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Originally published In Press as doi:10.1074/jbc.M507130200 on December 22, 2005

J. Biol. Chem., Vol. 281, Issue 9, 5982-5991, March 3, 2006
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Identification and Functions of Chondroitin Sulfate in the Milieu of Neural Stem Cells*

Michiru Ida{ddagger}, Takuya Shuo§, Kanako Hirano{ddagger}, Yoshihito Tokita{ddagger}, Keiko Nakanishi{ddagger}, Fumiko Matsui{ddagger}, Sachiko Aono{ddagger}, Hiroshi Fujita||, Yasuyuki Fujiwara, Toshiyuki Kaji, and Atsuhiko Oohira{ddagger}§1

From the {ddagger}Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan, the §Department of Neurochemistry, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan, the Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan, and ||Seikagaku Corporation, Yokosuka, Kanagawa 239-0831, Japan

Received for publication, June 30, 2005 , and in revised form, December 13, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Self-renewal and multidifferentiation activity are two major characteristics of neural stem cells (1, 2). Their proliferation and differentiation are regulated by both intrinsic factors such as transcription factors and extrinsic factors present in the cell environment (3). It is well known that epidermal growth factor (EGF)2 and fibroblast growth factor (FGF)-2 stimulate the proliferation of neural stem/progenitor cells. Other typical extrinsic factors reported are bone morphogenetic proteins (4) and Wnt (5). In addition, extracellular and cell surface glycoconjugates that participate in the construction of specialized cell microenvironments, so-called "niches," could be involved in the proliferation and differentiation of neural stem/progenitor cells.

Proteoglycans are a group of proteins bearing covalently bound sulfated glycosaminoglycans (GAGs) and are typical glycoconjugates in the extracellular matrix and at the surface of cells of the central nervous system (6, 7). In the embryonic day 13 (E13) rat brain, which is abundant in neural stem cells, two sulfated GAGs, heparan sulfate (HS) and chondroitin sulfate (CS), are detectable (8). HS has been shown to regulate FGF-mediated cell proliferation by forming a ternary complex with FGF-2 and FGF receptor 1 (9). In fact, a heparan sulfate proteoglycan preparation isolated from the early embryonic mouse telencephalon promoted the FGF-2-mediated proliferation of neuroepithelial cells (10). Heparan sulfate proteoglycans, such as glypican-4 (11), syndecan-1 (12), and perlecan-related molecule (13), are expressed in the neuroepithelium and are considered modulators for the proliferation of neural stem cells.

Many molecular species of chondroitin sulfate proteoglycans (CSPGs) are expressed in the central nervous system and have been shown to be involved in various cellular events in the formation and maintenance of the neural network (6, 7). However, very few studies have been done on the structure and function of CS-GAG/CSPGs in the brain primordium. Recently, neural precursor cells have been shown to synthesize and secrete CSPGs, including some lectican family members (14). In addition, it has been reported that cultured tissue segments of E13 mouse brains synthesize CS rich in a disaccharide unit containing 4,6-disulfated N-acetylgalactosamine (E-unit) and that the CS preparation has an affinity for midkine, a heparin-binding growth factor (15). A commercial preparation including chondroitin sulfate E (CS-E) from the squid cartilage binds with a high affinity to various heparin-binding growth factors such as FGFs, pleiotrophin, and midkine (16). CS rich in a disaccharide unit composed of iduronic acid and 4-sulfated N-acetylgalactosamine (B-unit) from embryonic pig brain also binds to them (17, 18). These observations led us to hypothesize that neural stem/progenitor cells synthesize and deposit some CSPGs with B/E-unit-rich CS chains in their surroundings and that these CSPGs modulate the proliferation and differentiation of neural stem cells through the molecular interaction of their CS chains with these growth factors. To substantiate the hypothesis, we first characterized CS-GAG/CSPGs in the milieu of neural stem cells and then examined the involvement of the CS-GAG in growth factor-mediated proliferation of neural stem cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The following materials were purchased from Seikagaku Corp. (Tokyo, Japan); protease-free chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4 [EC] ), chondroitinase ACII from Arthrobacter aurescens (EC 4.2.2.5 [EC] ), heparitinase I (EC 4.2.2.8 [EC] ) and II from Flavobacterium heparinum, hyaluronidase from Streptomyces hyalurolyticus (EC 4.2.2.1 [EC] ), GAGs (chondroitin sulfate A (CS-A) from whale cartilage, chondroitin sulfate B (CS-B/dermatan sulfate (DS)) from pig skin, chondroitin sulfate C (CS-C) from shark cartilage, chondroitin sulfate D (CS-D) from shark cartilage, chondroitin sulfate E (CS-E) from squid cartilage, HS from bovine kidney, keratan sulfate from bovine cornea, and hyaluronic acid (HA) from human umbilical cords), antibodies to HS (10E4, mouse IgM), CS (CS-56, mouse IgM), unsulfated CS stub (1-B-5, mouse IgG), and 6-O-sulfated CS stub (3-B-3, mouse IgM). Antibodies to nestin (mouse IgG1) and NG2 (rabbit IgG) were obtained from Chemicon (Temecula, CA). A polyclonal antibody to receptor type protein-tyrosine phosphatase beta (RPTPbeta) was obtained from BD Biosciences. Alexa Fluor 488-conjugated streptavidin, Alexa Fluor 594-conjugated goat anti-mouse IgG1 ({gamma}1), and 2-aminoacridone hydrochloride were obtained from Molecular Probes, Inc. (Eugene, OR). Biotinylated secondary antibodies and a Vectastain ABC elite kit were obtained from Vector Laboratories (Burlingame, CA). The following antibodies were prepared in our laboratory: a monoclonal anti-neurocan antibody (1G2, mouse IgG1 (19)), a monoclonal anti-phosphacan antibody (6B4, mouse IgM (20)), and a polyclonal anti-neuroglycan C (NGC) antibody (21). Heparin from porcine intestinal mucosa and EGF were purchased from Sigma. FGF-2 was obtained from Roche Applied Science.

Neurosphere Culture—The treatment of animals was performed according to the ethical rules of our institution. All efforts were made to minimize the number of animals used and their suffering. Pregnant Sprague-Dawley rats at a gestational age of 14 days were killed with an overdose of diethyl ether, and fetuses were transferred in Petri dishes containing ice-cold Hanks' balanced salt solution. Telencephalons were excised from the fetuses and mechanically dissociated by pipetting into a single cell suspension in Hanks' balanced salt solution without Mg2+ and Ca2+ in the presence of 0.01% DNase I (Roche Applied Science). Cells were plated at a density of 1.2 x 105 cells/ml/well in Falcon non-tissue culture-treated 24-well plates (catalog no. 35-1147; BD Biosciences) in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (1:1) mixture medium (Invitrogen) containing two supplements, N2 and B27 (both purchased from Invitrogen (22-24)), 10 ng/ml FGF-2, and 10 ng/ml EGF. FGF-2 and EGF were added at the same concentration every 2 days. On day 4, 0.5 ml of the culture medium was added to each well. On day 6, floating neurospheres were collected and used for further examinations as follows.

Immunohistochemistry—Pregnant (E14) dams were killed under ether anesthesia as described above, and their fetuses were removed and immersed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C. The fetuses were embedded in paraffin and cut to serial 6-µm-thick sagittal sections. The sections were deparaffinized and rehydrated. For the digestion of CS, sections were preincubated in 0.1 M Tris-HCl, pH 7.5, containing 0.03 M sodium acetate (chondroitinase buffer) for 15 min at 37 °C and digested with 0.5 units/ml protease-free chondroitinase ABC in digestion buffer for 2 h at 37 °C. After three washes with 10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl (Tris-buffered saline), sections were incubated in blocking solution containing 2% bovine serum albumin, 2% horse serum, and 2% goat serum for 1 h at room temperature. Primary antibodies were diluted with 0.1% bovine serum albumin in Tris-buffered saline and applied for 1 h. After three washes with Tris-buffered saline, sections were treated sequentially with biotinylated secondary antibodies for 1 h, with Alexa Fluor 488-conjugated streptavidin or Alexa Fluor 594-conjugated anti-mouse IgG1 ({gamma}1) antibody for 1 h. After being mounted with Permafluor (Thermo Shandon, Pittsburgh, PA), sections were examined with a laser-scanning confocal microscope (Fluoview; Olympus, Tokyo, Japan).

Neurospheres floating in the culture medium were fixed by adding the same volume of 4% paraformaldehyde in PBS containing 1% sucrose, 1 mM MgCl2, and 1 mM CaCl2 at 4 °C overnight and collected by centrifugation at 50 x g for 5 min. The collected neurospheres were washed with PBS and incubated overnight at 4 °C in 15% sucrose in PBS. They were embedded in a drop of Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA). Specimens were immediately frozen in liquid nitrogen and stored at -80 °C. Frozen sections, cut at 15 µm, were collected on MAS-coated slide glasses (Matsunami Glass, Osaka, Japan) and processed for immunocytochemistry as described above for embryo sections after permeabilization with 0.1% Triton X-100 in the blocking solution.

Preparation of Samples for Western Blotting—Brain homogenates were prepared as described previously (25). In brief, four E14 fetal rat telencephalons were homogenized on ice in 100 µl of PBS containing 20 mM EDTA, 10 mM N-ethylmaleimide (NEM), and 2 mM phenylmethylsulfonyl fluoride. To the homogenate, 400 µl of 2% SDS in PBS containing 20 mM EDTA, 10 mM NEM, and 2 mM phenylmethylsulfonyl fluoride was added. The homogenate was then solubilized by boiling for 5 min. To analyze CSPGs in neurospheres and the culture medium, neurospheres were separated from the culture medium on day 6 by centrifugation at 200 x g for 10 min. CSPGs in the neurospheres were extracted by stirring in PBS containing 0.2% Nonidet P-40, 0.2% sodium deoxycholate, 20 mM EDTA, 10 mM NEM, and 2 mM phenylmethylsulfonyl fluoride at 4 °C overnight. To analyze CSPGs in the neurosphere culture medium (60 ml), it was centrifuged at 12,000 x g for 10 min at 4 °C. Solid NaCl was added to the supernatant to a final concentration of 0.3 M. CSPGs in this solution were adsorbed to 4 ml of DEAE-Sephacel (Amersham Biosciences) equilibrated with 0.3 M NaCl in 50 mM Tris-HCl, pH 7.5, by gentle agitation for 2 days at 4 °C and then were eluted from the DEAE-Sephacel resin with 0.7 M NaCl in 50 mM Tris-HCl, pH 7.5. The eluate is hereafter referred to as the medium-PG fraction. Virtually no proteoglycans were obtained in the wash of the residual DEAE-Sephacel resin with 2 M NaCl.

Western Blotting—Proteins were precipitated from the brain homogenates, neurosphere extracts, and medium-PG fractions by adding 3 volumes of 95% ethanol containing 1.3% potassium acetate at 0 °C, and the precipitates were digested with protease-free chondroitinase ABC as described previously (25). The samples were then resolved by SDS-PAGE on a 3% stacking gel and a 6% separating gel. After electrophoresis, proteins in gels were electrotransferred onto polyvinylidene difluoride membranes (Millipore Corp.), and immunoreactive materials were detected by sequential treatments of the membranes with primary antibodies, biotinylated secondary antibodies, and a Vectastain ABC elite kit. For the detection of neurocan, phosphacan, and RPTPbeta, Konica immunostain HRP-1000 (Konica, Tokyo, Japan) was used to visualize the bands. For the detection of NG2, goat anti-rabbit IgG(H + L)-horse-radish peroxidase conjugate (Bio-Rad) and Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) were used.

Immunoprecipitation—Telencephalons of five E14 fetal rats or neurospheres from one plate of a 12-well plate were homogenized in 1 ml of PBS containing 0.2% Nonidet P-40, 0.2% sodium deoxycholate, 20 mM EDTA, 10 mM NEM, and 2 mM phenylmethylsulfonyl fluoride on ice, and the homogenates were stirred overnight at 4 °C. After centrifugation of the homogenate at 15,000 x g for 5 min at 4 °C, the supernatant was incubated with 2 µg of anti-NGC antibody (rabbit IgG) at 4 °C for 1 h. To the mixture, 2 µl of Protein A-Sepharose CL-4B (Amersham Biosciences) was added, and the suspension was stirred gently overnight at 4 °C. The immunocomplex was recovered as a pellet by centrifugation and washed with 0.2% Nonidet P-40 in PBS three times and then with chondroitinase buffer. The immunocomplex was digested with 10 milliunits of chondroitinase ABC in 25 µl of chondroitinase buffer at 37 °C for 1 h. The enzymatic reaction was stopped by adding 3 volumes of 95% ethanol, 1.3% potassium acetate at 0 °C. Proteins were precipitated by centrifugation and dissolved in 50 µl of the sample buffer for SDS-PAGE. Western blot analyses were performed as described above.

Isolation of GAGs—A GAG mixture was prepared from the telencephalon essentially as described previously (8). In brief, E14 fetal rat telencephalons were dissected free of adherent connective tissues from 251 fetuses under a dissecting microscope and placed in ice-cold Hanks' balanced salt solution. After centrifugation of the brain tissue suspension at 800 x g for 10 min at 4 °C, the brain pellet (wet weight, 3.1 g) was homogenized in 31 ml of ice-cold acetone. The homogenate was centrifuged at 7,000 x g for 20 min at 4 °C. After a wash with ice-cold ether, the final precipitate was dried under reduced pressure. The dried brain powder was hydrated by adding 4 ml of 50 mM Tris-HCl, pH 7.5, containing 3% ethanol. After the addition of Pronase-E (Kaken Seiyaku, Tokyo, Japan) at a concentration of 0.1 mg/ml, the mixture was incubated with shaking at 50 °C for 24 h. The digested material was then treated sequentially with 0.2 M NaOH and 5% trichloroacetic acid and precipitated with 75% ethanol containing 1% potassium acetate to obtain crude GAGs. Postnatal day 8 (P8) rat telencephalons were dissected from 13 rats in ice-cold Hanks' balanced salt solution. The brains were homogenized in 2.6 ml of distilled water and additionally homogenized by the addition of 26 ml of methanol/chloroform (2:1). The homogenate was centrifuged at 7,000 x g for 30 min at 4 °C. The pellet was homogenized in 26 ml of methanol/chloroform/distilled water (2:1: 0.8), and the homogenate was centrifuged at 7,000 x g for 30 min at 4 °C. After a wash with ice-cold ether, the final precipitate was dried and processed for the preparation of crude GAGs similar to the preparation of E14 GAGs described above.

The crude GAG preparation contaminated with nucleic acids was digested by RNase T1 and DNase I (both purchased from Roche Applied Science). GAGs precipitated from the digest by the ethanol treatment were dissolved in 1 ml of 2 M urea containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA (urea buffer), and the solution was centrifuged at 12,000 x g for 10 min at 4 °C. The supernatant was incubated with 1 ml of DEAE-Sephacel with gentle agitation overnight at 4 °C, and GAGs were separated by DEAE-Sephacel column chromatography with 10 ml of a linear salt gradient (0.15-0.7 M NaCl in urea buffer) and 5 ml of an additional 2 M NaCl in urea buffer. The amount of hexuronate in each fraction was determined by the method of Bitter and Muir (26). GAGs in peak fractions were precipitated with ethanol/potassium acetate, and the precipitate was redissolved in distilled water at a hexuronate concentration of 2 nmol/µl. Aliquots (2 nmol of hexuronate) of the solution were analyzed by cellulose acetate electrophoresis using a buffer system composed of 0.1 M pyridine and 0.47 M formic acid (8). GAGs on the cellulose acetate strip were stained with 0.5% Alcian blue in 3% acetic acid, and excess stain was removed by rinsing in distilled water.

Fluorophore-assisted Carbohydrate Electrophoresis—Obtained GAG fractions (3 nmol as uronic acid/tube) were lyophilized and dissolved in 0.1 M ammonium acetate, pH 7.3, and incubated at 37 °C for 18 h with chondroitinase ACII (0.96 units/ml) alone or with both chondroitinase ABC and ACII (0.18 and 0.88 units/ml, respectively); the digests were recovered with Microcon YM-3 ultrafiltration devices (Mr 3,000 cut-off; Millipore) to obtain the lyase products of HA, CS, and DS. The undigested materials on the filter of Microcon YM-3 were recovered in 0.1 M ammonium acetate, pH 7.0, containing 0.01% bovine serum albumin, and digested at 37 °C for 8 h with both heparitinase I and II (0.03 units/ml each); the HS lyase products were recovered with Microcon YM-3. The recovered samples were dried and then fluorotagged at 90 °C for 30 min with 2-aminoacridone hydrochloride (27). The fluorotagged disaccharide units of HA, CS, and DS were immediately separated on separating gels (19.5% acrylamide, 0.52% N,N'-methylenebisacrylamide, 2.5% glycerol, 0.05% ammonium persulfate, 0.6% agarose, 0.1% TEMED, and 0.045 M Tris acetate buffer, pH 7.0) with a stacking gel (7.5% acrylamide, 0.2% N,N'-methylenebisacrylamide, 2.5% glycerol, 0.05% ammonium persulfate, 0.6% agarose, 0.1% TEMED, and 0.045 M Tris acetate buffer, pH 7.0). Similarly, the fluorotagged disaccharide units of HS were separated on separating gels (19.5% acrylamide, 0.52% N,N'-methylenebisacrylamide, 2.5% glycerol, 0.1 M Tris borate, pH 8.3, 0.05% ammonium persulfate, and 0.1% TEMED) with a stacking gel (7.5% acrylamide, 0.2% N,N'-methylenebisacrylamide, 2.5% glycerol, 0.1 M Tris borate, pH 8.3, 0.05% ammonium persulfate, 0.6% agarose, and 0.1% TEMED). The fluorescent images were displayed in a gel documentation system (AE-6914; Atto, Tokyo, Japan). The bands of unsaturated disaccharide units were quantitatively analyzed with the NIH Image Analysis software using the bands of standards, D-galactose 6-sulfate (100, 200, 300, and 400 pmol/lane) in CS/DS and unsaturated glucuronic acid-6-O-sulfated N-acetylgalactosamine (50, 100, 200, and 400 pmol/lane) in HS, which were purchased from Sigma. Amounts of iduronic acid-containing disaccharide units from DS were estimated by subtracting the amount of disaccharide units generated by digestion with chondroitinase ACII alone from that of the corresponding disaccharide units generated by digestion with both chondroitinase ABC and ACII.

Molecular Weight Analysis—To remove HS and HA from the GAG fractions, an aliquot (200 nmol of hexuronate) was digested with hyaluronidase (5 turbidity-reducing units/ml) in 400 µl of 25 mM sodium acetate, pH 6.0, at 60 °C for 1 h. The enzyme reaction was stopped by adding 3 volumes of 95% ethanol containing 1.3% potassium acetate. The ethanol-precipitated material was additionally digested with heparitinase I (16 milliunits/ml) in 300 µl of 5 mM calcium acetate and 50 mM sodium acetate, pH 7.0, at 40 °C for 1 h. The enzyme reaction was stopped by the ethanol precipitation, and the material was separated by DEAE-Sephacel column chromatography as described above. CS recovered in peak fractions was precipitated with ethanol/potassium acetate, and the precipitate was redissolved in distilled water at a hexuronate concentration of 2 nmol/µl (about 0.9 µg of CS/µl).

To estimate the molecular size of CS, an aliquot (4 µg) was applied to a gel permeation chromatography system consisting of a TSK-gel G4000PWXL column and a TSK-gel G3000PWXL column (TOSOH Corp., Tokyo, Japan). CS was eluted from the column with 0.2 M NaCl, and its molecular size was calculated from the position of its elution with Borwin-gel permeation chromatography software (Japan Spectroscopic Co., Ltd., Tokyo, Japan) using a calibration curve depicted for a standard CS mixture (52.2, 31.4, 20.0, 10.2, and 6.6 kDa).

Effects of GAGs on Growth Factor-mediated Proliferation of Neural Stem Cells—Primary neurospheres, which were largely composed of neural stem cells, were collected and washed twice with the medium in the absence of EGF and FGF-2. The neurospheres were triturated with a 1000-µl micropipette tip in growth factor-free medium, and the cell suspension was filtered through a Falcon cell strainer (BD Biosciences) to obtain a single cell suspension. Cells (1.0 x 104 cells/well) in 0.1 ml of medium containing either EGF (10 ng/ml), FGF-2 (10 ng/ml), or a mixture of EGF and FGF-2 (each 10 ng/ml) were plated onto 96-well plates.

Commercial GAGs (1.0 mg/ml PBS) were added to the cultures at a final concentration of 10 µg/ml just after the plating of cells. In the case of telencephalon-derived CS, an aliquot of the GAG preparation (50 nmol of hexuronate) was digested with a mixture of heparitinase I and II (10 milliunits/ml) in 50 µl of 5 mM calcium acetate and 50 mM sodium acetate, pH 7.0, at 40 °C for 2 h in order to remove HS from the GAG preparation. The enzyme reaction was stopped by adding 3 volumes of 95% ethanol containing 1.3% potassium acetate. This enzymatic digestion removed 43, 13, and 8% of total hexuronate from fraction I, II, and III (see Fig. 4), respectively. The ethanol-precipitated material was lyophilized and then dissolved in sterile PBS at a final concentration of 0.5 nmol (fraction I; see Fig. 4) or 1 nmol (fractions II and III) of hexuronate/µl, about 0.25 µg (fraction I) or 0.5 µg (fractions II and III) of GAG/µl, respectively. Since our preliminary experiment confirmed the complete inactivation of both heparitinases by ethanol precipitation and lyophilization, the telencephalon-derived CSs were added without removal of these denatured enzymes to the cultures at a final concentration of 10 µg/ml (fractions II and III) or 5 µg/ml (fraction I) just after the plating of cells.

The growth factors and GAGs were again added to the cultures on day 2. On day 4, 50 µl of fresh medium was added to each well, and then the cultures were supplemented with the growth factors and GAGs at the final concentration described above. On day 6, growth of the cells in cultures was determined by measuring the activity of mitochondrial succinate-tetrazolium reductase detectable only in live cells with a Cell Proliferation Assay System (Premix WST-1; Takara, Kyoto, Japan). Four wells were used for a single experimental condition, and the data were expressed as the mean ± S.D. Statistical comparisons of data between samples with and without GAGs were performed using the Mann-Whitney U test. To estimate the extent of cell death in cultures, lactate dehydrogenase activity released into culture media from dead cells was measured with a lactate dehydrogenase-cytotoxic test Wako (Wako Pure Chemical Industries, Osaka, Japan).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of CSPGs to the Niche of Neural Stem Cells—To identify molecular components of the microenvironment, or niche, of neural stem cells, we performed immunohistochemistry in the E14 fetal rat telencephalon. Immunoreactivity with the monoclonal anti-CS antibody CS-56 was detectable all over the cerebral tissue, strongly in the ventricular zone and the outer zone (Fig. 1, A and C). This immunoreactivity completely disappeared from the section after digestion with protease-free chondroitinase ABC (Fig. 1E), and immunoreactivity with a CS stub antibody, 1-B-5, which recognizes the unsulfated CS stub produced on a CSPG core protein by chondroitinase ABC digestion, appeared instead (Fig. 1G). These observations indicate that CSPGs exist in the E14 fetal rat telencephalon, and the distribution pattern of CS agrees with the results of previous reports (28, 29). The cells with immunoreactivity for nestin, a molecular marker of neural stem/progenitor cells, were preferentially distributed in the ventricular zone, where CS is abundant, and elongated their fibers to the outer zone (Fig. 1, B and F). To identify the molecular species of CSPGs present in the ventricular zone, we performed immunohistochemistry using various anti-CSPG antibodies. Neurocan, phosphacan, and NGC were detected with monoclonal antibody 1G2 (Fig. 1H), monoclonal antibody 6B4 (Fig. 1I), and the anti-NGC antibody (Fig. 1J), respectively, indicating that the niche for neural stem/progenitor cells contains these brain-specific CSPGs. RPTPbeta was not detected with the anti-RPTPbeta monoclonal antibody that we used.


Figure 1
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  		FIGURE 1.
Localization of CSPGs in the E14 rat telencephalon. A, paraffin section of E14 fetal rat telencephalon stained with hematoxylin-eosin (HE). VZ, ventricular zone; OZ, outer zone. B-F, immunostaining with the anti-nestin (nes) antibody (B) and anti-chondroitin sulfate (CS) antibody CS-56 (C). A merged image is shown in D. Shown is an enlargement of the boxed area in F. The immunostains observed in C disappear almost completely after treatment with protease-free chondroitinase ABC (CHase +) as shown in E. G-J, immunostaining with anti-CSPG antibodies: anti-CS stub antibody 1-B-5 (G), anti-neurocan (NC), antibody 1G2 (H), anti-phosphacan (PC), antibody 6B4 (I), and an anti-neuroglycan C (NGC) antibody (J). Scale bar, 50 µm (A-E) and 10 µm (F-J).

 
Neural stem cells proliferate and form floating neurospheres in culture on non-tissue culture-treated plates in the presence of EGF and FGF-2 (30). We performed immunocytochemistry in neurospheres to examine whether these CSPGs were detectable in the neurospheres as well as in the ventricular zone. Most cells composing neurospheres were nestin-positive (Fig. 2A). CS immunoreactivity was observed all over the neurosphere (Fig. 2B). By digestion of the tissue section with protease-free chondroitinase ABC, the immunoreactivity for CS almost disappeared (Fig. 2D), and CS stub antigens appeared (Fig. 2, E and F). Using various anti-CSPG antibodies, we detected neurocan, phosphacan, NGC, and NG2 (Fig. 2, G-J). These results suggest that neural stem/progenitor cells participate in the construction of their niches by synthesizing and depositing these CSPGs in their surroundings.

Western Blot Analyses for CSPGs—To confirm the existence of the CSPGs detected immunohistochemically, not only in the E14 fetal rat telencephalon but also in neurospheres, we performed immunoblot analyses for these CSPGs in E14 fetal rat brain homogenates, lysates of neurospheres, and PG-rich fractions prepared from neurosphere-conditioned media. Treatment of the brain homogenate with protease-free chondroitinase ABC produced two monoclonal antibody 1G2-reactive bands with molecular sizes of 220 and 150 kDa, representing full-length neurocan and the C-terminal half of neurocan, respectively (Fig. 3A, lane 2). Similarly, the enzymatic treatment produced a 300-kDa band recognized by monoclonal antibody 6B4 (Fig. 3A, lane 4), confirming the existence of phosphacan. Phosphacan is a secreted variant of RPTPbeta. Besides the soluble variants, there are two transmembrane variants of RPTPbeta, long and short receptor forms (31, 32). The anti-RPTPbeta antibody recognized a narrow band of 270 kDa in the homogenate of the E14 telencephalon, the mobility of which did not change after treatment with chondroitinase ABC (Fig. 3A, lanes 5 and 6). This indicates that the protein band does not bear any CS chain. Since the calculated molecular size of the long receptor form is 254.5 kDa (33), the protein band would be a long RPTPbeta with less post-translational modification. NGC was also detected with immunoprecipitation followed by immunoblotting. The anti-NGC antibody recognized a broad band of 150 kDa and two narrow bands of 120 and 110 kDa (Fig. 3A, lane 7). Digestion of the sample with chondroitinase ABC resulted in disappearance of the broad band, intensification of the 120-kDa band, and appearance of a narrow band of 220 kDa (Fig. 3A, lane 8). It has been reported that the molecular sizes of the CSPG form of NGC and the NGC core glycoprotein, estimated by SDS-PAGE, are 150 and 120 kDa, respectively (34). Taken together, it can be concluded that a significant part of NGCs exist in a non-CSPG form. The 220-kDa band may be a dimer of the NGC core protein, because NGC tends to polymerize during preparation or enzymatic digestion (35). The 110-kDa band may be an immature form of NGC with less post-translational modification or a degradation product.


Figure 2
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  		FIGURE 2.
Localization of CSPGs in neurospheres. A-C, frozen sections of a neurosphere immunostained with the anti-nestin (nes) antibody (A) and anti-chondroitin sulfate (CS) antibody CS-56 (B). A merged image is shown in C. D, a neurosphere section immunostained with anti-CS antibody CS-56 after treatment with protease-free chondroitinase ABC (CHase +). The immunostains observed in B disappear. E-J, neurosphere sections immunostained with anti-CSPG antibodies. E and F, anti-CS stub antibody 3-B-3; G, anti-neurocan (NC) antibody 1G2; H, anti-phosphacan (PC) antibody 6B4; I, the anti-neuroglycan C (NGC) antibody; J, an anti-NG2 antibody. Immunostains with 3-B-3 appear after treatment with protease-free chondroitinase ABC (CHase +) in F. Scale bar, 50 µm.

 


Figure 3
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  		FIGURE 3.
Identification of CSPGs by immunoblotting. Western blot analyses were performed to detect brain-specific CSPGs in homogenates of E14 fetal rat telencephalon (A), and culture medium (B-1) and lysate (B-2) of neurospheres using various anti-CSPG antibodies. Samples were subjected to SDS-PAGE before (-) and after (+) digestion with protease-free chondroitinase ABC (CHase). To detect NGC, immunoprecipitated samples were analyzed by Western blotting. IP, immunoprecipitation; NC, neurocan; PC, phosphacan; NGC, neuroglycan C. The positions of molecular mass markers are indicated at the left. The arrowheads indicate the corresponding CSPG core proteins.

 
In the neurosphere culture, soluble CSPGs such as neurocan and phosphacan should be largely released into the culture medium. As expected, both secretory CSPGs could be detected in the medium (Fig. 3B, B-1, lanes 1 and 2 for neurocan and lanes 3 and 4 for phosphacan). The anti-RPTPbeta antibody recognized a 270-kDa band and a 210-kDa band in the lysate of neurospheres, and the mobilities of these bands did not change after chondroitinase ABC digestion (Fig. 3B, B-2, lanes 1 and 2). The enzymatic digestion of the lysate produced two immunopositive bands of 310 and 240 kDa (lane 2). Thus, both a non-CSPG type and a CSPG type of RPTPbeta existed in the neurospheres. NGC was detected with immunoprecipitation followed by immunoblotting in the lysate of neurospheres (Fig. 3B, B-2, lanes 3 and 4), as is the case in the developing telencephalon. Although NG2 could not be detected in homogenates of the developing telencephalon (data not shown), this CSPG was present in the lysate of neurospheres (Fig. 3B, B-2, lanes 5 and 6). This finding is consistent with the report that NG2 can be used as a molecular marker of neural stem/progenitor cells in particular cases (53). Thus, we demonstrated that neural stem/progenitor cells expressed some CSPGs and constructed their niche by depositing these CSPGs in their surroundings.

Characterization of GAGs Isolated from E14 Fetal Rat Telencephalons—To characterize CS polysaccharides in the developing telencephalon, we prepared crude GAG mixtures from E14 and P8 rat telencephalons. The GAG mixtures were first separated by DEAE-Sephacel column chromatography. The GAG preparation from E14 telencephalons was divided into three fractions eluted at different NaCl concentrations (Fig. 4A). These GAG fractions were subjected separately to electrophoresis on a cellulose acetate membrane. In the fraction (designated a in Fig. 4A) eluted at 0.3 M NaCl, only one narrow band with a mobility identical to that of authentic HA was detected (data not shown). In the other two fractions (designated I and II in Fig. 4A), a broad GAG band was observed between the positions for authentic CS and HS (Fig. 4C). Treatment of these GAG fractions with either chondroitinase ABC or heparitinase resulted in a partial reduction of the broad GAG band, and treatment with a mixture of both GAG lyases resulted in the disappearance of the GAG band (data not shown), indicating that fractions I and II contain both CS and HS. In the GAG preparation from P8 telencephalons, there were only two fractions eluted at a different salt concentration from the DEAE-Sephacel column (Fig. 4B). The fraction corresponding to fraction II in the E14 sample was not detectable at this developmental stage. Electrophoresis on cellulose acetate membranes again showed that the fraction (designated a in Fig. 4B) eluted at 0.3 M NaCl contained only a GAG spot corresponding to HA (data not shown) and that the other fraction (designated III in Fig. 4B) contained a broad spot recovered between the positions for CS and HS (Fig. 4C). The broad spot was shown to contain both CS and HS by the GAG lyase treatments described above.


Figure 4
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  		FIGURE 4.
Preparation of GAGs from rat telencephalon. DEAE-Sephacel column chromatography of crude GAG mixtures prepared from E14 (A) and P8 (B) rat telencephalons. The amount of hexuronic acid was measured in each fraction as described under "Experimental Procedures." Peak fraction a contained hyaluronic acid. Peak fractions I-III shown by solid horizontal bars were pooled and used for further analyses. The broken lines represent the concentration of NaCl. Cellulose acetate electrophoresis of the GAG fractions I-III separated by DEAE-Sephacel column chromatography (C). GAGs were visualized by staining with Alcian blue. A mixture of standard GAG markers (M) was run in both outside lanes. HA, hyaluronic acid; HS, heparan sulfate; CS, chondroitin sulfate; HP, heparin.

 
The unsaturated disaccharide compositions of CS and HS in these three fractions were determined by fluorophore-assisted carbohydrate electrophoresis as described under "Experimental Procedures." The major CS-disaccharide was {Delta}Di-4S (or A-unit; {Delta}HexUA-GalNAc(4S) derived from CS) in the CS of all of these fractions (Table 1). Fraction II had the smallest proportion of {Delta}Di-0S (or 0-unit; {Delta}HexUA-GalNAc) of the three fractions, and this could be why it is eluted at a higher concentration of salt than the other fractions, as shown in Fig. 4, A and B. Although fraction III from the P8 sample did not contain {Delta}Di-4SDS (or B-unit; {Delta}HexUA-GalNAc(4S) derived from DS) at all, both fractions from the E14 sample contained a significant amount of B-unit. These results are consistent with the finding that CS/DS obtained from the embryonic pig brain contains a significant proportion of iduronic acid-bearing disaccharides (B-unit; 8-9%), whereas such disaccharides accounted for less than 1% of CS/DS obtained from the adult pig brain (18). {Delta}Di-diSD (or D-unit; {Delta}HexUA(2-O-sulfate)-GalNAc(6S)) and {Delta}Di-diSE (or E-unit; {Delta}HexUA-GalNAc(4,6S)), which are both oversulfated CS-disaccharide units, could be detected in all of these fractions (Table 1). Furthermore, molecular sizes of CS in these fractions were determined by a gel permeation chromatography method. It should be noted that the molecular size of CS was considerably larger in fraction II than the other fractions (Table 1). This indicates that CS in the E14 rat telencephalon can be separated into two species differing in extent of sulfation and in molecular size: a small CS with low sulfation in fraction I and a large CS with high sulfation in fraction II.


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  		TABLE 1
Disaccharide composition and molecular size of CS in GAG fractions prepared from developing rat telencephalons The disaccharide composition and molecular size of CS in fractions I, II, and III prepared from E14 and P8 rat telencephalons were determined by fluorophore-assisted carbohydrate electrophoresis analysis and gel permeation chromatography analysis, respectively, as described under "Experimental Procedures." {Delta}Di-0S,2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose; {Delta}Di-6S, 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; {Delta}Di-4S, 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; {Delta}Di-4SDS, 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose from DS only; {Delta}Di-diSD, 2-acetamide-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; {Delta}Di-diSE, 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4,6-bis-O-sulfo-D-galactose.

 
The unsaturated disaccharide compositions of HS in these GAG fractions were also determined by fluorophore-assisted carbohydrate electrophoresis (Table 2), and were compared with those in the adult rat brain reported by others (36). The proportion (69%) of the unsulfated disaccharide, {Delta}DiHS-0S, was significantly higher in the adult sample than in the samples from the developing telencephalon, and the proportions of sulfated disaccharides, with the exception of {Delta}DiHS-6S, were reduced in the adult sample. This indicates that the sulfation of HS in the brain decreases as the brain matures.


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  		TABLE 2
Disaccharide composition of HS in GAG fractions prepared from developing rat telencephalons The disaccharide composition of HS in fractions I, II and III prepared from E14 and P8 rat telencephalons was analyzed by fluorophore-assisted carbohydrate electrophoresis analysis as described in "Experimental Procedures" and compared with that of the HS in adult rat brain reported by Tekotte et al. (36). {Delta}DiHS-0S, 2-acetamide-2-deoxy-4-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose; {Delta}DiHS-NS, 2-deoxy-2-sulfamino-4-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose; {Delta}DiHS-6S, 2-acetamide-2-deoxy-4-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; {Delta}DiHS-di(U,N)S, 2-deoxy-2-sulfamino-4-O-(beta-D-gluco-2-O-sulfo-4-enepyranosyluronic acid)-D-glucose; {Delta}DiHS-di(N,6)S, 2-deoxy-2-sulfamino-4-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; {Delta}DiHS-tri(U,N,6)S, 2-deoxy-2-sulfamino-4-O-(beta-D-gluco-2-O-sulfo-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose. tr., trace.

 
Effects of Commercial Preparations of CS on FGF-2-mediated Proliferation of Neural Stem Cells—To clarify the functional role of CS in the physiology of neural stem/progenitor cells, we first examined its effects on cell proliferation. Our preliminary experiments demonstrated that neurospheres formed in the presence of some GAGs tended to be larger than those formed under the standard culture conditions described above. Therefore, we tried to quantify the growth of neural stem/progenitor cells in the presence of various GAGs using a cell proliferation assay system (Fig. 5). Under the standard conditions of neurosphere culture, namely in culture with both EGF and FGF-2, cell proliferation was stimulated significantly by the addition of heparin to about 50% more than the control level (Fig. 5A). HS also stimulated cell proliferation to an extent almost identical to that of heparin (data not shown). Both CS-D and CS-E, the commercial preparations of highly sulfated CS, potentiated the activity of the growth factors slightly (about 20% more than the control) but significantly (Fig. 5A). Other commercial GAG preparations, including keratan sulfate and HA, did not exert any effects on the growth of neural stem cells under the standard culture conditions (data not shown).

Both heparin and HS potentiate the activity of FGF-2 (37, 38). Therefore, it can be considered that CS-D and CS-E also potentiate the activity of FGF-2, not the activity of EGF. As shown in Fig. 5B, EGF-mediated proliferation of neural stem/progenitor cells was not stimulated by any of the commercial GAGs examined. However, interestingly, the cell number in the culture treated with FGF-2 and a particular CS preparation such as CS-B or CS-E was significantly larger than that treated with FGF-2 alone (Fig. 5C). CS-D treatment also promoted slightly the FGF-2-mediated cell proliferation. The cell numbers of the CS-B and CS-E samples were almost identical to that of the heparin- or HS-containing sample. Since cell numbers are increased in cultures by stimulation of cell proliferation and/or by inhibition of cell death, the contribution of cell death was estimated by the lactate dehydrogenase method described under "Experimental Procedures." The level of cell death in cultures treated with any CS preparation was closely similar to that treated with FGF-2 alone (data not shown), indicating that stimulation of FGF-2-mediated cell proliferation, not inhibition of cell death, occurs in cultures treated with these CS preparations. This is the first evidence that CS is involved in the proliferation of neural stem/progenitor cells. The other GAGs, including CS-A and CS-C, examined in this study did not have any effect on either EGF- or FGF-2-mediated cell proliferation (data not shown).

The stimulation by CS-B and CS-E of the FGF-2-mediated proliferation of neural stem/progenitor cells was dose-dependent (Fig. 6). The stimulative activity of CS-B increased with dose to 10 µg/ml medium and reached a plateau at around 50 µg/ml. CS-E also had a stimulative activity for FGF-2-mediated cell proliferation, but its activity was lower than that of CS-B. The half-effective concentrations (EC50) of CS-B and CS-E estimated from data shown in Fig. 6 were about 5 and 15 µg/ml, respectively. At these doses, CS-C showed no stimulative activity at all, but it showed a slight activity at concentrations higher than 50 µg/ml.

Effects of Telencephalon-derived CS on FGF-2-mediated Proliferation of Neural Stem Cells—The results described above led us to examine whether CS preparations from the developing telencephalon also potentiate the activity of FGF-2. HS-free CS preparations were prepared from these three GAG fractions (fractions I, II, and III in Fig. 4, A and B) by complete digestion with a mixture of heparitinase I and II as described under "Experimental Procedures," and the CS preparations were added separately to neurosphere cultures in the presence of 10 ng/ml EGF (Fig. 7A) or FGF-2 (Fig. 7B). The preparation from fraction I slightly (about 20% more than the control) stimulated the EGF-mediated proliferation of neural stem/progenitor cells, but the other preparations did not. In contrast, all three preparations stimulated significantly (about 2-fold) the FGF-2-mediated cell proliferation. Thus, we demonstrated for the first time that CS has the capability to regulate the growth factor-mediated proliferation of cells. These observations suggest that at least some of the CS molecules in the niche of neural stem/progenitor cells participate in regulating the growth of these cells in the developing central nervous system.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, immunohistochemistry and Western blotting revealed the existence of CSPGs including neurocan, phosphacan/RPTPbeta, and NGC in the ventricular zone of the E14 fetal rat telencephalon, where nestin-positive cells, namely neural stem/progenitor cells, are abundant. These brain-specific CSPGs were also detectable in cultures of neurospheres, which are floating aggregates of nestin-positive cells prepared from the developing telencephalon. CS polysaccharides purified from the E14 fetal rat telencephalon were separated into two classes varying in molecular size and extent of sulfation. Both CS preparations promoted the FGF-2-mediated proliferation of neural stem/ progenitor cells, as the commercial preparations of heparin, HS, CS-B and CS-E did. We thus demonstrate for the first time that a CS preparation from the brain at an early developmental stage can modulate the growth of neural stem/progenitor cells.


Figure 5
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  		FIGURE 5.
Effect of commercial GAGs on proliferation of neural stem/progenitor cells. Neural stem/progenitor cells prepared from primary neurospheres were cultured in the presence of a mixture of FGF-2 and EGF (each 10 ng/ml; A), EGF (10 ng/ml; B), or FGF-2 (10 ng/ml; C), with various commercial GAGs such as CS-A, CS-B, CS-D, CS-E, and heparin. Cell number was estimated at 6 days using a Premix WST-1 cell proliferation assay system. Data represent percentages of the cell number in the sample (None) only with the growth factor(s) indicated (means ± S.D., n = 4). *, p < 0.05 versus none.

 


Figure 6
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  		FIGURE 6.
Dose-dependent activation of neural stem/progenitor cell proliferation by CS. Neural stem/progenitor cells prepared from primary neurospheres were cultured in the presence of 10 ng/ml of FGF-2 with CS-B, CS-C, or CS-E at concentrations ranging from 0 to 200 µg/ml. Cell number was estimated on day 6 using a Premix WST-1 cell proliferation assay system. Data represent percentages of the cell number in the sample without CS (means ± S.D., n = 4).

 
The existence of many molecular species of CSPGs is characteristic of the central nervous system, and the spatiotemporal expression of each CSPG is strictly regulated in a development-related manner (6, 39, 40). The functional roles of these CSPGs have been investigated mainly in relation to the formation of the neural network (33, 41-45). However, little information is available on the identification, localization, and function of CSPGs in the neuroepithelium or the cerebral primordium at early developmental stages such as E14 or earlier in the rat.


Figure 7
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  		FIGURE 7.
Effect of telencephalon-derived CSs on proliferation of neural stem cells. Neural stem/progenitor cells prepared from primary neurospheres were cultured, in the presence of 10 ng/ml EGF (A) or FGF-2 (B), with 5 µg/ml of CS purified from fraction I shown in Fig. 4 (I) or 10 µg/ml CS purified from fraction II (II) or from fraction III (III). Cell number was estimated on day 6 using a Premix WST-1 cell proliferation assay system. Data represent percentages of the cell number in the sample (None) without any CS preparations (means ± S.D., n = 3). *, p < 0.05 versus none.

 
The cerebral primordium contains a relatively large number of neural stem cells (1, 2), and it is feasible that CSPGs participate in the regulation of their functions as major constituents of their milieu, or the niche. To date, phosphacan has been detected in the neuroepithelium and in the ventricular zone of the fetal rat cerebrum (46, 47), but it has been shown that immunoreactivities for aggrecan and versican were very low, if any, in the ventricular zone of the developing rat cerebrum (40). We demonstrated the existence of neurocan, RPTPbeta, and NGC in the ventricular zone rich in nestin-positive cells, or neural stem/progenitor cells (Figs. 1 and 3). The cellular source of these CSPGs could be at least in part neural stem/progenitor cells, because all of these CSPGs were detected in the cultures of neurospheres (Figs. 2 and 3). NG2, a large transmembrane CSPG, could also be found in neurospheres (Figs. 2 and 3). Recently, Kabos et al. (14) reported that aggrecan proteins as well as phosphacan protein was detected in the culture medium of neurospheres. These results suggest that neural stem/progenitor cells participate in the construction of their own milieu by synthesizing these CSPGs and depositing them in their surroundings. The reason for the existence of multiple species of CSPGs and the function of each CSPG in the niche of neural stem/progenitor cells are not known at present. RNA interference technology and the addition of an antibody specific to each CSPG to cultures of neural stem/progenitor cells could help to solve these problems.

Individual CSPGs have CS side chains with different structural features although they are isolated from the same tissue at the same developmental stage (48). Therefore, it is of interest to determine and compare the structural features of the CS side chains among these individual CSPGs identified in the niche of neural stem/progenitor cells. However, this was practically impossible mainly because of the small amounts of CSPGs purified from such a small tissue primordium. Then we tried to characterize CS polysaccharides isolated from the telencephalon of the E14 rat fetus.

We obtained two CS preparations from the E14 rat telencephalon, a short CS with low sulfation (I) and a long CS with high sulfation (II), but only one (III) from the P8 rat brain (Table 1). CS-III was similar in size to CS-I and had a disaccharide composition almost identical to that of two major CSPGs, neurocan and phosphacan, purified from the P10 rat brain (48). It should be noted that, although CS-III did not contain any B-units, both CS-I and CS-II contained significant amounts (Table 1). B-unit is a disaccharide unit composed of iduronic acid and 4-sulfated N-acetylgalactosamine that is produced from DS by digestion with chondroitinase ABC, not with chondroitinase ACII. Bao et al. (18) reported that the pig brain contained CS/DS hybrid chains and that the embryonic pig brain contained CS/DS hybrid chains more abundant in B-units than the adult brain. They also reported that the B-unit-rich CS/DS hybrid chains had a higher affinity for some heparin-binding growth factors, including FGF-2, than the B-unit-poor chains. The present results together with their results suggest that the B-unit-containing CS polysaccharide regulates the proliferation of neural stem/progenitor cells through interaction with FGF-2.

HS has a high affinity for FGF-2 and promotes the FGF-2-mediated proliferation of various cells through the formation of a ternary complex with FGF-2 and FGF receptor (9). Recently, besides HS and heparin, some CS preparations have been shown to bind to FGF-2 with a high affinity (16, 18, 49, 50), suggesting that CS with a particular sulfated carbohydrate sequence modulates FGF-2-mediated cell proliferation. In fact, it has been reported that that CS-B/DS promotes proliferation of various cell lines in the presence of FGF-2 such as C2C12 myoblasts (51) and F32 HS-deficient lymphoid cells (49). It is well known that proliferation of neural stem/progenitor cells is promoted with growth factors, FGF-2 and EGF. In the present study, both CS-B and CS-E promoted the FGF-2-mediated proliferation of neural stem/progenitor cells to an extent almost identical to that of HS and heparin (Fig. 5). Neither CS-B nor CS-E promoted EGF-mediated cell proliferation, indicating that they do not have a promotive effect on the growth of neural stem/progenitor cells in themselves. Thus, we showed for the first time that CS-E as well as CS-B potentiated the activity of FGF-2 for cell proliferation.

The results obtained from the cell proliferation assay using the commercial CS preparations suggest that the CS preparations from the E14 fetal rat telencephalon can also modulate the FGF-2-mediated proliferation of neural stem/progenitor cells, because they contained both E-units and B-units (Table 1), although in small amounts. Since CS-E and CS-B-unit-containing CSs, like HS and heparin, have been reported to bind to FGF-2 (16, 18), we expected that CS containing both E- and B-units would modulate FGF-2-mediated cell proliferation. As expected, both the CS-I and CS-II preparations from the E14 fetal rat telencephalon promoted the FGF-2-mediated cell proliferation (Fig. 7B). Additionally, CS-III from the P8 rat brain also promoted cell proliferation. None of these CS fractions promoted the EGF-mediated cell proliferation at all (Fig. 7A), indicating that they themselves do not cause cells to proliferate. Although B-units were found only in CS-I and CS-II, a similar amount of E-unit existed in all of the CS preparations (Table 1). Therefore, it is possible to speculate that CS polysaccharides in the developing brain contain a cluster of E-units as a functional domain to modulate FGF-2-mediated neural stem cell proliferation.

Recently, it was reported that DS promotes FGF-2-dependent HS-deficient lymphoid cell proliferation, and the minimal size required for activation of FGF-2 was an octasaccharide (52). To examine whether any functional domain exists in the CS polysaccharides, an inhibition assay for their activities to promote FGF-2-mediated cell proliferation would be useful using CS-E-derived oligosaccharides with various structural features. The present study is the first to provide direct evidence that CSPGs, in addition to heparan sulfate proteoglycans, in the niche of neural stem/progenitor cells are also involved in modulation of cell proliferation through interaction with FGF-2.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Japan Society for the Promotion of Science. 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. Back

1 To whom correspondence should be addressed: Dept. of Perinatology and Neuroglycoscience, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan. Tel.: 81-568-88-0811; Fax: 81-568-88-0829; E-mail: oohira{at}inst-hsc.jp.

2 The abbreviations used are: EGF, epidermal growth factor; 4S, 4-O-sulfate; 6S, 6-O-sulfate; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; PG, proteoglycan; {Delta}HexUA, 4,5-unsaturated hexuronic acid; DS, dermatan sulfate; E13 and E14, embryonic day 13 and 14, respectively; EC50, half-effective concentrations; FGF, fibroblast growth factor; GAG, glycosaminoglycan; HA, hyaluronic acid; HS, heparan sulfate; NEM, N-ethylmaleimide; NGC, neuroglycan C; P8 and P10, postnatal day 8 and 10, respectively; PBS, phosphate-buffered saline; RPTPbeta, receptor-type protein-tyrosine phosphatase beta; TEMED, N,N,N',N'-tetramethylethylenediamine. Back


   ACKNOWLEDGMENTS
 
We thank A. Iida for preparation of paraffin sections.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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