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progenitor cells
Neuroscience Research 52 (2005) 75–82 www.elsevier.com/locate/neures
Functional expression of ABCG2 transporter in human neural stem/progenitor cells Mohammed Omedul Islam a,b, Yonehiro Kanemura a,c,*, Jesmin Tajria a, Hideki Mori a, Satoshi Kobayashi a, Masayuki Hara d, Mami Yamasaki c,e, Hideyuki Okano f,g, Jun Miyake a a
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Nakoji 3-11-46, Amagasaki, Hyogo 661-0974, Japan b Institute of Biomedical Research and Innovation, Kobe, Hyogo 650-0047, Japan c Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan d Department of Applied Bioscience, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Osaka 599-8570, Japan e Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan f Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan g Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Saitama 332-0012, Japan Received 15 October 2004; accepted 28 January 2005
Abstract We have studied the expression, localization, and function of the ABCG2 transporter, a universal stem cell marker, at the protein level in human cultured neural stem/progenitor cells (hNSPCs) using immunoblotting, immunofluorescence, and ATPase assays. Human NSPCs were isolated from human fetal brain and propagated in vitro as neurospheres. Both the cells in neurospheres and single cells dissociated from neurospheres showed high levels of ABCG2, and about 63% of the cells in neurospheres were ABCG2-positive, similar to the proportion of nestin-positive cells, and in most cases the ABCG2 and nestin staining co-localized in the same cells. Both the three-dimensional structure of single hNSPCs stained with anti-ABCG2 antibodies and an examination using a biochemical marker for the plasma membrane indicated that ABCG2 was localized to the plasma membrane of hNSPCs. The ABCG2 expressed in hNSPCs had prazosin-sensitive ATP hydrolysis activity, and the ABCG2 level was sharply down-regulated during hNSPC differentiation. All these results suggested that ABCG2, was functionally expressed in hNSPCs. ABCG2 might play a significant role in maintaining human neural stem cells in an undifferentiated state and in protecting hNSPCs from xenobiotics or other toxic substances in vivo. # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: ABC transporter; Human neural stem/progenitor cells; ABCG2
1. Introduction ABCG2, a half-transporter also called MXR/BCRP/ ABCP, is a member of the ATP-binding cassette (ABC) family of cell-surface transporter proteins. It is expressed in a variety of malignant and normal tissues, and promotes the efflux of a variety of anti-neoplastic drugs, including anthracyclines and campothecins, from the cell (Doyle et al., * Corresponding author. Tel.: +81 6 6494 7839; fax: +81 6 6494 7862. E-mail address: [email protected] (Y. Kanemura).
1998; Ross et al., 2000; Miyake et al., 1999). Unlike other ABC half-transporters, which are localized to intracellular membranes, ABCG2 is expressed exclusively in the plasma membrane (Rocchi et al., 2000). Recently, ABCG2 was found in the primitive stem cells of different tissues, which led to enthusiasm for the idea that it might play a significant role in maintaining stem cells in an undifferentiated state (Zhou et al., 2001). ABCG2 is highly expressed in a specific population of hematopoietic stem cells, the so-called ‘SP’ (side population) subset, which is isolated by the cells’ ability to promote the efflux of Hoechst
0168-0102/$ – see front matter # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2005.01.013
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33342 fluorescent dye (Zhou et al., 2001; Murayama et al., 2002; Goodell et al., 1996; Matsuzaki et al., 2004). During the differentiation of hematopoietic stem cells, the level of ABCG2 is sharply down-regulated, underscoring the gene’s potential use as a stem cell marker (Scharenberg et al., 2002). Semi-quantitative RT-PCR results indicated that ABCG2 mRNA was expressed at higher levels in SP cells than in non-SP cells in human, rhesus monkey, and mouse hematopoietic tissues (Zhou et al., 2001). ABCG2 mRNA was also expressed in the SP cell population of murine skeletal muscle progenitor cells, nestin-positive pancreatic islet-derived progenitor cells, and mouse embryonic stem cells (Scharenberg et al., 2002; Bunting, 2002; Kim et al., 2002). Therefore, in addition to its important use as a determinant of the SP phenotype, the ABCG2 gene is an attractive candidate marker for use in isolating stem cells, including neural stem cells, from a variety of sources, even though the function of ABCG2 in stem cells is not completely understood. In the central nervous system (CNS), neural stem cells (NSCs) differentiate into neurons and glial cells (Gage, 2000; Okano, 2002). Since the discovery of NSCs, many researchers have sought to improve the function of the damaged mammalian CNS using a population of ex vivo-expanded immature neural cells that contains NSCs (neural stem/ progenitor cells; NSPCs) instead of fetal brain tissues, and some studies have already reported hopeful results using human NSPCs (hNSPCs) in clinical applications (Bjorklund and Lindvall, 2000; Svendsen and Smith, 1999; Ishibashi et al., 2004). To use hNSPCs as a cellular therapy to regenerate damaged CNS, it is very important to investigate and characterize selective or specific phenotypic markers for these cells, so they can be manipulated and the quality of the donor cells improved. Although ABCG2 is considered a universal marker of stem cells (Bunting, 2002) and expression of the ABCG2 gene in mouse NSPCs using ‘SP’ and ‘non-SP’ (Kim and Morshead, 2003) subtractive hybridization (Terskikh et al., 2001) and array (Geschwind et al., 2001), and immunocytochemical (Cai et al., 2004) analyses has been reported, its protein level, localization, and function in NSPCs, including hNSPCs, are totally unknown. In this study, we analyzed the ABCG2 protein expression, determined the ABCG2-positive cell population in human neurospheres compared with the nestin-positive population, and studied the intracellular localization of ABCG2 in human fetal NSPCs. To determine whether the ABCG2 in hNSPCs was functionally active, we analyzed its ATPhydrolysis activity. Finally, we investigated the ABCG2 expression pattern during hNSPC differentiation. The present results demonstrated that ABCG2 was highly expressed in human fetal NSPCs, the ABCG2-positive cell population in neurospheres was similar to that of the nestinpositive cells, and ABCG2 and nestin were often colocalized in the same cells. The ABCG2 in hNSPCs was functionally active and its expression was sharply downregulated during differentiation, which suggested that the
ABCG2 in was functional. These findings provide some basic information that will be useful for clinical applications of hNSPCs and its possible use as a marker for human neural stem cells.
2. Materials and methods 2.1. Antibodies and chemicals Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and DMEM/Ham’s F-12 (1:1) were purchased from Sigma (St. Louis, MO, USA). Human recombinant epidermal growth factor (hr-EGF), fibroblast growth factor 2 (hr-FGF2), and leukemia inhibitory factor (hr-LIF) were from Invitrogen Corp. (Carlsbad, CA), PeproTech Inc. (Rocky Hill, NJ), and Chemicon International Inc. (Temecula, CA), respectively. Anti-ABCG2 (rabbit polyclonal, MXR8740) was a generous gift from Dr. Susan E. Bates (NIH, USA). Anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal and mouse monoclonal (clone G-A-5) antibodies were purchased from Sigma (St. Louis, MO) while anti-human nestin rabbit polyclonal and mouse monoclonal antibodies were purchased from Chemicon International Inc. (Temecula, CA), respectively. All other materials were of the finest grade commercially available. 2.2. Human NSPC culture and differentiation Approval to use human fetal neural tissues was obtained from the ethical committees of both Osaka National Hospital and the National Institute of Advanced Industrial Science and Technology. Tissue procurement was in accordance with the declaration of Helsinki and in agreement with the ethical guidelines of the European Network for Transplantation (NECTA) and the Japan Society of Obstetrics and Gynecology. Three human fetal forebrain samples, from fetuses at 7, 9, and 10 weeks gestational age (GW), were obtained from routine legal terminations performed at the Osaka National Hospital. Fetal brain tissue samples were mechanically dissected in DMEM/Ham’s F-12 (1:1). After dissection, the tissue samples were enzymatically digested with 0.05% trypsin/0.53 mM EDTA. After three washes in DMEM/Ham’s F-12 (1:1), the tissue samples were triturated using a fine polished Pasteur pipette and then passed through a 40 mm nylon mesh, to achieve single-cell suspensions. Cell counts and viability were assessed by trypan blue dye exclusion using a hemocytometer. NSPCs were cultured using the neurosphere culture technique as described previously (Kanemura et al., 2002, 2005). Briefly, cell suspensions were grown in DMEM/F-12 (1:1)-based defined medium supplemented with hr-EGF (20 ng/ml), hr-FGF2 (20 ng/ml), hr-LIF (10 ng/ml), heparin (5 mg/ml), B27 supplement, 15 mM HEPES, penicillin (100 units/ml), streptomycin (100 mg/ml), and amphotericin B (250 ng/ml). Viable single cells at a density of
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2 106 cells to 4 106 cells/15 ml were seeded into uncoated T75 culture flasks and incubated at 37 8C in 5% CO2–95% air. Half the volume of the culture medium was replaced by fresh growth medium every 7 days. For passaging, every 14–21 days, neurospheres were dissociated to single cells by digestion with 0.05% trypsin/0.53 mM EDTA at 37 8C for 20 min, and re-suspended in 50% fresh growth medium plus 50% neurosphere-conditioned medium. To induce glial differentiation, neurospheres cultured for 4 days were plated either on a poly-ornithine-coated glass disk (PLO-disk) or in a coated T25 primaria culture flask (Becton-Dickinson, Franklin Lakes, NJ) and cultured for 15 days in DMEM-high glucose medium plus 10% fetal bovine serum (FBS). The differentiated cells on the PLOdisk were used for immunocytochemistry and those in the T25 flask were used for immunoblotting. 2.3. Immunohistochemistry and immunocytochemistry The neurospheres were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 20 min at room temperature. After fixation, the neurospheres were dipped in 30% sucrose in PBS for 30 min at room temperature, embedded in OCT-compound (Sakura Finetechnical, Tokyo, Japan) and sectioned at 12 mm on a cryotome; single cells from dissociated neurospheres (1 h post-plating on glass disk) and the differentiated cells were fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature. The fixed neurosphere sections, and the single and differentiated cells on glass disks were washed in PBS and blocked with 10% goat serum for 1 h at room temperature, and then the single cells were incubated overnight with anti-nestin (monoclonal, 1:500) and antiABCG2 (1:500) in PBS containing 10% normal goat serum at 4 8C while the differentiated cells were incubated with anti-GFAP (monoclonal, 1:80) and anti-ABCG2 (1:500) antibodies using the same procedure. After washing, the neurosphere sections and the cells on glass disks were incubated with secondary antibodies (Alexa Fluor1 568 goat anti-rabbit IgG and Alexa Fluor1 488 goat anti-mouse IgG, Molecular Probes Inc., OR, USA) at room temperature for 1 h. Nuclear staining was performed using TO-PRO-31 (Molecular Probes Inc.). The quantification of the different phenotypes of the single cells was accomplished by counting the immunolabelled cells of four randomly chosen fields from every coverslip of total three coverslips. Fluorescent signals were detected with a confocal scanning laser microscope (LSM510, Carl Zeiss). The polyclonal ABCG2 antibody (MXR87405) was developed against a synthetic peptide corresponding to an epitope located in the cytoplasmic region of ABCG2 (Litman et al., 2002). 2.4. Microsome preparation and subcellular fractionation Microsomes from neurospheres, differentiated cells, and the A549 human cell line (human lung carcinoma cell line,
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RIKEN cell bank, Tsukuba, Japan), as a positive control for ABCG2, were prepared as described previously (Sarkadi et al., 1992) with slight modification. The neurospheres, differentiated astrocytes, and A549 cells were collected by centrifugation and washed three times with ice-cold PBS. The cell pellets were then homogenized using a glass–teflon homogenizer in TEMP (50 mM Tris–HCl, pH 7.0, containing 50 mM mannitol, 2 mM EGTA, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, 0.5 mM PMSF, and 2 mM 2mercaptoethanol), and the undisrupted cells and nuclear debris were removed by centrifugation at 500 g for 10 min. The supernatant fluid was then spun for 60 min at 100,000 g, and the pellet was suspended in TEMP at a protein concentration of 2–3 mg/ml; this preparation contained the microsomes. For membrane fractionations, microsomes from the neurospheres and the A549 cell line were fractionated by sucrose density gradient using 8, 25 and 45% sucrose and centrifugation at 100,000 g for 60 min. The three fractions (upper [U], medium [M], and lower [L]; from top to bottom) were collected and used for Na+/K+ATPase assays and ABCG2 immunoblotting. All procedures were carried out at 4 8C, and the samples were stored at 808C until use. 2.5. Immunoblotting Aliquots containing 30 mg microsomal proteins were diluted in SDS sample buffer (60 mM Tris–HCl, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.02% bromophenol blue [pH 6.8]), denatured, and separated on 6 or 7.5% acrylamide gels (SDS-PAGE). Proteins were transferred electrophoretically to PVDF membranes, and the membranes were blocked in 5% skim milk overnight. The blotted proteins were probed with antibodies against ABCG2 (1:500), nestin (polyclonal, 1:500), and GFAP (polyclonal, 1:1000) for 2 h at room temperature. After several washes, the membranes were incubated with an anti-rabbit HRPconjugated secondary antibody (Amersham, UK), and finally, the proteins were visualized with enhanced chemiluminescence (ECL system Amersham, UK). 2.6. ATPase assay Membrane ATPase activity was measured by the colorimetric detection of inorganic phosphate liberation as described (Chen and Lin-Shiau, 1988) with minor modifications. The reaction mixture contained 40 mM MOPS–Tris (pH 7.0), 50 mM KCl, 2 mM DTT, 5 mM EGTA, 5 mM sodium azide, and 30 mg microsomes. The Na+/K+-ATPase activity was determined as the difference between the activity in the absence and presence of 1 mM ouabain, and the Mg-ATPase (ABCG2) activity was determined by performing the assays with 1 mM ouabain in the absence or presence of 100 mm sodium-orthovanadate. The reaction was started by adding 3 mM MgATP, and after 30 min was stopped with 2 ml malachite
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Fig. 1. Immunoblot detection of the ABCG2 transporter expressed in hNSPCs and A549 cells. Microsomal proteins (30 mg/lane) from three different preparations (7, 9, and 10 GW) of hNSPCs and from A549 cells (human lung carcinoma cell line) were separated by polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and probed with a polyclonal anti-ABCG2 (MXR87405) antibody. The bound antibody was detected by ECL.
green. To determine the effect of prazosin on the MgATPase, the assay was carried out under basal conditions alone or with increasing concentrations of prazosin (0, 1, 5, 10, and 50 mM).
3. Results 3.1. Expression and localization of ABCG2 in hNSPCs The expression of ABCG2 at the protein level was ascertained by immunoblotting with the ABCG2-specific polyclonal antibody 87405 (Fig. 1). A band of approximately 72 kDa, corresponding to the expected molecular mass for ABCG2, was detected in all of three lines of hNSPCs and the A549 cell line, which has been reported to express ABCG2, MDR1, and MRPs (Kim et al., 2002). These findings indicated that ABCG2 was highly expressed in human cultured NSPCs at the protein level.
Several lines of evidence have suggested that ABCG2 acts as an efflux pump to reduce the intracellular drug concentration (Ross et al., 2000; Miyake et al., 1999). Therefore, it was of interest to determine whether the ABCG2 in hNSPCs was localized to the plasma membrane, in agreement with its function as a drug efflux pump, or to intracellular membranes, as expected if it acted as an ABC half-transporter. Immunofluorescence analysis using intact neurospheres showed that many of the cells in the neurospheres expressed ABCG2 (Fig. 2A). To determine its intracellular localization, we dissociated single cells from neurospheres and immunostained them with the antiABCG2 antibody. The three-dimensional structure revealed by confocal microscopy of the single cells stained with the anti-ABCG2 antibody showed that ABCG2 was localized to the plasma membrane (Fig. 2B). To confirm the localization of ABCG2 biochemically, we separated hNSPC microsomes into three factions by sucrose density gradient (U, M, and L fractions from top to bottom) and determined the Na+/K+ATPase activity and ABCG2 expression in all three fractions. The highest activity was observed in the M fraction and the next highest was in the U fraction, whereas the highest level of ABCG2 protein was detected in the U fraction (by immunoblotting) and the next highest was in the M fraction (Fig. 3). The lowest Na+/K+-ATPase activity and ABCG2 expression were observed in the L fraction. The Na+/K+-ATPase activity is used as a marker of the plasma membrane (Cai et al., 1999). Because we found the lowest Na+/K+-ATPase activity and ABCG2 expression in the L fraction, which is the ER fraction, of hNSPCs we proposed that the ABCG2 in hNSPCs was localized to the plasma membrane but not to the ER, unlike other half-transporters. We also fractioned A549 cells using the same procedure and found the same localization of ABCG2 as in hNSPCs (Fig. 3). We next determined the cell population in neurospheres with the ABCG2 phenotype. We dissociated single cells from neurospheres, stained them with anti-ABCG2 and antinestin (monoclonal) antibodies, and counted the ABCG2-
Fig. 2. Cellular localization of ABCG2 in human neurospheres and single cells. Human neurospheres (9 GW) (A) and single cells (B) dissociated from neurospheres were stained with the rabbit polyclonal anti-ABCG2 (MXR87405) antibody followed by Alexa Fluor1 568 goat anti-rabbit IgG and visualized (green) by fluorescence microscopy. The three-dimensional structure of single cells stained with the anti-ABCG2 antibody showed its localization to the plasma membrane (green). Nuclear staining was performed with TO-PRO-31 (scale bar = 50 mm).
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nestin-positive cells were also ABCG2 positive; therefore, most ABCG2-positive cells present in neurospheres were likely to be stem or progenitor cells. 3.2. ATPase activity
Fig. 3. Expression pattern of ABCG2 in different sub-cellular fractions of hNSPCs and A549 cells. Microsomes prepared from human NSPCs (9 GW) and A549 cells were separated into upper, medium, and lower fractions (from the top to bottom of the tube) by sucrose density gradient. The level of ABCG2 in the three fractions was detected with the anti-ABCG2 antibody (upper panel), and the corresponding Na+/K+-ATPase activity in the three fractions was also measured (lower panel). Values represent the means S.D. of three independent experiments in duplicate.
positive, nestin-positive, and ABCG2/nestin double-positive cells. Of the total neurosphere cell population, 62.9 7.8% of the cells were ABCG2 positive, 51.3 9.5% were nestin positive, and 49.4 8.6% expressed both ABCG2 and nestin (Fig. 4). These data showed that the number of ABCG2-positive cells present in neurospheres was slightly higher than that of nestin-positive cells, but most of the
Next, to investigate the functional significance of the ABCG2 expressed in hNSPCs, its ability to hydrolyze ATP was analyzed. ABCG2 transporter has previously been shown to exhibit drug-stimulated, vanadate-sensitive MgATPase activity, especially in the presence of prazosin (Polgar et al., 2004). We determined the vanadate-sensitive Mg-ATPase activity of the ABCG2 protein derived from 9 GW hNSPCs, in the presence of 0, 1, 5, 10, and 50 mM prazosin, and found that 50 mM prazosin increased the ATP hydrolysis approximately 2.5-fold over the basal level (Fig. 5). These findings suggested that hNSPCs expressed functionally active ABCG2. 3.3. Regulation of ABCG2 expression during hNSPCs differentiation ABCG2 is sharply down-regulated during hematopoietic stem cell differentiation and expressed at a low level in mature cells compared with progenitor cells (Scharenberg et al., 2002). To investigate whether the same phenomenon occurs in hNSPCs, we induced the differentiation of hNSPCs with 10% FBS and compared the ABCG2 expression with that of the astrocyte marker GFAP in the differentiated human normal astrocytes. Western blotting
Fig. 4. Phenotype analysis of the ABCG2- and nestin-positive cells in neurospheres. Neurospheres were dissociated into single cells and double-immunostained with anti-ABCG2 (polyclonal; red) and anti-nestin (green; monoclonal) antibodies. The individual population of ABCG2- and nestin-positive cells, and of ABCG2/nestin double positive cells were counted. Nuclear staining was performed with TO-PRO-31 (scale bar = 20 mm).
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did not express ABCG2, and a very few GFAP-positive cells also expressed ABCG2 (Fig. 6B, round). Therefore, most of the differentiated astrocytes were ABCG2 negative and only a few astrocytes were ABCG2 and GFAP double positive. This finding suggested that, as in hematopoietic stem cells, the ABCG2 of hNSPCs was down-regulated during differentiation or maturation, indicating that its expression might be related to the physiology of stem/progenitor cells.
4. Discussion
Fig. 5. Substrate-stimulated Mg-ATP hydrolysis of hNSPCs. The vanadatesensitive Mg-ATP hydrolysis (ABCG2 activity) in the presence of the indicated concentrations of prazosin in the crude membranes of 9 GW hNSPCs expressing ABCG2. The line represents the average of three measurements using two different membrane preparations.
(Fig. 6A) of human neurospheres and the differentiated astrocytes from hNSPCs showed that ABCG2 was sharply down-regulated in the astrocytes, whereas GFAP was upregulated slightly. During the differentiation of hNSPCs, their morphology changed and the expression of GFAP increased, which might have been due to the maturation of the hNSPCs. Although some GFAP-negative cells expressed ABCG2 after differentiation, but most GFAP-positive cells
The present study is the first to show ABCG2 protein expression in hNSPCs. We also showed direct evidence that cultured NSPCs derived from human fetal brain expressed a higher level of ABCG2 than did the malignant cell line A549, and that ABCG2 was localized to the plasma membrane of hNSPCs. The ABCG2 level in hNSPCs was sharply downregulated during astrocyte differentiation, similar to the sharp down-regulation of ABCG2 in hematopoietic stem cells at the stage of lineage commitment (Scharenberg et al., 2002), suggesting that ABCG2 might play an important role in the unique physiology of stem/progenitor cells. Generally, full-transporters are found in the plasma membrane, whereas half-transporters are found in intracellular membranes, such as the mitochondria and endoplasmic reticulum. By both three-dimensional structure analysis and biochemical examination we found that the localization of ABCG2 was to the plasma membrane of hNSPCs, similar to
Fig. 6. Regulation of ABCG2 expression during hNSPC differentiation. (A) Human neurospheres were induced to differentiate into astrocytes with 10% FBS for 15 days, then the expression patterns of ABCG2 and GFAP in the neurospheres and in the differentiated astrocytes were quantified by western blotting ABCG2 (polyclonal) and GFAP (polyclonal) antibodies. (B) The expression pattern of ABCG2- and GFAP-positive cells in the differentiated astrocytes was detected by immunochemistry with ABCG2 (polyclonal) and GFAP (monoclonal) antibodies (scale bar = 20 mm).
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its expression in ABCG2-over-expressing MCF7 cells (Rocchi et al., 2000). The most important finding of the present study was that about 63% of the cells within neurospheres were ABCG2 positive, similar to the proportion of nestin-positive cells, and the ABCG2 and nestin staining in most cases labeled the same cells, which is evidence that, like nestin, ABCG2 might be a stem/ progenitor cell marker for hNSPCs. This is the first report of the co-localization of ABCG2- and nestin-positive cells among hNSPCs, and this finding indicates that ABCG2 may have a functional role in hNSPCs. Consistent with the idea that ABCG2 might be expressed in its native form and have physiological functions in hNSPCs, we found that the ABCG2 in hNSPCs had a high substrate-stimulated ATPhydrolysis activity. This result suggested that the ABCG2 expressed in hNSPCs was functionally active, possibly playing a regulatory role in the maintenance of the undifferentiated state. The sharp down-regulation of ABCG2 levels during hNSPC differentiation has implications for hNSPC maturation. Although mature astrocytes expressed a high level of the astrocyte marker GFAP, they expressed only a low level of ABCG2, and most of the GFAP-positive cells did not show ABCG2 labeling, although co-localization was observed in a few of the mature astrocytes. The differences in ABCG2 expression between neurospheres and differentiated astrocytes support the idea that ABCG2 is expressed in progenitor cells rather than in differentiated cells. Although we found functional ABCG2 in hNSPCs, similar to the findings of other investigators studying other stem cells, its functional role in these cells, including NSPCs, has not been clear. The ABC transporters are known to transport a variety of toxic lipophilic compounds; thus, ABCG2 and other ABC transporters may act to protect stem cells from cytotoxic agents. It is also possible that a high expression of ABC transporters, as suggested by Bunting et al. (2000), may be critical to maintaining stem cells in a quiescent state. Perhaps the expression of transporters that are similar to ABCG2 is necessary for stem cells to regulate the uptake of small hydrophobic molecules involved in cell differentiation (Zhou et al., 2002). In conclusion, we propose that ABCG2, which is known as a universal stem cell marker, is also functionally expressed in hNSPCs, and may protect human NSPCs from xenobiotics and other toxic substances, as proposed previously (Zhou et al., 2002). Moreover, the anti-ABCG2 antibody might be used to separate neural stem/progenitor cells from the cell populations in neurospheres, and we are presently investigating this possibility.
Acknowledgments We thank Dr. Chiaki Ban at Osaka National Hospital for providing human fetal tissues and Dr. Susan E. Bates of the National Cancer Institute, NIH, USA, for providing the
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anti-ABCG2 (MXR87405) antibody. This study was supported in part by the Millennium Project from the Ministry of Economy, Trade and Industry of Japan, and by the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) project from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Kim, M., Morshead, C.M., 2003. Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis. J. Neurosci. 23, 10703–10709. Litman, T., Jensen, U., Hansen, A., Covitz, K.M., Zhan, Z., Fetsch, P., Abati, A., Hansen, P.R., Horn, R., Skovsgaard, T., Bates, S.E., 2002. Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2. Biochim. Biophys. Acta 1565, 6–16. Matsuzaki, Y., Kinjo, K., Mulligan, R.C., Okano, H., 2004. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 20, 87–93. Miyake, K., Mickley, L., Litman, T., Zhan, Z., Robey, R., Cristensen, B., Brangi, M., Greenberger, L., Dean, T., Fojo, M., Bates, S.E., 1999. Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res. 59, 8–13. Murayama, A., Matsuzaki, Y., Kawaguti, A., Shimazaki, T., Okano, H., 2002. Flow cytometric analysis of stem cells in the developing and adult mouse brain. J. Neurosci. Res. 69, 837–847. Okano, H., 2002. The stem cell biology of the central nervous system. J. Neurosci. Res. 69, 698–707. Polgar, O., Robey, R.W., Morisaki, K., Dean, M., Michejda, C., Sauna, Z.E., Ambudkar, S.V., Tarasova, N., Bates, S.E., 2004. Mutational analysis of ABCG2: role of the GXXXG motif. Biochemistry 43, 9448– 9456. Rocchi, E., Khodjakov, A., Volk, E.L., Yang, C.H., Litman, T., Bates, S.E., Schneider, E., 2000. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem. Biophys. Res. Commun. 271, 42–46.
Ross, D.D., Karp, J.E., Chen, T.T., Doyle, L.A., 2000. Expression of breast cancer resistance protein in blast cells from patients with acute leukemia. Blood 96, 365–368. Sarkadi, B., Price, E.M., Boucher, R.C., Germann, U.A., Scarborough, G.A., 1992. Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J. Biol. Chem. 267, 4854–4858. Scharenberg, C.W., Harkey, M.A., Torok-Storb, B., 2002. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 99, 507–512. Svendsen, C.N., Smith, A.G., 1999. New prospects for human stem-cell therapy in the nervous system. Trends Neurosci. 22, 357–364. Terskikh, A.V., Easterday, M.C., Hood, L., Kornblum, H.I., Geschwind, D.H., Weissman, I.L., 2001. From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc. Natl. Acad. Sci. U.S.A. 98, 7934–7939. Zhou, S., Morris, J.J., Barnes, Y., Lan, L., Schuetz, D., Sorrentino, B.P., 2002. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc. Natl. Acad. Sci. U.S.A. 99, 12339–12344. Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, M., Sorrentino, B.P., 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med. 7, 1028–1034.
Functional expression of ABCG2 transporter in human neural stem/progenitor cells Mohammed Omedul Islam a,b, Yonehiro Kanemura a,c,*, Jesmin Tajria a, Hideki Mori a, Satoshi Kobayashi a, Masayuki Hara d, Mami Yamasaki c,e, Hideyuki Okano f,g, Jun Miyake a a
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Nakoji 3-11-46, Amagasaki, Hyogo 661-0974, Japan b Institute of Biomedical Research and Innovation, Kobe, Hyogo 650-0047, Japan c Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan d Department of Applied Bioscience, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Osaka 599-8570, Japan e Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan f Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan g Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Saitama 332-0012, Japan Received 15 October 2004; accepted 28 January 2005
Abstract We have studied the expression, localization, and function of the ABCG2 transporter, a universal stem cell marker, at the protein level in human cultured neural stem/progenitor cells (hNSPCs) using immunoblotting, immunofluorescence, and ATPase assays. Human NSPCs were isolated from human fetal brain and propagated in vitro as neurospheres. Both the cells in neurospheres and single cells dissociated from neurospheres showed high levels of ABCG2, and about 63% of the cells in neurospheres were ABCG2-positive, similar to the proportion of nestin-positive cells, and in most cases the ABCG2 and nestin staining co-localized in the same cells. Both the three-dimensional structure of single hNSPCs stained with anti-ABCG2 antibodies and an examination using a biochemical marker for the plasma membrane indicated that ABCG2 was localized to the plasma membrane of hNSPCs. The ABCG2 expressed in hNSPCs had prazosin-sensitive ATP hydrolysis activity, and the ABCG2 level was sharply down-regulated during hNSPC differentiation. All these results suggested that ABCG2, was functionally expressed in hNSPCs. ABCG2 might play a significant role in maintaining human neural stem cells in an undifferentiated state and in protecting hNSPCs from xenobiotics or other toxic substances in vivo. # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: ABC transporter; Human neural stem/progenitor cells; ABCG2
1. Introduction ABCG2, a half-transporter also called MXR/BCRP/ ABCP, is a member of the ATP-binding cassette (ABC) family of cell-surface transporter proteins. It is expressed in a variety of malignant and normal tissues, and promotes the efflux of a variety of anti-neoplastic drugs, including anthracyclines and campothecins, from the cell (Doyle et al., * Corresponding author. Tel.: +81 6 6494 7839; fax: +81 6 6494 7862. E-mail address: [email protected] (Y. Kanemura).
1998; Ross et al., 2000; Miyake et al., 1999). Unlike other ABC half-transporters, which are localized to intracellular membranes, ABCG2 is expressed exclusively in the plasma membrane (Rocchi et al., 2000). Recently, ABCG2 was found in the primitive stem cells of different tissues, which led to enthusiasm for the idea that it might play a significant role in maintaining stem cells in an undifferentiated state (Zhou et al., 2001). ABCG2 is highly expressed in a specific population of hematopoietic stem cells, the so-called ‘SP’ (side population) subset, which is isolated by the cells’ ability to promote the efflux of Hoechst
0168-0102/$ – see front matter # 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2005.01.013
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33342 fluorescent dye (Zhou et al., 2001; Murayama et al., 2002; Goodell et al., 1996; Matsuzaki et al., 2004). During the differentiation of hematopoietic stem cells, the level of ABCG2 is sharply down-regulated, underscoring the gene’s potential use as a stem cell marker (Scharenberg et al., 2002). Semi-quantitative RT-PCR results indicated that ABCG2 mRNA was expressed at higher levels in SP cells than in non-SP cells in human, rhesus monkey, and mouse hematopoietic tissues (Zhou et al., 2001). ABCG2 mRNA was also expressed in the SP cell population of murine skeletal muscle progenitor cells, nestin-positive pancreatic islet-derived progenitor cells, and mouse embryonic stem cells (Scharenberg et al., 2002; Bunting, 2002; Kim et al., 2002). Therefore, in addition to its important use as a determinant of the SP phenotype, the ABCG2 gene is an attractive candidate marker for use in isolating stem cells, including neural stem cells, from a variety of sources, even though the function of ABCG2 in stem cells is not completely understood. In the central nervous system (CNS), neural stem cells (NSCs) differentiate into neurons and glial cells (Gage, 2000; Okano, 2002). Since the discovery of NSCs, many researchers have sought to improve the function of the damaged mammalian CNS using a population of ex vivo-expanded immature neural cells that contains NSCs (neural stem/ progenitor cells; NSPCs) instead of fetal brain tissues, and some studies have already reported hopeful results using human NSPCs (hNSPCs) in clinical applications (Bjorklund and Lindvall, 2000; Svendsen and Smith, 1999; Ishibashi et al., 2004). To use hNSPCs as a cellular therapy to regenerate damaged CNS, it is very important to investigate and characterize selective or specific phenotypic markers for these cells, so they can be manipulated and the quality of the donor cells improved. Although ABCG2 is considered a universal marker of stem cells (Bunting, 2002) and expression of the ABCG2 gene in mouse NSPCs using ‘SP’ and ‘non-SP’ (Kim and Morshead, 2003) subtractive hybridization (Terskikh et al., 2001) and array (Geschwind et al., 2001), and immunocytochemical (Cai et al., 2004) analyses has been reported, its protein level, localization, and function in NSPCs, including hNSPCs, are totally unknown. In this study, we analyzed the ABCG2 protein expression, determined the ABCG2-positive cell population in human neurospheres compared with the nestin-positive population, and studied the intracellular localization of ABCG2 in human fetal NSPCs. To determine whether the ABCG2 in hNSPCs was functionally active, we analyzed its ATPhydrolysis activity. Finally, we investigated the ABCG2 expression pattern during hNSPC differentiation. The present results demonstrated that ABCG2 was highly expressed in human fetal NSPCs, the ABCG2-positive cell population in neurospheres was similar to that of the nestinpositive cells, and ABCG2 and nestin were often colocalized in the same cells. The ABCG2 in hNSPCs was functionally active and its expression was sharply downregulated during differentiation, which suggested that the
ABCG2 in was functional. These findings provide some basic information that will be useful for clinical applications of hNSPCs and its possible use as a marker for human neural stem cells.
2. Materials and methods 2.1. Antibodies and chemicals Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and DMEM/Ham’s F-12 (1:1) were purchased from Sigma (St. Louis, MO, USA). Human recombinant epidermal growth factor (hr-EGF), fibroblast growth factor 2 (hr-FGF2), and leukemia inhibitory factor (hr-LIF) were from Invitrogen Corp. (Carlsbad, CA), PeproTech Inc. (Rocky Hill, NJ), and Chemicon International Inc. (Temecula, CA), respectively. Anti-ABCG2 (rabbit polyclonal, MXR8740) was a generous gift from Dr. Susan E. Bates (NIH, USA). Anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal and mouse monoclonal (clone G-A-5) antibodies were purchased from Sigma (St. Louis, MO) while anti-human nestin rabbit polyclonal and mouse monoclonal antibodies were purchased from Chemicon International Inc. (Temecula, CA), respectively. All other materials were of the finest grade commercially available. 2.2. Human NSPC culture and differentiation Approval to use human fetal neural tissues was obtained from the ethical committees of both Osaka National Hospital and the National Institute of Advanced Industrial Science and Technology. Tissue procurement was in accordance with the declaration of Helsinki and in agreement with the ethical guidelines of the European Network for Transplantation (NECTA) and the Japan Society of Obstetrics and Gynecology. Three human fetal forebrain samples, from fetuses at 7, 9, and 10 weeks gestational age (GW), were obtained from routine legal terminations performed at the Osaka National Hospital. Fetal brain tissue samples were mechanically dissected in DMEM/Ham’s F-12 (1:1). After dissection, the tissue samples were enzymatically digested with 0.05% trypsin/0.53 mM EDTA. After three washes in DMEM/Ham’s F-12 (1:1), the tissue samples were triturated using a fine polished Pasteur pipette and then passed through a 40 mm nylon mesh, to achieve single-cell suspensions. Cell counts and viability were assessed by trypan blue dye exclusion using a hemocytometer. NSPCs were cultured using the neurosphere culture technique as described previously (Kanemura et al., 2002, 2005). Briefly, cell suspensions were grown in DMEM/F-12 (1:1)-based defined medium supplemented with hr-EGF (20 ng/ml), hr-FGF2 (20 ng/ml), hr-LIF (10 ng/ml), heparin (5 mg/ml), B27 supplement, 15 mM HEPES, penicillin (100 units/ml), streptomycin (100 mg/ml), and amphotericin B (250 ng/ml). Viable single cells at a density of
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2 106 cells to 4 106 cells/15 ml were seeded into uncoated T75 culture flasks and incubated at 37 8C in 5% CO2–95% air. Half the volume of the culture medium was replaced by fresh growth medium every 7 days. For passaging, every 14–21 days, neurospheres were dissociated to single cells by digestion with 0.05% trypsin/0.53 mM EDTA at 37 8C for 20 min, and re-suspended in 50% fresh growth medium plus 50% neurosphere-conditioned medium. To induce glial differentiation, neurospheres cultured for 4 days were plated either on a poly-ornithine-coated glass disk (PLO-disk) or in a coated T25 primaria culture flask (Becton-Dickinson, Franklin Lakes, NJ) and cultured for 15 days in DMEM-high glucose medium plus 10% fetal bovine serum (FBS). The differentiated cells on the PLOdisk were used for immunocytochemistry and those in the T25 flask were used for immunoblotting. 2.3. Immunohistochemistry and immunocytochemistry The neurospheres were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 20 min at room temperature. After fixation, the neurospheres were dipped in 30% sucrose in PBS for 30 min at room temperature, embedded in OCT-compound (Sakura Finetechnical, Tokyo, Japan) and sectioned at 12 mm on a cryotome; single cells from dissociated neurospheres (1 h post-plating on glass disk) and the differentiated cells were fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature. The fixed neurosphere sections, and the single and differentiated cells on glass disks were washed in PBS and blocked with 10% goat serum for 1 h at room temperature, and then the single cells were incubated overnight with anti-nestin (monoclonal, 1:500) and antiABCG2 (1:500) in PBS containing 10% normal goat serum at 4 8C while the differentiated cells were incubated with anti-GFAP (monoclonal, 1:80) and anti-ABCG2 (1:500) antibodies using the same procedure. After washing, the neurosphere sections and the cells on glass disks were incubated with secondary antibodies (Alexa Fluor1 568 goat anti-rabbit IgG and Alexa Fluor1 488 goat anti-mouse IgG, Molecular Probes Inc., OR, USA) at room temperature for 1 h. Nuclear staining was performed using TO-PRO-31 (Molecular Probes Inc.). The quantification of the different phenotypes of the single cells was accomplished by counting the immunolabelled cells of four randomly chosen fields from every coverslip of total three coverslips. Fluorescent signals were detected with a confocal scanning laser microscope (LSM510, Carl Zeiss). The polyclonal ABCG2 antibody (MXR87405) was developed against a synthetic peptide corresponding to an epitope located in the cytoplasmic region of ABCG2 (Litman et al., 2002). 2.4. Microsome preparation and subcellular fractionation Microsomes from neurospheres, differentiated cells, and the A549 human cell line (human lung carcinoma cell line,
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RIKEN cell bank, Tsukuba, Japan), as a positive control for ABCG2, were prepared as described previously (Sarkadi et al., 1992) with slight modification. The neurospheres, differentiated astrocytes, and A549 cells were collected by centrifugation and washed three times with ice-cold PBS. The cell pellets were then homogenized using a glass–teflon homogenizer in TEMP (50 mM Tris–HCl, pH 7.0, containing 50 mM mannitol, 2 mM EGTA, 10 mg/ml leupeptin, 2 mg/ml pepstatin A, 0.5 mM PMSF, and 2 mM 2mercaptoethanol), and the undisrupted cells and nuclear debris were removed by centrifugation at 500 g for 10 min. The supernatant fluid was then spun for 60 min at 100,000 g, and the pellet was suspended in TEMP at a protein concentration of 2–3 mg/ml; this preparation contained the microsomes. For membrane fractionations, microsomes from the neurospheres and the A549 cell line were fractionated by sucrose density gradient using 8, 25 and 45% sucrose and centrifugation at 100,000 g for 60 min. The three fractions (upper [U], medium [M], and lower [L]; from top to bottom) were collected and used for Na+/K+ATPase assays and ABCG2 immunoblotting. All procedures were carried out at 4 8C, and the samples were stored at 808C until use. 2.5. Immunoblotting Aliquots containing 30 mg microsomal proteins were diluted in SDS sample buffer (60 mM Tris–HCl, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.02% bromophenol blue [pH 6.8]), denatured, and separated on 6 or 7.5% acrylamide gels (SDS-PAGE). Proteins were transferred electrophoretically to PVDF membranes, and the membranes were blocked in 5% skim milk overnight. The blotted proteins were probed with antibodies against ABCG2 (1:500), nestin (polyclonal, 1:500), and GFAP (polyclonal, 1:1000) for 2 h at room temperature. After several washes, the membranes were incubated with an anti-rabbit HRPconjugated secondary antibody (Amersham, UK), and finally, the proteins were visualized with enhanced chemiluminescence (ECL system Amersham, UK). 2.6. ATPase assay Membrane ATPase activity was measured by the colorimetric detection of inorganic phosphate liberation as described (Chen and Lin-Shiau, 1988) with minor modifications. The reaction mixture contained 40 mM MOPS–Tris (pH 7.0), 50 mM KCl, 2 mM DTT, 5 mM EGTA, 5 mM sodium azide, and 30 mg microsomes. The Na+/K+-ATPase activity was determined as the difference between the activity in the absence and presence of 1 mM ouabain, and the Mg-ATPase (ABCG2) activity was determined by performing the assays with 1 mM ouabain in the absence or presence of 100 mm sodium-orthovanadate. The reaction was started by adding 3 mM MgATP, and after 30 min was stopped with 2 ml malachite
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Fig. 1. Immunoblot detection of the ABCG2 transporter expressed in hNSPCs and A549 cells. Microsomal proteins (30 mg/lane) from three different preparations (7, 9, and 10 GW) of hNSPCs and from A549 cells (human lung carcinoma cell line) were separated by polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and probed with a polyclonal anti-ABCG2 (MXR87405) antibody. The bound antibody was detected by ECL.
green. To determine the effect of prazosin on the MgATPase, the assay was carried out under basal conditions alone or with increasing concentrations of prazosin (0, 1, 5, 10, and 50 mM).
3. Results 3.1. Expression and localization of ABCG2 in hNSPCs The expression of ABCG2 at the protein level was ascertained by immunoblotting with the ABCG2-specific polyclonal antibody 87405 (Fig. 1). A band of approximately 72 kDa, corresponding to the expected molecular mass for ABCG2, was detected in all of three lines of hNSPCs and the A549 cell line, which has been reported to express ABCG2, MDR1, and MRPs (Kim et al., 2002). These findings indicated that ABCG2 was highly expressed in human cultured NSPCs at the protein level.
Several lines of evidence have suggested that ABCG2 acts as an efflux pump to reduce the intracellular drug concentration (Ross et al., 2000; Miyake et al., 1999). Therefore, it was of interest to determine whether the ABCG2 in hNSPCs was localized to the plasma membrane, in agreement with its function as a drug efflux pump, or to intracellular membranes, as expected if it acted as an ABC half-transporter. Immunofluorescence analysis using intact neurospheres showed that many of the cells in the neurospheres expressed ABCG2 (Fig. 2A). To determine its intracellular localization, we dissociated single cells from neurospheres and immunostained them with the antiABCG2 antibody. The three-dimensional structure revealed by confocal microscopy of the single cells stained with the anti-ABCG2 antibody showed that ABCG2 was localized to the plasma membrane (Fig. 2B). To confirm the localization of ABCG2 biochemically, we separated hNSPC microsomes into three factions by sucrose density gradient (U, M, and L fractions from top to bottom) and determined the Na+/K+ATPase activity and ABCG2 expression in all three fractions. The highest activity was observed in the M fraction and the next highest was in the U fraction, whereas the highest level of ABCG2 protein was detected in the U fraction (by immunoblotting) and the next highest was in the M fraction (Fig. 3). The lowest Na+/K+-ATPase activity and ABCG2 expression were observed in the L fraction. The Na+/K+-ATPase activity is used as a marker of the plasma membrane (Cai et al., 1999). Because we found the lowest Na+/K+-ATPase activity and ABCG2 expression in the L fraction, which is the ER fraction, of hNSPCs we proposed that the ABCG2 in hNSPCs was localized to the plasma membrane but not to the ER, unlike other half-transporters. We also fractioned A549 cells using the same procedure and found the same localization of ABCG2 as in hNSPCs (Fig. 3). We next determined the cell population in neurospheres with the ABCG2 phenotype. We dissociated single cells from neurospheres, stained them with anti-ABCG2 and antinestin (monoclonal) antibodies, and counted the ABCG2-
Fig. 2. Cellular localization of ABCG2 in human neurospheres and single cells. Human neurospheres (9 GW) (A) and single cells (B) dissociated from neurospheres were stained with the rabbit polyclonal anti-ABCG2 (MXR87405) antibody followed by Alexa Fluor1 568 goat anti-rabbit IgG and visualized (green) by fluorescence microscopy. The three-dimensional structure of single cells stained with the anti-ABCG2 antibody showed its localization to the plasma membrane (green). Nuclear staining was performed with TO-PRO-31 (scale bar = 50 mm).
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nestin-positive cells were also ABCG2 positive; therefore, most ABCG2-positive cells present in neurospheres were likely to be stem or progenitor cells. 3.2. ATPase activity
Fig. 3. Expression pattern of ABCG2 in different sub-cellular fractions of hNSPCs and A549 cells. Microsomes prepared from human NSPCs (9 GW) and A549 cells were separated into upper, medium, and lower fractions (from the top to bottom of the tube) by sucrose density gradient. The level of ABCG2 in the three fractions was detected with the anti-ABCG2 antibody (upper panel), and the corresponding Na+/K+-ATPase activity in the three fractions was also measured (lower panel). Values represent the means S.D. of three independent experiments in duplicate.
positive, nestin-positive, and ABCG2/nestin double-positive cells. Of the total neurosphere cell population, 62.9 7.8% of the cells were ABCG2 positive, 51.3 9.5% were nestin positive, and 49.4 8.6% expressed both ABCG2 and nestin (Fig. 4). These data showed that the number of ABCG2-positive cells present in neurospheres was slightly higher than that of nestin-positive cells, but most of the
Next, to investigate the functional significance of the ABCG2 expressed in hNSPCs, its ability to hydrolyze ATP was analyzed. ABCG2 transporter has previously been shown to exhibit drug-stimulated, vanadate-sensitive MgATPase activity, especially in the presence of prazosin (Polgar et al., 2004). We determined the vanadate-sensitive Mg-ATPase activity of the ABCG2 protein derived from 9 GW hNSPCs, in the presence of 0, 1, 5, 10, and 50 mM prazosin, and found that 50 mM prazosin increased the ATP hydrolysis approximately 2.5-fold over the basal level (Fig. 5). These findings suggested that hNSPCs expressed functionally active ABCG2. 3.3. Regulation of ABCG2 expression during hNSPCs differentiation ABCG2 is sharply down-regulated during hematopoietic stem cell differentiation and expressed at a low level in mature cells compared with progenitor cells (Scharenberg et al., 2002). To investigate whether the same phenomenon occurs in hNSPCs, we induced the differentiation of hNSPCs with 10% FBS and compared the ABCG2 expression with that of the astrocyte marker GFAP in the differentiated human normal astrocytes. Western blotting
Fig. 4. Phenotype analysis of the ABCG2- and nestin-positive cells in neurospheres. Neurospheres were dissociated into single cells and double-immunostained with anti-ABCG2 (polyclonal; red) and anti-nestin (green; monoclonal) antibodies. The individual population of ABCG2- and nestin-positive cells, and of ABCG2/nestin double positive cells were counted. Nuclear staining was performed with TO-PRO-31 (scale bar = 20 mm).
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did not express ABCG2, and a very few GFAP-positive cells also expressed ABCG2 (Fig. 6B, round). Therefore, most of the differentiated astrocytes were ABCG2 negative and only a few astrocytes were ABCG2 and GFAP double positive. This finding suggested that, as in hematopoietic stem cells, the ABCG2 of hNSPCs was down-regulated during differentiation or maturation, indicating that its expression might be related to the physiology of stem/progenitor cells.
4. Discussion
Fig. 5. Substrate-stimulated Mg-ATP hydrolysis of hNSPCs. The vanadatesensitive Mg-ATP hydrolysis (ABCG2 activity) in the presence of the indicated concentrations of prazosin in the crude membranes of 9 GW hNSPCs expressing ABCG2. The line represents the average of three measurements using two different membrane preparations.
(Fig. 6A) of human neurospheres and the differentiated astrocytes from hNSPCs showed that ABCG2 was sharply down-regulated in the astrocytes, whereas GFAP was upregulated slightly. During the differentiation of hNSPCs, their morphology changed and the expression of GFAP increased, which might have been due to the maturation of the hNSPCs. Although some GFAP-negative cells expressed ABCG2 after differentiation, but most GFAP-positive cells
The present study is the first to show ABCG2 protein expression in hNSPCs. We also showed direct evidence that cultured NSPCs derived from human fetal brain expressed a higher level of ABCG2 than did the malignant cell line A549, and that ABCG2 was localized to the plasma membrane of hNSPCs. The ABCG2 level in hNSPCs was sharply downregulated during astrocyte differentiation, similar to the sharp down-regulation of ABCG2 in hematopoietic stem cells at the stage of lineage commitment (Scharenberg et al., 2002), suggesting that ABCG2 might play an important role in the unique physiology of stem/progenitor cells. Generally, full-transporters are found in the plasma membrane, whereas half-transporters are found in intracellular membranes, such as the mitochondria and endoplasmic reticulum. By both three-dimensional structure analysis and biochemical examination we found that the localization of ABCG2 was to the plasma membrane of hNSPCs, similar to
Fig. 6. Regulation of ABCG2 expression during hNSPC differentiation. (A) Human neurospheres were induced to differentiate into astrocytes with 10% FBS for 15 days, then the expression patterns of ABCG2 and GFAP in the neurospheres and in the differentiated astrocytes were quantified by western blotting ABCG2 (polyclonal) and GFAP (polyclonal) antibodies. (B) The expression pattern of ABCG2- and GFAP-positive cells in the differentiated astrocytes was detected by immunochemistry with ABCG2 (polyclonal) and GFAP (monoclonal) antibodies (scale bar = 20 mm).
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its expression in ABCG2-over-expressing MCF7 cells (Rocchi et al., 2000). The most important finding of the present study was that about 63% of the cells within neurospheres were ABCG2 positive, similar to the proportion of nestin-positive cells, and the ABCG2 and nestin staining in most cases labeled the same cells, which is evidence that, like nestin, ABCG2 might be a stem/ progenitor cell marker for hNSPCs. This is the first report of the co-localization of ABCG2- and nestin-positive cells among hNSPCs, and this finding indicates that ABCG2 may have a functional role in hNSPCs. Consistent with the idea that ABCG2 might be expressed in its native form and have physiological functions in hNSPCs, we found that the ABCG2 in hNSPCs had a high substrate-stimulated ATPhydrolysis activity. This result suggested that the ABCG2 expressed in hNSPCs was functionally active, possibly playing a regulatory role in the maintenance of the undifferentiated state. The sharp down-regulation of ABCG2 levels during hNSPC differentiation has implications for hNSPC maturation. Although mature astrocytes expressed a high level of the astrocyte marker GFAP, they expressed only a low level of ABCG2, and most of the GFAP-positive cells did not show ABCG2 labeling, although co-localization was observed in a few of the mature astrocytes. The differences in ABCG2 expression between neurospheres and differentiated astrocytes support the idea that ABCG2 is expressed in progenitor cells rather than in differentiated cells. Although we found functional ABCG2 in hNSPCs, similar to the findings of other investigators studying other stem cells, its functional role in these cells, including NSPCs, has not been clear. The ABC transporters are known to transport a variety of toxic lipophilic compounds; thus, ABCG2 and other ABC transporters may act to protect stem cells from cytotoxic agents. It is also possible that a high expression of ABC transporters, as suggested by Bunting et al. (2000), may be critical to maintaining stem cells in a quiescent state. Perhaps the expression of transporters that are similar to ABCG2 is necessary for stem cells to regulate the uptake of small hydrophobic molecules involved in cell differentiation (Zhou et al., 2002). In conclusion, we propose that ABCG2, which is known as a universal stem cell marker, is also functionally expressed in hNSPCs, and may protect human NSPCs from xenobiotics and other toxic substances, as proposed previously (Zhou et al., 2002). Moreover, the anti-ABCG2 antibody might be used to separate neural stem/progenitor cells from the cell populations in neurospheres, and we are presently investigating this possibility.
Acknowledgments We thank Dr. Chiaki Ban at Osaka National Hospital for providing human fetal tissues and Dr. Susan E. Bates of the National Cancer Institute, NIH, USA, for providing the
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anti-ABCG2 (MXR87405) antibody. This study was supported in part by the Millennium Project from the Ministry of Economy, Trade and Industry of Japan, and by the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) project from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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