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The Journal of Immunology, 2006, 177: 7733-7739.
Copyright © 2006 by The American Association of Immunologists, Inc.

Expression of Integrin beta3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype1

Terumasa Umemoto*, Masayuki Yamato*, Yoshiko Shiratsuchi{dagger}, Masao Terasawa{dagger}, Joseph Yang*, Kohji Nishida{ddagger}, Yoshiro Kobayashi{dagger} and Teruo Okano2,*

* Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; {dagger} Division of Molecular Medicine, Department of Biomolecular Science, Toho University, Chiba, Japan; and {ddagger} Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Miyagi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 beta3 (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta3 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, Lineage– (Lin–)/integrin beta3high (beta3high) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit+/Sca-1+/Lin– SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta3 subunit that is present in SP cells having the properties of HSCs, is associated with integrin {alpha}v (CD51). Therefore, our results demonstrate that high expression of integrin beta3 is correlated to the properties of quiescent HSCs and suggest that the integrin beta3 subunit is available as a common surface marker of tissue-specific stem cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In 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, 4, 5, 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 of the limbal epithelium (7, 8, 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 {alpha}- and beta-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 beta1 (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 {alpha}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 {alpha}1 (CD49a), integrin {alpha}4 (CD49d), and integrin beta3 (CD61) when compared with non-SP (NSP) cells (data not shown).

Integrin beta3 is commonly associated with either integrin {alpha}V (CD51) or integrin {alpha}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 {alpha}IIbbeta3 is expressed in mouse embryonic and neonatal HSCs (15, 16), the expression of the integrin beta3 subunit has not been previously correlated to adult stem cells. In this study, we show that expression of integrin beta3 is correlated to the properties of quiescent HSCs that possess the SP phenotype. We also demonstrate that this expression of integrin beta3 is correlated to the presence of integrin {alpha}V within this population of HSCs with the ability for long-term multilineage reconstitution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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 beta3 (2C9.G2), anti-CD51/integrin {alpha}V (RMV-7), anti-CD41/integrin {alpha}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 (RB6-8C5), 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, anti-integrin beta3 (C-20; Santa Cruz Biotechnology) and anti-integrin beta3 (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 µg/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 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 µg/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.

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-beta-D-glucosidase, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/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 x 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mouse bone marrow SP cells have high expression of integrin beta3

Because integrin subunit profiling showed that limbal epithelial SP cells had higher expression of integrins {alpha}1, {alpha}4, and beta3 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 beta3 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 beta3 was expressed (Fig. 1B).


Figure 1
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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 {alpha}1 (b), {alpha}4 (c), and beta3 (d). Data are presented as mean ± SD (*, p < 0.01). B, Expression of integrin beta3 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 beta3 Abs (b). White histogram, Isotype control; gray histogram, integrin beta3. After immunoprecipitation for integrin beta3 in Lin– SP cells, Western blotting with another anti-integrin beta3 Ab was performed (c). C, Expression of integrin beta3 is correlated to the SP phenotype in mouse bone marrow. Lin– cells were stained with Hoechst 33342, followed by staining with anti-integrin beta3 Abs. Cells were sorted based on the strength of Hoechst 33342 efflux (a), and integrin beta3 expression was analyzed in Tip-SP (b), Middle-SP (c), and Upper-SP (d) cells. White histogram, Isotype control; gray histogram, integrin beta3. The graph represents the percentage of beta3+ cells in each fraction (e). Data are presented as mean ± SD (*, p < 0.01).

 
Expression of integrin beta3 is correlated to the relative strength of the SP phenotype

To further investigate the relationship between integrin beta3 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 beta3+ cells also increased (Fig. 1C). In particular, nearly all cells within the Tip-SP fraction, which has the highest expression of ABCG2 (2), were beta3+ cells (Fig. 1C). Furthermore, we investigated whether SP cells possessing stronger expression of integrin beta3 also show high expression of ABCG2, which is the molecular determinant of the SP phenotype. To completely prevent contamination by integrin beta3– cells, "high" and "low" gates containing the highest 30% and the lowest 30% of fluorescence intensity were isolated based on integrin beta3 staining plots (Fig. 2, A and B). Significantly higher mRNA higher expression of ABCG2 was observed in Lin–/beta3high SP cells compared with the Lin– NSP, Lin– SP, and Lin– beta3low SP fractions (Fig. 2D). Interestingly, the Lin–/beta3high 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).


Figure 2
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  		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–/beta3high or Lin–/beta3low 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 real-time quantitative RT-PCR. Data are presented as mean ± SD (**, p < 0.05).

 
Lin–/integrin beta3high 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 Lin–/beta3high 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).


Figure 3
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  		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–/ beta3low SP cells (C), Lin–/beta3high 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 (**, p < 0.05).

 
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–/beta3high SP cells had increased p57Kip2 expression compared with the Lin– NSP, Lin– SP, and Lin– beta3low SP fractions, and have mRNA levels as high as KSL SP cells (Fig. 2E). We also confirmed that p21Cip1, another cyclin-dependent kinase inhibitor related to HSC quiescence (19), was expressed in Lin–/beta3high SP cells, but without a significant difference compared with Lin–/beta3low SP cells (data not shown). Thus, nearly all of Lin–/beta3high 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 beta3 is directly correlated to increased strength of the quiescent SP phenotype.

Lin–/integrin beta3high SP cells have stem cell properties

To confirm that quiescent Lin–/beta3high SP cells possessed stem cell-like properties, several markers of HSCs were examined. Analysis demonstrated that Lin–/beta3high SP cells contained a significantly higher percentage of c-Kit+/Sca-1+, endoglin+, and CD150+ cells, compared with Lin–/beta3low SP cells (Fig. 4A). Endoglin, a homolog of the type III TGF-beta 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 self-renewing stem cells from more differentiated lineages, including transiently renewing multipotent progenitors (MPPs) (22). Together, these results indicate that Lin–/beta3high SP cells account for a greater proportion of HSCs within the mouse bone marrow SP fraction than Lin–/beta3low SP cells. Moreover, using competitive reconstruction assays, we confirmed that Lin–/beta3high SP cells have a significantly higher potential for long-term hemopoietic reconstitution compared with Lin–/beta3low SP cells (Fig. 4B). Three months after reconstitution with 100 donor cells, Lin–/beta3high SP cells showed an increased ability to reconstruct all hemopoietic lineages in the transplanted animals (Table I). Additionally, Lin–/beta3high SP cells also had as high ability for hemopoiesis as KSL SP cells (Fig. 4B), indicating that expression of integrin beta3 can distinguish HSCs from other cell types within the SP fraction as effectively as the combination of Sca-1 and c-Kit.


Figure 4
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  		FIGURE 4. Lin–/integrin beta3high SP cells have hemopoietic stem cell properties. A, Mouse bone marrow Lin–/beta3low SP and Lin–/beta3high SP cells were analyzed for expression of hemopoietic stem cell markers. Lin–/beta3low SP and Lin–/beta3high 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–/beta3low SP cells and Lin–/beta3high 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 (*, p < 0.01). B, Long-term competitive repopulation assay performed with Lin–/beta3low SP and Lin–/beta3high SP cells. One hundred Lin–/beta3low SP, Lin–/beta3high SP, or KSL SP cells derived from C57BL/6-Ly5.1 mice, and 2 x 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 (*, p < 0.01).

 

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  		Table I. Multilineage repopulation by Lin–/beta3high SP cellsa

 
Integrin beta3 is associated with integrin {alpha}V in hemopoietic stem cells with the SP phenotype

In general, integrins function as receptors by forming heterodimers with the association of {alpha} and beta subunits, and integrin beta3 has commonly been associated with either integrin {alpha}V or integrin {alpha}IIb. Although previous reports have suggested that that integrin {alpha}IIbbeta3 is expressed in mouse embryonic and neonatal HSCs (15, 16), the presence of the integrin beta3 subunit has not been related to adult stem cells. To examine which {alpha} subunit was associated with integrin beta3 in SP cells, Hoechst exclusion assays were performed using Lin–/beta3+/integrin {alpha}V+ ({alpha}V+) or Lin–/beta3+/integrin {alpha}IIb+ ({alpha}IIb+) fractions. Results showed that the Lin–/beta3+/{alpha}V+ fraction contained a much higher percentage of SP cells, compared with Lin–/beta3+/{alpha}IIb+ cells or Lin–/beta3+/integrin {alpha}V– ({alpha}V–) (Fig. 5A). Moreover, Hoechst exclusion assays using Lin–/{alpha}V+ or Lin–/{alpha}IIb+ fractions also showed that Lin–/{alpha}V+ fraction not only contained a higher percentage of SP cells than Lin–/{alpha}IIb+ cells, but also closely resembled the distribution plots for the Lin–/beta3+ fraction (data not shown). When expression of Sca-1, endoglin, and CD150 were examined, Lin–/beta3+/{alpha}V+ SP cells also demonstrated significantly higher expression of these stem cell markers, in comparison to Lin–/beta3+/{alpha}IIb+ SP cells or Lin–/beta3+/{alpha}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 beta3 was associated with integrin {alpha}V. To further confirm this hypothesis, bone marrow cells triple-stained for integrins {alpha}V, {alpha}IIb, and beta3 were examined. Flow cytometric analysis showed that only Lin–/beta3+/{alpha}V+/integrin {alpha}IIb– ({alpha}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–/beta3+/{alpha}V+/{alpha}IIb– SP fraction (Fig. 6E). These results confirmed that SP cells possessing the features of HSCs, expressed integrin {alpha}V and not integrin {alpha}IIb within the Lin–/beta3+ gate. Previous reports have shown that integrin {alpha}IIb is not expressed by adult HSCs and that integrin {alpha}IIb+ cells have no ability for hemopoiesis (15, 16, 22). Therefore, it appeared likely that integrin beta3 is associated with integrin {alpha}v in the portion of SP cells that have the properties of adult stem cells.


Figure 5
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  		FIGURE 5. Hemopoietic stem cell properties in Lin–/integrin beta3+/integrin {alpha}V + SP cells and Lin–/integrin beta3+/integrin {alpha}llb+ SP cells. A, Analysis of integrin {alpha}-chain expression in Lin–/beta3+ SP cells. Lin– bone marrow cells were double-stained with Abs for integrin beta3 and integrin {alpha}V (a); integrin beta3 and integrin {alpha}IIb (e); isotype controls (ICs) for integrin beta3 and integrin {alpha}V (b) or ICs for integrin beta3 and integrin {alpha}IIb (f), followed by analysis of Hoechst 33342 exclusion assays in Lin–/beta3+/{alpha}V+ cells (c), Lin–/beta3+/{alpha}V– cells (d) and Lin–/beta3+/{alpha}IIb+ cells (g). B, Analysis of stem cell markers expressed in Lin–/beta3+/{alpha}V+ SP cells, Lin–/beta3+/{alpha}V+ SP cells, and Lin–/beta3+/{alpha}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 (*, p < 0.01).

 

Figure 6
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  		FIGURE 6. Integrin beta3 is coupled to integrin {alpha}V in hemopoietic stem cells with the SP phenotype. Lin– beta3+ cells were stained with Abs for integrin {alpha}V and integrin {alpha}IIb (A), or isotype controls (ICs) for these integrin {alpha}-chains (B) followed by analysis with Hoechst 33342 exclusion assays in Lin–/beta3+/{alpha}V+/{alpha}IIblow cells (C), Lin–/beta3+/{alpha}V+/{alpha}IIb+ cells (D), and Lin–/beta3+/{alpha}V–/{alpha}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 (*, p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we present strong evidence that quiescent HSCs possessing the SP phenotype express integrin {alpha}Vbeta3. Integrin {alpha}Vbeta3 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 {alpha}Vbeta3 in SP cells could not be directly investigated, integrin beta3 has previously only been associated with integrins {alpha}V and {alpha}llb, and adult HSCs have much lower or no expression of the integrin {alpha}llb subunit (Figs. 5 and 6) (15, 16, 22). Therefore, although the possibilities that integrin beta3 may be heterodimerized with other integrin {alpha} 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 {alpha}Vbeta3. In addition, at the very least, our results definitively show that expression of integrin beta3 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 beta3 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 beta3, with most of these differentiated cells likely present within NSP gate. Therefore, in bone marrow SP cells, the relationship between integrin beta3 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 beta3 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 beta3 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 beta3 may be a common feature of SP cells that possess stem cell-like properties. Therefore, it is suggested that the integrin beta3 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 beta3 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 beta3-endonexin, known as integrin beta3 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). beta3-endonexin inhibits retinoblastoma protein (pRB) kinase activity associated with cyclin A-Cdk2, while leaving histone H1 kinase activity unaffected (31). Interestingly, many of these molecules that associate with integrin beta3 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–/beta3+/{alpha}v+ SP cells also suggests that TGF-beta signaling may play a key role in the maintenance of HSCs that may be linked to integrin {alpha}Vbeta3. Interestingly, TGF-beta also induces the expression of integrin {alpha}Vbeta3 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 beta3 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 beta3. These results suggest that integrin beta3 is exploitable as common marker of tissue-specific stem cells and may contribute to the general determination of stemness such as niche interactions, quiescence, and the manifestation of the SP phenotype.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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. Back

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{at}abmes.twmu.ac.jp Back

3 Abbreviations used in this paper: SP, side population; NSP, non-SP; ABCG2, ATP-binding cassette transporter G2; HSC, hemopoietic stem cell; PY, pyronin Y; Lin, lineage. Back

Received for publication July 17, 2006. Accepted for publication September 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Goodell, M. A., K. Brose, G. Paradis, A. S. Conner, R. C. Mulligan. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183: 1797-1806. [Abstract/Free Full Text]
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