cells continue to proliferate up to day 25 of culture [28]. This observation suggests a strategy to
reduce the volume of the media required to produce an ex-vivo RBC transfusion product by sequential depletion/storage of basoerythroblasts.
Erythroblasts obtained in expansion cultures progress through the stage of orthochromatic erythroblast in 3–4 days when transferred into maturation
cultures stimulated with EPO and IGF-1 (or insulin). The low (5–18% depending on the donor) frequency of erythroblasts that will undergo enucleation
in these cultures can be greatly enhanced by the presence of the murine MS-5 cell line [29,33]. (b) FSC analyses of cells at different stages of
maturation indicating the significant size reduction associated with maturation of erythroblasts. Each curve corresponds to a different population, as
indicated on the right. Proerythroblasts (in red) were obtained in expansion cultures, whereas basoerythroblasts and orthoerythroblasts and RBCs were
at day 4 of maturation. (c) Time in culture and changes in morphology and size of human erythroblasts as they mature. The length of time and the
changes in size are compared with those observed during the maturation of adult erythroblasts in the marrow. Cell sizes are expressed as cell diameter
and were determined by comparison of the FSCs of the different populations presented in (b) with those of commercial beads of defined size. Ex-vivo
generated proerythroblasts range in size from 30 to 50 mm. These cells significantly reduce their diameter upon exposure to EPO from 40.1  1.4 to
28.5  2.0, 21.2  0.7 and 11.6  0.3 mm by 24, 48 and 96 h (P < 0.01, in all cases). Transition during the early phases of maturation occurs more
rapidly in vitro than in vivo, although later on maturation in vitro appears delayed (possibly because of absence of MS-5 cells). The diameter of ex-vivo
generated proerythroblasts is three times greater than that of the corresponding cells generated in vivo. With maturation, the size of ex-vivo generated
erythroblasts becomes similar to that of in-vivo generated cells, although they remain macrocytic (11.6 mm) (the size of RBCs in vivo decreases during
ontogenesis from 20 mm of embryonic erythroblasts to 12.5 mm of fetal erythroblasts and 8 mm of the adult normocytic RBCs) [20]. DXM,
dexamethasone; EPCs, erythroid progenitor cells; EPO, erythropoietin; FSC(s), forward size scatters; HSCs, hematopoietic stem cells; IGF,
insulin-like growth factor; IL, interleukin; MNCs, mononuclear cells; RBC(s), red blood cell(s); SCF, stem cell factor.

addressable with current knowledge. The major obstacles
in defining an ex-vivo RBC production process, however,
cannot be surmounted with current knowledge and
derive from the fact that cultured erythroblasts release
in the supernatant a host of negative regulatory factors
[50], including transforming growth factor beta and tumor
necrosis factor-related apoptosis-inducing ligand, that
inhibit the growth of additional erythroblasts by accel-
erating maturation [51], inducing apoptosis [52] or both
(Fig. 4 [52–60]). To reduce the effects of this paracrine
loop, erythroblasts must be maintained in culture at
densities lower than 106 cells/ml. Therefore, ex-vivo

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 Erythroid cells in vitro Migliaccio et al. 265

Table 4 Design considerations for an erythroblasts production process

Parameter

Cell source Cord blood, adult blood, hESCs, iPCs, and others
Number of cells in the transfusion product
RBCs 2.5
1012 per transfusion
Erythroblasts 5
1010 per transfusion
Culture volumes required (l)
RBCs 2500
Erythroblasts 50
Costs (/l) 1000 $
Time required for production (days)
RBCs 30
Erythroblasts 10
The number of RBCs for transfusion is by comparison with the number of RBCs present in a blood unit. The hypothetic number of erythroblasts to be
transfused has been calculated on the basis of the conservative estimate that each erythroblast would undergo at least 5–6 duplications in vivo
(an erythroblast may undergo from four to 64 times before becoming a RBC). The volumes required to produce a transfusion product are calculated on
the basis of the assumption that the cell density within the expansion culture must be kept at 106/ml. Costs are calculated on the basis of the market
price of growth factors considering that the source of EPO may be represented by relatively economic preparations used in the clinic. The time required
for production is calculated on the basis of the speed of expansions in cultures depleted of basoerythroblasts reported in [28]. EPO, erythropoietin;
hESCs, human embryonic stem cells; iPCs, induced pluripotency cells; RBC(s), red blood cell(s).

generation of a unit of blood (2.5
1012 erythroblasts)
requires one cubic meter of media (Table 4). The identi-
fication of manipulations that increase the density at
which erythroblasts can be cultured (i.e. inhibition of
the paracrine loop, removal/storage of erythroblasts or
both as they mature) will play a major role in developing
an ex-vivo RBC production process.
Definition of biomarkers
In the case of ex-vivo RBC transfusion products, bio-
markers must be developed to predict potency of EPCs
from different donors to generate erythroblasts, the pro-
gression of the production process and the potency of
ex-vivo generated erythroblasts as transfusion product
in vivo.
Potency of erythroid progenitor cells from different donors to
generate erythroblasts
In contrast to the constant number of erythroblasts that
are generated in repeated cultures from the same donor,
great variability exists in the number of erythroblasts
generated in cultures from different donors. Therefore,
biomarkers must be developed to predict the potency of a
donor to generate erythroblasts ex vivo. Studies are
necessary to establish whether these biomarkers may
be represented by the frequency of EPCs present in cord
blood and adult blood (Table 3), by genetic polymorph-
isms that affect the ability of the EPCs to generate
erythroblasts in culture or both. Candidate polymorphic
loci include glucocorticoid receptor [61] and ESR [62]
(Fig. 4) or loci linked to the length of the telomeres
[63,64]. As both DXM [59] and estradiol [60] target the
levels of GATA1 activity in erythroblasts, different glu-
cocorticoid receptor and ESR expression patterns in
EPCs from donors may establish a range of alternative
synergistic–antagonistic interactions that affects the effi-
ciency of the erythroblasts production process (Fig. 4). In
addition, telomere length of CD34þ cells has been
reported to predict the numbers of erythroblasts that
are generated by different cord blood units in cultures
[63,64] and telomerase expression, low in CD34þ cells,
increases by several fold with erythroid maturation [65].
Progression of the production process
Biomarkers that can be used to monitor erythroblasts
expansion during production are represented by anti-
genic, expression profiling or both (Fig. 2). Antigenic
profiling can be performed using two-color flow cytome-
try and the levels of CD253a (glycophorin A) and either
CD71 (transferrin receptor type I) [66] or CD36 (throm-
bospondin receptor, a receptor for malarial parasites) [38]
expression (Fig. 3). Expression profiling may be based on
levels of GATA1 expression. In precultures, GATA1/
PU1 and GATA2/GATA1 expression ratios may be used
as indexes, respectively, of the frequency of EPCs within
the progenitor cells and of the numbers of divisions
allowed for each EPC. The progeny of HSCs become
EPCs when expression of GATA1 is higher than that
of PU1, a transcription factor essential for myeloid differ-
entiation [56]. EPCs retain proliferation activity when
expression of GATA2, another member of the GATA
family, is higher than that of GATA1 [67]. On the con-
trary, the level of GATA1 activity may predict if eryth-
roblasts in expansion cultures will expand, mature or
enter apoptosis (Fig. 4).
The potency of the transfusion product in vivo
The purpose of the product potency assay is to compare
potencies of products obtained from different prep-
arations. A potency assay must be based on xenogenetic
animal models, and human erythroblasts have been
shown to generate RBCs, although at low frequency,
in nonobese diabetic/severe combined immunodefi-
ciency mice. As the low efficiency may be related to
the inefficient binding of murine EPO to the human
EPO receptor [68], transfusion of ex-vivo generated

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 266 Hematopoiesis
Figure 4 Growth factors and hormones control the fate of the
erythroid progenitor cells (proliferation, maturation or apopto-
sis) through discrete changes of GATA1 activity
Genetic evidence in mice indicates that progression along the erythroid
maturation pathway is controlled by discrete changes in the levels of
GATA1 activity. In fact, both mice carrying the hypomorphic GATA1
mutations [53] and those overexpressing the GATA1 gene [54] die of
anemia. In the case of the hypomorphic mutations, the anemia is induced
by increased apoptosis rates at the basophilic erythroblasts stage.
Increased apoptotic rates are due to the fact that in erythroblasts GATA1
controls the expression of the antiapoptotic gene BCLXL [55]. Therefore,
if the GATA1 activity decreases below the thresholds necessary to
sustain BCLXL expression, erythroblasts will undergo apoptosis. On the
contrary, the anemia in mice overexpressing GATA1 is due to insufficient
RBC production because of accelerated maturation. GATA1, by forming
complexes with numerous other proteins (FOG1, Lmo2 and many
others) binds to multiple sites present on the promoter of all the
erythroid-specific genes, activating their expression [56]. Maturation
occurs when the concentration of GATA1 is sufficiently high to form
these complexes and to saturate all binding sites. Therefore, the rate at
which GATA1 activity increases determines the speed with which
maturation is completed. The anemia observed in mice overexpressing
GATA1 is due to the fact that in the erythroblasts of these mice GATA1
concentration increases too rapidly, limiting expansion. These obser-
vations in mice suggests that factors that would either decrease or
increase too rapidly the levels of GATA1 activity will both reduce the
numbers of erythroblasts generated in vitro by either promoting apop-
tosis or accelerating maturation. Both types of factors are present in
expansion cultures. The major positive control on GATA1 activity is
exerted by EPO. EPO stimulates GATA1 biosynthesis by regulating the
transcription rate of the gene and the stability (by inhibiting the caspase
pathway) [57] and phosphorylation state (via the ERK/MAP kinase
pathway) of the protein [58]. Both glucocorticoid receptor and ESR,
instead, negatively regulate GATA1 activity in erythroblasts, glucocorti-
coid receptor by destabilizing the protein [59], ESR by binding GATA1
into transcriptionally inactive complexes [60]. In addition, the paracrine
production of TGF-b and TRAIL during the culture may also affect
GATA1 activity. TGF-b, by increasing GATA1 activity, may accelerate
maturation, whereas TRAIL, by activating the caspase 8-dependent
degradation of GATA1, may induce apoptosis [52]. Generation of
optimal numbers of erythroblasts in cultures depends on the establish-
ment of a delicate equilibrium among all these factors that ensures that
the level of GATA1 activity remains within the ranges that allow pro-
liferation. EPC, erythroid progenitor cell; EPO, erythropoietin; ESR,
estrogen receptor; MAP, mitogen-activated protein; RBC, red blood
cell; TGF-b, transforming growth factor beta; TRAIL, tumor necrosis
factor-related apoptosis-inducing ligand.
erythroblasts in immunocompromised mouse models
treated with human EPO may represent a more sensitive
potency assay for these products (Fig. 2).
Safety of the product
Ongoing clinical trials indicate that immunization of
recipients to xenogeneic products used for cell pro-
duction represents a major risk of cell therapy [69,70].
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
This risk is higher for products that will be administered
multiple times such as ex-vivo generated RBCs. There-
fore, completely humanized media must be developed
for use in production. Transmission of known and
unknown adventitious agents [71] and neoplastic trans-
formation are also concerns [72–74]. Infection risk can be
addressed following guidelines already developed for
RBC blood products. Although ex-vivo generated eryth-
roblasts stored for 8 years remain chromosomally normal
[75], the safety of ex-vivo generated erythroblasts must
be further evaluated by cytogenetic/genotoxicity assays
in compliance with FDA guidelines (http://www.fda.gov/
cber/ind/ind.htm).
Conclusion
Substantial progress has been made in expanding eryth-
roblasts ex vivo, suggesting that ex-vivo generation of RBC
transfusion products may became a reality in the near
future. Much of the research performed in this field is
directed toward developing hESCs and iPCs that would
generate erythroblasts customized for the recipient.
Development of a clinical grade transfusion product also
requires better definition of the identity of the product
(EPC, erythroblasts or RBCs) and the development of
production processes that permit safe ex-vivo expansion of
human erythroblasts in a cost-efficient manner.
Acknowledgements
This study was supported by Ministero per la Ricerca Scientifica, grant
no. RBNE0189JJ_003, and RBNE015P72_003, the National Cancer
Institute, grant no. P01-CA108671 and a New York State STAR grant,
USA.
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