Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells.
ABSTRACT Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.
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ABSTRACT: To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.Yonsei medical journal 03/2014; 55(2):304-9. · 0.77 Impact Factor
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ABSTRACT: The generation of red blood cells (RBCs) from stem cells provides a solution for deficiencies in blood transfusion. Currently, primary hematopoietic stem cells, embryonic stem cells and induced pluripotent stem cells have shown the potential to produce fully mature RBCs. Here, we discuss the advantages, induction protocols, progress and possible clinical applications of stem cells in RBC production.Science China. Life sciences 05/2014; · 2.02 Impact Factor
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ABSTRACT: Red blood cell (RBC) supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs) from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB) and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB) are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.BioMed Research International 01/2014; 2014:435215. · 2.88 Impact Factor
Efficient enucleation of
in vitro from hematopoietic
stem and progenitor cells
Kenichi Miharada1,2, Takashi Hiroyama1,
Kazuhiro Sudo1, Toshiro Nagasawa2&
Erythroblast enucleation is thought to be largely dependent on
signals mediated by other cells, such as macrophages. In an
attempt to improve the in vitro production of red blood cells
(RBCs) from immature hematopoietic progenitor cells, we have
developed a method to produce enucleated RBCs efficiently in
the absence of feeder cells. Our method may represent an
efficient way to produce transfusable RBCs on a large scale
from hematopoietic progenitors.
The mechanism of erythroblast enucleation, one of the more critical
steps of RBC production, remains to be fully elucidated1,2. The role of
the interaction of erythroblasts with other cells such as macrophages is
a controversial topic in the study of erythroblast enucleation3–7. In
retinoblastoma-deficient (Rb?/?) embryos, macrophages are unable
to physically interact with erythroblasts and RBC production is
impaired6. In addition, in vitro production of enucleated RBCs
from immature hematopoietic progenitor cells proceeds efficiently
in the presence8but not in the absence9of feeder cells. However,
enucleation appears to initiate in vitro in the absence of feeder cells in
erythroblasts induced to differentiate in vivo to a developmental stage
that is competent for nuclear self-extrusion7,10.
Because the supply of transfusable RBCs for medical use is
insufficient in many countries, we have attempted to improve upon
existing methods for producing enucleated RBCs from human hema-
topoietic progenitor cells8,9. We used human CD34+cells comprising
hematopoietic stem and progenitor cells as the starting material8,9. We
developed a four-passage culture protocol. In the first step (passage I),
we cultured CD34+cells collected by magnetic cell sorting (MACS)
from human umbilical cord blood (approximate purity 95%) (Sup-
plementary Fig. 1 online) in erythroid differentiation medium (EDM;
Supplementary Methods online) in the presence either of stem cell
factor (SCF), erythropoietin (EPO) and interleukin-3 (IL-3) (protocol A)
or of SCF, EPO, IL-3, vascular endothelial growth factor (VEGF)
and insulin-like growth factor-II (IGF-II) (protocol B; Table 1). In
passages II and III, we cultured the cells in EDM in the presence of
SCF and EPO alone (Table 1).
We observed that a greater number of cells were generated by
protocol B than by protocol A after passage I (Table 1; Supplementary
Fig. 2 online). This result is consistent with the fact that the majority
of the human CD34+hematopoietic stem and progenitor cells used as
the starting material expressed VEGF receptor-2 (a functional receptor
for VEGF), at a low level, and also the IGF-I receptor (a functional
receptor for both IGF-I and IGF-II) (Fig. 1a). The relative increases in
cell numbers after passage III of protocol B were similar to earlier
results obtained by a method that avoided the use of feeder cells9
(Table 1; Supplementary Fig. 3 online).
Two lines of evidence suggested that protocol B led to efficient
induction of erythroid differentiation. First, although the majority of
cells produced by protocols A and B expressed both CD71 (the
transferrin receptor) and glycophorin-A (Gly-A, a specific marker of
mature erythroid cells) after passage II, CD71 expression was lower in
the cells cultured by protocol B (Fig. 1b). This implied that the cells
cultured by protocol B had differentiated to more mature stages.
As immature stem and progenitor cells mature into erythroid lineages,
their phenotype progresses from CD71–Gly-A–to CD71+Gly-A–
© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology
Table 1 Characteristics of cultured cells
Day 6Day 10 Day 16Day 20
% Ortho. cells
% Enucleated cells
133.8 ± 5.1
55.2 ± 3.3
29.3 ± 2.2
167.5 ± 14.9
64.5 ± 4.2
34.1 ± 3.6
61.8 ± 9.6
84.5 ± 3.2
42.2 ± 1.2
5.4 ± 2.3
1.6 ± 1.3
86.4 ± 10.1
86.2 ± 4.6
47.3 ± 4.6
11.5 ± 5.2
1.9 ± 1.7
40.4 ± 5.3
90.3 ± 5.3
73.9 ± 3.4
10.8 ± 4.4
6.2 ± 2.9
49.6 ± 7.4
87.1 ± 3.6
78.1 ± 2.0
20.6 ± 5.5
14.0 ± 2.3
1.1 ± 0.1
92.1 ± 4.8
94.6 ± 4.7
45.4 ± 4.2
50.0 ± 2.8
1.0 ± 0.1
93.6 ± 2.5
95.8 ± 3.2
21.2 ± 3.2
77.5 ± 2.8
Days 6, 10, 16 and 20 represent cells after passages I, II, III and IV, respectively. Stem cell factor (SCF), erythropoietin (EPO), interleukin-3 (IL-3), vascular endothelial growth factor (VEGF) and
insulin-like growth factor-II (IGF-II) were used as follows: in protocol A, SCF+EPO+IL-3 in passage I and SCF+EPO in passages II and III; in protocol B, SCF+EPO+IL-3+VEGF+IGF-II in passage I
and SCF+EPO in passages II and III. The culture method for passage IV was common to both protocols (see Supplementary Methods). Proliferation is defined as the relative increase in cell number
after each passage. Gly-A, glycophorin-A; Ortho. cells, orthochromatic erythroblasts. Values are mean ± s.d. (n ¼ 3).
Received 24 January; accepted 1 August; published online 17 September 2006; doi:10.1038/nbt1245
1Cell Engineering Division, RIKEN BioResource Center, Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074, Japan.2Division of Hematology, Institute of Clinical Medicine,
University of Tsukuba, Ten-nodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. Correspondence should be addressed to Y.N. (firstname.lastname@example.org).
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to CD71+Gly-A+and finally to CD71–Gly-A+(Supplementary Fig. 4
online). Second, protocol B gave rise to a greater number of ortho-
chromatic erythroblasts (mature erythroblasts) after the completion of
passage III, and we observed a greater number of enucleated cells in the
cells produced by protocol B (Table 1; Supplementary Fig. 5 online).
In passage IV (the enucleation step), the cells expanded in passage
III were cultured in enucleation medium (Supplementary Methods)
for 4 d. To increase the stability of the RBCs, we added to the medium
mannitol, adenine and phosphate11(MAP; Supplementary Methods),
a mixture that is commonly used to preserve RBCs in Japan.
We observed many aggregated cells in cultures grown in the absence
but not in the presence of MAP (Supplementary Fig. 6 online),
suggesting that MAP functioned to prevent the loss of viability of
erythroblasts, cells undergoing enucleation and/or enucleated RBCs.
Glucocorticoids promote proliferation and inhibit differentiation of
erythroid progenitors12. Glucocorticoids are usually present to some
degree in serum, suggesting that the use of serum-free medium may be
preferable for inducing terminal differentiation of cultured cells. How-
ever, removal of serum from the medium may also impair cell viability.
In fact, serum was necessary in passage IV to maintain cell viability.
(Because we found that Plasmanate, a commercially available human
serum, could be successfully substituted for fetal bovine serum, we used
Plasmanate for this purpose.) Hence, we added mifepristone, an
antagonist of glucocorticoid function, to the medium in passage IV,
which accelerated the process of enucleation (Fig. 1c). Mifepristone was
more effective at inducing enucleation in the presence of a lower (0.5%)
rather than a higher (10%) concentration of Plasmanate (Fig. 1c),
strongly suggesting that the Plasmanate contained glucocorticoids.
With both protocols, the vast majority of cells produced after pass-
age IV expressed Rh-D antigen, a specific marker of erythroid pro-
genitors and terminally differentiated RBCs (Table 1; Supplementary
Fig. 7 online). Notably, protocol B produced a much larger proportion
of enucleated cells (nearly 80%) than did protocol A (Fig. 1d; Table 1).
Given that reticulocytes and even erythroblasts produced in vitro can
differentiate into fully mature RBCs in vivo8,9, we believe that mature
erythroid cells produced by protocol B are also likely to do so.
In contrast to previously published methods8,9, the method des-
cribed here included VEGF and IGF-II in the culture medium. These
two factors have been reported to promote the survival, proliferation
and/or differentiation of hematopoietic progenitors13–15. Consistent
with these findings, these factors promoted the expansion of erythroid
progenitors (Table 1). However, a much more important feature of our
culture system is that it allows erythroid cells to differentiate to a
developmental stage competent for nuclear self-extrusion. In fact, we
observed many enucleated cells after the completion of our culture
process (Fig. 1d; Table 1).
It has generally been thought that efficient enucleation of erythro-
blasts depends on cells in their local environment3–6. However, our
findings demonstrate that the interaction of erythroblasts with other
cells is not necessary and that signals mediated by humoral factors
seem to be sufficient for efficient autonomous completion of erythro-
Blood transfusion is indispensable for many clinical procedures,
yet the supply of transfusable materials is insufficient in many
countries. Because culture without feeder cells is technically easier
and less expensive than culture with feeder cells, our method may
represent an efficient way to produce transfusable RBCs on a large scale
from immature hematopoietic progenitors in umbilical cord blood.
Note: Supplementary information is available on the Nature Biotechnology website.
We thank the Stem Cell Resource Network for providing cord blood.
K.M. designed and performed research and wrote the manuscript; T.H. and K.S.
provided expertise; T.N. provided useful discussion; Y.N. coordinated research,
analyzed data and wrote the manuscript.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturebiotechnology/
Reprints and permissions information is available online at http://npg.nature.com/
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© 2006 Nature Publishing Group http://www.nature.com/naturebiotechnology
Day 0Day 6
50 µm 50 µm
Figure 1 Production of erythroid cells from CD34+hematopoietic stem
and progenitor cells isolated from human umbilical cord blood. (a) Flow
cytometric analysis of IGF-I and VEGF receptor-2 expression. Cells were
stained with anti-IGF-I receptor and anti-VEGF receptor-2 monoclonal
antibodies and analyzed by flow cytometry. (a) Day 0, cells before culture.
Day 6, cells cultured by protocol B for 6 d (cells following passage I).
Results with isotype controls are also shown. (b) Flow cytometric analysis of
CD71 expression. Cells following passage II (day 10) were stained with anti-
CD71 monoclonal antibody. Solid line with gray shadow, staining with an
isotype control antibody. Solid lines with blue and red shadow, staining of
the cells cultured by protocol A and protocol B, respectively. (c) Effect
of mifepristone and Plasmanate on enucleation. Cells were cultured
by protocol B. The percentage of enucleated cells was calculated by the
formula 100 ? (number of enucleated cells/number of enucleated cells
plus nucleated cells). Values are mean ± s.d. (n ¼ 3). (d) Morphology
of the cells produced after passage IV. Enucleated RBCs were more
abundant in the cells cultured by protocol B: that is, nearly 80% of
RBCs were enucleated. Results shown in a,b,d are representative of
three independent experiments.
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