Phenotype Cells Possessing the Side Population Properties of Quiescent

Expression of Integrin β3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype This information is current as of September 26, 2021. Terumasa Umemoto, Masayuki Yamato, Yoshiko Shiratsuchi, Masao Terasawa, Joseph Yang, Kohji Nishida, Yoshiro Kobayashi and Teruo Okano J Immunol 2006; 177:7733-7739; ; doi: 10.4049/jimmunol.177.11.7733 Downloaded from http://www.jimmunol.org/content/177/11/7733

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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. 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*

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

␤ Downloaded from 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, Lin- ؊ ؊ ␤ high ␤ high ؉ ؉ ؊ eage (Lin )/integrin 3 ( 3 ) 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 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. http://www.jimmunol.org/

n stem cell biology, the identification of markers that distin- of the limbal epithelium (7–9), which is located at the transitional guish stem cells from their differentiated progeny is most zone between the cornea and the peripheral bulbar conjunctiva. We essential for a clear understanding of stem cell-related prop- have previously demonstrated that limbal epithelial SP cells have I 3 erties. The side population (SP) phenotype is characterized by stem cell-like phenotypes and, similar to HSCs, are maintained in cells with the capacity to efflux the DNA-binding dye Hoechst the quiescent state (10). However, while the ability for Hoechst 33342 via the ATP-binding cassette transporter G2 (ABCG2), and dye efflux via ABCG2 is now considered a general characteristic of is a reliable marker of hemopoietic stem cells (HSCs) with the adult stem cells, other molecules that may be common markers of by guest on September 26, 2021 ability for long-term multilineage reconstitution (1, 2). More re- various tissue-specific stem cells remain unidentified. cently, SP cells from numerous other adult tissues and species have Integrin receptors are heterodimers composed of ␣- and also demonstrated stem cell-like properties (3–6), suggesting that ␤-chains and function by binding to ligands that are components of the SP phenotype is a common feature of tissue-specific stem cells. the extracellular matrix, as well as some soluble ligands such as the The enrichment of adult stem cells based on the SP phenotype has ICAMs, which can lead to aggregation of cells. In addition to therefore proven to be an extremely useful tool in the isolation of mediating cell adhesion and cytoskeletal organization, integrins stem cells from a variety of tissue systems. can also function as cell-signaling receptors. Signal transduction In the corneal epithelial system, a model tissue for epithelial pathways involving integrins play a role in many biological pro- stem cell biology, stem cells are thought to reside in the basal layer cesses, 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 *Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Med- related to stem cell niches and may be available as surface markers ical University, Tokyo, Japan; †Division of Molecular Medicine, Department of Bio- ‡ of these stem cells. molecular Science, Toho University, Chiba, Japan; and Department of Ophthalmol- ␤ ogy and Visual Science, Tohoku University Graduate School of Medicine, Miyagi, Previously, integrin 1 (CD29) has been shown to be highly Japan expressed and able to induce adhesion to niche cells such as os- Received for publication July 17, 2006. Accepted for publication September 12, 2006. teoblasts in quiescent HSCs treated with Tie-2 (11). Recently, ex- ␣ The costs of publication of this article were defrayed in part by the payment of page pression of integrin 2 (CD49b) has also been used to distinguish charges. This article must therefore be hereby marked advertisement in accordance HSCs possessing the capacity for long-term hemopoiesis (12). with 18 U.S.C. Section 1734 solely to indicate this fact. Moreover, from integrin subunit profiling, we have observed that 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 Educa- limbal epithelial SP cells, which closely resemble HSCs, have a tion, Culture, Sports, Science, and Technology, Japan; the Core Research for Evolu- ␣ ␣ significantly higher expression of integrin 1 (CD49a), integrin 4 tion 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. (CD49d), and integrin 3 (CD61) when compared with non-SP 2 (NSP) cells (data not shown). Address correspondence and reprint requests to Dr. Teruo Okano, Institute of Ad- ␤ ␣ vanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Integrin 3 is commonly associated with either integrin V 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail address: tokano@ (CD51) or integrin ␣ (CD41), and participates in many biolog- abmes.twmu.ac.jp IIb ical process including cell adhesion, aggregation, and migration in 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, several cells (13, 14). Although it has been reported that integrin ␣ ␤ lineage. IIb 3 is expressed in mouse embryonic and neonatal HSCs (15,

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 Ͻ ␤ Ϫ Ϫ ء Ϯ are presented as mean SD ( , p 0.01). B, Expression of integrin 3 at the protein level in Lin SP cells. Upon staining of Lin cells (obtained by MACS) Ϫ ␤ ␤ with Hoechst 33342 (a), Lin SP cells were stained with anti-integrin 3 Abs (b). White histogram, Isotype control; gray histogram, integrin 3. After immu- http://www.jimmunol.org/ ␤ Ϫ ␤ ␤ noprecipitation 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 Ϫ ␤ 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 Ͻ ء ϩ Ϯ ␤ ␤ control; gray histogram, integrin 3. The graph represents the percentage of 3 cells in each fraction (e). Data are presented as mean SD ( , p 0.01).

␤ ␣ 16), the expression of the integrin 3 subunit has not been previ- anti-CD41/integrin IIb (MWReg30), anti-Sca-1 (E13-161.7), anti-c-Kit ously correlated to adult stem cells. In this study, we show that (2B8), anti-endoglin (MJ7/18; Santa Cruz Biotechnology), anti-CD150 expression of integrin ␤ is correlated to the properties of (TC15-12F12.2; BioLegend), anti-CD45.2 (104), anti-CD45.1 (A20), 3 anti-B220/CD45R (RA3-6B2), anti-Mac-1 (M1/70), anti-Gr-1 (RB6- quiescent HSCs that possess the SP phenotype. We also demon- 8C5), anti-CD4 (L3T4), and anti-CD8 (53-6.72). All Abs were obtained by guest on September 26, 2021 ␤ strate that this expression of integrin 3 is correlated to the pres- from BD Biosciences/BD Pharmingen unless otherwise noted. ence of integrin ␣ within this population of HSCs with the ability For immunoprecipitation and Western blotting experiments, anti- V ␤ ␤ for long-term multilineage reconstitution. integrin 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 Materials and Methods Ab for Western blotting. All animal experiments were performed according to the Guidelines of Tokyo Women’s Medical University on Animal Use, the Principles of Cell preparation Laboratory Animal Care formulated by the National Society for Medical Cell suspensions of bone marrow from C57BL/6 and C57BL/6-Ly5.1 con- Research, and the Guide for the Care and Use of Laboratory Animals genic mice were prepared as described previously (17). prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (National Institutes of Health Publication FACS and flow cytometric analysis No. 86-23, revised 1985). Analysis and sorting of SP cells were performed as described previously Antibodies (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 stain- The following mAbs were used for FACS and flow cytometric analy- ing medium (DMEM containing 2% FBS (Moregate Biotech) and 10 mM ␤ ␣ sis: anti-CD61/integrin 3 (2C9.G2), anti-CD51/integrin V (RMV-7), HEPES) for 90 min at 37°C. After staining, cells were resuspended in

FIGURE 2. mRNA expression of markers for quiescent SP cells. LinϪ cells were stained with Hoechst 33342 and sorted into SP and NSP cells, respectively (A). LinϪ SP cells were then analyzed and separated into Ϫ ␤ high Ϫ ␤ low either Lin / 3 or Lin / 3 SP cells, respectively (B), or c-Kitϩ/Sca-1ϩ/LinϪ (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 .(p Ͻ 0.05 ,ءء) mean Ϯ SD 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 Ϫ Ϫ Ϫ 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 / ␤ low Ϫ ␤ high Ϫ 3 SP cells (C), Lin / 3 SP cells (D), and KSL SP cells (E). The graph represents percentage of PY cells (cells in G0 phase) in each fraction (F). .(p Ͻ 0.05 ,ءء) Data are presented as mean Ϯ SD Downloaded from

Dulbecco’s PBS containing 2% FBS and 1 mM HEPES. Before analysis Results and cell sorting, propidium iodide (Sigma-Aldrich) was added at a final ␮ Mouse bone marrow SP cells have high expression of concentration of 2 g/ml, to distinguish between live and nonviable cells. ␤ Analysis and cell sorting were then performed using a dual laser fluores- integrin 3 cence-activated cell sorter (Epics Altra FACS analysis system; Beckman Because integrin subunit profiling showed that limbal epithelial SP http://www.jimmunol.org/ Coulter). ␣ ␣ ␤ For the isolation of specific cell populations, Lineage marker-positive cells had higher expression of integrins 1, 4, and 3 when com- cells were first eliminated by magnetic cell sorting (Auto MACS system; pared with NSP cells at the mRNA level (data not shown), we Miltenyi Biotec) using the Lineage Cell Depletion kit (Miltenyi Biotec), speculated that the high expression of these integrin chains may be Ϫ Ϫ before staining with Hoechst 33342. Lineage (Lin ) cells were stained conserved among SP cells isolated from various tissues. When with Hoechst 33342 and were then incubated with the corresponding Abs gene expression of these integrin mRNAs was examined in mouse 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 bone marrow, only integrin 3 showed significantly higher expres- before analysis. Stained cells were then subjected to sorting by FACS. sion in SP cells compared with NSP cells (Fig. 1A). Moreover, flow cytometric analysis and immunoprecipitation followed by

Ϫ by guest on September 26, 2021 Gene expression analysis Western blotting also showed that within Lin SP cells, integrin ␤ 3 was expressed (Fig. 1B). For gene expression assays, total RNA was obtained from 10,000 cells of each population, using Isogen (Nippongene) according to the manufactur- ␤ Expression of integrin 3 is correlated to the relative strength of er’s suggested protocol. Single-stranded cDNA was created with the Su- the SP phenotype perscript First-strand System for RT-PCR (Invitrogen Life Technologies), ␤ and used as PCR templates. Primer pairs and TaqMan MGB probes labeled To further investigate the relationship between integrin 3 and the with FAM at the 5Ј end and nonfluorescent quencher at the 3Ј end, were SP phenotype, mouse bone marrow cells were subjected to designed with the TaqMan gene expression assay (Applied Biosystems). Quantitative PCR was performed with the 7300 Real Time PCR System Hoechst exclusion assay and sorted into finer SP gates (Fig. 1Ca). (Applied Biosystems). Thermocycling programs consisted of an initial cy- As the strength of the SP phenotype was amplified (indicated by cle at 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C lower Hoechst fluorescence intensity due to greater dye efflux), the for 15 s and 60°C for 1 min. All assays were run in duplicate for more than ␤ ϩ percentage of integrin 3 cells also increased (Fig. 1C). In par- four individual samples. mRNA expression levels were normalized with ticular, nearly all cells within the Tip-SP fraction, which has the the expression level of GAPDH. To compare mRNA expression between ␤ ϩ cell populations, the Mann-Whitney rank sum test was applied. Statistics highest expression of ABCG2 (2), were 3 cells (Fig. 1C). Fur- were calculated using SigmaStat 2.0 (SPSS). thermore, we investigated whether SP cells possessing stronger ␤ expression of integrin 3 also show high expression of ABCG2, Immunoprecipitation and Western blotting which is the molecular determinant of the SP phenotype. To com- pletely prevent contamination by integrin ␤ Ϫ cells, “high” and ␤ 3 Sorted cells were lysed in buffer (TBS containing 50 mM n-octyl- -D- “low” gates containing the highest 30% and the lowest 30% of glucosidase, 1 mM CaCl , 1 mM MgCl , 0.1 mM PMSF, 10 ␮g/ml leu- 2 2 ␤ peptin, and 10 ␮g/ml aprotinin), followed by immunoprecipitation with fluorescence intensity were isolated based on integrin 3 staining specific Ab and protein G Sepharose (Amersham Biosciences) for3hat plots (Fig. 2, A and B). Significantly higher mRNA higher expres- Ϫ ␤ high 4°C. Immunoprecipitated proteins were separated on 7.5% polyacrylamide sion of ABCG2 was observed in Lin / 3 SP cells compared gels and transferred to Immobilon-P membranes (Millipore). After block- Ϫ Ϫ Ϫ ␤ low with the Lin NSP, Lin SP, and Lin 3 SP fractions (Fig. ing, membranes were sequentially incubated with primary and secondary Ϫ ␤ high 2D). Interestingly, the Lin / 3 SP fraction also had compara- Abs followed by development with ECL (ECL Advance; Amersham ϩ ϩ Ϫ Biosciences). ble ABCG2 mRNA levels compared with c-Kit /Sca-1 /Lin (KSL) SP cells (Fig. 2, C and D), which are considered one of the Long-term competitive repopulation assay most highly enriched HSC populations (11). Ϫ ␤ high C57BL/6 mice irradiated at 11 Gy total, were transplanted with 100 test Lin /integrin 3 SP cells reside in the quiescent state cells prepared from C57BL/6-Ly5.1 mice and 2 ϫ 105 mononuclear bone marrow cells obtained from C57BL/6-Ly5.2 mice. Three months after Currently, the SP phenotype is considered the single characteristic transplantation, mononuclear cells isolated from the peripheral blood were that most accurately identifies quiescent stem cells in the bone analyzed. marrow niche (11). Pyronin Y (PY) staining demonstrated that the ␤ 7736 QUIESCENT HSCs EXPRESS INTEGRIN 3

Ϫ ␤ high a Table I. Multilineage repopulation by Lin / 3 SP cells

T Cell B Cell Monocyte/Granulocyte

51.04 Ϯ 0.88 28.54 Ϯ 2.51 35.71 Ϯ 1.80

a The percentages of CD4ϩ or CD8ϩ (T cell lineage), B220/CD45Rϩ (B cell lineage), and Mac-1ϩ or Gr-1ϩ (monocyte/granulocyte lineage) cells in Ly-5.1ϩ (do- Ϫ ␤ high nor-derived cells) of mice transplanted with Lin / 3 SP fractions. Data are presented as mean Ϯ SD.

Ϫ ␤ high Lin /integrin 3 SP cells have stem cell properties Ϫ ␤ high To conﬁrm that quiescent Lin / 3 SP cells possessed stem cell-like properties, several markers of HSCs were examined. Ϫ ␤ high Analysis demonstrated that Lin / 3 SP cells contained a signiﬁcantly higher percentage of c-Kitϩ/Sca-1ϩ, endoglinϩ, and ϩ Ϫ ␤ low CD150 cells, compared with Lin / 3 SP cells (Fig. 4A). En- doglin, a homolog of the type III TGF-␤ receptor, is differentially

expressed by HSCs (20) and believed to account for all long-term Downloaded from 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 Ϫ high

␤ http://www.jimmunol.org/ FIGURE 4. Lin /integrin 3 SP cells have hemopoietic stem cell transiently renewing multipotent progenitors (MPPs) (22). To- Ϫ ␤ low Ϫ ␤ high Ϫ ␤ high properties. A, Mouse bone marrow Lin / 3 SP and Lin / 3 SP gether, these results indicate that Lin / 3 SP cells account for cells were analyzed for expression of hemopoietic stem cell markers. a greater proportion of HSCs within the mouse bone marrow SP LinϪ/␤ low SP and LinϪ/␤ high SP cells were double-stained for Sca-1 Ϫ ␤ low 3 3 fraction than Lin / 3 SP cells. Moreover, using competitive and c-Kit (a and b), stained for endoglin (d and e), or stained for CD150 Ϫ ␤ high reconstruction assays, we confirmed that Lin / 3 SP cells have (g and h). (a, d, and g) and (b, e, and h) represent LinϪ/␤ low SP cells 3 a significantly higher potential for long-term hemopoietic recon- Ϫ ␤ high and Lin / 3 SP cells, respectively. Graphs show the percentage of Ϫ low ϩ ϩ stitution compared with Lin /␤ SP cells (Fig. 4B). Three KSL cells (c), endoglin cells (f), and CD150 cells (i), respectively. Data are 3 Ϫ ␤ high p Ͻ 0.01). B, Long-term competitive repopulation months after reconstitution with 100 donor cells, Lin / 3 SP ,ء) presented as mean Ϯ SD Ϫ ␤ low Ϫ ␤ high cells showed an increased ability to reconstruct all hemopoietic assay performed with Lin / 3 SP and Lin / 3 SP cells. One hundred LinϪ/␤ low SP, LinϪ/␤ high SP, or KSL SP cells derived from C57BL/ lineages in the transplanted animals (Table I). Additionally, by guest on September 26, 2021 3 3 Ϫ 5 ␤ high 6-Ly5.1 mice, and 2 ϫ 10 mononuclear bone marrow cells from C57BL/ Lin / 3 SP cells also had as high ability for hemopoiesis as ␤ 6-Ly5.2 mice were transplanted into lethally irradiated C57BL/6-Ly5.2 KSL SP cells (Fig. 4B), indicating that expression of integrin 3 mice. The plot represents the percentage of donor-derived cells (percentage can distinguish HSCs from other cell types within the SP fraction ϩ of Ly5.1 cells) in the peripheral blood of each recipient animal, 3 mo after as effectively as the combination of Sca-1 and c-Kit. .(p Ͻ 0.01 ,ء) bone marrow transplantation. Bars represent mean values ␤ ␣ Integrin 3 is associated with integrin V in hemopoietic stem cells with the SP phenotype In general, integrins function as receptors by forming heterodimers LinϪ/␤ high SP fraction possessed a significantly higher number of ␣ ␤ ␤ 3 with the association of and subunits, and integrin 3 has com- cells that resided in the G phase (PYϪ cells) compared with all cell ␣ ␣ 0 monly been associated with either integrin V or integrin IIb. populations and also showed the same percentage as KSL SP cells ␣ ␤ Although previous reports have suggested that that integrin IIb 3 (Fig. 3). is expressed in mouse embryonic and neonatal HSCs (15, 16), the p57Kip2 is a member of the Cip/Kip family of cyclin-dependent ␤ presence of the integrin 3 subunit has not been related to adult kinase inhibitors and is a potent negative regulator of the cell cycle stem cells. To examine which ␣ subunit was associated with in- Kip2 ␤ (18). In bone marrow SP cells, increased expression of p57 has tegrin 3 in SP cells, Hoechst exclusion assays were performed Ϫ ␤ ϩ ␣ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ ϩ been previously observed, with significantly higher levels in KSL using Lin / 3 /integrin V ( V )orLin / 3 /integrin IIb Kip2 ␣ ϩ Ϫ ␤ ϩ ␣ ϩ SP cells (17). Therefore, it appears that the expression of p57 ( IIb ) fractions. Results showed that the Lin / 3 / V fraction may be closely involved in the maintenance of stem cell quies- contained a much higher percentage of SP cells, compared with Ϫ ␤ high Ϫ ␤ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ Ϫ ␣ Ϫ cence. Our results showed that Lin / 3 SP cells had increased Lin / 3 / IIb cells or Lin / 3 /integrin V ( V ) (Fig. 5A). Kip2 Ϫ Ϫ Ϫ ␣ ϩ p57 expression compared with the Lin NSP, Lin SP, and Moreover, Hoechst exclusion assays using Lin / V or Ϫ ␤ low Ϫ ␣ ϩ Ϫ ␣ ϩ Lin 3 SP fractions, and have mRNA levels as high as KSL Lin / IIb fractions also showed that Lin / V fraction not only Cip1 Ϫ ␣ ϩ SP cells (Fig. 2E). We also confirmed that p21 , another cyclin- contained a higher percentage of SP cells than Lin / IIb cells, Ϫ ␤ ϩ dependent kinase inhibitor related to HSC quiescence (19), was but also closely resembled the distribution plots for the Lin / 3 Ϫ ␤ high expressed in Lin / 3 SP cells, but without a significant differ- fraction (data not shown). When expression of Sca-1, endoglin, Ϫ ␤ low Ϫ ␤ ϩ ␣ ϩ ence compared with Lin / 3 SP cells (data not shown). Thus, and CD150 were examined, Lin / 3 / V SP cells also demon- Ϫ ␤ high nearly all of Lin / 3 SP cells appear to be cell cycle arrested, strated significantly higher expression of these stem cell markers, Ϫ ␤ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ Ϫ with a percentage of G0 cells that is comparable to the KSL SP in comparison to Lin / 3 / IIb SP cells or Lin / 3 / V SP ␤ fraction. These results indicate that the expression of integrin 3 is cells (Fig. 5B). These results implied that in the subfraction of SP directly correlated to increased strength of the quiescent SP cells that possessed the properties of HSCs, the expression of in- ␤ ␣ phenotype. tegrin 3 was associated with integrin V. To further confirm this The Journal of Immunology 7737

FIGURE 5. Hemopoietic stem cell properties in Ϫ ␤ ϩ ␣ ϩ Ϫ Lin /integrin 3 /integrin V SP cells and Lin /in- ␤ ϩ ␣ ϩ tegrin 3 /integrin llb SP cells. A, Analysis of inte- ␣ Ϫ ␤ ϩ Ϫ grin -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 inte- ␣ ␤ ␣ grin V (b) or ICs for integrin 3 and integrin IIb (f), followed by analysis of Hoechst 33342 exclusion assays Ϫ ␤ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ Ϫ in Lin / 3 / V cells (c), Lin / 3 / V cells (d) and Ϫ ␤ ϩ ␣ ϩ Lin / 3 / IIb cells (g). B, Analysis of stem cell mark- Ϫ ␤ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ ϩ ers expressed in Lin / 3 / V SP cells, Lin / 3 / V Ϫ ␤ ϩ ␣ ϩ SP cells, and Lin / 3 / IIb SP cells. Graphs show the ϩ ϩ percentages of Sca-1 cells (a), endoglin cells (b), and Downloaded from CD150ϩ cells (c), respectively. Data are presented as .(p Ͻ 0.01 ,ء) mean Ϯ SD http://www.jimmunol.org/

␣ ␣ ␤ ␣ hypothesis, bone marrow cells triple-stained for integrins V, IIb, integrin 3 is associated with integrin v in the portion of SP cells ␤ and 3 were examined. Flow cytometric analysis showed that only that have the properties of adult stem cells. Ϫ ␤ ϩ ␣ ϩ ␣ Ϫ ␣ Ϫ Lin / 3 / V /integrin IIb ( IIb ) SP cells had enhanced Hoechst exclusion capabilities with localization to the Tip-SP frac- Discussion by guest on September 26, 2021 tion that possesses the highest strength of the SP phenotype (Fig. In this study, we present strong evidence that quiescent HSCs pos- ␣ ␤ ␣ ␤ 6, A–D). Moreover, long-term repopulation assays also showed sessing the SP phenotype express integrin V 3. Integrin V 3 that the strong ability for long-term hemopoiesis was demonstrated also known as CD51/CD61 or the vitronectin receptor; is related to Ϫ ␤ ϩ ␣ ϩ ␣ Ϫ only with the Lin / 3 / V / IIb SP fraction (Fig. 6E). These adhesion, movement, and apoptosis in several cell types (13, 14), results conﬁrmed that SP cells possessing the features of HSCs, but has not been previously correlated to adult stem cells. Al- ␣ ␣ Ϫ ␤ ϩ ␣ ␤ expressed integrin V and not integrin IIb within the Lin / 3 though the expression of integrin V 3 in SP cells could not be ␣ ␤ gate. Previous reports have shown that integrin IIb is not ex- directly investigated, integrin 3 has previously only been associ- ␣ ϩ ␣ ␣ pressed by adult HSCs and that integrin IIb cells have no ability ated with integrins V and llb, and adult HSCs have much lower ␣ for hemopoiesis (15, 16, 22). Therefore, it appeared likely that 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. LinϪ ␤ ϩ ␣ 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 Ϫ ␤ ϩ ␣ ϩ ␣ low 33342 exclusion assays in Lin / 3 / V / IIb cells (C), Ϫ ␤ ϩ ␣ ϩ ␣ ϩ Ϫ ␤ ϩ ␣ Ϫ ␣ ϩ 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 Ly5.1ϩ cells) in the peripheral blood of each recipient mouse, 3 mo after bone marrow p Ͻ ,ء) transplantation (F). Bars represent mean values 0.01). ␤ 7738 QUIESCENT HSCs EXPRESS INTEGRIN 3

␤ 22). Therefore, although the possibilities that integrin 3 may be interactions, quiescence, and the manifestation of the SP heterodimerized with other integrin ␣ subunits, associated with phenotype. other molecules, or even exist independently in SP cells, cannot be excluded; our result strongly suggest that HSCs possessing the SP Disclosures ␣ ␤ phenotype showed high expression of integrin V 3. In addition, The authors have no financial conflict of interest. at the very least, our results definitively show that expression of ␤ integrin 3 is correlated to the features of HSCs and is as effective References as Sca-1 and c-Kit for identifying HSCs within bone marrow SP 1. Goodell, M. A., K. Brose, G. Paradis, A. S. Conner, and R. C. Mulligan. 1996. ␤ cells. This expression of integrin 3 has never been previously Isolation and functional properties of murine hematopoietic stem cells that are correlated to adult tissue-specific stem cells, including HSCs. replicating in vivo. J. Exp. Med. 183: 1797–1806. 2. Zhou, S., J. D. Schuetz, K. D. Bunting, A. M. Colapietro, J. Sampath, J. J. Morris, In the hemopoietic system, several cell types such as mega- I. Lagutina, G. C. Grosveld, M. Osawa, H. Nakauchi, and B. P. Sorrentino. 2001. ␤ karyocytes and macrophages, also express integrin 3, with most The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells of these differentiated cells likely present within NSP gate. There- and is a molecular determinant of the side-population phenotype. Nat. Med. 7: 1028–1034. ␤ fore, in bone marrow SP cells, the relationship between integrin 3 3. Jackson, K. A., T. Mi, and M. A. Goodell. 1999. Hematopoietic potential of stem expression and stem cell properties may have previously gone un- cells isolated from murine skeletal muscle. Proc. Natl. Acad. Sci. USA 96: 14482–14486. noticed. Via screening of integrin chain mRNA expression in lim- 4. Shimano, K., M. Satake, A. Okaya, J. Kitanaka, N. Kitanaka, M. Takemura, bal epithelial SP cells that closely resemble quiescent HSCs, sig- M. Sakagami, N. Terada, and T. Tsujimura. 2003. Hepatic oval cells have the side nificant insight into the relationship between integrin ␤ and stem population phenotype defined by expression of ATP-binding cassette transporter 3 ABCG2/BCRP1. Am. J. Pathol. 163: 3–9. cell properties in the SP fraction could thus be identified. 5. Summer, R., D. N. Kotton, X. Sun, B. Ma, K. Fitzsimmons, and A. Fine. 2003. Downloaded from Although only compared in the limbal epithelium via mRNA Side population cells and Bcrp1 expression in lung. Am. J. Physiol. 285: expression, the requirements for enzymatic dissociation of epithe- L97–L104. 6. Uchida, N., F. Y. Leung, and C. J. Eaves. 2002. Liver and marrow of adult lia into single cells, as well as the lack of an accepted and estab- mdr-1a/1bϪ/Ϫ mice show normal generation, function, and multi-tissue traffick- lished reconstitution assay, currently remain limitations of epithe- ing of primitive hematopoietic cells. Exp. Hematol. 30: 862–869. lial stem cell biology that must be overcome. Nevertheless, our 7. Thoft, R. A., and J. Friend. 1983. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest. Ophthalmol. Vis. Sci. 24: 1442–1443.

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