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Expression of Integrin β3 Is Correlated to the
Properties of Quiescent Hemopoietic Stem
Cells Possessing the Side Population
Phenotype
Terumasa Umemoto, Masayuki Yamato, Yoshiko
Shiratsuchi, Masao Terasawa, Joseph Yang, Kohji Nishida,
Yoshiro Kobayashi and Teruo Okano
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 177:7733-7739; ;
doi: 10.4049/jimmunol.177.11.7733
http://www.jimmunol.org/content/177/11/7733
The Journal of Immunology
Expression of Integrin ␤3 Is Correlated to the Properties
of Quiescent Hemopoietic Stem Cells Possessing the Side
Population Phenotype1
Terumasa Umemoto,* Masayuki Yamato,* Yoshiko Shiratsuchi,† Masao Terasawa,†
Joseph Yang,* Kohji Nishida,‡ Yoshiro Kobayashi,† and Teruo Okano2*
I
n stem cell biology, the identification of markers that distinguish stem cells from their differentiated progeny is most
essential for a clear understanding of stem cell-related properties. The side population (SP)3 phenotype is characterized by
cells with the capacity to efflux the DNA-binding dye Hoechst
33342 via the ATP-binding cassette transporter G2 (ABCG2), and
is a reliable marker of hemopoietic stem cells (HSCs) with the
ability for long-term multilineage reconstitution (1, 2). More recently, SP cells from numerous other adult tissues and species have
also demonstrated stem cell-like properties (3– 6), suggesting that
the SP phenotype is a common feature of tissue-specific stem cells.
The enrichment of adult stem cells based on the SP phenotype has
therefore proven to be an extremely useful tool in the isolation of
stem cells from a variety of tissue systems.
In the corneal epithelial system, a model tissue for epithelial
stem cell biology, stem cells are thought to reside in the basal layer
*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; †Division of Molecular Medicine, Department of Biomolecular Science, Toho University, Chiba, Japan; and ‡Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Miyagi,
Japan
Received for publication July 17, 2006. Accepted for publication September 12, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the Center of Excellence Program for the 21st
Century and the High-Tech Research Center Program, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; the Core Research for Evolution Science and Technology Program by the Japan Science and Technology Agency;
and the Core to Core Program from the Japan Society for the Promotion of Science.
2
Address correspondence and reprint requests to Dr. Teruo Okano, Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University,
8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail address: tokano@
abmes.twmu.ac.jp
3
Abbreviations used in this paper: SP, side population; NSP, non-SP; ABCG2, ATPbinding cassette transporter G2; HSC, hemopoietic stem cell; PY, pyronin Y; Lin,
lineage.
Copyright © 2006 by The American Association of Immunologists, Inc.
of the limbal epithelium (7–9), which is located at the transitional
zone between the cornea and the peripheral bulbar conjunctiva. We
have previously demonstrated that limbal epithelial SP cells have
stem cell-like phenotypes and, similar to HSCs, are maintained in
the quiescent state (10). However, while the ability for Hoechst
dye efflux via ABCG2 is now considered a general characteristic of
adult stem cells, other molecules that may be common markers of
various tissue-specific stem cells remain unidentified.
Integrin receptors are heterodimers composed of ␣- and
␤-chains and function by binding to ligands that are components of
the extracellular matrix, as well as some soluble ligands such as the
ICAMs, which can lead to aggregation of cells. In addition to
mediating cell adhesion and cytoskeletal organization, integrins
can also function as cell-signaling receptors. Signal transduction
pathways involving integrins play a role in many biological processes, including cell growth, differentiation, migration, and apoptosis. Based on the numerous roles of the integrin family of receptors, it can be reasoned that specific integrin subunits are
related to stem cell niches and may be available as surface markers
of these stem cells.
Previously, integrin ␤1 (CD29) has been shown to be highly
expressed and able to induce adhesion to niche cells such as osteoblasts in quiescent HSCs treated with Tie-2 (11). Recently, expression of integrin ␣2 (CD49b) has also been used to distinguish
HSCs possessing the capacity for long-term hemopoiesis (12).
Moreover, from integrin subunit profiling, we have observed that
limbal epithelial SP cells, which closely resemble HSCs, have a
significantly higher expression of integrin ␣1 (CD49a), integrin ␣4
(CD49d), and integrin ␤3 (CD61) when compared with non-SP
(NSP) cells (data not shown).
Integrin ␤3 is commonly associated with either integrin ␣V
(CD51) or integrin ␣IIb (CD41), and participates in many biological process including cell adhesion, aggregation, and migration in
several cells (13, 14). Although it has been reported that integrin
␣IIb␤3 is expressed in mouse embryonic and neonatal HSCs (15,
0022-1767/06/$02.00
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With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of
considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye
Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high
expression of integrin ␤3 (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed
that the expression of integrin ␤3 is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the
strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineageⴚ (Linⴚ)/integrin ␤3high (␤3high) SP cells have as strong a capacity for long-term hemopoiesis as c-Kitⴙ/Sca-1ⴙ/Linⴚ SP
cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin ␤3 subunit that is present
in SP cells having the properties of HSCs, is associated with integrin ␣v (CD51). Therefore, our results demonstrate that high
expression of integrin ␤3 is correlated to the properties of quiescent HSCs and suggest that the integrin ␤3 subunit is
available as a common surface marker of tissue-specific stem cells. The Journal of Immunology, 2006, 177: 7733–7739.
QUIESCENT HSCs EXPRESS INTEGRIN ␤3
7734
16), the expression of the integrin ␤3 subunit has not been previously correlated to adult stem cells. In this study, we show that
expression of integrin ␤3 is correlated to the properties of
quiescent HSCs that possess the SP phenotype. We also demonstrate that this expression of integrin ␤3 is correlated to the presence of integrin ␣V within this population of HSCs with the ability
for long-term multilineage reconstitution.
Materials and Methods
All animal experiments were performed according to the Guidelines of
Tokyo Women’s Medical University on Animal Use, the Principles of
Laboratory Animal Care formulated by the National Society for Medical
Research, and the Guide for the Care and Use of Laboratory Animals
prepared by the Institute of Laboratory Animal Resources and published by
the National Institutes of Health (National Institutes of Health Publication
No. 86-23, revised 1985).
Antibodies
The following mAbs were used for FACS and flow cytometric analysis: anti-CD61/integrin ␤3 (2C9.G2), anti-CD51/integrin ␣V (RMV-7),
FIGURE 2. mRNA expression of markers for quiescent SP cells. Lin⫺ cells were stained with Hoechst
33342 and sorted into SP and NSP cells, respectively
(A). Lin⫺ SP cells were then analyzed and separated into
either Lin⫺/␤3high or Lin⫺/␤3low SP cells, respectively
(B), or c-Kit⫹/Sca-1⫹/Lin⫺ (KSL) SP cells (C). Graphs
represent mRNA expression of ABCG2 (D) and p57Kip2
(E) in the various cell fractions, as determined by realtime quantitative RT-PCR. Data are presented as
mean ⫾ SD (ⴱⴱ, p ⬍ 0.05).
anti-CD41/integrin ␣IIb (MWReg30), anti-Sca-1 (E13-161.7), anti-c-Kit
(2B8), anti-endoglin (MJ7/18; Santa Cruz Biotechnology), anti-CD150
(TC15-12F12.2; BioLegend), anti-CD45.2 (104), anti-CD45.1 (A20),
anti-B220/CD45R (RA3-6B2), anti-Mac-1 (M1/70), anti-Gr-1 (RB68C5), anti-CD4 (L3T4), and anti-CD8 (53-6.72). All Abs were obtained
from BD Biosciences/BD Pharmingen unless otherwise noted.
For immunoprecipitation and Western blotting experiments, antiintegrin ␤3 (C-20; Santa Cruz Biotechnology) and anti-integrin ␤3 (N-20;
Santa Cruz Biotechnology) Abs were used, respectively. Peroxidase-linked
polyclonal anti-goat IgG (Amersham Biosciences) was used as secondary
Ab for Western blotting.
Cell preparation
Cell suspensions of bone marrow from C57BL/6 and C57BL/6-Ly5.1 congenic mice were prepared as described previously (17).
FACS and flow cytometric analysis
Analysis and sorting of SP cells were performed as described previously
(10, 17). Briefly, isolated bone marrow cells were stained with 5 ␮g/ml
Hoechst 33342 (Sigma-Aldrich) at a concentration of 106 cells/ml in staining medium (DMEM containing 2% FBS (Moregate Biotech) and 10 mM
HEPES) for 90 min at 37°C. After staining, cells were resuspended in
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FIGURE 1. Expression of integrin chains in SP cells derived from mouse bone marrow. A, mRNA expression of integrin subunits in mouse bone marrow cells.
The dot plot of Hoechst staining denotes sorting gates for NSP and SP cells (a). Graphs represent mRNA expression of integrins ␣1 (b), ␣4 (c), and ␤3 (d). Data
are presented as mean ⫾ SD (ⴱ, p ⬍ 0.01). B, Expression of integrin ␤3 at the protein level in Lin⫺ SP cells. Upon staining of Lin⫺ cells (obtained by MACS)
with Hoechst 33342 (a), Lin⫺ SP cells were stained with anti-integrin ␤3 Abs (b). White histogram, Isotype control; gray histogram, integrin ␤3. After immunoprecipitation for integrin ␤3 in Lin⫺ SP cells, Western blotting with another anti-integrin ␤3 Ab was performed (c). C, Expression of integrin ␤3 is correlated
to the SP phenotype in mouse bone marrow. Lin⫺ cells were stained with Hoechst 33342, followed by staining with anti-integrin ␤3 Abs. Cells were sorted based
on the strength of Hoechst 33342 efflux (a), and integrin ␤3 expression was analyzed in Tip-SP (b), Middle-SP (c), and Upper-SP (d) cells. White histogram, Isotype
control; gray histogram, integrin ␤3. The graph represents the percentage of ␤3⫹ cells in each fraction (e). Data are presented as mean ⫾ SD (ⴱ, p ⬍ 0.01).
The Journal of Immunology
7735
FIGURE 3. PY staining in mouse bone marrow cells. The cell cycle state of cells present in each sorting gate was then analyzed by PY staining, with
PY⫺ cells indicating cells in the G0 state. Dot plots of PY staining in mouse bone marrow cells are presented for NSP cells (A), Lin⫺ SP cells (B), Lin⫺/
␤3low SP cells (C), Lin⫺/␤3high SP cells (D), and KSL SP cells (E). The graph represents percentage of PY⫺ cells (cells in G0 phase) in each fraction (F).
Data are presented as mean ⫾ SD (ⴱⴱ, p ⬍ 0.05).
Gene expression analysis
For gene expression assays, total RNA was obtained from 10,000 cells of
each population, using Isogen (Nippongene) according to the manufacturer’s suggested protocol. Single-stranded cDNA was created with the Superscript First-strand System for RT-PCR (Invitrogen Life Technologies),
and used as PCR templates. Primer pairs and TaqMan MGB probes labeled
with FAM at the 5⬘ end and nonfluorescent quencher at the 3⬘ end, were
designed with the TaqMan gene expression assay (Applied Biosystems).
Quantitative PCR was performed with the 7300 Real Time PCR System
(Applied Biosystems). Thermocycling programs consisted of an initial cycle at 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C
for 15 s and 60°C for 1 min. All assays were run in duplicate for more than
four individual samples. mRNA expression levels were normalized with
the expression level of GAPDH. To compare mRNA expression between
cell populations, the Mann-Whitney rank sum test was applied. Statistics
were calculated using SigmaStat 2.0 (SPSS).
Immunoprecipitation and Western blotting
Sorted cells were lysed in buffer (TBS containing 50 mM n-octyl-␤-Dglucosidase, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM PMSF, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin), followed by immunoprecipitation with
specific Ab and protein G Sepharose (Amersham Biosciences) for 3 h at
4°C. Immunoprecipitated proteins were separated on 7.5% polyacrylamide
gels and transferred to Immobilon-P membranes (Millipore). After blocking, membranes were sequentially incubated with primary and secondary
Abs followed by development with ECL (ECL Advance; Amersham
Biosciences).
Long-term competitive repopulation assay
C57BL/6 mice irradiated at 11 Gy total, were transplanted with 100 test
cells prepared from C57BL/6-Ly5.1 mice and 2 ⫻ 105 mononuclear bone
marrow cells obtained from C57BL/6-Ly5.2 mice. Three months after
transplantation, mononuclear cells isolated from the peripheral blood were
analyzed.
Results
Mouse bone marrow SP cells have high expression of
integrin ␤3
Because integrin subunit profiling showed that limbal epithelial SP
cells had higher expression of integrins ␣1, ␣4, and ␤3 when compared with NSP cells at the mRNA level (data not shown), we
speculated that the high expression of these integrin chains may be
conserved among SP cells isolated from various tissues. When
gene expression of these integrin mRNAs was examined in mouse
bone marrow, only integrin ␤3 showed significantly higher expression in SP cells compared with NSP cells (Fig. 1A). Moreover,
flow cytometric analysis and immunoprecipitation followed by
Western blotting also showed that within Lin⫺ SP cells, integrin
␤3 was expressed (Fig. 1B).
Expression of integrin ␤3 is correlated to the relative strength of
the SP phenotype
To further investigate the relationship between integrin ␤3 and the
SP phenotype, mouse bone marrow cells were subjected to
Hoechst exclusion assay and sorted into finer SP gates (Fig. 1Ca).
As the strength of the SP phenotype was amplified (indicated by
lower Hoechst fluorescence intensity due to greater dye efflux), the
percentage of integrin ␤3⫹ cells also increased (Fig. 1C). In particular, nearly all cells within the Tip-SP fraction, which has the
highest expression of ABCG2 (2), were ␤3⫹ cells (Fig. 1C). Furthermore, we investigated whether SP cells possessing stronger
expression of integrin ␤3 also show high expression of ABCG2,
which is the molecular determinant of the SP phenotype. To completely prevent contamination by integrin ␤3⫺ cells, “high” and
“low” gates containing the highest 30% and the lowest 30% of
fluorescence intensity were isolated based on integrin ␤3 staining
plots (Fig. 2, A and B). Significantly higher mRNA higher expression of ABCG2 was observed in Lin⫺/␤3high SP cells compared
with the Lin⫺ NSP, Lin⫺ SP, and Lin⫺ ␤3low SP fractions (Fig.
2D). Interestingly, the Lin⫺/␤3high SP fraction also had comparable ABCG2 mRNA levels compared with c-Kit⫹/Sca-1⫹/Lin⫺
(KSL) SP cells (Fig. 2, C and D), which are considered one of the
most highly enriched HSC populations (11).
Lin⫺/integrin ␤3high SP cells reside in the quiescent state
Currently, the SP phenotype is considered the single characteristic
that most accurately identifies quiescent stem cells in the bone
marrow niche (11). Pyronin Y (PY) staining demonstrated that the
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Dulbecco’s PBS containing 2% FBS and 1 mM HEPES. Before analysis
and cell sorting, propidium iodide (Sigma-Aldrich) was added at a final
concentration of 2 ␮g/ml, to distinguish between live and nonviable cells.
Analysis and cell sorting were then performed using a dual laser fluorescence-activated cell sorter (Epics Altra FACS analysis system; Beckman
Coulter).
For the isolation of specific cell populations, Lineage marker-positive
cells were first eliminated by magnetic cell sorting (Auto MACS system;
Miltenyi Biotec) using the Lineage Cell Depletion kit (Miltenyi Biotec),
before staining with Hoechst 33342. Lineage⫺ (Lin⫺) cells were stained
with Hoechst 33342 and were then incubated with the corresponding Abs
for 30 min on ice. In cases of biotinylated Abs, cells were stained with
streptavidin-conjugated CyChrome (BD Pharmingen) for 30 min at 4°C
before analysis. Stained cells were then subjected to sorting by FACS.
QUIESCENT HSCs EXPRESS INTEGRIN ␤3
7736
Table I. Multilineage repopulation by Lin⫺/␤3high SP cellsa
T Cell
51.04 ⫾ 0.88
B Cell
Monocyte/Granulocyte
28.54 ⫾ 2.51
35.71 ⫾ 1.80
The percentages of CD4 or CD8 (T cell lineage), B220/CD45R⫹ (B cell
lineage), and Mac-1⫹ or Gr-1⫹ (monocyte/granulocyte lineage) cells in Ly-5.1⫹ (donor-derived cells) of mice transplanted with Lin⫺/␤3high SP fractions. Data are presented as mean ⫾ SD.
a
⫹
⫹
Lin⫺/integrin ␤3high SP cells have stem cell properties
Lin⫺/␤3high SP fraction possessed a significantly higher number of
cells that resided in the G0 phase (PY⫺ cells) compared with all cell
populations and also showed the same percentage as KSL SP cells
(Fig. 3).
p57Kip2 is a member of the Cip/Kip family of cyclin-dependent
kinase inhibitors and is a potent negative regulator of the cell cycle
(18). In bone marrow SP cells, increased expression of p57Kip2 has
been previously observed, with significantly higher levels in KSL
SP cells (17). Therefore, it appears that the expression of p57Kip2
may be closely involved in the maintenance of stem cell quiescence. Our results showed that Lin⫺/␤3high SP cells had increased
p57Kip2 expression compared with the Lin⫺ NSP, Lin⫺ SP, and
Lin⫺ ␤3low SP fractions, and have mRNA levels as high as KSL
SP cells (Fig. 2E). We also confirmed that p21Cip1, another cyclindependent kinase inhibitor related to HSC quiescence (19), was
expressed in Lin⫺/␤3high SP cells, but without a significant difference compared with Lin⫺/␤3low SP cells (data not shown). Thus,
nearly all of Lin⫺/␤3high SP cells appear to be cell cycle arrested,
with a percentage of G0 cells that is comparable to the KSL SP
fraction. These results indicate that the expression of integrin ␤3 is
directly correlated to increased strength of the quiescent SP
phenotype.
Integrin ␤3 is associated with integrin ␣V in hemopoietic stem
cells with the SP phenotype
In general, integrins function as receptors by forming heterodimers
with the association of ␣ and ␤ subunits, and integrin ␤3 has commonly been associated with either integrin ␣V or integrin ␣IIb.
Although previous reports have suggested that that integrin ␣IIb␤3
is expressed in mouse embryonic and neonatal HSCs (15, 16), the
presence of the integrin ␤3 subunit has not been related to adult
stem cells. To examine which ␣ subunit was associated with integrin ␤3 in SP cells, Hoechst exclusion assays were performed
using Lin⫺/␤3⫹/integrin ␣V⫹ (␣V⫹) or Lin⫺/␤3⫹/integrin ␣IIb⫹
(␣IIb⫹) fractions. Results showed that the Lin⫺/␤3⫹/␣V⫹ fraction
contained a much higher percentage of SP cells, compared with
Lin⫺/␤3⫹/␣IIb⫹ cells or Lin⫺/␤3⫹/integrin ␣V⫺ (␣V⫺) (Fig. 5A).
Moreover, Hoechst exclusion assays using Lin⫺/␣V⫹ or
Lin⫺/␣IIb⫹ fractions also showed that Lin⫺/␣V⫹ fraction not only
contained a higher percentage of SP cells than Lin⫺/␣IIb⫹ cells,
but also closely resembled the distribution plots for the Lin⫺/␤3⫹
fraction (data not shown). When expression of Sca-1, endoglin,
and CD150 were examined, Lin⫺/␤3⫹/␣V⫹ SP cells also demonstrated significantly higher expression of these stem cell markers,
in comparison to Lin⫺/␤3⫹/␣IIb⫹ SP cells or Lin⫺/␤3⫹/␣V⫺ SP
cells (Fig. 5B). These results implied that in the subfraction of SP
cells that possessed the properties of HSCs, the expression of integrin ␤3 was associated with integrin ␣V. To further confirm this
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FIGURE 4. Lin⫺/integrin ␤3high SP cells have hemopoietic stem cell
properties. A, Mouse bone marrow Lin⫺/␤3low SP and Lin⫺/␤3high SP
cells were analyzed for expression of hemopoietic stem cell markers.
Lin⫺/␤3low SP and Lin⫺/␤3high SP cells were double-stained for Sca-1
and c-Kit (a and b), stained for endoglin (d and e), or stained for CD150
(g and h). (a, d, and g) and (b, e, and h) represent Lin⫺/␤3low SP cells
and Lin⫺/␤3high SP cells, respectively. Graphs show the percentage of
KSL cells (c), endoglin⫹ cells (f ), and CD150⫹ cells (i), respectively. Data are
presented as mean ⫾ SD (ⴱ, p ⬍ 0.01). B, Long-term competitive repopulation
assay performed with Lin⫺/␤3low SP and Lin⫺/␤3high SP cells. One hundred Lin⫺/␤3low SP, Lin⫺/␤3high SP, or KSL SP cells derived from C57BL/
6-Ly5.1 mice, and 2 ⫻ 105 mononuclear bone marrow cells from C57BL/
6-Ly5.2 mice were transplanted into lethally irradiated C57BL/6-Ly5.2
mice. The plot represents the percentage of donor-derived cells (percentage
of Ly5.1⫹ cells) in the peripheral blood of each recipient animal, 3 mo after
bone marrow transplantation. Bars represent mean values (ⴱ, p ⬍ 0.01).
To confirm that quiescent Lin⫺/␤3high SP cells possessed stem
cell-like properties, several markers of HSCs were examined.
Analysis demonstrated that Lin⫺/␤3high SP cells contained a
significantly higher percentage of c-Kit⫹/Sca-1⫹, endoglin⫹, and
CD150⫹ cells, compared with Lin⫺/␤3low SP cells (Fig. 4A). Endoglin, a homolog of the type III TGF-␤ receptor, is differentially
expressed by HSCs (20) and believed to account for all long-term
repopulating HSCs within bone marrow SP cells (20, 21). CD150,
a member of the signaling lymphocytic activation molecule
(SLAM) family of receptors that regulate lymphocyte signaling, is
specifically expressed by HSCs, and can be used to isolate selfrenewing stem cells from more differentiated lineages, including
transiently renewing multipotent progenitors (MPPs) (22). Together, these results indicate that Lin⫺/␤3high SP cells account for
a greater proportion of HSCs within the mouse bone marrow SP
fraction than Lin⫺/␤3low SP cells. Moreover, using competitive
reconstruction assays, we confirmed that Lin⫺/␤3high SP cells have
a significantly higher potential for long-term hemopoietic reconstitution compared with Lin⫺/␤3low SP cells (Fig. 4B). Three
months after reconstitution with 100 donor cells, Lin⫺/␤3high SP
cells showed an increased ability to reconstruct all hemopoietic
lineages in the transplanted animals (Table I). Additionally,
Lin⫺/␤3high SP cells also had as high ability for hemopoiesis as
KSL SP cells (Fig. 4B), indicating that expression of integrin ␤3
can distinguish HSCs from other cell types within the SP fraction
as effectively as the combination of Sca-1 and c-Kit.
The Journal of Immunology
7737
hypothesis, bone marrow cells triple-stained for integrins ␣V, ␣IIb,
and ␤3 were examined. Flow cytometric analysis showed that only
Lin⫺/␤3⫹/␣V⫹/integrin ␣IIb⫺ (␣IIb⫺) SP cells had enhanced
Hoechst exclusion capabilities with localization to the Tip-SP fraction that possesses the highest strength of the SP phenotype (Fig.
6, A–D). Moreover, long-term repopulation assays also showed
that the strong ability for long-term hemopoiesis was demonstrated
only with the Lin⫺/␤3⫹/␣V⫹/␣IIb⫺ SP fraction (Fig. 6E). These
results confirmed that SP cells possessing the features of HSCs,
expressed integrin ␣V and not integrin ␣IIb within the Lin⫺/␤3⫹
gate. Previous reports have shown that integrin ␣IIb is not expressed by adult HSCs and that integrin ␣IIb⫹ cells have no ability
for hemopoiesis (15, 16, 22). Therefore, it appeared likely that
integrin ␤3 is associated with integrin ␣v in the portion of SP cells
that have the properties of adult stem cells.
Discussion
In this study, we present strong evidence that quiescent HSCs possessing the SP phenotype express integrin ␣V␤3. Integrin ␣V␤3
also known as CD51/CD61 or the vitronectin receptor; is related to
adhesion, movement, and apoptosis in several cell types (13, 14),
but has not been previously correlated to adult stem cells. Although the expression of integrin ␣V␤3 in SP cells could not be
directly investigated, integrin ␤3 has previously only been associated with integrins ␣V and ␣llb, and adult HSCs have much lower
or no expression of the integrin ␣llb subunit (Figs. 5 and 6) (15, 16,
FIGURE 6. Integrin ␤3 is coupled to integrin ␣V in
hemopoietic stem cells with the SP phenotype. Lin⫺
␤3⫹ cells were stained with Abs for integrin ␣V and
integrin ␣IIb (A), or isotype controls (ICs) for these integrin ␣-chains (B) followed by analysis with Hoechst
33342 exclusion assays in Lin⫺/␤3⫹/␣V⫹/␣IIblow cells (C),
Lin⫺/␤3⫹/␣V⫹/␣IIb⫹ cells (D), and Lin⫺/␤3⫹/␣V⫺/␣IIb⫹
cells (E). Long-term competitive repopulation assay
performed with test cells isolated from the corresponding sorting gates. One hundred test cells derived from
each population were then injected into C57BL/6-Ly5.2
mice. The plot represents the percentage of donor-derived cells (percentage of Ly5.1⫹ cells) in the peripheral
blood of each recipient mouse, 3 mo after bone marrow
transplantation (F ). Bars represent mean values (ⴱ, p ⬍
0.01).
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FIGURE 5. Hemopoietic stem cell properties in
Lin⫺/integrin ␤3⫹/integrin ␣V ⫹ SP cells and Lin⫺/integrin ␤3⫹/integrin ␣llb⫹ SP cells. A, Analysis of integrin ␣-chain expression in Lin⫺/␤3⫹ SP cells. Lin⫺
bone marrow cells were double-stained with Abs for
integrin ␤3 and integrin ␣V (a); integrin ␤3 and integrin
␣IIb (e); isotype controls (ICs) for integrin ␤3 and integrin ␣V (b) or ICs for integrin ␤3 and integrin ␣IIb (f ),
followed by analysis of Hoechst 33342 exclusion assays
in Lin⫺/␤3⫹/␣V⫹ cells (c), Lin⫺/␤3⫹/␣V⫺ cells (d) and
Lin⫺/␤3⫹/␣IIb⫹ cells (g). B, Analysis of stem cell markers expressed in Lin⫺/␤3⫹/␣V⫹ SP cells, Lin⫺/␤3⫹/␣V⫹
SP cells, and Lin⫺/␤3⫹/␣IIb⫹ SP cells. Graphs show the
percentages of Sca-1⫹ cells (a), endoglin⫹ cells (b), and
CD150⫹ cells (c), respectively. Data are presented as
mean ⫾ SD (ⴱ, p ⬍ 0.01).
QUIESCENT HSCs EXPRESS INTEGRIN ␤3
7738
interactions, quiescence, and the manifestation of the SP
phenotype.
Disclosures
The authors have no financial conflict of interest.
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22). Therefore, although the possibilities that integrin ␤3 may be
heterodimerized with other integrin ␣ subunits, associated with
other molecules, or even exist independently in SP cells, cannot be
excluded; our result strongly suggest that HSCs possessing the SP
phenotype showed high expression of integrin ␣V␤3. In addition,
at the very least, our results definitively show that expression of
integrin ␤3 is correlated to the features of HSCs and is as effective
as Sca-1 and c-Kit for identifying HSCs within bone marrow SP
cells. This expression of integrin ␤3 has never been previously
correlated to adult tissue-specific stem cells, including HSCs.
In the hemopoietic system, several cell types such as megakaryocytes and macrophages, also express integrin ␤3, with most
of these differentiated cells likely present within NSP gate. Therefore, in bone marrow SP cells, the relationship between integrin ␤3
expression and stem cell properties may have previously gone unnoticed. Via screening of integrin chain mRNA expression in limbal epithelial SP cells that closely resemble quiescent HSCs, significant insight into the relationship between integrin ␤3 and stem
cell properties in the SP fraction could thus be identified.
Although only compared in the limbal epithelium via mRNA
expression, the requirements for enzymatic dissociation of epithelia into single cells, as well as the lack of an accepted and established reconstitution assay, currently remain limitations of epithelial stem cell biology that must be overcome. Nevertheless, our
results also demonstrated that high expression of integrin ␤3 was
commonly observed in SP cells derived from both the rabbit limbal
epithelium (data not shown) and mouse bone marrow, indicating
that high expression of integrin ␤3 may be a common feature of SP
cells that possess stem cell-like properties. Therefore, it is suggested that the integrin ␤3 subunit is available as a common marker
of tissue-specific stem cells, which may contribute to simple purification and isolation, when used in combination with the SP
phenotype.
Integrin ␤3 is known to associate with various signaling molecules such as CD47, also known as integrin-associated protein
(IAP) (23); the nectin and necl family of molecules (24, 25); and
␤3-endonexin, known as integrin ␤3 binding protein (26). It has
been reported that CD47 binding to SH2 domain-bearing protein
tyrosine phosphate substrate 1 (SHPS-1), also known as ligand of
CD47, is followed by inhibition of cell migration (27). The nectin
and necl family contribute to the formation of adherence junctions
composed of cadherins (28, 29), of which N-cadherin is a known
niche molecule of HSCs (11, 30). ␤3-endonexin inhibits retinoblastoma protein (pRB) kinase activity associated with cyclin ACdk2, while leaving histone H1 kinase activity unaffected (31).
Interestingly, many of these molecules that associate with integrin
␤3 are related to the negative regulation of various biological processes of cells, suggesting that some of these ligands may be related to the quiescence and maintenance of adult stem cells.
The expression of endoglin by Lin⫺/␤3⫹/␣v⫹ SP cells also suggests that TGF-␤ signaling may play a key role in the maintenance
of HSCs that may be linked to integrin ␣V␤3. Interestingly, TGF-␤
also induces the expression of integrin ␣V␤3 in human lung fibroblasts (32), as well as p57Kip2 in hemopoietic cells (33), both of
which are highly expressed by SP cells possessing the properties of
HSCs. Although further investigation into the specific mechanisms
involved is still required, our results indicate that signal transduction pathways associated to integrin ␤3 may likely be related to the
SP phenotype in adult stem cells.
Nevertheless, our findings demonstrate for the first time, that
quiescent HSCs with the SP phenotype demonstrate high expression of integrin ␤3. These results suggest that integrin ␤3 is exploitable as common marker of tissue-specific stem cells and may
contribute to the general determination of stemness such as niche
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