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STEM CELLS IN HEMATOLOGY
Chromatin-modifying agents permit human hematopoietic stem cells to undergo
multiple cell divisions while retaining their repopulating potential
Hiroto Araki,1 Kazumi Yoshinaga,1 Piernicola Boccuni,1 Yan Zhao,1 Ronald Hoffman,1,2 and Nadim Mahmud1,2
1Section
of Hematology/Oncology, Department of Medicine, University of Illinois at Chicago, IL; 2University of Illinois Cancer Center, Chicago, IL
Human hematopoietic stem cells (HSCs)
exposed to cytokines in vitro rapidly
divide and lose their characteristic functional properties presumably due to the
alteration of a genetic program that
determines the properties of an HSC.
We have attempted to reverse the silencing of this HSC genetic program by the
sequential treatment of human cord
blood CD34ⴙ cells with the chromatinmodifying agents, 5-aza-2ⴕ-deoxycytidine (5azaD) and trichostatin A (TSA).
We determined that all CD34ⴙCD90ⴙ
cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide,
but to a lesser degree than cells exposed to only cytokines. When
CD34ⴙCD90ⴙ cells that have undergone
extensive number of cell divisions (510) in the presence of cytokines alone
were transplanted into immunodeficient
mice, donor cell chimerism was not
detectable. By contrast, 5azaD/TSAtreated cells that have undergone similar numbers of cell divisions retained
their marrow repopulating potential. The
expression of several genes and their
products previously implicated in HSC
self-renewal were up-regulated in the
cells treated with 5azaD/TSA as compared to cells exposed to cytokines
alone. These data indicate that HSC
treated with chromatin-modifying agents
are capable of undergoing repeated cell
divisions in vitro while retaining their
marrow-repopulating potential. (Blood.
2007;109:3570-3578)
© 2007 by The American Society of Hematology
Introduction
Primitive hematopoietic stem cells (HSCs) are relatively quiescent
cells that reside in the G0/G1 phase of the cell cycle.1,2 Recently,
HSCs have been shown to slowly cycle in vivo during steady-state
hematopoiesis.3-10 Self-renewal of HSCs has been tracked in mice
by marking HSCs with retroviral vectors.11 Quantitative studies
have also documented HSC expansion following stem cell transplantation.12 The maintenance of the size of the HSC pool during adult
life is a consequence of HSCs undergoing asymmetrical cell
divisions, whereas the expansion of the HSC compartment that
occurs during fetal life or following HSC transplantation likely
requires symmetrical HSC divisions.8,13-15
Numerous investigators have attempted to create ex vivo
conditions that favor HSC self-renewal.16-23 The clinical use of
such ex vivo expanded grafts could theoretically shorten the time
required for successful hematopoietic engraftment to occur following HSC transplantation. Widespread use of such expanded HSC
grafts has been limited due to the lack of the detailed understanding
of factors that regulate symmetrical HSC division as well as access
to culture conditions that maintain HSCs in an uncommitted state.16
Previous attempts to create an in vitro microenvironment that
would allow HSC symmetrical cell division to occur have,
however, resulted in the progressive loss of the numbers of
marrow-repopulating cells and generation of large numbers of
committed hematopoietic progenitor cells.20,21
The ability of human cells to engraft immunodeficient mice has
been used as a surrogate assay for human HSCs. In our earlier
studies we have demonstrated that a 10-fold expansion of severe
combined immunodeficiency (SCID) mouse repopulating cells
(SRCs) can be achieved when cord blood (CB) CD34⫹ cells are
treated with 5-aza-2⬘-deoxycytidine (5azaD)/trichostatin A (TSA)
in the presence of an optimal cytokine combination.24 Extensive
(⬎ 10-fold) expansion of human HSCs, which retain their in
vivo marrow-repopulating potential has not been previously
possible.20,21 The loss of HSC function observed following
previous attempts to expand human HSCs in vitro has been
related to the transit of HSCs through specific phases of the cell
cycle.25 Gothot et al have shown that the transition of human
HSCs from G0 into G1 is associated with the rapid loss of HSC
transplantability.6 We have shown that the expansion of CB
SRCs in the presence of 5azaD/TSA treatment is not due to
selection of a nondividing primitive HSC population retained in
the culture but is rather associated with the active cellular
division of SRCs present within a CD34⫹CD90⫹ cell subpopulation.24,26 In our current studies we have used the same culture
strategy to explore the functional potential of reisolated
CD34⫹CD90⫹ cells that have undergone progressively greater
numbers of cell divisions.
We have previously shown that 5azaD/TSA treatment affects
methylation patterns of CpG sites of ␥-globin promoter26 and
induces changes in the acetylation status at the histone H4 region.24
Both of these events are likely to affect the gene expression patterns
of SRCs present within the CD34⫹CD90⫹ cell population, which
possibly favors expansion of in vivo repopulating HSCs. Recent
studies have identified candidate intracellular factors that might
regulate self-renewal of HSCs.27-41 In our current studies we have
examined whether chromatin-modifying agents permit expansion
of SRCs in vitro by promoting their division and the expression of
the genes required for HSC self-renewal.
Submitted July 13, 2006; accepted December 8, 2006. Prepublished online as Blood
First Edition Paper, December 21, 2006; DOI 10.1182/blood-2006-07-035287.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
Materials and methods
Isolation of CB CD34ⴙ cells
Fresh CB collections were obtained from the Placental Blood Program of
the New York Blood Center (New York, NY) according to guidelines
established by the University of Illinois at Chicago Institutional Review
Board. CB cells were isolated by density-gradient centrifugation on
Ficoll-Paque (⬍ 1.077 g/mL) (Amersham Biosciences, Uppsala, Sweden).
CD34⫹ cells were immunomagnetically enriched using the magneticactivated cell sorting (MACS) CD34 progenitor kit (Miltenyi Biotech,
Auburn, CA) as previously described.22,24 The purity of CB CD34⫹ cells
ranged between 90% and 99%.
Ex vivo cultures
The human CB CD34⫹ cells (5 ⫻ 104/well) were cultured in Iscove
modified Dulbecco medium (IMDM; BioWhittaker, Walkersville, MD)
containing 30% fetal bovine serum (FBS; HyClone Laboratories, Logan,
UT) supplemented with 100 ng/mL stem cell factor (SCF), 100 ng/mL
FLT-3 ligand (FL), 100 ng/mL megakaryocyte growth and development
factor (MGDF), and 50 ng/mL interleukin 3 (IL-3). The cytokines were a
gift of Amgen (Thousand Oak, CA) and incubated as described previously.24 After an initial 16 hours of incubation, the cells were exposed to
5azaD (Pharmachemie, Haarlem, The Netherlands). After 48 hours the cells
were washed and then distributed to new culture plates containing SCF, FL
and MGDF, and TSA (Sigma, St Louis, MO). The cultures were then
continued for an additional 7 days. The cultures lacking 5azaD/TSA
treatment were incubated in the identical culture condition supplemented
with cytokines (IL-3, SCF, FL, MGDF for the initial 48 hours and SCF, FL,
MGDF for the terminal 7 days) only.
Flow cytometric analysis
Briefly, cells were stained with anti–human CD34 monoclonal antibody
(mAb) conjugated to fluorescein isothiocyanate (FITC) and anti–human
CD90 conjugated to phycoerythrin (PE). Cells were analyzed on a
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and at least
10 000 live cells were acquired. All mAbs were purchased from Becton
Dickinson PharMingen (San Diego, CA).
CFSE labeling to assess cell division
Primary CB CD34⫹ cells were labeled for 10 minutes at 37°C with 0.5 ␮M
carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes,
Eugene, OR) in DPBS as described previously.24,26,42 After 9 days of culture
the cells were labeled with anti–CD34-allophycocyanin and anti–CD90-PE
and analyzed for a progressive decline of fluorescence intensity of CFSE,
using flow cytometry as described earlier.24,26,42 The cells were sorted based
on their cell division history using FACSVantage (Becton Dickinson).
Cell cycle analysis
Cell cycle kinetics of the CB CD34⫹ cells in the expansion culture with or
without 5azaD/TSA treatment was determined by using a thymidine analog,
BrdU (Sigma) in conjunction with propidium iodide (PI; Sigma) as
described previously with minor modifications.5,43 After 5 days of initial
culture with or without 5azaD/TSA, reisolated CD34⫹ cells were pulsed
with 5 ␮g/mL BrdU for 2 hours, and then cultured in the presence of
cytokines (SCF, FL, MGDF) without BrdU for an additional 48 hours. After
the identified intervals, cells were harvested and fixed in 70% ethanol. BrdU
quantitation and DNA contents of the cells were analyzed by flow
cytometry as described previously.5,43 The fraction of CD34⫹ cells in S
phase marked with BrdU was traced during the subsequent 48 hours in the
culture without any additional BrdU or 5azaD/TSA.
Colony-forming cell assays
Colony-forming cells (CFCs) were assayed in semisolid media as previously described.24,26 Briefly, 5 ⫻ 102 cells were plated per dish in duplicate
SELF-RENEWAL OF HSCs AND EPIGENETICS
3571
cultures containing 1 mL IMDM with 1.1% methylcellulose supplemented
with 30% FBS, 5 ⫻ 10⫺5 M 2-ME (StemCell Technologies, Vancouver,
BC, Canada), to which 100 ng/mL SCF, 100 ng/mL FL, 50 ng/mL IL-3,
50 ng/mL IL-6, 50 ng/mL granulocyte-macrophage colony-stimulating
factor, and 5 U/mL erythropoietin (EPO; Amgen) was added as
described previously.24 The colonies were enumerated after 14 days
using standard criteria.24,26
Cobblestone area-forming cell assays
To quantitate the number of cobblestone area-forming cells (CAFCs),
primary CD34⫹ cells and ex vivo cultured cells were plated in limiting
dilution onto irradiated monolayer of the murine stromal fibroblast line
M2-10B4 as previously described.24,26
NOD/SCID assay for in vivo marrow repopulating potential of
ex vivo expanded CB HSCs
NOD/SCID mice were purchased from the Jackson Laboratories (Bar
Harbor, ME). The NOD/SCID assay was performed as previously described.24,26 CD34⫹ cells cultured in the presence of cytokines with or
without 5azaD/TSA treatment were harvested after 9 days, CD34⫹CD90⫹
cells that have undergone 4 or fewer divisions or 5 or more divisions were
reisolated by flow cytometry and then injected (5 ⫻ 104 CD34⫹CD90⫹
cells/mouse) through the tail vein intravenously to nonobese diabetic/SCID
(NOD/SCID) mice 12 to 16 hours after a sublethal dose of total body
irradiation (300 cGy). Bone marrow (BM) cells from NOD/SCID mice
were analyzed flow cytometrically to detect human cell engraftment after 8
weeks of transplantation as previously described.24,26,44,45
Secondary transplantation
NOD/SCID mice were treated with a single intraperitoneal injection (200
␮g/mouse) of TM-␤1 (BD Biosciences PharMingen) within 4 hours after
total body irradiation as a conditioning for the stem cell transplantation
assay. TM-␤1 is a mAb directed against the murine IL-2R␤ to eliminate
residual natural killer cell activity. BM from chimeric primary recipient
mice engrafted with 5azaD/TSA-expanded cells were harvested and injected into
secondary NOD/SCID recipients without further reisolation of human cells. BM
from a single chimeric mouse was transplanted into a single secondary
mouse. Mouse BM cells were stained for human cell chimerism after 7
weeks of transplantation using a panel of mAbs as described earlier to
assess multilineage human hematopoietic engraftment. 24,26
Differentiation potential of 5azaD/TSA-treated expanded CB
cells in subsequent in vitro culture
After 9 days of initial 5azaD/TSA treatment in culture, cells were placed in
a secondary culture supplemented with GM-CSF, G-CSF, SCF, IL-3, IL-6,
and EPO in the absence of 5azaD/TSA treatment. At the end of 7 days of
secondary culture (total 16 days), cytospin preparations (Shandon, Pittsburgh, PA) of cultured cells were stained with Wright-Giemsa stain and
viewed under a light microscope (objective used 40⫻/0.75 NA). The
phenotype of cells following secondary culture (day 16) was determined by
staining with mAbs directed toward CD34, CD90, CD14, CD15, CD36, and
lineage markers (CD2, CD14, CD15, CD19, glycophorin A).
RNA extraction and real-time PCR
Total RNA was extracted from CB cells cultured in the presence of
cytokines with or without 5azaD/TSA treatment at the indicated time points
(day 5 and day 9) using RNeasy Mini kit (Qiagen, Valencia, CA) or TRIzol
(Invitrogen, Carlsbad, CA). Total RNA was subjected to DNase digestion to
remove gDNA contaminant. Approximately 1 ␮g total RNA was reversetranscribed by use of a GeneAmp cDNA synthesis kit with random hexamer
primers (Roche Molecular Systems, Applied Biosystems, Branchburg, NJ).
To quantitate the level of mRNAs expression, we carried out polymerase
chain reaction (PCR) amplification using the 7700 Sequence detector (PE
Applied Biosystems, Foster City, CA), and the PCR products were detected
by use of SYBR green technology (ABI, Foster City, CA). PCR cycling
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
ARAKI et al
Western blotting analysis
Whole-cell lysates were prepared from CB cells cultured for 9 days with or
without 5azaD/TSA in the presence of cytokines using the Mammalian Cell
Extraction Kit (BioVision, Mountain View, CA) as described previously.24
Equal amounts of protein were subjected to Western blotting analysis.
Samples were separated by 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene diflouride membranes. The membranes were probed with HOXB4 (Santa Cruz
Biotechnology, Santa Cruz, CA), Bmi-1 (Upstate Biotechnology, Lake
Placid, NY), P21 (Santa Cruz Biotechnology), and ␤-actin (Sigma) and
developed using the enhanced chemiluminescence system with horseradish
peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) as described previously.24
Statistical analysis
Results are expressed as mean ⫾ SE when appropriate. Statistical differences
were evaluated using the student t test with significance at P of .05 or less.
Results
5azaD/TSA affects the division of CD34ⴙCD90ⴙ cells
The optimal combination of cytokines to be used for in vitro HSC
expansion remains the topic of continued investigation. Most
investigators have concluded that a cytokine cocktail including the
early-acting cytokines, SCF, FL, and MGDF, is effective or at least
serves as a core cytokine combination to which additional cytokines may be added for these purposes.49-51 After 9 days of culture
of CB CD34⫹ cells, the addition of 5azaD/TSA to the combination
of SCF, FL, and MGDF promoted far greater expansion of the
number of CD34⫹CD90⫹ cells than observed with the addition of
the same cytokines alone lacking 5azaD/TSA treatment.24
We have previously demonstrated that 5azaD/TSA-treated
CD34⫹CD90⫹ cells, but not CD34⫹CD90⫺ cells are responsible
for in vivo SRC potential of such expanded cell products.24 The cell
division history of CB CD34⫹CD90⫹ cells after in vitro culture
was assessed using CFSE staining, a fluorescent cytoplasmic dye,
which is equally distributed between daughter cells after each cell
division. Irrespective of the treatment with 5azaD/TSA, virtually
no CD34⫹CD90⫹ cells (0.1%) remained quiescent after 9 days of
Day 5
CD34+CD90+
Day 9
CD34+CD90+
89.2%
5-10 div.
26.2%
5-10 div.
Day 0
CD34+CD90+
72.8%
1-4 div.
10.7%
1-4 div.
1%
0 div.
Cytokines alone
0.1%
0 div.
100%
0 div.
58.3%
5-10 div.
98.0%
1-4 div.
41.6%
1-4 div.
2%
0 div.
Cytokines
+5azaD/TSA
0.1%
0 div.
CFSE
Figure 1. The cell division history of CD34ⴙCD90ⴙ cells during ex vivo culture.
CFSE-labeled CD34⫹ cells were cultured in the presence of cytokines with or without
5azaD/TSA treatment for 9 days. The panel shows a representative (1 of 3
experiments) flow cytometric profile of CFSE fluorescence intensity after 5 days and
9 days of culture. The arrow indicates the fraction of cells that have not undergone cell
division.
culture. In the absence of 5azaD/TSA treatment, about 73% of the
CD34⫹CD90⫹ cells had divided up to 4 times and 26% had divided
5 to 10 times after 5 days of culture, whereas 98% of 5azaD/TSAtreated culture of CD34⫹CD90⫹ cells had divided only 1 to 4 times
(Figure 1). After 9 days of culture in the absence of 5azaD/TSA
treatment, 90% of CD34⫹CD90⫹ cells already has divided 5 to 10
times, whereas in the presence of 5azaD/TSA treatment 42% of
CD34⫹CD90⫹ had divided 1 to 4 times and 58% of CD34⫹CD90⫹
cells had undergone 5 to 10 cell divisions (Figure 1). These findings
suggest that treatment with 5azaD/TSA is associated with
CD34⫹CD90⫹ cell division occurring at a rate much slower than
that observed in the presence of cytokines alone.
5azaD/TSA-treated CD34ⴙ cells cycle relatively slowly
For precise examination of the rate of cycling of 5azaD/TSAexpanded CD34⫹ cells, we have reisolated CD34⫹ cells from
cultures with or without 5azaD/TSA treatment and pulsed with
BrdU. The BrdU positively marked cells from both cultures were
chased for the subsequent 48 hours at different time intervals in
identical cultures (SCF, FL, TPO). The populations of cells that
divide faster are expected to re-enter into S phase faster than cells
cycling relatively slower possessing a longer doubling time.43 The
passage of the cells from 5azaD/TSA-treated culture during the
G2/M though the G1 phase paralleled that of the cells obtained from
culture lacking 5azaD/TSA treatment (Figure 2). However, re-entry
into the S phase of BrdU⫹ cells cultured in the presence of
5azaD/TSA was significantly delayed by approximately 6 hours in
G2/M, G1
Re-entry to S phase
100
Cytokines alone
BrdU+ S phase cells (%)
conditions were standard except for annealing/elongation temperature,
which ranged between 57°C and 62°C and was chosen based on preliminary
primer optimization experiments. GAPDH mRNA quantification was used
as internal calibrator and the standard curve method was used for relative
mRNA quantitation. Measurements were done in triplicate and a negative
control (lacking cDNA template) was included in each assay.46-48
The primer sequences used in real-time PCR assays are as follows:
GATA 2: forward primer: 5⬘-GATACCCACCTATCCCTCCTATGTG-3⬘,
reverse primer: 5⬘-GTGGCACCACAGTTGACACACTC-3⬘; NOTCH 1:
forward primer: 5⬘-GAGGCGTGGCAGACTATGC-3⬘, reverse primer
5⬘-CTTGTACTCCGTCAGCGTGA-3⬘; BMI 1: forward primer: 5⬘TGGCTCTAATGAAGATAGAGG-3⬘, reverse primer: 5⬘-TTCCGATCCAATCTGTTCTG-3⬘; HOXB4: forward primer: 5⬘- TCCCACTCCGCGTGCAAAGA-3⬘, reverse primer: 5⬘-GCCGGCGTAATTGGGGTTTA- 3⬘;
P21: forward primer: 5⬘-GTCTTGTACCCTTGTGCCTC-3⬘, reverse
primer: 5⬘-GGTAGAAATCTGTCATGCTGG-3⬘; P27: forward primer:
5⬘-TTTAATTGGGTCTCAGGCAAACTCT-3⬘, reverse primer: 5⬘CCGTCTGAAACATTTTCTTCTGTTC-3⬘; C-MYC: forward primer:
5⬘-TCCTCGGATTCTCTGCTCTC-3⬘, reverse primer: 5⬘-CTTGTTCCTCCTCAGAGTCG-3⬘; MPO: forward primer: 5⬘-ACCCTCATCCAACCCTTC-3⬘, reverse primer: 5⬘-GTCAATGCCACCTTCCAG-3⬘;
GATA 1: forward primer: 5⬘-ACAAGATGAATGGGCAGAAC-3⬘, reverse
primer: 5⬘-TACTGACAATCAGCGCTTC-3⬘.
Cell number
3572
Cytokines
+5azaD/TSA
80
60
6h
40
20
0
6
12
18
24
30
36
42
48
Time after BrdU pulse (h)
Figure 2. Effect of 5azaD/TSA treatment on cell cycle progression. After 5 days
of initial culture, CD34⫹ cells were reisolated from culture containing 5azaD/TSA
treatment or cytokines alone. Reisolated CD34⫹ cells were pulse-labeled with BrdU
and then cultured without BrdU or additional 5azaD/TSA treatment for an additional
48 hours. The harvested cells were stained with FITC-anti-BrdU mAb and PI, and
BrdU⫹ S phase cells quantitated every 6 to 12 hours by flow cytometry. The results
show the percentage of BrdU⫹ cells in the S phase at indicated time intervals.
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
SELF-RENEWAL OF HSCs AND EPIGENETICS
3573
Figure 3. The influence of cell division history of CD34ⴙCD90ⴙ
cells on frequency and absolute numbers of CFUs-Mix and
CAFCs following ex vivo culture. CD34⫹ cells cultured in the
presence of cytokines with or without 5azaD/TSA treatment after
9 days were harvested and CD34⫹CD90⫹ cells that have undergone 4 or fewer divisions or 5 or more divisions were reisolated
and assayed for CFCs and CAFCs. (A) Frequency of CFUs-Mix
generated from populations of day 0 CD34⫹CD90⫹ cells and
CD34⫹CD90⫹ cells undergoing 4 or fewer divisions or 5 or more
divisions (day 9) was determined. The bar graph indicates the
numbers of CFUs-Mix assayed in 500 CD34⫹CD90⫹ cells plated.
(B) Numbers of CAFCs/104 cells assayed from day 0
CD34⫹CD90⫹ cells and CD34⫹CD90⫹ cells having undergone 4
or fewer divisions or 5 or more cell divisions (day 9). (C) The
absolute numbers of CFUs-Mix/well generated from populations
of day 0 CD34⫹CD90⫹ cells and CD34⫹CD90⫹ cells undergoing
4 or fewer divisions or 5 or more divisions (day 9) present in an
individual well was determined by the following calculation:
(numbers of CFUs-Mix assayed in 500 CD34⫹CD90⫹ cells plated
that have undergone ⱕ 4 or ⱖ 5 cell divisions) ⫻ (total number of
CD34⫹CD90⫹ cells having undergone ⱕ 4 divisions or ⱖ 5 cell
divisions present in an individual well). (D) The absolute number
of CAFCs/well was determined by the following calculation:
(number of CAFCs/104 cells assayed from reisolated
CD34⫹CD90⫹ cells having undergone ⱕ 4 divisions or ⱖ 5 cell
divisions in an individual well after 9 days of culture) ⫻ (total
number of CD34⫹CD90⫹ cells having undergone ⱕ 4 divisions or
ⱖ 5 cell divisions present in an individual well). The bar graphs
represent mean ⫾ SE of 3 independent experiments. *Previously
published data, reprinted with permission.24
comparison to cells in the absence of 5azaD/TSA treatment. These
results indicate that cells expanded in the presence of chromatinmodifying agents cycle relatively slowly in comparison to untreated cells.
CD34ⴙCD90ⴙ
In vitro behavior of
multiple cell divisions
cells undergoing
Reisolated CD34⫹CD90⫹ cells that had undergone various numbers of cell divisions (1-4 or 5-10 cell divisions) were assayed for
mixed colony-forming units (CFUs-Mix) as well as CAFCs. Cells
undergoing 1 to 4 divisions contained greater numbers of these
primitive hematopoietic progenitors than cells undergoing 5 to 10
cell divisions irrespective of the culture conditions used (Figure
3A-B). Cells undergoing 5 to 10 cell divisions in the presence of
chromatin-modifying agents contained statistically greater numbers of assayable CFUs-Mix and CAFCs than CD34⫹CD90⫹ cells
that had undergone similar numbers of cell divisions in the
presence of cytokines alone after 9 days of culture (P ⱕ .05; Figure
3A-B). The absolute numbers of CFUs-Mix and CAFCs generated
under each culture condition after 5 and 9 days were also
determined. The absolute numbers of CFUs-Mix and CAFCs
assayed from CD34⫹CD90⫹ cells that had undergone 1 to 4 or 5 to
10 cell divisions were far greater in the 5azaD/TSA-treated cultures
than similar cell populations exposed to cytokines alone (Figure
3C-D). Absolute numbers of CD34⫹CD90⫹ cells from 5azaD/TSAtreated cultures that have undergone 1 to 4 divisions was 38-fold
greater than the CD34⫹CD90⫹ cells undergoing equal numbers of
divisions from cultures receiving cytokines alone. Furthermore,
absolute numbers of CD34⫹CD90⫹ cells from cultures receiving
the 5azaD/TSA that have undergone 5 to 10 divisions was 6.4-fold
higher than the CD34⫹CD90⫹ cells from cultures receiving
cytokines alone (Table 1). These findings indicate that 5azaD/TSA
treatment promotes the retention of functional potential of
CD34⫹CD90⫹ cells even after they have undergone extensive
rounds of cell division.
Effects of 5azaD/TSA treatment on the marrow-repopulating
potential of reisolated CD34ⴙCD90ⴙ cells based
on their cell division history
To further examine the functional potential of CD34⫹CD90⫹ cells
cultured in presence or absence of 5azaD/TSA, CD34⫹CD90⫹ cells
were reisolated based on their cell division history after 9 days of
culture and were transplanted into NOD/SCID mice following total
body irradiation. When 5 ⫻ 104 CD34⫹CD90⫹ cells that had
undergone 1 to 4 cell divisions after 5azaD/TSA treatment were
injected into NOD/SCID mice, all mice displayed human multilineage hematopoietic cell engraftment after 8 weeks of transplantation (Figure 4A). In addition, 50% of mice receiving CD34⫹CD90⫹
cells treated with 5azaD/TSA in the culture that had undergone 5 to
10 cellular divisions still possessed evidence of human multilineage hematopoietic cell engraftment (Figure 4A-B). By contrast,
when an equivalent number of CD34⫹CD90⫹ cells that had
undergone 5 to 10 cellular divisions isolated from cultures receiving cytokines alone were transplanted, none of the recipient mice
had any evidence of detectable human hematopoietic cell engraftment (Figure 4A). Since 90% of CD34⫹CD90⫹ cells in the cultures
exposed to cytokines alone lacking 5azaD/TSA treatment had
undergone 5 or more cell divisions by day 9 (Figure 1), only a small
Table 1. Absolute number of CD34ⴙCD90ⴙ cells per well
Absolute no. CD34ⴙCD90ⴙ
cells/well, ⴛ 104
Day 0
1.46 ⫾ 0.1
Day 9
4 or fewer divisions
Cytokines alone
Cytokines and 5azaD/TSA
0.24 ⫾ 0.1
9.2 ⫾ 0.8
5 or more divisions
Cytokines alone
Cytokines and 5azaD/TSA
2.0 ⫾ 0.4
12.8 ⫾ 1.1
Absolute number of day 0 CD34⫹CD90⫹ cells and CD34⫹CD90⫹ cells having
undergone 4 or fewer divisions or 5 or more divisions (day 9) present in an
individual well.
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
ARAKI et al
Figure 5. The cells treated with 5azaD/TSA retain long-term repopulating ability
after serial transplantation into secondary NOD/SCID recipients. Representative
flow cytometric analysis of multilineage hematopoietic differentiation potential of
engrafted human hematopoietic cells in secondary NOD/SCID mice (no. 2 secondary
mouse BM as shown in Table 2).
Figure 4. The percentage of human cell chimerism following transplantation of
reisolated CD34ⴙCD90ⴙ cells that have undergone 5 or more cell divisions
following ex vivo culture. CD34⫹ cells cultured in the presence of cytokines with or
without 5azaD/TSA treatment were harvested after 9 days; the CD34⫹CD90⫹ cells
that have undergone 4 or fewer divisions and 5 or more divisions were reisolated,
then injected into NOD/SCID mice. (A) NOD/SCID (n ⫽ 21, data pooled from 2
independent experiments) engraftment achieved with CD34⫹CD90⫹ cells ( ⱕ 4
divisions and ⱖ 5 divisions). The percent of human hematopoietic cell chimerism is
plotted as dots and their mean values indicated as horizontal bars. (B) Representative flow cytometric analysis of multilineage hematopoietic differentiation potential of
engrafted human hematopoietic cells in NOD/SCID mice given transplants with
reisolated CD34⫹CD90⫹ cells ( ⱖ 5 divisions) is shown. *Previously published data,
reprinted with permission.24
fraction of CD34⫹CD90⫹ cells (0.15% of total cells) remained
within the range of 1 to 4 divisions (Table 1), making it technically
challenging to directly assess their marrow-repopulating potential.
The transplantation of CD34⫹CD90⫹ cells cultured in the presence
of 5azaD/TSA treatment that had undergone 1 to 4 cell divisions led
to a greater degree of human chimerism than that observed
following the transplantation of CD34⫹CD90⫹ cells that had
undergone 5 to 10 cell divisions (Figure 4A), although the
difference was not statistically significant (P ⫽ .13).
Self-renewal and long-term reconstitution function of HSCs is
generally demonstrated by reconstitution of secondary recipients
after serial transplanrtation.52 To evaluate whether 5azaD/TSAtreated expanded CB cells still retain their self-renewal capacity
after primary transplantation, BM cells from the primary NOD/
SCID recipients were transplanted into secondary NOD/SCID
recipients. Five of 6 secondary NOD/SCID mice receiving BM
from primary mice engrafted with the cells treated with 5azaD/TSA
resulted in human cell engraftment (Table 2). Furthermore, these
cells were capable of differentiating into cells belonging to multiple
hematopoietic lineages in secondary recipients (Figure 5).
5azaD/TSA-treated cells possess the multiple lineage
differentiation potential in subsequent in vitro culture
Previously we have demonstrated that human BM CD34⫹ cells
treated with 5azaD/TSA results in an expansion of HSCs capable of
differentiating into multiple hematopoietic lineages, both in vitro
and in vivo like primary BM CD34⫹ cells.26 To evaluate the
differentiation potential of the CB cells expanded following
treatment with 5azaD/TSA, the cells were placed in a secondary
culture containing GM-CSF, G-CSF, SCF, IL-3, IL-6, and EPO in
the absence of 5azaD/TSA for an additional 7 days to assess their
ability to terminally differentiate. After an additional 7 days of
culture (total 16 days), the cells were found to be capable of
differentiating into multiple blood cell lineages as evident by
examining both morphology as well as their immunophenotype
Table 2. Secondary NOD/SCID mice repopulation of 5azaD/TSA treated ex vivo expanded cord blood cells
Primary transplants
Human cell dose*
Secondary transplants
Primary mouse BM
chimerism, % human
Human cell dose†
Secondary mouse BM
chimerism, % human
No. 1
1.0 ⫻ 106
5.2
5.0 ⫻ 105
0.21
No. 2
1.0 ⫻ 106
13.1
1.4 ⫻ 106
1.5
No. 3
5.0 ⫻ 105
2.6
4.2 ⫻ 105
ND
No. 4
5.0 ⫻ 105
48.4
5.0 ⫻ 106
0.60
No. 5
5.0 ⫻ 105
11.2
1.4 ⫻ 106
0.20
No. 6
2.5 ⫻ 105
22.8
2.6 ⫻ 106
0.12
Murine BM from engrafted primary recipients was harvested, and unfractionated murine BM cells were injected into secondary NOD/SCID recipients. BM from primary and
secondary recipients was stained for human CD45, CD71, CD19, CD33, CD34, and CD41 to assess multilineage human hematopoietic engraftment after 7 weeks of
transplantation.
ND indicates not detectable.
*Cells injected in the primary mouse were cultured for 9 days pretreated with 5azaD/TSA (approximately 50% of these cells express CD34).
†Cell dose is based on human CD45⫹ cells.
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
SELF-RENEWAL OF HSCs AND EPIGENETICS
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Figure 6. Differentiation potential of the cells expanded following 5azaD/TSA treatment in the ex vivo culture. After 9 days of initial culture (as described in “Material and
methods”), 5azaD/TSA-treated cells were placed in a secondary culture supplemented with GM-CSF, G-CSF, SCF, IL-3, IL-6, and EPO in the absence of additional 5azaD/TSA
treatment. Cytospin preparations were stained with Giemsa and Wright stains and viewed with a light microscope (objective used 40⫻/0.75 NA) equipped with an Axiocam
camera (Zeiss, Thornwood, NY). Images were processed using Zeiss Axiovision software version 4.1. The phenotype of cells was determined by staining with mAb directed
toward CD34, CD90, CD14, CD15, CD36, and lineage markers (CD2, CD14, CD15, CD19, glycophorin A).
(Figure 6). Furthermore, we show that 5azaD/TSA-treated cells are
capable of differentiating into both myeloid and lymphoid lineages
including CD41⫹ megakaryocytes following transplantation in
vivo (Figure 4B). These findings thus fur suggest that the 5azaD/
TSA-treated CB cells are likely to function normally for possible
therapeutic use.
Treatment of CB cells with 5azaD/TSA modulates expression of
genes implicated in HSC self-renewal
To understand the molecular mechanism responsible for the
expansion of functional HSCs observed following 5azaD/TSA
treatment, we examined transcription levels of several genes
implicated in self-renewal of HSCs using real-time quantitative
PCR. Primers specific for HOXB4, BMI 1, GATA 2, NOTCH 1, P21,
P27, C-MYC, GATA 1, and myeloperoxidase (Mpo) were used.
Total RNA from cells after 5 and 9 days of culture was extracted.
We observed relatively higher levels of transcripts for HOXB4,
Bmi-1, and GATA-2 genes in cells treated with 5azaD/TSA in the
culture. In addition, the transcript levels of the genes regulating cell
cycle, such as P21 and P27 were increased to a greater degree in
5azaD/TSA-treated cells after 5 days but not after 9 days of culture
(Figure 7A). The higher transcript level of P21 and P27 in
5azaD/TSA-treated cells is consistent with the proposed role of
these gene products in regulating the cycling behavior of HSCs.35,36
Furthermore, the expression of another transcription factor, CMYC, which is involved in the regulation of cellular proliferation
was also examined. A significantly lower level of C-MYC transcript
was observed in the 5azaD/TSA-treated cells in comparison to the
cells cultured with cytokines alone. The expression of NOTCH 1,
which has been implicated in promoting stem cell expansion, was
similarly examined.53 Surprisingly Notch expression was increased
in cells exposed to cytokines alone but not the chromatinmodifying agents.
The expression of lineage-specific genes such as MPO and
GATA-1 were also examined by real-time quantitative PCR. GATA1
is a transcription factor, important for commitment of HSCs to
adult erythroid and megakaryocytic lineages,54,55 whereas the
expression of MPO is associated with granulocytic differentiation.56 The GATA1 transcript level progressively increased to a
similar degree in cultures receiving cytokines alone as well as
culture receiving the chromatin-modifying agents. Higher transcript levels of MPO were observed in cultures exposed to
cytokines alone as compared to cells cultured in the presence of
5azaD/TSA.
To confirm the role of higher transcript levels of genes
observed, protein levels of HOXB4, BMI 1, and P21 were
examined in the CB cell cultures treated with and without
5azaD/TSA. A significantly higher degree of HOXB4, Bmi-1, and
P21 protein expression was detected in the cells treated with
5azaD/TSA in comparison to the cells treated with cytokines alone
(Figure 7B).
Discussion
Previous attempts to promote the expansion of human HSCs in
vitro have led either to HSC differentiation resulting in HSC
exhaustion or, at best, asymmetrical HSC divisions and maintenance of the numbers of HSCs.20,21 We hypothesize that previously
used ex vivo culture conditions for HSC expansion result in the
silencing of pivotal genes that are crucial for HSCs to undergo
symmetrical cell division. In this study chromatin-modifying
agents were used to alter the methylation and acetylation status of
promoters of critical genes that are pivotal for the retention of the
characteristic biological properties of dividing HSCs. Our data
suggest that chromatin-modifying agents not only prevent the loss
of HSCs in the culture but promote symmetrical division of SRCs
resulting in the expansion of the numbers of marrow-repopulating
cells.24,26,57 Although a more careful safety assessment of 5azaD/
TSA-treated expanded CB cells is required before proceeding with
clinical development of this technology, the expanded CB cells
show preliminary therapeutic potential as an alternative graft for
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3576
ARAKI et al
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
Figure 7. Treatment of CB cells with 5azaD/TSA
modifies the expression of transcription levels of
genes and their products implicated in HSC selfrenewal. (A) Effects of 5azaD/TSA treatment on the
relative transcript levels of genes (HOXB4, BMI 1, GATA 2,
NOTCH 1, P21, P27, C-MYC, GATA 1, and MPO) were
measured by real-time quantitative PCR. Total RNA was
extracted from CB CD34⫹ cells (day 0) or cells obtained
after 5 and 9 days of culture in the presence of cytokines
with or without 5azaD/TSA treatment. Relative mRNA
levels in cultured cells (day 5 and day 9) to primary CB
cells (day 0) was determined by real-time quantitative
PCR. GAPDH was used as internal calibrator (control
gene), the standard curve method was used for relative
mRNA quantitation. Measurements were obtained in
triplicate and a negative control (lacking the cDNA template) was included for each assay. (B) Detection of
HOXB4, Bmi-1, and P21 proteins in the cells cultured
with or without 5azaD/TSA treatment in the presence of
cytokines after 9 days using Western blotting analyses as
described in “Materials and methods.” Equal loading of
protein was verified with anti–␤-actin antibody on the
same membrane.
adult recipients. The 5azaD/TSA-treated cells are not only capable
of differentiating into myeloid and lymphoid lineages like unmanipulated primary CB CD34⫹ cells in vivo but they can also
terminally differentiate in vitro when further cultured in an
appropriate environment.
HSCs slowly cycle in vivo; however, in the presence of a
variety of cytokine combinations in vitro CD34⫹ cells rapidly cycle
and undergo repeated cell divisions resulting frequently in the
generation of large numbers of differentiated progenitor cells but a
decline in the number of marrow-repopulating cells.5,6,9,43 The
residual marrow-repopulating potential of such ex vivo generated
grafts has been reported to reside either within a population of
HSCs that had either remained quiescent or had undergone a
limited numbers of divisions.58-60 In the present studies, we confirm
CB CD34⫹CD90⫹ cells that have undergone 5 to 10 cell divisions
in vitro in response to a cytokine combination lack in vivo
marrow-repopulating potential. By contrast, CD34⫹CD90⫹ cells
exposed to 5azaD/TSA, which also had undergone 5 to 10 cell
divisions, still contained assayable SRCs. We conclude that exposure of the SRCs within the CD34⫹CD90⫹ cell population to
cytokines alone results in their rapid cell division and their terminal
differentiation, whereas exposure of SRCs to 5azaD/TSA in the
culture results in these same cells dividing at a slower rate and
retention of their marrow-repopulating potential. In an additional
experiment we have demonstrated that 5azaD/TSA-treated cells
re-enter into S phase significantly slower in contrast to the cells
cultured in cytokines alone. These findings support the concept that
the kinetics of HSC progression through the cell cycle is associated
with or determined by the decision of an HSC to undergo
self-renewal or differentiation. The chromatin-modifying agents
might influence the doubling time by regulating cell cycle machinery, but it is more likely that chromatin-modifying agents in our
culture system result in the expansion of more primitive HSCs,
which cycle slowly. It has been suggested by several investigators
that primitive HSCs divide more slowly than the relatively
committed progenitor cells.3,4,43,57
The expressions of a number of genes have been implicated in
determining the function and fate of HSCs. These genes include
HOXB4, Bmi-1, GATA-2, Notch-1, P21, P27, GATA 2, and possibly
other as of now unidentified genes. GATA 2 is a transcription factor
expressed by HSCs, which has been reported to be necessary for
maintenance of HSCs and primitive hematopoietic progenitor
cells.33 In this report significantly higher transcript levels of
GATA 2 were observed exclusively in 5azaD/TSA-treated cells
after 5 and 9 days of culture. The increased transcript level of
GATA 2 observed in cells expanded with chromatin-modifying
agents indicates that GATA-2 is likely one of the contributing
factors that determines the characteristic properties of an HSC.
Members of the Polycomb group of genes such as BMI 1 and
members of the homeobox family of genes such as HOXB4 may
bias HSCs to undergo self-renewal rather than differentiation.27
Many of these pathways interact with the basic cell cycle machinery.27 We have detected a high transcript level of both HOXB4 and
BMI 1 in human CB CD34⫹ cells treated with chromatinmodifying agents. HOXB4 requires a functional BMI 1 to execute
its function as an HSC activator.32 Overexpression of HOXB4 and
Bmi-1 genes has been shown to induce self-renewal of long-term
multilineage repopulating HSCs without causing leukemia.27-32
The greater expression of HOXB4 and BMI 1 transcripts present in
CD34⫹ cells treated with chromatin-modifying agents is therefore
consistent with the proposed roles of these genes in HSC
self-renewal.
The activity of these cyclin-dependent kinases is regulated by a
number of inhibitors, including P21 and P27, which have been
shown to play a role in determining HSC behavior. Cheng and
coworkers have indicated that the absence of P21 leads to
expansion of the stem cell pool, more active HSC cycling, and
greater sensitivity of the HSC pool to exhaustion in response to a
variety of challenges.35 By contrast P27 has been shown not to
affect stem cell kinetics but rather to determine progenitor cell
proliferation and the size of HSC pool.36 The transcript level of
both P21 and P27 were increased after 5 days in the cultures
exposed to the chromatin-modifying agents. Although the transcript levels of P21 and P27 in 5azaD/TSA-treated cells declined
after 9 days of culture, the expression of P21 protein level in the
chromatin-modifying agents treated cells remained higher in
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BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
SELF-RENEWAL OF HSCs AND EPIGENETICS
comparison to the untreated cells. These findings could be partially
responsible for the expansion of CD34⫹CD90⫹ cells after 9 days of
culture but the decline of CD34⫹CD90⫹ cell numbers observed
after 14 days of culture (H.A. and N.M., unpublished observation,
2006).
Studies of the effect of C-MYC on stem cell behavior has
resulted in conflicting information.37,61 Our studies revealed progressively greater amounts of C-MYC transcripts in cells exposed to
cytokines alone, which did not contain assayable SRCs as compared to cells exposed to the chromatin-modifying agents. These
findings indicate that C-MYC expression alone is unlikely to define
the functionality of HSCs after in vitro division but might affect in
vivo HSC self-renewal by altering the interaction between an HSC
and its niche within the marrow microenvironment as suggested by
Wilson et al.37 In our current studies we detected greater transcript
levels of NOTCH 1 in the cultures performed in the absence of
5azaD/TSA treatment than observed in cultures exposed to the
chromatin-modifying agents. These findings are similar to those
reported by DeFelice et al who demonstrated that Notch-1 pathway
was not associated with ex vivo expansion of primitive hematopoietic progenitor cells treated with another HDAC inhibitor, valproic
acid.62 GATA 1 expression is associated with HSC commitment to
erythropoiesis and megakaryocytopoiesis,54,55 whereas MPO expression is associated with terminal myeloid maturation.56 GATA 1
transcript levels increased progressively in cells exposed to cytokines alone to a similar degree as cells receiving the chromatinmodifying agents. MPO transcript levels peaked after 5 days of
incubation in cultures exposed to cytokines alone but maximal
expression was delayed in cultures containing the chromatinmodifying agents until day 9 of incubation. The methylation of the
MPO promoter in primitive hematopoietic progenitor cells may be
sensitive to the action of chromatin modifying agents.56
Collectively, our data suggest that epigenetic mechanisms likely
result in the loss of in vivo marrow-repopulating potential of CB
HSCs following ex vivo culture in the presence of cytokines alone.
This loss of HSC function is at least in part accounted for by a faster
rate of HSC division occurring in the CD34⫹ cells exposed to
cytokines alone. However, this loss of SRCs can be circumvented
by the use of chromatin-modifying agents, which results in a
slower rate of cell division associated with higher expressions of a
group of HSC regulatory genes including HOXB4, BMI 1, GATA 2,
3577
P21, as well as P27. The exact functional roles of these genes for
maintaining self-renewal of HSCs have yet to be fully elucidated.
The possibility remains where the expression of additional or yet to
be identified HSC regulatory genes may also contribute to the
retention of the marrow-repopulating potential of 5azaD/TSAtreated CB CD34⫹ cells. The global gene expression pattern of
CD34⫹ cells following treatment with chromatin-modifying agents
is of immense interest and currently being sought in our laboratory.
Acknowledgments
This work was supported in part by grants from the State of Illinois
through Illinois Regenerative Medicine Institute (N.M.) and grants
from the Prudence Cole Foundation (N.M.) and start-up funding
from the Department of Medicine and the University of Illinois
Cancer Center (N.M.).
We would like to thank Edward Bruno, Rifat Rahman, and
Sakina M. Petiwala for their excellent technical assistance. We
gratefully acknowledge the helpful comments of Joseph DeSimone
and Donald Lavelle. Dolores Mahmud is acknowledged for critical
reading of the manuscript. We wish to thank Amgen Inc (Thousand
Oaks, CA) for providing cytokines. We are indebted to Pablo
Rubenstein and Luda Dobrila of New York Blood Center (New
York, NY) for providing CB units.
Authorship
Contribution: H.A. designed and performed research, interpreted
the data, and wrote the paper; K.Y. performed research; P.B. and
Y.Z. performed and analyzed PCR data; R.H. was responsible for
critical reading of the manuscript for important intellectual content;
and N.M. was responsible for the study concept, design, execution
of the research, interpretation of data, and writing and revising the
draft paper.
Conflict-of-interest disclosure: The authors have no competing
financial interests.
Correspondence: Nadim Mahmud, University of Illinois, 909 S
Wolcott Ave, COMRB, Rm 3095, M/C 734 Chicago, IL 60612;
e-mail: [email protected].
References
1. Lajtha LG, Pozzi LV, Schofield R, Fox M. Kinetic
properties of hematopoietic stem cells. Cell Tissue Kinet. 1969;2:39-49.
2. Traycoff CM, Orazi A, Ladd AC, Rice S, McMahel
J, Srour EF. Proliferation-induced decline of primitive hematopoietic progenitor cell activity is
coupled with an increase in apoptosis of ex vivo
expanded CD34⫹ cells. Exp Hematol. 1998;26:
53-62.
3. Bradford GB, Williams B, Rossi R, Bertoncello I.
Quiescence, cycling, and turnover in the primitive
hematopoietic stem cell compartment. Exp Hematol. 1997;25:445-453.
4. Cheshier SH, Morrison SJ, Liao X, Weissman IL.
In vivo proliferation and cell cycle kinetics of longterm self-renewing hematopoietic stem cells.
Proc Natl Acad Sci U S A. 1999;96:3120-3125.
5. Mahmud N, Devine SM, Weller KP, et al. The
relative quiescence of hematopoietic stem cells in
non-human primates. Blood. 2001;97:3061-3068.
6. Gothot A, van der Loo JC, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34⫹
cells in non-obese diabetic/severe combined immune-deficient mice. Blood. 1998;92:2641-2649.
differentiation commitment and maturation in haemopoietic cells. Nature. 1989;339:27-30.
7. Mcculloch EA, Till JE. Proliferation of hematopoietic colony-forming cells transplanted into irradiated mice. Radiat Res. 1964;22:383-397.
14. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The
repopulation potential of fetal liver hematopoietic
stem cells in mice exceeds that of their adult marrow counterparts. Blood. 1996;87:3500-3507.
8. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood. 1993;81:28442853.
9. Wagner W, Ansorge A, Wirkner U, et al. Molecular
evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis. Blood.
2004;104:675-686.
10. Attar EC, Scadden DT. Regulation of hematopoietic stem cell growth. Leukemia. 2004;18:17601768.
11. Jordan CT, McKearn JP, Lemischka IR. Cellular
and developmental properties of fetal hematopoietic stem cells. Cell. 1990;61:953-963.
12. Pawliuk R, Eaves C, Humphries RK. Evidence of
both ontogeny and transplant dose regulated expansion of hematopoietic stem cells in vivo.
Blood. 1996;88:2852-2858.
13. Metcalf D. The molecular control of cell division,
15. Iscove NN, Nawa K. Hematopoietic stem cells
expand during serial transplantation in vivo without apparent exhaustion. Curr Biol. 1997;7:805808.
16. Goff JP, Shields DS, Greenberger JS. Influence
of cytokines on the growth kinetics and immunophenotype of daughter cells resulting from the
first division of single CD34⫹Thy⫹lin⫺ cells.
Blood. 1998;92:4098-4107.
17. Robinson SN, Ng J, Niu T, et al. Superior ex vivo
cord blood expansion following co-culture with
bone marrow-derived mesenchymal stem cells.
Bone Marrow Transplant. 2006;37:359-366.
18. Williams DA. Ex vivo expansion of hematopoietic
stem and progenitor cells—robbing Peter to pay
Paul? Blood. 1993;81:3169-3172.
19. Stiff P, Chen B, Franklin W, et al. Autologous
transplantation of ex vivo expanded bone marrow
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3578
BLOOD, 15 APRIL 2007 䡠 VOLUME 109, NUMBER 8
ARAKI et al
cells grown from small aliquots after high-dose
chemotherapy for breast cancer. Blood. 2000;95:
2169-2174.
35. Cheng T, Rodrigues N, Shen H, et al. Hematopoietic stem cell quiescence maintained by p21cip1/
waf1. Science. 2000;287:1804-1808.
20. Hoffman R. Progress in the development of systems for in vitro expansion of human hematopoietic stem cells. Curr Opin Hematol. 1999;6:184191.
36. Cheng T, Rodrigues N, Dombkowski D, Stier S,
Scadden DT. Stem cell repopulation efficiency but
not pool size is governed by p27(kip1). Nat Med.
2000;6:1235-1240.
21. McNiece I. Ex vivo expansion of hematopoietic
cells. Exp Hematol. 2004;32:409-410.
37. Wilson A, Murphy MJ, Oskarsson T, et al. c-Myc
controls the balance between hematopoietic stem
cell self-renewal and differentiation. Genes Dev.
2004;18:2747-2763.
22. Rosler E, Brandt J, Chute J, Hoffman R. Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34⫹CD38- cells. Exp Hematol. 2000;28:
841-852.
23. Brandt JE, Bartholomew AM, Fortman JD, et al.
Ex vivo expansion of autologous bone marrow
CD34⫹ cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons. Blood. 1999;94:106113.
38. Steinman R, Yaroslavskiy B, Goff JP, Alber SM,
Watkins SC. Cdk-inhibitors and exit from quiescence in primitive haematopoietic cell subsets.
Br J Haematol. 2004;124:358-365.
immunodeficient mice of human CD34⫹ cord
blood cells after ex vivo expansion: evidence for
the amplification and self-renewal of repopulating
stem cells. Blood. 1999;93:3736-3749.
50. Tanavde VM, Malehorn MT, Lumkul R, et al. Human stem-progenitor cells from neonatal cord
blood have greater hematopoietic expansion capacity than those from mobilized adult blood. Exp
Hematol. 2002;30:816-823.
51. Ueda T, Tsuji K, Yoshino H, et al. Expansion of
human NOD/SCID-repopulating cells by stem cell
factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and
soluble IL-6 receptor. J Clin Invest. 2000;105:
1013-1021.
39. Stier S, Cheng T, Forkert R, et al. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of
human hematopoietic stem cells. Blood. 2003;
102:1260-1266.
52. Hess DA, Wirthlin L, Craft TP, et al. Selection
based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells. Blood. 2006;107:
2162-2169.
24. Araki H, Mahmud N, Milhem M, et al. Expansion
of human umbilical cord blood SCID-repopulating
cells using chromatin modifying agents. Exp Hematol. 2006;34:140-149.
40. Miyake N, Brun AC, Magnusson M, Miyake K,
Scadden DT, Karlsson S. HOXB4-induced selfrenewal of hematopoietic stem cells is significantly enhanced by p21 deficiency. Stem Cells.
2006;24:653-661.
53. Ohishi K, Varnum-Finney B, Bernstein ID. The
notch pathway: modulation of cell fate decisions
in hematopoiesis. Int J Hematol. 2002;75:449459.
25. Habibian HK, Petersm SO, Hsieh CC, et al. The
fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med.
1998;188:393-398.
41. Klump H, Schiedlmeier B. Baum C. Control of
self-renewal and differentiation of hematopoietic
stem cells: HOXB4 on the threshold. Ann N Y
Acad Sci. 2005;1044:6-15.
54. Pevny L, Simon MC, Robertson E, et al. Erythroid
differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor
GATA-1. Nature. 1991;349:257-260.
26. Milhem M, Mahmud N, Lavelle D, et al. Modification of hematopoietic stem cell fate by 5aza2⬘ deoxycytidine and trichostatin A. Blood. 2004;103:
4102-4110.
42. Araki H, Katayama N, Yamashita Y, et al. Reprogramming of human postmitotic neutrophils into
macrophages by growth factor. Blood. 2004;103:
2973-2980.
55. Visvader JE, Elefanty AG, Strasser A, Adams JM.
GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J.
1992;11:4557-4564
27. Stein MI, Zhu J, Emerson SG. Molecular pathways regulating the self-renewal of hematopoietic
stem cells. Exp Hematol. 2004;32:1129-1136.
43. Mahmud N, Katayama N, Itoh R, et al. A possible
change in doubling time of haemopoietic progenitor cells with stem cell development. Br J Haematol. 1996;94:242-249.
56. Guo Y, Engelhardt M, Wider D, Abdelkarim M,
Lubbert M. Effects of 5-aza-2⬘-deoxycytidine on
proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells.
Leukemia. 2006;20:115-121.
28. Amsellem S, Pflumio F, Bardinet D, et al. Ex vivo
expansion of human hematopoietic stem cells by
direct delivery of the HOXB4 homeoprotein. Nat
Med. 2003;9:1423-1427.
29. Krosl J, Austin P, Beslu N, Kroon E, Humphries
RK, Sauvageau G. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4
protein. Nat Med. 2003;9:1428-1432.
30. Park IK, Qian D, Kiel M, et al. Bmi-1 is required
for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003;423:302-305.
31. Iwama A, Oguro H, Negishi M, et al. Enhanced
self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity. 2004;21:843-851.
32. Iwama A, Oguro H, Negishi M, Kato Y, Nakauchia
H. Epigenetic regulation of hematopoietic stem
cell self-renewal by polycomb group genes. Int
J Hematol. 2005;81:294-300.
33. Tsai FY, Keller G, Kuo FC, et al. An early haematopoietic defect in mice lacking the transcription
factor GATA-2. Nature. 1994;371:221-226.
34. Varnum-Finney B, Brashem-Stein C, Bernstein
ID. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors
with lymphoid and myeloid reconstituting ability.
Blood. 2003;101:1784-1789.
44. Coneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord
blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating
activity in nonobese diabetic-scid/scid mice. Proc
Natl Acad Sci U S A. 1997;94:9836-9841.
45. Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch
B, Dick JE. Quantitative analysis reveals expansion of human hematopoietic repopulating cells
after short-term ex vivo culture. J Exp Med. 1997;
186:619-624.
46. Scandura JM, Boccuni P, Massague J, Nimer SD.
Transforming growth factor beta-induced cell
cycle arrest of human hematopoietic cells requires p57KIP2 up-regulation. Proc Natl Acad Sci
U S A. 2004;101:15231-15236.
47. Mahmud N, Rose D, Pang W, Walker R, Patil V,
Weich, N, et al. Characterization of primitive marrow CD34⫹ cells that persist after a sublethal
dose of total body irradiation. Exp Hematol. 2005;
33:1388-1401.
48. Heid CA, Stevens J, Livak KL, Williams PM. Real
time quantitative PCR. Genome Res. 1996;6:
986-994.
49. Piacibello W, Sanavio F, Severino A, et al. Engraftment in nonobese diabetic severe combined
57. Mahmud N, Milhem M, Araki H, Hoffman R. Alteration of hematopoietic stem cell fates by chromatin-modifying agents. In: Ho AD, Hoffman R, Zanjani ED, eds. Frontiers in Stem Cell Transplantation.
Weinheim, Germany: Wiley-VCH; 2006:27-42.
58. Ho AD. Kinetics and symmetry of divisions of hematopoietic stem cells. Exp Hematol. 2005;33:
1-8.
59. Srour EF. Proliferative history and hematopoietic
function of ex vivo expanded human CD34⫹ cells.
Blood. 2000;96:1609-1612.
60. Young JC, Lin K, Hansteen G, et al. CD34⫹ cells
from mobilized peripheral blood retain fetal bone
marrow repopulating capacity within the Thy-1⫹
subset following cell division ex vivo. Exp Hematol. 1999;27:994-1003.
61. Satoh Y, Matsumura I, Tanaka H, et al. Roles for
c-Myc in self-renewal of hematopoietic stem cells.
J Biol Chem. 2004;279:24986-24993.
62. De Felice L, Tatarelli C, Mascolo MG, et al. Histone deacetylase inhibitor valproic acid enhances
the cytokine-induced expansion of human hematopoietic stem cells. Cancer Res. 2005;65:15051513.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2007 109: 3570-3578
doi:10.1182/blood-2006-07-035287 originally published
online December 21, 2006
Chromatin-modifying agents permit human hematopoietic stem cells to
undergo multiple cell divisions while retaining their repopulating
potential
Hiroto Araki, Kazumi Yoshinaga, Piernicola Boccuni, Yan Zhao, Ronald Hoffman and Nadim Mahmud
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