Human Hematopoietic Stem Cells Can Survive In Vitro for Several Months

Abstract

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

Full text

Hindawi Publishing Corporation
Advances in Hematology
Volume 2009, Article ID 936761, 7pages
doi:10.1155/2009/936761
Research Article
Human Hematopoietic Stem Cells Can
Survive In Vitro for Several Months
Tar o I s hi g ak i ,1, 2 Kazuhiro Sudo,1Takashi Hiroyama,1Kenichi Miharada,1
Haruhiko Ninomiya,2Shigeru Chiba,2Toshiro Nagasawa,2and Yukio Nakamura1
1Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
2Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
Correspondence should be addressed to Yukio Nakamura, yukionak@brc.riken.jp
Received 22 September 2008; Revised 8 December 2008; Accepted 15 December 2008
Recommended by N. Chao
We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate
embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting
in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain
long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due
to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to
nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that
even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge,
this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.
Copyright © 2009 Taro Ishigaki et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
1. Introduction
Identification of in vitro culture protocols that enable
somatic stem cells to survive and proliferate will be of value
not only for basic research but also clinical applications that
require somatic stem cells. The development of an efficient
method for in vitro proliferation of mesenchymal stem cells,
for example, has allowed cultured mesenchymal stem cells to
be used in clinical applications [1].
Although hematopoietic stem cells have been extensively
analyzed and characterized [2], in vitro proliferation of these
cells remains problematic using established culture methods
[3,4]. In addition, the length of time that hematopoietic
stem cells can survive in an in vitro culture system remains
uncertain. CD34-positive (CD34+) cells have been identified
in long-term in vitro cultures of hematopoietic stem cells [5–
9]. However, as none of these previous studies performed an
in vivo assay of the cultured cells, such as transplantation into
mice, it is uncertain whether hematopoietic stem cells with
the capacity to reconstitute long-term in vivo hematopoiesis
were present in these prolonged in vitro cultures.
We previously described a culture method that pro-
duced long-lasting in vitro hematopoiesis using non-human
primate embryonic stem (ES) cells [10]. We speculated
that hematopoietic stem cells derived from ES cells could
sustain long-lasting in vitro hematopoiesis. To test this
hypothesis, we initiated long-term in vitro cultures of human
hematopoietic stem cells using the same culture method
as previously [10]. In addition, we evaluated the in vivo
function of cells cultured in vitro for several months by
transplanting them into immunodeficient mice.
2. Materials and Methods
2.1. Cell Culture. We purchased human umbilical cord blood
samples from the Cell Engineering Division of RIKEN
BioResource Center (Tsukuba, Ibaraki, Japan). The ethical
committee of the RIKEN Tsukuba Institute approved the
use of human umbilical cord blood before the study was
initiated. CD34+hematopoietic stem/progenitor cells were
collected from human umbilical cord blood using a magnetic
cell sorting system, MACS CD34 Isolation kit (Miltenyi

2Advances in Hematology
Biotec Inc., Sunnyvale, Calif, USA), according to the man-
ufacturer’s instructions.
Mouse-derived cell lines (OP9 and C3H10T1/2) were
purchased from the Cell Engineering Division of RIKEN
BioResource Center (Tsukuba, Ibaraki, Japan) and were
cultured as follows: OP9 in Minimum Essential Medium-
α(MEM-α; Invitrogen, Carlsbad, Calif, USA) containing
20% fetal bovine serum (FBS; Invitrogen, Calif, USA);
C3H10T1/2 in Basal Medium Eagle (BME; Invitrogen)
containing 10% FBS (BioWest, Miami, Fla, USA). The cell
lines were γ-irradiated (50 Gy) before use as feeder cells.
CD34+cells were cultured on feeder cells in a 100 mm
dish in Iscove’s modified Dulbecco’s medium (IMDM;
SIGMA, St Louis, Mo, USA) containing 10% FBS (BioW-
est), 10 μg/mL bovine insulin, 5.5 μg/mL human transfer-
rin, 5 ng/mL sodium selenite (ITS liquid MEDIA supple-
ment; SIGMA-Aldrich, Mass, USA), 100unit/mL penicillin,
100 μg/mL streptomycin, 2 mM L-glutamine (PSQ; Invit-
rogen), 50 ng/mL stem cell factor (SCF; R&D Systems,
Minneapolis, Minn, USA), 50 ng/mL Flt-3 ligand (Flt-
3L; R&D Systems), and 50 ng/mL thrombopoietin (TPO;
R&D Systems). The initial number of CD34+cells placed
in culture varied between experiments: 5 ×103cells in
Exp-OP9-A, Exp-10T1/2-A, and Exp-OP9-B; 4 ×104cells
in Exp-OP9-F and Exp-10T1/2-F; 5 ×104cells in Exp-
OP9-C, Exp-OP9-E, Exp-10T1/2-E, Exp-OP9-H, and Exp-
10T1/2-H; 8 ×104cells in Exp-OP9-G and Exp-10T1/2-
G; 1 ×105cells in Exp-10T1/2-I; 2 ×105cells in Exp-
OP9-D and Exp-10T1/2-D. The letters “A” to “I” after
Exp-OP9 or Exp-10T1/2 indicate 9 different umbilical cord
blood samples derived from 9 different neonates. Samples
A, B, C, F, G, H, and I were frozen after collection,
while samples D and E were used immediately as fresh
samples.
Twenty-four hours after initiation of culture, the
medium together with any detached cells was removed
and fresh medium was added to the culture. Thereafter,
the medium was changed every 3-4 days (twice a week).
The number of cells attached to the feeder cells increased
gradually. Approximately four weeks after initiation of
culture, attached cells were dissociated using a 0.25% trypsin
EDTA solution (SIGMA-Aldrich) and cultured again on new
feeder cells. Thereafter, similar passages of attached cells were
performed every 3-4 weeks.
The number of viable cells was assessed using an
automated cell counter and an assay based on the trypan
blue dye exclusion method, ViCell (BECKMAN COULTER,
Fullerton, Calif, USA). The morphology of the cells was
analyzed by microscopic examination after Wright staining
(Muto Pure Chemicals, Tokyo, Japan).
2.2. Flow Cytometry. Cells were stained with monoclonal
antibodies (MoAbs) and analyzed using a FACS Calibur
(BD Biosciences, San Jose, Calif, USA). The following
MoAbs were purchased from BD Biosciences: fluores-
cein isothiocyanate- (FITC-) conjugated MoAb against
human CD14 (FITC-CD14), FITC-CD34, FITC-CD41a,
and FITC-CD45; phycoerythrin-conjugated MoAb against
human CD4(PE-CD4), PE-CD11b, PE-CD13, PE-CD34,
PE-CD45, PE-CD56, and PE-CD235a (Glycophorin A);
allophycocyanin-conjugated MoAb against human CD3
(APC-CD3), APC-CD8, APC-CD19, APC-CD33, and APC-
CD45. PE-CD33 was purchased from eBiosciences (San
Diego, Calif, USA). FITC-mouse IgG1, PE-mouse IgG1,
APC-mouse IgG1, FITC-mouse IgG2a, and PE-mouse IgG2b
were also purchased from BD Biosciences and were used as
isotype controls. Cell viability was monitored by staining
with propidium iodide (SIGMA-Aldrich). Flow cytometry
data were analyzed using CellQuest (BD Biosciences) analysis
software.
2.3. Colony Formation Assay. Cells (1 ×104)werecultured
in a 35 mm dish with 1 mL of Methocult (H4435; Stem cell
technology, Vancouver, British Columbia, Canada) for 10–14
days, and the number of separate colonies was determined
by macroscopic morphology. Representative colonies were
picked up and cell morphology was analyzed by microscopic
examination after Wright staining (Muto Pure Chemicals).
2.4. Cell Transplantation into Mice. Eight-week-old male
NOD/Shi-scid IL-2Rγnull (NOG) mice were purchased from
the Central Institute for Experimental Animals (Kawasaki,
Kanagawa, Japan) and used within two weeks of delivery
in all experiments. Prior to cell transplantation, the mice
were given a sublethal dose of γ-rays (3.0 Gy). A 200 μL
cell suspension in phosphate-buffered saline (PBS; SIGMA)
containing 5% FBS (BioWest) was injected intravenously
into the tail vein of each mouse. All experimental procedures
on the mice were approved by the Institutional Animal Care
and Use Committee of the RIKEN Tsukuba Institute.
3. Results and Discussion
3.1. Long-Lasting In Vitro Hematopoiesis from Human
Hematopoietic Stem Cells. Human CD34+cells were cultured
on feeder cells in the presence of SCF, Flt-3L, and TPO. We
used the mouse-derived cell lines, OP9 and C3H10T1/2, as
feeder cells. Both OP9 [10–15] and C3H10T1/2 [10,16–
18] have been used in many studies to maintain in vitro
hematopoiesis. As we show below, both OP9 and C3H10T1/2
cells supported long-lasting in vitro hematopoiesis from
human hematopoietic stem cells.
About one week after initiation of culture, cobblestone
areas were observed on the feeder cells (Figures 1(a)
and 1(b)), indicating that human hematopoietic cells were
proliferating. The numbers of cells attached to the feeder
cells increased gradually (Figures 1(c) and 1(d)) and the
numbers of detached cells, derived from the attached cells,
also increased gradually. When the medium was changed,
the numbers of detached cells in the medium were counted
and the cells were subjected to analyses such as flow
cytometric analysis. Detached cells were removed during the
medium changes and were not cultured further in any of the
experiments.
Detached cells were continuously produced for several
months in experiments Exp-OP9-A, Exp-OP9-F, Exp-OP9-
H, Exp-10T1/2-A, and Exp-10T1/2-H (Figure 2). As we

Advances in Hematology 3
(a) (b) (c) (d)
Figure 1: Appearance of the cells attached to feeder cells. Representative examples of CD34(+) human hematopoietic stem/progenitor cells
cultured on either OP9 feeder cells ((a), (c)) or C3H10T1/2 feeder cells ((b), (d)) for 7 days ((a), (b)) or 22 days ((c), (d)).
0 70 140 210 280
Days of culture
Exp-OP9-A
0 70 140 210 280
Days of culture
Exp-OP9-B
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-OP9-C
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-OP9-D
0.001
0.01
0.1
1
10
×103
0.001
0.01
0.1
1
10
×103
#
Numbers of detached cells produced from CD34(+)cells
0 70 140 210 280
Days of culture
Exp-OP9-E
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-OP9-F
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-OP9-G
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-OP9-H
0.001
0.01
0.1
1
10
×103
Numbers of detached cells produced from CD34(+)cells
(a)
0 70 140 210 280
Days of culture
Exp-10T1/2-A
0 70 140 210 280
Days of culture
Exp-10T1/2-D
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-10T1/2-E
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-10T1/2-F
0.001
0.01
0.1
1
10
×103
0.001
0.01
0.1
1
10
×103
#
Numbers of detached cells produced from CD34(+)cells
0 70 140 210 280
Days of culture
Exp-10T1/2-G
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-10T1/2-H
0.001
0.01
0.1
1
10
×103
0 70 140 210 280
Days of culture
Exp-10T1/2-I
0.001
0.01
0.1
1
10
×103
Numbers of detached cells produced from CD34(+)cells
(b)
Figure 2: Production of hematopoietic cells in long-term cultures of human hematopoietic stem cells. (a) Eight independent experiments
were performed using eight different umbilical cord blood samples and OP9 cells as feeder cells. (b) Seven independent experiments were
performed using seven different umbilical cord blood samples and C3H10T1/2 cells as feeder cells. ((a), (b)) The number of detached cells
in the overlying medium was counted at each medium change (approximately half weekly). The data are shown as the mean number of
detached cells produced from a single CD34(+) cell, that is, the total number of detached cells divided by the number of CD34(+) cells used
to initiate the culture. Exp: experiment. A to I after Exp-OP9 and Exp-10T1/2 indicate 9 different umbilical cord blood samples derived from
9different neonates. #: Cultures Exp-OP9-A and Exp-10T1/2-F were terminated because of fungal infection.
detail below, flow cytometric analysis and a transplantation
assay demonstrated that the detached cells produced in
this culture method included both mature and imma-
ture hematopoietic cells, such as colony-forming cells and
hematopoietic stem cells. We found that production of
detached cells eventually ceased in all experiments except
for Exp-OP9-A. Unfortunately, we were forced to halt Exp-
OP9-A because of fungal infection although the cells in this
culture proliferated efficiently and robustly before fungal
contamination (Figure 2).
The numbers of detached cells varied among the exper-
iments (Figure 2) and, notably, showed no correlation with
the initial number of CD34+cells used in each experiment.
Thus, 5×103CD34+cellswereusedinExp-OP9-AandExp-
10T1/2-A and both cultures produced substantial numbers
of detached cells over a prolonged period (Figure 2). In
contrast, a larger number of CD34+cells (2 ×105)wasused
to initiate the Exp-OP9-D and Exp-10T1/2-D cultures, but
they produced considerably fewer detached cells (Figure 2).
These results indicate that the rate of production of detached

4Advances in Hematology
CD33
CD34
On OP9 On C3H10T1/2
CD33
CD34
(a)
Day 57
0
20
40
Numberof colonies
Day 60
0
20
40
Numberof colonies
Day 127
0
20
40
Numberof colonies
On OP9
Day 134
0
20
40
Numberof colonies
On C3H10T1/2
(b)
Figure 3: Characterization of cultured cells. (a) Flow cytometric analysis of detached cells produced in cultures on OP9 (Exp-OP9-A) and
C3H10T1/2 (Exp-10T1/2-A) feeder cells and collected on Day 218 of culture. The detached cells were stained for CD33, a marker specific
for granulocyte/macrophage lineage cells, and CD34, a marker specific for hematopoietic stem/progenitor cells. Flow cytometric analyses
of detached cells from other experiments showed similar results. (b) Colony-formation assays. Detached cells produced in culture on OP9
feeder cells (Exp-OP9-A) were collected on Days 57 and 127 of culture. Similarly, detached cells produced in culture on C3H10T1/2 feeder
cells (Exp-10T1/2-D) were collected on Days 60 and 134 of culture. The cell samples were used in a standard colony-formation assay. Black
bars: colony-forming unit of monocyte/macrophage lineage cells, CFU-M. Blue bars: colony-forming unit of granulocyte lineage cells, CFU-
G. Yellow bars: colony-forming unit of granulocyte and monocyte/macrophage lineage cells, CFU-GM. Red bars: burst-forming unit of
erythroid cells, BFU-E. Similar results were obtained in colony-formation assays using detached cells from other cultures.
cells depended on the quality rather than the number of
CD34+cells used in each experiment. In other words, when
the quality of CD34+cells was high, 5 ×103CD34+cells
were sufficient to generate efficient in vitro hematopoiesis as
shown in Exp-OP9-A and Exp-10T1/2-A (Figure 2).
The majority of detached cells had the morphological
characteristics of granulocyte/macrophage lineage cells (Sup-
plementary Figure S1, available at doi 10.1155/2009/936761)
although some blast-like cells were also present. Con-
sistent with their morphological phenotype, the major-
ity of the detached cells expressed CD33, a marker
of granulocyte/macrophage lineage cells (Figure 3(a)). Of
note, CD34+CD33+cells, which are less mature than
CD34−CD33+cells, were abundant among the detached cells
even at 7 months after initiation of culture (Figure 3(a)).
A colony-formation assay demonstrated that granulo-
cyte, macrophage, and erythrocyte progenitor cells were
present among the detached cells (Figure 3(b)). As men-
tioned above, when the culture medium was changed,
detached cells were removed and were not cultured further.
Instead, they were either used in experimental analyses or
discarded. As is shown in Figure 2, detached cells were
continuously produced, and they included abundant colony-
forming cells even at Day 127 and Day 134. As one
example, the calculated total number of colony-forming
cells present in detached cells at Day 127 (upper right,
Figure 3(b)) was 13 311, which corresponded to a greater
than ten-fold increase in the numbers of colony-forming cells
compared to the starting material of this culture, that is,
983 colony-forming cells in 5 ×103CD34+cells. Thus, the
culture method we describe here could continuously produce
abundant colony-forming cells for several months.
Although a mixed colony (a colony derived from
very immature hematopoietic cells) was not observed in
the colony formation assay (Figure 3(b)), it nevertheless
remained possible that hematopoietic stem cells were present
at a very low frequency among the detached cells. Therefore,
we performed a transplantation assay in which detached cells
were injected into an immunodeficient NOD/Shi-scid IL-
2Rγnull (NOG) mouse (mentioned hereafter).
3.2. Hematopoietic Stem Cells Cultured In Vitro for Several
Months Give Rise to Long-Lasting In Vivo Hematopoiesis
after Transplantation into Mice. Detachedcellswerecollected
from Exp-OP9-A on Day 169 after initiation of culture
and transplanted into an NOG mouse (3.9×106cells)
(Figure 4(a)). Peripheral blood samples from the mouse
were subjected to flow cytometric analysis on Days 56 and

Advances in Hematology 5
CD33
CD34(+) cells (Exp-OP9-A)
In vitro culture for 169 days
Detached cells produced for 3.5 days
Transplantation into mouse
In vivo hematopoiesis for 126 days
Sacrificed and analyzed
CD13
(a)
CD45
CD33
(b)
CD13
CD33
(c)
CD13
CD33
CD14
CD11b
CD45
CD19
(d)
CD34(+) cells (Exp-OP9-F)
In vitro culture for 125 days
Detached cells produced for 3.5 days
Transplantation into mouse
In vivo hematopoiesis for 184 days
Sacrificed and analyzed
CD34
CD33
(e)
CD33
CD19
Gly-A
CD3
CD14
CD11b
CD4
CD56
CD41a
CD8
(f)
Figure 4: Flow cytometric analysis of hematopoietic cells of the mouse that had been transplanted with human hematopoietic cells
produced by in vitro culture. ((a), (e)) Schema of the experimental procedure and flow cytometric analysis of transplanted cells. ((a)–
(d)) Detached cells (3.9×106cells) produced on OP9 feeder cells (Exp-OP9-A) were collected on Day 169 of culture and transplanted
into an immunodeficient NOG mouse. Peripheral blood was collected on Days 56 (b) and 112 (c) after transplantation, and bone marrow
cells were collected on Day 126 after transplantation (d). ((e), (f)) Detached cells (2.4×106cells) produced on OP9 feeder cells (Exp-
OP9-F) were collected on day 125 of culture and transplanted into an immunodeficient NOG mouse. The bone marrow cells were
collected on Day 184 after transplantation and were analyzed. ((a)–(f)) The cells were stained using monoclonal antibodies against CD45, a
leukocyte common antigen, CD34, a marker specific for hematopoietic stem/progenitor cells, CD33 and CD13, markers of granulocyte and
monocyte/macrophage lineage cells, CD11b and CD14, markers of monocyte/macrophage lineage cells, CD19, a marker of B lymphocyte
lineage cells, CD3, CD4, and CD8, markers of T lymphocyte lineage cells, Gly-A (Glycophorin A), a marker of erythroid cells, CD56, a
marker of large granular lymphocytes and natural killer cells, and CD41a, a marker of megakaryocyte/platelet lineage cells.

6Advances in Hematology
112 after transplantation. Human hematopoietic cells were
clearly present in the peripheral bloods (Figures 4(b), 4(c)).
The mouse was sacrificed on Day 126 after transplantation,
and bone marrow and spleen cells were subjected to flow
cytometric analysis. Human hematopoietic cells were present
in the bone marrow (Figure 4(d)) but were present at a
very low rate in spleen (data not shown). The estimated
rate of chimerism of human CD45+cells in bone marrow
was 2.6% when compared to the number of mouse CD45+
cells. The human hematopoietic cells detected in the bone
marrow included cells of the myeloid lineage (CD13+CD33+:
9.7% of the human CD45+cells), the monocyte/macrophage
lineage (CD11b+CD14+: 3.8% of the human CD45+cells),
the B cell lineage (CD19+: 80.5% of the human CD45+cells)
(Figure 4(d)), and other lineages at very low levels (data not
shown).
Detached cells were collected from Exp-OP9-F on
day 125 after initiation of culture and transplanted into
an NOG mouse (2.4×106cells) (Figure 4(e)). CD45+
human hematopoietic cells were present in peripheral blood
from the mouse one month after transplantation (data
not shown). The mouse was sacrificed on Day 184 after
transplantation, and the bone marrow cells were subjected
to flow cytometric analysis. The rate of chimerism of human
CD45+cells in the bone marrow was 0.2% when compared
to the number of mouse CD45+cells.Thebonemarrow
contained human hematopoietic cells, which included cells
of the myeloid lineage (CD33+: 23.5% of the human CD45+
cells), the monocyte/macrophage lineage (CD11b+CD14+:
6.7% of the human CD45+cells), as well as the B (CD19+:
53.7% of the human CD45+cells) and T cell lineages
(CD3+,CD4
+,CD8
+, and CD4+CD8+: 10.7% of the human
CD45+cells) (Figure 4(f)). Erythroid (Glycophorin A+)and
megakaryocyte (CD41a+)lineagecellswerepresentatvery
low rates (Figure 4(f)).
In the transplantation assay described above, human
hematopoietic cells of various lineages were present in mice
up to six months after cell transplantation. In general,
it is impossible that in vivo hematopoiesis derived from
transplanted cells is maintained for several months solely by
committed progenitor cells. Therefore, the transplanted cells
appeared to include hematopoietic stem cells, that is, even
after in vitro culture for several months, hematopoietic stem
cells were still present in cultures Exp-OP9-A and Exp-OP9-
F. In light of the numbers of cells produced in culture and of
the duration of this production, the hematopoietic stem cells
also appeared to have survived for several months in Exp-
OP9-H, Exp-10T1/2-A, and Exp-10T1/2-H.
The in vitro expansion of human hematopoietic stem
cells is known to be very difficult [3]. In agreement with
this, a mixed colony (a colony derived from very imma-
ture hematopoietic cells) was not observed in the colony
formation assay in this study (Figure 3(b)), indicating that
hematopoietic stem cells did not expand to any great extent
in our culture method. However, since it was highly unlikely
that in vitro hematopoiesis could be maintained for several
months solely by committed progenitor cells present in the
starting materials, the long-lasting in vitro hematopoiesis
was likely maintained by hematopoietic stem cells.
On the basis of a previous estimation of the numbers
of hematopoietic stem cells capable of repopulating in
NOD/SCID mice [19], the starting materials we used in
Exp-OP9-A and Exp-OP9-F should have included a very
low number of NOD/SCID-repopulating cells. However,
the transplantation assay demonstrated that NOD/SCID-
repopulating cells were present among the detached cells
that were continuously produced in our culture method
(Figure 4), strongly suggesting that our culture method
continuously produced small numbers of new NOD/SCID-
repopulating cells throughout the long-term culture period.
Hence, the total number of NOD/SCID-repopulating cells
that were produced as detached cells throughout the whole
long-term in vitro culture was likely greater than the number
of such cells that were present in the starting materials.
Taken together, the hematopoietic stem cells capable
of repopulating in NOD/SCID mice in our culture system
appeared to be maintained by asymmetric cell division, that
is, one of the daughter cells retained the characteristics of
hematopoietic stem cells and another did not.
Leukemic stem cells might also survive and/or proliferate
in our culture method for a prolonged period, enabling basic
research or screening for effective anticancer drugs to be
performed on these cultured cells. In addition, basic research
on specific diseases, such as aplastic anemia or paroxysmal
nocturnal hemoglobinuria, might benefit from a long-term
culture system for hematopoietic stem cells derived from
patients.
4. Conclusions
To the best of our knowledge, this is the first report to show
that human hematopoietic stem cells can survive in vitro for
several months. Since the duration of in vitro hematopoiesis
appeared to depend on the quality of hematopoietic stem
cells present in each sample, our culture method may be of
value for assessing the quality of hematopoietic stem cells
prior to their use in the clinic. In particular, our method
could be used for the evaluation of umbilical cord bloods
since these samples are routinely used in the clinic following
preservation for several months. For example, in this study
the quality of hematopoietic stem cells derived from samples
A, F, and H appeared to be higher compared to other
samples.
Acknowledgments
The authors obtained human umbilical cord blood from the
Cell Engineering Division of RIKEN BioResource Center,
which was supported by the National Bio-Resources Project
and the Stem Cell Resource Network (Banks at Miyagi,
Tokyo, Kanagawa, Aichi, and Hyogo) of the Ministry of
Education, Culture, Sports, Science, and Technology in Japan
(MEXT), and from Dr. Isamu Ishiwata (Ishiwata Hospital,
Mito, Ibaraki, Japan). This work was supported by grants
from MEXT. The authors thank all members in the Cell
Engineering Division for help, discussion, and secretarial
assistance.

Advances in Hematology 7
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