Journal of Stem Cells
Volume 11, Number 3
Stage-Specific Regulation of Erythropoiesis and Its
Implications in Ex-Vivo RBCs Generation
Vimal Kishor Singh1,, Abhishek Saini1,
Manisha Kalsan1, Neeraj Kumar1, and
Ramesh Chandra2
1 more than a decade now, and all the distinct types of stem
Stem Cell Research Laboratory, Department of
Biotechnology, Delhi Technological University,
Delhi, India
2
Dr. B. R. Ambedkar Center for Biomedical Research,
University of Delhi, Delhi, India
Correspondence: Dr. Vimal Kishor Singh: O/I, Stem cell
Research Laboratory, Department of Biotechnology,
Delhi Technological University, Shahbad Daulatpur,
Bawana road, Delhi-110042.
E-mails: vim_kissor@yahoo.co.in;
vimalkishorsingh@gmail.com, vimalksingh@dce.ac.in
ISSN: 1556-8539
2016 Nova Science Publishers, Inc.
Abstract
Ex vivo erythropoiesis methods are being developed for
cells (such as CD34+ HSCs, ESCs, IPSCs, and extensively
proliferating erythropoietic progenitor cells) are defined to
bear the potential for large scale RBC production shortly.
The various regulating factors at different levels of RBCs
production are being explored. Since most of the ex-vivo
erythropoiesis protocols mimic the dogma followed by
hematopoietic stem cells in vivo to give rise to mature
RBCs which essentially deals with the intermediate stages
of erythropoiesis such as burst forming unit-erythroid
(BFU-E) and committed erythroid colony forming unit-
erythroid (CFU-E). In vivo generation of erythroid
progenitors (BFU-E/CFU-E) is essentially controlled by
several factors including glucocorticoids, inflammation, and
stress. Furthermore, regular production of functionally
mature /transfusable units of RBCs is possible only through
the coordinated regulation of terminal proliferation and
differentiation of erythroid progenitors by external signals,
such as erythropoietin, SCF, IL-3 and interaction to
extracellular matrix protein(s) in a 3D culture system. We
discuss these complex intracellular networks of coordinated
factors and try to understand their molecular mechanism
through gene regulation by transcription factors, and
miRNAs that might be helpful in developing the optimal
RBCs production protocols for commercial production.
Abbreviations
EPO Erythropoietin
SCF Stem Cell Factor
IL3 Interleukin-3
TPO Thrombopoietin
Flt3 Fms related Tyrosine Kinase 3
IGF1 Insulin like Growth Factor 1
Sox2 SRY (Sex determining region Y)-
box-2
miRNAs micro RNA(Ribose Nucleic Acid)
shRNA short hairpin RNA
3D Three Dimensional
150 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

WHO World Health Organization circulatory system and developed ways to study the
RBC Red Blood Cells cross circulatory effects in animals. Later on Dr.
HSCs Hematopoietic Stem Cells James Blundel (1818) reported first successful blood
ESCs Embryonic Stem Cells transfusion among humans. In 1901, Karl Landsteiner
hESCs human Embryonic Stem Cells discovered the blood group antigens and also
iPSCs induced Pluripotent Stem Cells demonstrated the adverse effects associated with the
UCB Umbilical Cord Blood transfusion of incompatible blood groups in human
PB Peripheral Blood recipients leading to severe immunological disorders
BM Bone Marrow causing death. This landmark study (Nobel Prize
CD34 Cluster of Differentiation 34
1930) laid the foundation for the identification of
CD235a Cluster of Differentiation 235a
various blood type antigens and paved the way for
(Glycophorin A)
safe and fruitful clinical blood transfusions. Later
CFU-E Colony Forming Unit Erythroid
studies demonstrated a number of different blood cell
BFU-E Burst Forming Unit Erythroid
MCSF Macrophage Colony Stimulating surface antigens playing a crucial role in determining
Factor the compatibility of donor-derived blood in clinics.
GCSF Granulocyte Colony Stimulating In 19401950s, a number of inventions made it
Factor possible to store the donor-derived human blood
Stat5 Signal Transducer and Activator of under appropriate conditions, and a vast network of
Transcription 5 blood banking system flourished during these years.
GATA 1 Globlin Transcription Factor 1 In modern medical modalities, blood bank system
GATA 2 Globlin Transcription Factor 2 remains axial to support all kinds of blood transfusion
SCL/Tal1 Stem Cell Leukemia/ T-cell Acute needs (Alter and Klein, 2008). There are about 108
Lymphocytic Leukemia 1 million blood units collected throughout the globe
LDB1 LIM Domain Binding 1 every year (www.who.int/worldblooddonorday/en/)
LMO2 LIM Domain Only 2 (Department of Health and Human Services, 2010,
Klf1 Kruppel Like Factor1 2013; World Health Organization, 2011). However,
Gfi-1b Growth Factor Independent 1b the accessibility to safe and adequate blood supplies is
transcription Repressor limited in developing countries as compared to the
MYB Myeloblastosis developed countries. Developed countries rather
ALK Anaplastic Lymphoma Kinase sparsely populated (15% of the global population) but
contribute to more than 50% of the total global blood
collection per annum. This is the reason that problem
Introduction of shortage of transfusable blood units is not so severe
in the economically developed countries, and the
In the17th century, Dr. Harvey established a blood collection seems to be sufficient for the time
breakthrough by reporting his findings regarding being. According to the WHO reports, there are
blood circulation system that eventually led to the approximately 8000 blood centers scattered in 159
blood transfusion experiments. Initial blood high-income countries which score >30,000 annual
transfusion experiments were done on animals that blood donations on an average per blood center
laid the foundation for human transfusion (Department of Health and Human Services, 2010,
experiments. In 1665, Dr. Jean-Baptiste Denys 2013). On the other hand, only 3700 blood collections
demonstrated first successful human transfusion from per center are reported in developing countries which
a sheep in 15 years old boy. Unfortunately, this have to support ~ 80% of the world population
experimentation of animal blood transfusion in residing in these countries. The WHO reports states
humans resulted in the death of most of the recipients that there are only ten donations per 1000 people in
as reported by him in successive studies. Another the 82 low income and middle-income countries
milestone was achieved by Dr. Richard Lower, who (World Health Organization, 2011) (Figure 1).
showed the effects of changes in blood volume in the
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation 151

Figure 1. Graphical presentation showing a total number of blood donations, blood donations in developed vs. developing
countries. The population of developed countries is about 18% who have access to approximately 50% of the total donated
blood. Whereas, the developing countries where more than 80% of the global population have access to the remaining 50%
of the donated blood.

What makes the situation much horrible is the
inefficient screening facilities available in most of the
undeveloped countries. A significant number of
countries (39 countries) are reported to lack efficient
screening facilities for most severe transfusion-
transmissible infections (TTIs) viz. HIV, hepatitis B,
hepatitis C, and syphilis (Table 1) (Department of
Health and Human Services, 2013). Further, only 106
countries have laid national guidelines for appropriate
blood transfusions, and only 13% of the low-income
countries are equipped with the national
haemovigilance system to ensure the safety of the
transfusion process. It is also interesting to be noticed
that most developed countries are having a rapidly
growing proportion of elderly people (>60 years old).
Moreover, it would be difficult to support the need of
burgeoning demand for blood transfusions for
surgical treatments by the year of 2050 (U.S. Census
Bureau, 2004; Ali et al., 2010). The situation would
be more difficult if one has to define the blood group
compatibility for more than 30 blood group system
(308 recognized antigens) including ABO & Rh
antigens (Daniels et al., 2009). Also, demand for
rare phenotypic blood, patients with various
hemoglobinopathies, polytransfusion patients, and
polyimmunization may put more constraints on the
donor derived blood supply system across the globe
(Table 1) (World Health Organization, 2011).
Although, there are reports showing methods to
develop universal blood group by using antigen
masking and/or enzymatic cleavage techniques but
these methods are yet to be elaborated in a detailed
way (Bagnis et al., 2011).
Another important issue is the association of
various types of adverse reactions with the donor
derived blood supplies. Despite a number of testing,
preclinical precautionary measures taken to avoid any
infectious agent [e.g., HIV-1, HIV-2, HTLV-1,
HTLV-2, Hepatitis B, Hepatitis C, Syphilis (T.
pallidum), Chagas disease (T. cruzi), and West Nile
Virus, Cytomegalovirus (CMV)], blood transfusions
are prone to several complicated situations (Alter and
Klein, 2008; World Health Organization, 2011).
These complications cause delayed cure rate with
prolonged hospitalization periods and may raise the
overall cost of treatment also (Department of Health
and Human Services, 2010).
152 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

Table 1. Risks associated with the blood transfusion

S.No. Agent Risk Factor Remarks

HIV 1 in 4 million
Hepatitis B attacks liver 1 in 1.2 million
1. Blood borne infections
Hepatitis C 1 in 3 million
West Nile Virus 1 in 1 million
Swelling
2. Allergies Can be treated with antihistamines
Itching
Maybe due to febrile transfusion
Fever
Body temperature reaction
3.
fluctuation Due to temperature difference of blood
Hypothermia
being transfused
The white cells in the transfused
4. Graft versus host disease Use of irradiated blood prevents it
blood attack patients tissue
Acute immune hemolytic Patients immune system destroys
5. Very rare but serious due to mismatch.
reaction the transfused blood cells

Figure 2. Timeline Schematic representation of the major events related to ex-vivo erythropoiesis generation by different
research groups highlighting there break-troughs.

For the last few years, various non-donor derived
sources have been proposed by different research
groups. However, the majority of artificial blood
molecules (hemoglobin-based oxygen carriers or
perfluorocarbon solutions) are inefficient oxygen
carriers and have limited functional application
(Winslow, 2006; HenkelHonke and Oleck, 2007;
Natanson et al., 2008; Silverman and Weiskopf, 2009;
Castro and Briceno, 2010). One of the most promising
approaches is ex vivo expansion of erythrocytes from
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation
stem cells. Initially, Nakamura and his colleague
demonstrated survival of lethally irradiated mice by
ex vivo expanded RBCs which were derived from
immobilized ESCs cell line (Hiroyama et al., 2008).
Following the Nakamuras findings, many people
have developed different protocols to derive
functionally mature erythrocytes from various types
of stem cells viz. CD34+ HSCs, Embryonic stem
cells, and IPSCs. The proof-of-concept study
demonstrated by Giarratana et al., shown first-in-
human blood transfusion of ex-vivo generated RBCs
(Giarratana et al., 2011). These studies indicated that
expansion of erythrocytes from stem cells could
become a powerful tool to solve the problems of
blood supply shortages and other associated issues.
However, there are several important matters
which need to be resolved to ensure the expected
yield of functionally mature RBCs through these
protocols suitable for clinical transfusions.
Fortunately, there are reports showing possibilities of
exponential expansion of erythrocyte precursors by
using similar techniques. Neildez-Nguyen et al.
demonstrated significant yield (up to 0.5 transfusion
unit) from UCB derived CD34+ cells. An
approximately 2 million fold expansions of
enucleated erythrocytes was reported in their studies
showing functional similarities between native RBCs
and cultured RBCs (Neildez-Nguyen et al., 2002). In
similar studies, UCB derived CD34+ are reported to
give rise to 10 transfusion unit by a technically
complex protocol (Fujimi et al., 2008) (Figure 2).
Despite their ill-suitedness for the large scale
commercial production, these methods precede the
potential use of stem cell-derived methods in near
future. Apart from overall yield arising from these
protocols, the enucleation efficiency of maturing
RBCs has been reported to be significantly low which
is a major indicator of their functional maturation.
However, it was demonstrated RBCs precursors,
cellular intermediates/nucleated erythroid precursors
generated during ex vivo expansion may also be of use
if transfused. It is proposed that these maturing
nucleated cells along with enucleated erythrocytes can
function like filler supporting the cellular content
during the transfusion and may give rise to
functionally matured cell by proliferating into 464
enucleated RBCs. This is reflected by studies showing
the functional maturation of cellular intermediates/
153
nucleated erythroid precursors in vivo through their
interaction with internal factors regulating their
maturation. This fact is well supported by the results
from studies reported both in human and animal
models (Ende and Ende, 1972; Neildez-Nguyen et al.,
2002). The significance of the maturing erythrocytic
precursor is also supported by several reports showing
the occasional clinical use of compatible Cord Blood
(40-80 ml/47 1010RBCs and 28 109 erythroblast
cells) for transfusion in emergent conditions (Ende
and Ende, 1972). It is evident from these reports that
ex vivo RBCs expansion can support the different
clinical short-comes that exist because of supply
constraints and many rare phenotypes related blood-
based disorder. However various technological
barriers exist, and a regular large scale production
would only be possible if one can find the most
suitable way to produce clinical transfusable units at a
reasonable cost. To enhance the present scale of
RBCs production more cost-effective and timely
automated ex vivo culture approaches are required.
This is possible if we have an in-depth knowledge of
the various regulatory elements and their mode of
action playing an axial role in almost all of these
methods demonstrated so far.
Here we present the various essential regulator
elements playing an important role during in vivo
erythropoiesis, and their optimal use may aid to
develop more efficient large-scale expansion
protocols.
Erythropoiesis in a Glance
Mammalian definitive erythropoiesis is initiated
in the fetal liver/yolk sac and later on it remains
limited to the adult bone marrow throughout the life.
This highly regulated process involves hematopoietic
progenitor cells and their subsequent differentiation
into committed erythroid progenitors. After that, these
progenitor cells proliferate further and give rise to
functional erythrocytes. Hematopoietic stem cells
(HSCs) proliferate in the medulla of the adult bone
marrow with high self-renew capabilities and
differentiate into myeloid and lymphoid progenitor
cells. Lymphoid progenitor cells undergo
differentiation and give rise to lymphocytes and
plasma. A myeloid progenitor cell is a multipotent
154 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

progenitor and further down the line differentiates
into bipotential progenitors which are restricted to
erythroid/megakaryocyte and granulocyte / macro-
phage pathways (Akashi et al., 2000). The hierarchy
of erythropoiesis may be defined in three distinct
stages (Figure 3). First, the commitment of HSCs into
erythropoietic lineages which result to the
development of erythroid-committed blast cells from
multipotent hematopoietic progenitors; second,
Division and differentiation of these morphologically
distinguishable erythroid progenitor cells; and third,
Terminal differentiation that includes a change in cell
morphology viz. removal of nucleus or enucleation to
produce mature RBCs. During erythroid commitment
phase, the burst forming unit erythroid (BFU-E) (slow
proliferating) are the earliest committed progenitors
that can be identified ex vivo. Early BFU-E cells get
matured and then differentiated into rapidly dividing
stage of colony forming unit-erythroid (CFU-E)
(Elliott et al., 2008). In the next few days, these CFU-
E progenitors divide further (3-5times) and
differentiate into later stages. This includes various
significant changes such as reduced cell size,
chromatin condensation, and synthesis of hemoglobin
that leads to their enucleation and expulsion of other
organelles (Fawcett et al., 1997).

Figure 3. In vivo erythropoiesis: This schematic diagram represents the three major phases of in-vivo erythropoiesis a)
Phase1- Hematopoietic stem cells differentiate into erythroid precursor BFU-E, b) Phase2- Expansion and differentiation of
BFU-E into erythroid progenitors CFU-E in the presence of IL3, EPO, c) Phase3- Maturation of erythrocytes through
enucleation the reticulocytes are transformed into mature enucleated erythrocytes by the expulsion of the nucleus.

Human RBCs turnover is approximately 1% per
day which gets substantially increased several times
under stress conditions such as trauma or hemolysis.
This stress-induced short term enhanced
erythropoiesis is majorly regulated by Erythropoietin
(EPO). EPO is secreted by kidney cells under hypoxic
conditions and stimulates the terminal proliferation
and differentiation of CFU-E progenitors (Molineux
et al., 2009). Under chronic stress conditions, more
CFU-E cells are required since they are not capable of
giving rise to such an extreme RBCs population even
in the presence of high concentration of Epo. BFU-E
cells which have receptors for Epo, Stem Cell Factor
(SCF), IL-3, insulin-like growth factor 1 (IGF-1),
glucocorticoids (GCs), and IL-6, get proliferated and
differentiated into a number of CFU-E cells to meet
the demand. However, their origin in fetal/bone
marrow has not been defined. The regulatory
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation
mechanism for the division of BFU-E cells, another
cytokine interaction essential to control their self-
renewal and differentiation into mature CFU-E
progenitors is not properly understood. There are
many intracellular signal transduction molecules
including protein-ligand to the above-mentioned
cytokines/ growth factor receptors, which may elicit
activation of various downstream transcription
factors, DNA-binding molecules and chromatin
modifiers to control cellular division and
differentiation. Besides, multiple noncoding
regulatory RNAs, (microRNAs); are essential for
these function.
Thus, the overall in vivo RBC generation process
is conceived to be a well-regulated system that relies
on cytokines, transcription factors, and cofactors,
post-translational modifications, and miRNAs at
specific stages. A detailed understanding of these
regulating elements could be helpful in designing
optimal protocols for large scale RBCs production by
manipulating ex vivo erythropoietic culture states
through these molecules.
Ex Vivo Expansion of RBCs
The methods for developing terminally
differentiated, fully functional RBCs in transfusable
amount have evolved enough during last decade
(Migliaccio et al., 2012). There have been significant
studies defining various regulatory elements to access
biological control over the erythropoietic process. As
discussed in the next section, studies to define in vivo
hematopoietic development process have shown that
the generation of erythroblast is regulated by various
growth factors (Lodish et al., 2010). These reports
helped researchers to explore the enormous potential
inherited by all stem cells types including (i) CD34+
HSPCs, (II) Embryonic stem cells/induced pluripotent
stem cells, and (III) immortalized erythroid
precursors.
All these cell types are shown to get
differentiated into erythroid lineages in different
protocols which all share almost similar growth
regulators (Figure 4) (Table 2). Despite significant
differences in their methodologies used, time of
culture and yield, all of these methods are categorized
in three basic/ major steps as follows: (1)
155
commitment, (2) expansion, and (3) maturation.
These protocols are well summarized by Singh et al.,
(2014), and various cytokines/growth factors (SCF
and EPO are most common) are demonstrated to
generate variable yields of immature progenitors or
mature RBCs (Singh et al., 2014). Similarly, studies
are done showing variable effects of animal/human
derived feeder co-culture system on the yield and
maturation level of cultured RBCs. Most of the
protocols demonstrated so far have followed the
dogma in which stem cells are converted into
committed erythroid lineages through hematopoietic
cell progenitors states but recently trans-
differentiation procedures are reported showing direct
differentiation of human fibroblast cells into
erythroblasts (Szabo et al., 2010).
Although promising all these processes reported
so far inherit almost similar problems including
inefficient enucleation of erythrocytes, less overall
yield, lack of expression of an adult form of Hb
predominantly, use of animal derived products such
as serum and feeder cell co-culture. Apart from them,
there is clinical transfusion related problems also such
as difficulty in matching appropriate blood type and
unavailability of sufficient number of RBCs for
transfusion.
Molecular Regulators
for Erythropoiesis
Many groups tried to categorize various key
regulators of ex-vivo RBC expansion in a certain way.
There are several factors which play pivotal roles
including cytokines (e.g., EPO, SCF, and IL3),
transcription factors, co-factors and non-coding
regulatory RNAs (Table 2, Figure 4). All these factors
affect different stages of ex-vivo erythropoiesis. Some
of them may regulate the initial growth and
differentiation of most primitive HSCs (e.g., SCF, IL-
3) which are the source in almost all protocols. The
addition of other molecules such as steroids,
erythropoietin may be essential for the maintenance of
growth and maturation of erythroid progenitors/
precursors and mature RBCs respectively. These
molecular regulators have a distinct role in these
ex vivo expansion approaches. The overall
erythropoiesis can be categorized under normal
156 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

regulatory conditions and the regulation of same
under stress condition. Like the first category, the
normal erythropoietic condition is regulated by a
number of different regulatory elements as discussed
below i.e., growth factors, proteins and miRNAs.
Whereas, stress conditions is defined in the second
category which involves a somewhat different
mechanism to maintain the sudden need of
hyperproliferative erythropoiesis and has been
discussed in the later part.

Figure 4. Insight to erythropoiesis with respect to ex-vivo erythropoiesis: This schematic diagram represents the site of
erythropoiesis i.e., bone marrow in adult human where differentiation of HSCs into erythroid progenitors takes place in an
organized manner under the regulation of various sets of cytokines, transcription factors, and miRNAs which finally matures
into erythrocytes through enucleation of reticulocytes and gets into the circulation. For ex-vivo erythropoiesis, the essentially
required are the source of HSCs which could be UCB, iPSCs, PB which has to be cultured in the presence of growth factors
(blue), chemical inducers (Green) in a span of 18 -21 days to get mature erythrocytes.
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation 157

Table 2. Factors that regulate ex-vivo erythropoiesis

S.
Factors Role/Effect in Ex-Vivo Erythropoiesis Receptor References
No.
1 Animal/ Human-Derived Growth Factors/Cytokines
Differentiation and proliferation of
a) EPO EPOR Gregory et al., 1978
erythroid
Differentiation and proliferation of Korpelainen et al.,
b) IL3 CD123/IL3RA,CD131/IL3RB
myeloid progenitor cells 1996
c) GM-CSF White blood cells growth factor CD116 Rosas et al., 2007
Inducer of HSCs mobilization from the
d) G-CSF CD114 Basu et al., 2002
bone marrow into the bloodstream
stimulate erythroid colony forming units in
e) IGF-1 IGF1 Receptor Brox et al.,1996
the mouse
f) SCF Regulates HSCs in the bone marrow CD117 Linden et al., 2004
2 Synthetic Molecules
a) Dex Anti inflammation Leberbauer et al., 2005
b) Poloxamer 88 Induces enucleation of RBCs Baek et al., 2009
Cholestrol
c) Induces proliferation rate Leberbauer et al., 2005
rich Lipid
d) Hydrocortisone Enhance the proliferation of EPCs Timmins et al., 2011
3 mi-RNAs
Differentiation towards the
a) miR-150 megakaryocytic lineage at the expense of MYB Lu et al., 2008
erythroid lineage
Downregulation of miR-221 and miR222
miR-221
b) is required for terminal erythroid KIT Felli et al., 2005
and miR-222
progenitor proliferation
Downregulation of miR-223 is required for
c) miR-223 ALK4 Felli et al., 2009
the terminal differentiation
Downregulation of miR-24 is required for
d) miR-24 LMO-2 Wang et al., 2008
terminal erythroid differentiation
4 Transcription Factors
Globin, erythroid specific
regulates p21 gene expression during
a) GATA1 membrane proteins, Papetti et al., 2010
erythroid differentiation
GATA-1, GATA-2
the cellular proliferation of non-committed Orkin et al., 1992 &
b) GATA2 GATA-1 and GATA-2 gene
and committed erythroid progenitors 2000
required for specification of HSCs and
Ravet et al., 2004; Hall
c) SCL/TAL1 maturation of the erythroid and Glycophorin A, p21 gene
et al., 2005
megakaryocytic lineages
d) LDB1/LMO2 Ldb1 complexes regulate Klf1 Love et al., 2014
Acts as global regulator of erythroid
e) Klf1 Globin Micheal et al., 2010
production and integrity
f) Gfi1B represses p21, SOCS1, 3 and its own genes p21, SOCS1-3, BclxL Kuo et al., 2007

Category-I: Erythropoiesis under Normal is considered as a regulator of erythroid lineage alone

Conditions is insufficient for large scale amplification of HSCs
(Munugalavadla et al., 2005; Arcasoy et al., 2005).
a. Role of Growth Factors Therefore, EPO-supplemented with different
Different research groups working on RBC cytokines viz. SCF, IL-3, Flt-3 & TPO seem to be
production reported that Erythropoietin (EPO) which most promising (Kawada et al., 1999; Jiang et al.,
2006; Drouet et al., 1999).
158 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

EPO response to EPO due to fewer receptors. Further,

Erythropoietin (EPO) a glycoprotein hormone
that is produced by peritubular capillary lining cells in
the kidney and liver. It acts on its target cells present
in the bone marrow to regulate the proliferation,
differentiation, and maturation of red blood cells
(Gregory et al., 1978). It is an approximately 30-
34kDa glycoprotein. The function of EPO depends on
the conditions such as availability of oxygen, iron and
cofactors, sufficient number of responsive progenitor
cells, and optimal microenvironment. It does not
specify the cell fate directly into the erythroid lineage
but supports the growth, differentiation and survival
of the erythroid committed progenitor cells (Koury et
al., 1990). EPO receptor (EPOR) is primarily
expressed on erythroid cells. Its expression is highest
from CFU-E to pro-erythroblast of erythroid cell
development; whereas BFU-E shows a weaker
down the erythroid differentiation line, EPOR per cell
expression is gradually reduced, and according to the
studies the reticulocyte and erythrocyte are devoid of
EPO receptors (James 2003). EPO binds EPOR to
activate dimerized EPOR and trigger a cascade of
events viz. change in EPORs conformation and trans-
phosphorylation of JAK2 resulting in phosphorylation
of multiple tyrosine residues in the cytoplasmic tail of
EPOR. These phosphorylated tyrosine molecules are
responsible for activation of downstream signaling
pathways such as STAT5, PI3-kinase/AKT, and Ras/
MAPK. All these pathways are essential for erythroid
expansion and maturation. The primitive erythroid
progenitor, i.e., BFU-E has a low sensitivity for EPO.
Therefore, additional growth factors (SCF, IL-3)
come in the play for survival and proliferation of cells
(Lindern et al., 2004) (Table 2, Figure 5).

Figure 5. Regulation of erythropoiesis by growth factors: Differentiation of common myeloid progenitor CFU-GEMM into
erythroid lineage is governed by IL3, GM-CSF, and SCF. First cell in erythroid lineage is burst forming unit- erythroid
(BFU-E) which is EPO responsive and thereafter EPO regulates the maturation of erythroblasts into mature erythrocytes.

BMP4 of BMP4/Smad-4 mediated signaling pathways.

Another important factor having an essential role
in stress erythropoiesis is Bone morphogenetic protein
4 (BMP4). Studies in mice models expressing a
dominant negative Smad5 mutant from that inhibits
BMP4 signaling have shown neonatal anemic
characteristics. That lasts for two weeks after birth
and then resolved (Heg et al., 2007; Lenox et al.,
2005; Subramanian et al., 2008).
This suggests that BMP4 signaling through
Smad5 is essential for stress-induced erythropoiesis
and adult or fetal liver hematopoiesis is independent
(Singbrant et al., 2010; Singbrant et al., 2006).
Furthermore, the BMP4 expression is induced by
hypoxia in spleen stromal cells (Wu et al. 2010;
Millot et al., 2010). BFU-E cells need priming with
Sonic Hedgehog to be responsive to BMP4 which is a
morphogen synthesized and secreted spleen cells
(Perry et al., 2009).
On their activation with Sonic Hedgehog the
transcription factor Smad5 gets activated through
BMP-4. BMP-4 also induces activities of Scl and
Gata2 as well and thus enhances the probability of
BFU-E self-renewal. (Lugus et al., 2007; Fuchs et al.,
2002; Harandi et al., 2010).
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation 159

Stem Cell Factor
SCF is the ligand for the c-kit cell-surface
receptor or the tyrosine kinase receptor. In anemic
mice, hemoglobin concentration increases to a modest
degree upon addition of exogenous EPO due of
defects in either SCF or c-kit (Pharr et al., 2000). SCF
works in coordination with other cytokines like GM-
CSF (Table 2, Figure 5) whose function is to
stimulate the growth and proliferation of granulocyte
and macrophage progenitor cells. It also influences
the differentiation, induce maturation in HSCs and
later stimulate the functional activity of mature HSCs
(Inaba et al., 1992; Metcalf et al., 1995; Rosas et al.,
2007). Another important molecule, IL-3 stimulates
the generation and functional activities of various
blood cell types (Korpelainen et al., 1996). MCSF
looks after the proliferation, differentiation, and
survival of macrophages, monocytes and bone
marrow progenitor cells (Stanley et al., 1997). G-CSF
is another glycoprotein playing major role bone
marrow stimulation and production of granulocytes. It
interaction is also important for their gradual release
into the bloodstream (Basu et al., 2002).
Now the major question arises how to deploy
these growth factors in ex-vivo erythropoiesis.
Various groups reported different concentrations and
combinations of cytokines. Neildez-Nguyen et al.,
(2002) reported FLT-3, SCF, TPO, EPO, IGF-1
(Neildez-Nguyen et al., 2002) in similar studies.
Later, Giarratana et al., (2005) shown that only SCF,
EPO, and hydrocortisone were sufficient. Leberbauer
et al., (2005) also reported a set of EPO, SCF, IGF-1
Dex and lipid mix (Leberbauer et al., 2005). Baek et
al. (2008-09) have shown similar effects by using
SCF, IL-3, EPO, TPO and Flt-3 in their experiments
(Baek et al., 2009). Other groups used a different set
of growth factors and certain hormones, steroids, and
chemicals which point out that cytokines alone are not
enough, and they need helping hand. A possible
explanation for that may be the effect of cytokine is
dependent on different cell stages. Therefore, it
becomes necessary to choose the most appropriate set
of cytokines for culturing the clinical grade
erythrocytes.

Figure 6. Regulation of erythropoiesis by different proteins and miRNAs. The intarcellular fators such as transcriptions
factors and miRNAs rgulate the formation of RBCs. GATA2 is activity is downregulated by GATA1 to regulate in
erythrocytic determination and terminal differentiation. Other proteins such as SCL/TAL1, Lmo-2, and Ldb1 aid this
regulation. miRNAs come into the action starting from the initial stage where miR-150 down-regulates expression of Myb
gene to inhibit erythroid lineage differentiation. During terminal maturation these miRNAs enhances terminal differentiation
by down regulating the KIT gene that regulates proliferation of erythroid progenitors.
160 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.
b. Regulation by Proteins
During erythropoiesis binding of most important
growth factor elicit pattern of intracellular proteins.
For example, several transcription factors are
activated like, Stat5 and EPO-regulated tran-
scriptional regulators present in erythroid cells
interact with few lineages restricted transcriptional
regulators such as GATA-1 (Globin Transcription
Factor 1), SCL/Tal1 (Stem cell leukemia/T-Cell
Acute Lymphocytic Leukemia 1), LDB1 (LIM
Domain Binding 1), LMO2 (LIM Domain Only 2),
Klf1(Kruppel-Like Factor 1 ), and Gfi-1b (Growth
Factor-Independent 1B Transcription Repressor),
responsible for the production of mRNAs that are
essential for erythropoiesis (Table 2, Figure 6).
GATA1 belongs to the GATA family of transcription
factors which regulates the switch of fetal hemoglobin
to adult hemoglobin in erythroid development. While
the stem cell leukemia (SCL) gene, which is also
known as Tal-1 encodes a critical regulator of both
hemopoiesis and vasculogenesis i.e., a basic helix
loophelix (bHLH) transcription factor and, therefore,
positively regulates erythroid differentiation. Next
LDB1 acts with LMO2 by binding to the LIM domain
of a transcription factor in order to regulate red blood
cell development by maintaining erythroid precursors
in their immature state. LMO2 is a cysteine-rich, two
LIM-domain protein that has a central and crucial role
in hematopoietic development along with TAL1/SCL
and is highly conserved. Klf1 is a hematopoietic-
specific transcription factor and induces high
expression of adult beta-globin and other erythroid
genes. Gfi-1b is an essential proto-oncogenic
transcriptional regulator which controls the expression
of genes involved in development and differentiation
of erythrocytes and megakaryocytes by forming
complexes with transcriptional regulatory proteins
including GATA-1, histone deacetylases, and runt-
related transcription factor-1.
The expression pattern of specific genes is
regulated in a precise time frame at the transcriptional
level during the differentiation of erythroid lineage.
Functional analysis of erythroid and megakaryocytic
specific genes has shown the importance of a
sequence, 5 A/T GATA A/G 3, now called the
GATA motif, in the lineage-specific expression of
these genes. This sequence is associated with a GT or
CACC-like sequence in erythroid-specific genes,
whereas it is associated with an ETS (E26
transformation-specific) binding site in mega-
karyocytic specific genes (Orkin et al., 2000; Orkin et
al., 1992; Papetti et al., 2010). The GATA family of
transcription factors consists of six transcription
factors, GATA- 1 to -6 that can bind the consensus
sequence 5 A/T GATA A/G 3. These proteins
contain two conserved zinc finger motifs (Cys-X2-
Cys-X17-Cys-X2-Cys) peculiar to the GATA family.
The carboxyl terminal zinc finger binds to DNA,
whereas the amino-terminal zinc finger stabilizes the
DNA/GATA interaction. Outside of the zinc finger
regions, the conservation between GATA factors is
low, but each factor is conserved between
species. GATA-1 and GATA- 2 reported to regulate
in erythrocytic determination and terminal
differentiation.
This is supported by motif analysis reports
showing activation of genes enriched consensus
sequences for SCL/Tal1 at GATA1-bound sites.
These studies suggested correlation between GATA1
activation and SCL/Tal1 activities. (Fujiwara et al.,
2009; Yu et al., 2009; Cheng et al., 2009; Kassouf
et al., 2010; Tripic et al., 2009; Ravet et al., 2004;
Hall et al., 2003). In erythroid cells, SCL/Tal1
interacts with basic helix loop helix protein E2A
(bHLH protein E2A) and makes an activation
complex by interacting with LIM domain of LMO2
and Ldb1. Along with GATA1, this complex binds to
the GATA1/E-box domains that are spaced ~9 to 11
nucleotides apart (Elliot et al., 2008). Components
other than GATA1 in the activation complex are also
necessary for RBC development. This is indicated by
studies in Lmo2, GATA1, and SCL/Tal1 knockout
mice where all such mice are anemic and die due to
lack of primitive erythropoiesis in fetal development
(Cantor et al., 2002). TAL-1 can assemble in a
pentameric complex (Wadman et al., 1997)
containing the ubiquitously expressed.
E proteins (E2A, HEB, E2-2), LMO-2, Ldb, and
GATA-1 in erythroid-committed cells. This complex
can lead to activation of erythroid-specific terminal
genes expression. GATA-2 variant of this TAL-1
complex is responsible for activation of c-Kit
transcription in CD34+ hematopoietic progenitors
(Lecuyer et al., 2002; Love et al., 2014; Michael et
al., 2010; Kuo et al., 2007). Therefore, to mimic
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation
erythropoiesis ex-vivo a better understanding at
molecular level becomes inevitable.
c. Regulation by miRNAs
In the last few years, a new class of small,
endogenous, non-coding RNAs called microRNAs
(miRNAs) is reported. miRNAs are important
regulators of gene expression at the post-
transcriptional level. Thousands of such miRNAs are
identified in various organisms including unicellular
microorganism and complex multicellular organisms
showing that regulatory miRNA pathway is conserved
in evolution (Listowski et al., 2013). miRNAs are
defined as a class of small regulatory RNAs that
down-regulates their target genes expression either
through degradation of mRNA or inhibiting their
translation (Bartel et al., 2009; Guo et al., 2010).
Erythropoiesis is also regulated by several miRNAs at
various stages which include lineage determination,
progenitor proliferation, terminal differentiation of
erythroid, and enucleation of reticulocytes (Table 2,
Figure-6). Out of them miR-24, 144, 150, 191, 221,
222, 223, & 451 is reported to have significant roles
in human erythropoiesis. miR-150 come into the
action at the initial stage and down-regulates
expression of Myb gene to inhibit erythroid lineage
differentiation (Lu et al., 2008). While miR-24
negatively regulates activin signaling, it down-
regulates the mRNA encoding the Activin receptor-
like kinase 4 (ALK4) (Wang et al., 2008). LMO2 is a
critical transcription factor which gets up-regulated
during erythroid differentiation of CD34- progenitor
cells and is required for erythroid differentiation, but
gets down-regulated due to overexpression of miR-
223 (Felli et al., 2009). During the terminal erythroid
progenitor proliferation miR-221 and miR-222 come
into action where these miRNAs enhances terminal
differentiation by down-regulating the KIT gene that
regulates proliferation of erythroid progenitors (Felli
et al., 2005)
Category-II: Regulation of Erythropoiesis
under Stress Conditions
As discussed above the erythropoiesis level is
several folds enhanced under stress conditions such as
hypoxia, chronic anemia, and acute blood loss. This
161
process is governed by several important factors
which tightly regulates survival, proliferation and
terminal differentiation of erythroid progenitors such
as CFU-E and produce more number of erythroblast
cells. Since limited proliferative potential (3-5cell
divisions) of early EPO-responding CFU-E even in
the presence of high level of EPO due to stress
conditions remain insufficient to support the extreme
demand of mature RBCs in a short span of time. Bone
marrow produces more number of CFU-E cells from
BFU-E cells. This process of generating more number
of BFU-/CFU-E is independent of EPO-mediated
activities. It is shown that single BFU-E cell can give
rise to hundreds of CFU-Es in vitro. BFU-E
progenitors may undergo limited self-renewal during
stress erythropoiesis supporting the survival of
lethally irradiated mice from anemia. Even more,
these BFU-Es can be re-transplanted and protects
secondary and tertiary recipients (Harandi et al.,
2010).
Under stress conditions, a significant number of
cell division, self-renewal, and differentiation
activities take place which are regulated by various
factors such as microenvironment growth factor/
Glucocorticoids and Hedgehog signaling along with
tissue hypoxia. This is evident from the studies
showing the movement of erythropoietic activities
from the bone marrow to the spleen during stress
conditions until optimal conditions are restored.
(McCulloch et al., 1964; Hara et al, 1976). Fetal liver
and spleen-derived stromal cell lines are demonstrated
to inherit better-supporting properties for in vitro fetal
BFU-E proliferation in comparison to bone marrow
stroma cells (Slaper-CortenbachI et al., 1987; Yanai
et al, 1989; Ohneda et al., 1990). Furthermore, the
microenvironment have a significant role in
sustaining erythroid progenitors self-renewal. This is
also evident from the mice studies showing resistance
to Friend erythroid leukemia virus-induced erythroid
leukemia (Kreja et al., 1985). A number of attempts
have been made by various researcher groups to
define various regulating factors in spleen and fetal
liver.
Another important regulatory factor is the
glucocorticoids (GC) which are released from the
adrenal glands during conditions of stress
erythropoiesis (sepsis/severe trauma). Prednisolone,
which is a GC analog, is often used to treat the
162 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

patients with the RBC progenitor disorder Diamond- al., 2010). That is how it may have positive effects on
Blackfan anemia. (Flygare et al, 2007; Vlachos et al., a rapid and long-lasting increase in RBC production.
2008). Studies in mice lacking GC receptors or The presence of Hypoxia-induced factor-1 (HIF-
expressing a mutated version GC (deficient in DNA 1) binding sites in the promoter regions of the most of
binding or transactivation) has shown a normal the genes which are induced through GCs mediated
steady-state erythropoiesis but severely impaired signals indicate a link between Hypoxia and GCs
stress erythropoiesis. (Reichardt et al., 1998; Bauer et signaling. It suggests that activation of HIF-1 may
al., 1999). Similarly, GC receptors deficiency resulted enhance or replace the effect of GCs on BFU-E self-
to loss of responsiveness to phenylhydrazine-induced renewal. This is evident from studies showing
hemolysis and failed to increase CFU-E numbers in enhanced production of CFU-E and then erythroblasts
the spleen. approximately through HIF-1 activation by a prolyl
There are reports showing antagonistic effects of hydroxylase inhibitor (Flygare et al., 2011).
p53 activation on GC receptor-induced erythropoiesis.
p53-/-mice shown the rapid growth of CFU-E and
c-Kit/CD34cells that is faster than normal mice in Other Important Issues Related to
response to hypoxic stress in the spleen.(Ganguli et al,
2002).
Ex Vivo RBCs production
It is reported that GCs activity is facilitated
Despite significant progress achieved by
through the induction of expression of Myb, Kit, and
researchers in the field of ex vivo expansion
Lmo2 and inhibition of Gata1 expression (Ganguli et
techniques for blood components during past several
al., 2002; von Lindern et al., 1999). The up-regulated
years, a number of substantial issues remain to be
expression of Kit mRNA is detected as early as 4
solved to ensure the production of manmade blood
hours after GC stimulation of BFU-E cells (Flygare et
components for clinical uses. Altogether these issues
al., 2011). The stress erythropoietic regulation by GCs
may be categorized as (1) initial source material
can be modeled in vitro by growing early erythroid
related issues defining their suitability and proper
progenitors in the presence of SCF, GCs, and Epo
availability for large scale production, (2) quantitative
(Bauer et al., 1999; Flygare et al., 2011; Kolbus et al.,
issues to ensure sufficient production of transfusable
2003; Wessely et al., 1997). These studies reported
units (3) optimal and standardized use of various
that disruption of GC receptor dimerization severely
growth factors in different protocols, (4) biophysical
reduces in vitro proliferation of fetal liver
and biochemical parameters assessment of the
erythroblasts (Reichardt et al., 1998; Bauer et al.,
cultured blood cells to ensure native blood cell-like
1999). This suggests a significant role of GC receptor
properties, (5) vastly defined antigenic profiles to
dimerization in GCs induced RBC production. GCs
avoid any mismatch and associated adverse effects,
dimerization is required for efficient transactivation of
(6) ethnicity, (7) enucleating efficiency of RBCs, (8)
promoters and/or gene repression (Reichardt et al.,
and most importantly production cost
1998; Bauer et al., 1999).
The source materials preferably should be a
GCs induce limited self-renewal of BFU-E cells,
discarded material to reduce the overall production
and not of CFU-E cells or erythroblasts as reported by
cost. To establish a consistent blood manufacturing-
studies using mouse fetal liver BFU-E/CFU-E cells
supplying chain, a regular availability of the source
grown in serum-free conditions along with SCF and
material would be necessary. Apart from that source
IGF-1. These studies have shown protection of BFU-
material should have huge potential to produce
E cells from exhaustion and enhanced formation of
functionally matured cells without eliciting any
CFU-E per BFU-E (Flygare et al., 2011). It is also
immunogenic reactions in the recipients on the
proposed by Lodish and Flygare that increased levels
infusion of the product. One of the most discussed
of GCs likely help in maintaining the earliest
and promising easily available clinical by-product is
erythroid progenitors, increase generation of CFU-E,
umbilical cord blood that is readily available in all the
and may stimulate terminal differentiation (Lodish et
maternity hospitals. The suitability of UCB is also
evident from the facts that significant number of
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation
transfusion are possible for the rare hemo-
globinopathies patients (1%) by using ex vivo
cultivated RBCs units that can be generated from
UCB (>90ml discarded regularly). It is demonstrated
that each discarded unit of UCB may give rise to 10
75 RBCs products which might be useful in
transfusions for rare hemoglobinopathies (Peyrard et
al., 2011). Another important source may be derived
from Leukopheresis, which is routinely done in
clinical centers. The cells obtained from leuko-
reduction buffy coat are demonstrated to have
significant amounts of RBCs producing cells
(equivalent to umbilical CB) (Zhou et al., 2009). The
clinical use was demonstrated by Migliaccio et al.
who shown successful transfusion of CD34+ cells
obtained from a G-CSF mobilized donor (Migliaccio
et al., 2010). Besides, ESCs, and hiPSCs based
methodology might serve as a potential source for
regular manufacturing processes with the
advancement in technologies.
Since RBCs are the most frequently transfused
blood component and are very high in population
(21012 cells/unit) and rich cell density (3106
cells/ml.). As WHO data states >100 million units of
blood are collected every year, and it remains largely
insufficient to support the global annual blood unit
demands. Obviously, ex vivo expansion of RBCs
alone would require a vast number of skilled
manpower along with huge amounts of essential
ingredients.
As per a hypothetical estimation, a cell density of
5107 cells/ml is critical to producing regularly to
meet this demand. It is also presumed that generation
of a single unit of blood (200 ml) through static
culture methods may need unexpectedly huge amount
of culturing media, growth factors and culturing
vessels (660 liters medium/ 9500 lab scale 175 cm2
culturing flask) (Timmins and Nielsen, 2009; Zeuner
et al., 2012). Development of automated methods
such as various types of bioreactors has been reported
in recent past few years. The advancement of their
design, culturing protocols could be helpful for the
researchers to achieve required cell density. Timmins
et al. have demonstrated the generation of high cell
density of RBCs in a wave bioreactor (Timmins et al.,
2011). Similarly, the cell density of 107 cells/ml is
reported in a stirred small scale bioreactor (Ratcliffe
et al., 2012). A massive yield of 2108 cells/ml was
163
reported by using hollow fiber bioreactor technique
(Housler et al., 2012) (Table 3). These reports provide
enough temptations, and it is believed that 3D
bioreactors with some scaling up designs / method-
ologies may be a suitable tool to produce 12 units of
RBCs in 34 weeks of time.
The clinical utility of cultured RBCs or other
blood components would definitely require the
development of stringent quality checks to ensure
their safety and native-like functional quality. The
determination of antigenic profiles and hemoglobin
contents and identification of physical progenies
derived from these culture processes would be of
utmost importance. Reports from many laboratories
have shown slightly macrocytic nature of the cultured
RBCs but normal in size. There are significant level
of -hemoglobin stabilizing proteins (AHSP)
expression, and heterogeneity in the expression of
BCL11A and globin genes (Keel et al., 2008). The ex
vivo generated erythroblasts express a slightly higher
level of -globin in comparison to native RBCs (Lu et
al., 2008).
The number of surface antigens plays vital role in
maintaining the function of RBCs. Many surface
antigens are known for the native mature RBCs. The
similar antigenic profile is demonstrated on ex vivo
generated RBCs such as expression of the normal
level of antigens present on both the Ankyrin A
(GPA, M/N, Ena FS, RhAO, and band 3/4.1 R) and
Glycophorin B complex. Similarly, other important
surface antigens are also expressed including the urea
transporter, the complement receptor, and receptors
that protect RBCs from complement-mediated lysis.
Satchwell et al. also reported an appropriate level of
surface antigens such as band 3 and Rh antigen by ex
vivo generated RBCs (Satchwell et al., 2011).
One of the important parameter to determine the
quality/functionality of cultured RBCs is to assess
their degree of maturation. Enucleation efficiency of
the cultured RBCs is an inevitable requirement for all
of these production methods. ESCs derived
erythroblasts may yield >60% enucleated RBCs (Lu
et al., 2008). Whereas, the nucleation efficiency of
maturing erythroblast from UCB is merely 410% of
RBCs produced (Lapillonne et al., 2010). The factors
regulating enucleation efficiency of RBCs are being
explored, and one such important protein is Histone
deacetylase (specifically HDAC-2 isoform) which
164 Vimal Kishor Singh, Abhishek Saini, Manisha Kalsan et al.

regulates chromatin condensation and enucleation as
reported in mice fetal erythroblast (Chang et al.,
2011). Interestingly, GCs are shown to function as
important negative regulators for these proteins as
mentioned above, and activation of HDACs through
an application of molecular agents may be useful to
enhance the enucleation yields. Similarly, enucleation
efficiency may also be improved by using methods of
co -culturing of maturating erythroblast along with
stromal cells.

Table 3. Bioreactors for ex vivo RBC production

S. No. Year Design and Key factors Limitations References

First stirred bioreactor with pH and DO control for Collins et al.,
1 1998 No data on cell maturation
expansion of HSCs Total cells about 400 times 1998
2 2006 Rotating wall vessel bioreactor Complex design Liu et al., 2006
Timmins et al.,
3 2011 High yield up to 500units/ UCB Inconsistent enucleation
2011
15ml minimal scale stirred and monitored Ratcliffe et al.,
5 2012 No data on cell maturation
bioreactor with 6 populations doublings in 10 days 2012
A complex system with poor cell
Custom made hollow fiber bioreactor with
expansion and maturation, with Housler et al.,
6 2012 compartmental Hollow Fiber Capillary Membrane.
only 40% CD235+ cells, Actual 2012
Maximal cell concentration.
size 8ml and 2ml.
Bio-mimetic co-culture platform using hollow fiber Xue et al.,
7 2014 Limited differentiation
bioreactor with 14288 fold expansion 2014

However, co-culture methods are not appreciated
much due to their inherent property to impose
improperly defined potentially immunogenic and/or
infectious agent and thus can compromise the quality
production of GMP for RBCs. The use of synthetic
drugs e.g., Mifepristone and plasmanate may provide
a better safe alternate (Miharada et al., 2006).
Besides, other molecules promoting the vesicle
trafficking such as vaccuoline may also be used to
enhance the enucleation efficiency if employed during
phase-3 cultures (Keerthivasan et al., 2010).
The most substantial factor which would define
the fate of commercial production of RBCs is cost of
production. It has been proposed that by using present
methodologies available for ex vivo expansion the
approximated cost for RBCs production would lie
$800015000 per unit (Timmins and Nielsen, 2009;
Zeuner et al., 2012). This may look too high if one
compares the hospital cost for leuco-reduced RBC,
which is only $225. Even more, the cost for the rare
phenotypically matched unit of RBCs is $7001200
that is significantly low in comparison to the proposed
synthetic version of RBCs (Table 4). Further,
commercial scale production would fetch a huge
monetary investment. It may not be easy to motivate
financial inflows in the direction of R&D in most of
the low-income grade countries. Identification of
immediately achievable goals from the outcomes of
the existing research endings may play a major role in
generating money for the same.
Prospective
Blood components can be produced in limited
quantities through different methods and types of
stem cells available to us. There are many important
issues including the quantity and quality of these
products, and a regular commercial production would
only be possible after advancements in the present
culturing technologies in near future.
The studies done so far regarding molecular
mechanism of various growth factors and their impact
on the various developmental stages of the
erythropoiesis have provided profound insights into
these processes, and we can regulate both the
quantitative and qualitative issues related to RBCs
production. Furthermore, the comprehensive
information about the various molecules and
development of their synthetic alternatives may be
helpful in finding novel treatments for anemia and
other RBC disorders. Recent findings in the
regulation mechanism of -hemoglobin switching
 Stage-Specific Regulation of Erythropoiesis and Its Implications in Ex-Vivo RBCs Generation 165

may lead to develop more efficient remedies for
related disorders, such as -thalassemia and sickle
cell disease (Sankaran et al., 2010). Similarly,
advancement in the understanding of different genetic
and epigenetic programs which essentially regulate
different stages of RBC development would enable
regular large-scale production of functional RBCs
from various types of stem cells for clinical blood
transfusions.

Table 4. Brief details of approximate cost 1L culture ingredients used in bioreactor

S. No. Parameter Concentration Quantity Approximate Cost in USD

1 Basal Medium (IMDM) - 1L $80
2 Media Supplements
a) Transferrin 330 ug/ml 330 mg $235
b) Insulin 10 ug/ml 10 mg $35
c) Human Serum 5% 50 ml $100
3 Growth Factors
a) Erythropoietin 2I U/ml 2000 IU $2650
b) Interleukin-3 5 ng/ml 5 ug $100
c) Stem Cell Factor 100 ng/ml 100 ug $1500
4 Chemicals
a) Dex/ Hydrocortisone 1 uM 36.2 mg $50

Acknowledgments
We are thankful to the Honorable Chairman and
the Honorable Vice- Chancellor of the Delhi
Technological University, Shahbad Daulatpur,
Bawana road, Delhi-42, for support. Dr. Vimal K.
Singh particularly thanks the Department of Science
and Technology and Indian National Science
Academy (INSA), INDIA, for the research grant.
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