- Email: [email protected]
Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro
Journal of Bioscience and Bioengineering VOL. 110 No. 4, 479 – 486, 2010 www.elsevier.com/locate/jbiosc
Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro Yuejie Zhang,1,3 Shuliang Song,2 Hao Liang,2 Yunshan Wang,2 Weili Wang,2 and Aiguo Ji1,2,⁎ School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China 1 International Biotechnology Research and Development Centre, Shandong University at Weihai, Weihai 264209, China 2 and School of Life Sciences, Shandong University of Technology, Zibo 250049, China 3 Received 15 April 2010; accepted 13 May 2010 Available online 11 June 2010
Neural stem/progenitor cells (NSPCs) exhibit therapeutic potential in neuronal diseases. Previously, we reported that a sulfated polysaccharide (HS) from the sea cucumber Stichopus japonicus increased the proliferation of NSPCs. Since the formation of neurospheres is related with NSPCs proliferation, we investigated the mechanism leading to neurosphere formation with and without HS. The results showed that HS significantly promoted neurosphere formation in a dosedependent manner at concentrations between 2 and 8 μg/ml. Cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. On the other hand, we observed a significantly rapid aggregation of NSPCs, resulting in formation of neurospheres as early as 2 h after HS treatment. However, the aggregation was not caused by chemotactic migration of NSPCs, as evidenced by the transwell chamber assay. Furthermore, the effect of HS on NSPCs was similar to the tumor necrosis factor-α (TNF-α) that activated nuclear factor NF-κB. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Neurosphere formation; Neural stem/progenitor cells; Sulfated polysaccharide; Nuclear factor NF-κB]
Neural stem/progenitor cells (NSPCs) are defined as undifferentiated neural cells that are endowed with a high potential for proliferation and the capacity for generating all the major lineages of the central nervous system (CNS). Currently, neurosphere culture system is considered to be the most widely used technique for expansion of NSPCs in vitro, which seems to be a less time-consuming, and more economical and accurate method. The formation of neurospheres is considered to be an efficient indicator for proliferation of NSPCs (1). Neurosphere formation is associated with (a) neurotrophic factors, (b) cell contacts, and (c) cytokines. Studies show that neurotrophic factors and glycosaminoglycans within the extracellular matrix milieu of CNS provide signals for cell proliferation, differentiation, adhesion and migration (2–6). It is known that cell contacts are important to cell survival and growth (especially in regard to primary culture of NSPCs), and aggregation of NSPCs could contribute to their own proliferation through the formation of neurospheres (7). Tumor necrosis factor-α (TNF-α) increases aggregation and promotes proliferation of isolated NSPCs by activating the upstream nuclear factor kappa-B (NF-κB) pathway (8,9). NF-κB is ubiquitously expressed throughout the nervous system, and the pathway is
⁎ Corresponding author. School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. Tel./fax: + 86 631 5685049. E-mail addresses: [email protected] (Y. Zhang), [email protected] (A. Ji).
activated by cell surface receptors that signal to degrade its inhibitor IκB, leading to NF-κB nuclear translocation (10). The NF-κB signaling pathway plays a central role in neuronal integrity, synaptic plasticity, neuroprotection and neurogenesis (11,12). Recently, we demonstrated that a sulfated polysaccharide (HS) extracted from the sea cucumber Stichopus japonicus, was able to promote NSPC proliferation in a dose-dependent manner, and it synergized with fibroblast growth factor, FGF-2 (13). Since the formation of neurospheres is important for NSPCs, the studies of optimization of culture systems and exploration on mechanism of neurosphere formation will provide a further understanding of neural development and their potential clinical use. The aim of the present study was to test the effect of HS on formation of neurospheres, and to define its underlying molecular mechanism. MATERIALS AND METHODS Preparation of sample Sulfated polysaccharide (HS) was extracted from the sea cucumber Stichopus japonicus by procedures described previously (13). The glycosaminoglycan consists of a homogenous fraction with an average molecular weight of 4.23 × 105 Da, as determined by analytical high-performance liquid chromatography. Its chemical composition is 38.12% fucose, 16.52% uronic acid, 32.64% sulfate group and small amounts of galactose. HS was dissolved in cell culture medium as described below. Primary culture of NSPCs and treatment Animal experiments were approved by the Ethics Committee of Shandong University. Isolation of rat NSPCs was accomplished by previously described methods (14) with minor modifications. Briefly, Wistar rats were sacrificed by cervical dislocation under anesthesia. Embryos were harvested at day 14 (E14), and the brains were removed using a dissection microscope.
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.05.009
480
ZHANG ET AL.
J. BIOSCI. BIOENG.,
Cerebral cortex tissues were carefully isolated by syringes and collected in cold serumfree medium consisting of DMEM/F-12 nutrient (1:1; Invitrogen, Carlsbad, CA, USA). The tissue was mechanically and enzymatically dissociated into cell suspensions, which were plated in 96-well plates or T75 flasks at the density of 2 × 105 cells/ml. To examine effects of HS on the formation of neurospheres, NSPCs were treated with different concentrations of HS (1, 2 , 4 , and 8 μg/ml) and were incubated for 96 h (h). After 48 h, half of the media was exchanged for fresh media, and the additives were supplemented accordingly. FGF-2 (20 ng/ml, Invitrogen) was used as the positive control. At the end of the incubation period, the number of neurospheres (with a diameter over 30 μm) was counted in five random microscopic areas at the center of each well under the microscope for use in further quantitative analysis.
Sigma) for 10 min at RT. The images were acquired using a fluorescence microscope with a high-resolution digital camera (DMRA, Q-Fish system, Leica, Germany). Statistical analysis Experiments were performed in triplicate. Results are shown as the mean ± standard deviations. Statistical differences between the control and experimental group were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test. P value less than 0.05 was considered statistically significant.
Cell cycle analysis NSPCs were plated in T75 flasks at a concentration of 3 × 105 cells/ml. The additives were added into the media 6 h after seeding the cells and supplemented every second day, thereafter. After incubation for 72 h, neurospheres were harvested and dissociated into single cells by incubation in 0.125% trypsin (Gibco, Grand Island, NY, USA). The enzymatic reaction was blocked with trypsin inhibitor (0.125%, Invitrogen) to inactivate the trypsin. Subsequently, cells were washed with ice-cold phosphate buffered saline (PBS), fixed by ice-cold 100% ethanol and resuspended in ice-cold PBS containing 2% fetal calf serum, RNase (100 μg/ml, Invitrogen), and propidium iodide (50 μg/ml) for 15 min. DNA analysis was then performed using FACS Calibur instrument (BD Biosciences). Dead cells and cell aggregates were eliminated from the analysis by gating. The cell cycle was evaluated by measuring the percentage of remaining cells at G0/G1-, S-, and G2/M phase cells by side scatter analysis (15).
Effects of sulfated polysaccharide (HS) on neurosphere formation The sulfated polysaccharide HS, extracted from sea cucumber Stichopus japonicus, has been shown to increase NSPC viability in a dose-dependent manner, and does not induce apoptosis of NSPCs (13). To examine the effects of HS on the formation of neurospheres, we counted the number of neurospheres present on day 4 so to avoid the interference of the coalescence of neurospheres in later culture (18). As shown in Fig. 1, NSPCs were inclined to differentiate and display some neurite outgrowth in the absence of HS or FGF-2 (Fig. 1A). HS alone promoted formation of neurospheres in a dose-dependent manner, and the effect peaked at 8 μg/ml, a level which was equivalent to cultures treated with 20 ng/ml FGF-2 (Fig. 1E, * P b 0.05). Furthermore, HS acted synergistically with FGF-2 to promote the formation of neurospheres (Fig. 1E, * P b 0.05). These data suggested that HS significantly increased neurosphere formation and synergistically promoted proliferation of NSPCs with FGF-2. Mechanism of neurosphere formation induced by sulfated polysaccharide (HS) Cell cycle analysis revealed that HS induced an increase in the percentage of cells in S phase and decreased those in G1 phase at 72 h (Fig. 2, * P b 0.05). There was an approximately 2.8-fold increase in the S phase cells in the culture incubated with HS (4 μg/ml), as compared to the untreated control cells. NSPCs treated with HS and FGF-2 (20 ng/ml) displayed an increase in the percentage of S phase cells higher than that obtained with FGF-2, suggesting that HS acted synergistically with FGF-2 to promote cell division. These findings suggest that the increased amount of cells in the S phase, promoted by HS, may attribute to formation of neurospheres and maintaining an undifferentiated potential in vitro. The movement of neural cells is important in the development of neurospheres and neurosphere properties involving cell–cell or cell– environment interactions (18). We used a time-lapse observation technique to investigate the behavior of cells from a single cell suspension (Fig. 3). As seen in Fig. 3, the cells began to aggregate as early as half an hour after HS treatment, and the aggregate of 4–6 cell clusters was observed at 2 h after HS treatment. The cells in the control also displayed some random motility, but did not aggregate into cell clusters as seen in the HS treatment group. Compared to a control culture, the aggregation level showed a 2.6-fold increase after HS treatment (Fig. 3, ** P b 0.01). Thus, HS treatment activated a faster motility and aggregation of NSPCs, resulting in a faster neurosphere formation. To verify the potential mechanism underlying the aggregation effects induced by HS, we examined the chemotactic migration of NSPCs using the transwell chamber assay. The results showed that the treatment with different concentrations of HS did not display any differences in the number of cells able to pass through the filter (Fig. 4, * P b 0.05). The positive control treatment, FGF-2 group, induced a weak but non-statistically significant increase in migratory cells compared with the control. These results suggested that NSPCs chemotactic migration properties were not stimulated by HS. Since NF-κB is known to control the proliferation and aggregation of NSPCs, we examined interaction between the effects of HS on NSPCs and the activation of nuclear factor NF-κB. We used an ELISA kit to measure the amount of p65 in the nucleus of NSPCs that indicated the extent of activation of NF-κB. As shown in Fig. 5, HS significantly
Cell aggregation assay Aggregation behavior of NSPCs, from the single cells in suspension culture, is likely to involve the regeneration and self-renewal of neurospheres (8). In order to investigate further the functions of HS, we tested the influence of HS on motility and/or movement behavior of NSPCs. NSPCs were plated in 96-well plates and stimulated with 50 μg/ml HS in the growth medium. Aggregation was recorded for up to 2 h after HS stimulation and analyzed using Image J software (National Institute of Health, USA; http://rsb.info.nih.gov/ij). Aggregation level was determined as the size of free space between cell aggregations, that is, the bigger the size of free space between cell aggregations was, the higher the aggregation level was. Maximal aggregation level was set as 100%. Cell migration assay NSPC migration was assessed by a modified Boyden's chamber assay in 24-well transwell chamber (Corning, NY, USA) (16). The upper surface was composed of polycarbonate filters with 8 μm pores. A suspension of cells (3000 cells/100 μl resuspended in serum-free DMEM/F-12 medium) was placed in the upper chambers. The lower chambers were filled with 600 μl of different concentrations of HS or growth factors. The NSPCs were allowed to migrate toward the indicated chemoattractant in the lower chamber. After 24 h of incubation at 37 °C under optimal conditions, the filters were fixed with 10% buffered formalin and stained with hematoxylin. After removal of the non-migrated cells by wiping with cotton swabs, the number of cells was counted in five random microscopic fields per filter at a magnification of 200×. NF-κB activation assay Nuclear extracts were prepared as described previously (17) with minor modifications. Neurospheres were stimulated with different concentrations of HS (1, 2, 4, 5, 30, and 50 μg/ml) for 45 min, then the cells were harvested and nuclear protein extracted to detect NF-κB p65 proteins. In brief, the cells were washed with ice-cold PBS and suspended in 800 ul of a lysis buffer and incubated on ice for 5 min. Cells were then lysed by adding 0.1% Nonidet P-40 and vortexed vigorously for 20 s. After incubation for 5 min at 4 °C, the samples were subjected to centrifugation at 2000 g for 10 min at 4 °C. The precipitate was resuspended in 100 μl of ice-cold nuclear extraction buffer and vortexed vigorously for 20 s. After incubation for 30 min at 4 °C, the samples were subjected to centrifugation at 12000 g for 2 min at 4 °C, and the resulting nuclear extract supernatant was collected in a chilled Eppendorf tube. An aliquot was taken and its protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). NF-κB p65 protein content was quantified using a commercial ELISA kit from R & D system (Minneapolis, MN, USA). All procedures were performed according to the manufacturer's instructions. Immunochemistry assay After incubation with HS (4 μg/ml) for 72 h in culture, neurospheres were harvested for immunostaining. For differentiation assay of the neurospheres stimulated by HS, dissociated cells were plated onto poly-D-lysine (0.1 mg/ml, Sigma) coated coverslips in 24-well plates in the medium containing 1% fetal bovine serum (FBS, Invitrogen) at the density of 1 × 105 cells/ml. The cultures were allowed to differentiate for 5 days in vitro before being fixed for immunostaining. Both neurospheres and cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT), then washed and blocked in 0.3% Triton X-100-containing 10% goat serum in PBS for 30 min at RT, and then incubated with the following primary antibodies overnight at 4 °C: the monoclonal mouse antibodies IgG against Nestin (1:800, Chemicon) for NSPCs, the monoclonal mouse antibodies IgM against O4 (1:600, R& D) for oligodendrocytes, glial fibrillary acidic protein (GFAP, 1:200, Sigma) for astrocytes and microtubule-associated protein 2 (MAP2, 1:200, Sigma) for neurons. After being washed with PBS, neurospheres or cells were incubated for 60 min at 37 °C with the appropriate secondary antibody: fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (1:100, Sigma), tetramethyl rhodamine iso-thiocyanate (TRITC)conjugated goat anti-mouse IgG (1:100, Sigma). After being washed with PBS, the coverslips were incubated with a fluorescent nuclear dye, Hoechst 33342 (10 μg/ml,
RESULTS
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
481
FIG. 1. HS promoted proliferation of neurospheres. Phase-contrast images of neurospheres in untreated controls (A), HS (4 μg/ml, B), FGF-2 (20 ng/ml, C), and FGF-2 (20 ng/ml) + HS (4 μg/ml, D). Scale bar = 100 μm. Histogram indicating dose-independent response of HS-treated neurospheres compared to FGF-2 used as a positive control (E). P b 0.05 (*) compared to positive control.
activated the translocation of NF-κB and the effects were dosedependent. HS (50 μg/ml) increased NF-κB nuclear translocation nearly 1.5-fold greater than the controls (Fig. 5, *P b 0.05). These findings suggest that HS stimulation significantly enhanced NF-κB activities.
Hoechst (blue). These findings suggest that HS stimulation might not influence stemness of neurospheres and their multi-lineage potential.
Multi-lineage potential of NSPCs HS-stimulated formation of neurospheres did not alter the lineage of after differentiation. Neurospheres were remained positive for intermediate filament protein (Nestin), a NSC marker, and the expression of the marker did not change after HS treatment (Fig. 6A and B). As expected, neurospheres were negative for the neuronal marker MAP2 and the astrocytes marker GFAP (date not shown). To elucidate whether neurosphere formed with HS had the ability of multi-lineage potential, the markers of three neural lineages were investigated. The neurospheres, which were cultured in the medium containing 4 μg/ml HS for 3 days, were dissociated and plated on coverslips with 1% FBS for 5 days. The cells were immunostained with oligodendrocytes marker, O4 (red) (Fig. 6C), astrocytes marker, GFAP (green) (Fig. 6D), neuronal marker, MAP2 (green) (Fig. 6E), and nuclear stain,
In the present study, we demonstrated that HS alone was able to promote the formation of neurospheres in a dose-dependent manner, and acted synergistically with FGF-2. Its proliferative effect was due to an increased number of cells existing in S phase. On the other hand, HS modulated the motility behavior of NSPCs and resulted in a rapid neurosphere formation. However, these effects were not due to chemotactic migration. The mechanism was related to the activation of NF-κB signaling pathway. It has been reported that some polysaccharides act as accessory molecules that regulate growth factors binding to their receptors and the activation of the cognate signaling pathways (19). A polysaccharide, fucoidan, extracted from brown seaweed, was shown to increase the presence of circulating mature white blood cells and mobilize hematopoietic progenitor/stem cells in a selectin-independent
DISCUSSION
482
ZHANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. HS promoted cell division and increased the number of the cells in the S phase. Images of flow cytometric analysis in untreated controls (A), HS (4 μg/ml, B), FGF-2 (20 ng/ml, C), and FGF-2 + HS (D). Histogram indicating an increase in the S phase and less in G1 phase in HS-treated culture and the synergism with FGF-2 to promote cell division (E). P b 0.05 (*) compared to untreated controls.
manner in mice and non-human primates (20). Other polysaccharides also have been shown to serve as signaling molecules in stem cell proliferation, homing, neurogenesis and neural network formation (21,22). It is possible that exogenous polysaccharides may be able to recruit minute amounts of endogenous growth factors. The application of polysaccharides, in lieu of growth factors, has been suggested as an alternative therapeutic strategy (23). Fucoidan composed of L-fucose and ester sulfate, is a good alternative to heparin due to its antithrombotic activity and its relative safety: being of invertebrate origin fucoidan is less likely to contain infectious agents, such as viruses or prions (24). The anticoagulant activity of fucoidan is lower than heparin (25), so that the hemorrhage risk is lowered for clinical use of transplanting NSPCs into the CNS if contaminated with fucoidan (26). In the present study, we found that HS promoted the formation
of neurospheres at the early culture stage and acted synergistically with FGF-2 in both neurosphere formation assay and cell cycle analysis. Moreover, HS-stimulated formation of neurospheres did not alter the neuronal lineage of NSPCs after differentiation. Thus, our finding indicated that HS could be a more effective and lower-risk growth co-adjuvant for clinical use in culturing NSPCs. We have shown that the most effective dose of HS to promote the formation of neurospheres was between 4 and 8 μg/ml. Thus, the concentration of 4 μg/ml HS was applied in the subsequent experiments, including cell cycle analysis and immunochemistry assay. In aggregation assay of NSPCs, if the concentration of HS in the medium was 5 μg/ml, the record was up to 24 h for appearance of 4–6 cell clusters (date not shown). Therefore, we carried out HS at a higher concentration 50 μg/ml in order to facilitate quantitative analysis of
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
483
FIG. 3. HS induced enhanced aggregation of NSPCs. NSPCs were stimulated with 50 μg/ml HS (upper panel) in growth medium; the lower panel corresponds to the control. (A) 30 min after HS treatment. (B) 120 min after HS treatment. (C) Control 30 min. (D) Control 120 min. Scale bar = 100 μm. Histogram indicating the quantification of aggregation level, and suggested that HS induced a fast aggregation of NSPCs and a rapid neurosphere formation at the early culture stage. P b 0.01 (**) compared to untreated controls.
aggregation level in this assay. In cell migration assay, we did not find a significant difference in migratory cells in all treatment, even though at a broad concentration ranging from 50 to 5000 ng/ml. Although we did not find a significant difference at a concentration ranging from 1 to 4 μg/ml in NF-κB activation assay, there was a good linear relationship at a higher concentration ranging from 5 to 50 μg/ml. Therefore, we carried out HS at a higher concentration in this assay. Isolation of NSPCs from the CNS using the neurosphere formation assay was first described in 1992 (27). Neurosphere culture system using FGF-2 and/or EGF as the mitogen is considered to be the most widely used technique for the in vitro expansion of NSPCs. However, the mechanism leading to neurosphere formation remains largely unclear. It is reported that FGF-2 not only promotes cell proliferation of embryonic cortical stem cells (28), but also controls their differentiation into neuronal and glial lineages under differentiation conditions (29). Thus, we think that FGF-2 is essential for proliferation of NSPCs in process of neurosphere formation. In general, neurospheres can form from dissociated cells after only 3 days of culture. Since NSPCs double approximately every 20 h, after
3 days neurospheres contain, on the average, eight cells, and are identified as neurosphere forming units (1). In the present study, we observed a significantly faster formation of neurosphere units on the same day of plating. Meanwhile, a larger free space between cells and/or neurosphere units appeared in HS cultures, as compared to the control group. Thus, we attempted to explore the mechanisms of the faster formation of neurosphere units from these two aspects: aggregation and proliferation of NSPCs. The results showed that HS induced an increase in the percentage of cells in S phase and promoted the proliferation of NSPCs, similar to reports of heparin and chondroitin sulfate glycosaminoglycans. On the other hand, a rapid aggregation occurred as early as 30 min after HS treatment, resulting in the formation of neurosphere units of 3–5 cells after 2 h treatment. Although HS had a less effect on proliferation than FGF-2 as indicated in neurosphere formation assay and cell cycle analysis, HS treatment activated a faster cell aggregates formed at the early culture stage. The cell aggregates might provide a favorable environment for the proliferation of NSPCs. Therefore, there was more neurospheres appeared in FGF-2 cultures with HS treatment than that of FGF-2
484
ZHANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Chemotactic migration of NSPCs of HS-enhanced aggregation of NSPCs by a modified transwell chamber assay. (A) no additive control. (B) 50 ng/ml HS. (C) 500 ng/ml HS. (D) 5000 ng/ml HS. (E) 20 ng/ml FGF-2. (F) 20 ng/ml FGF-2 + HS 500 ng/ml. Scale bar = 100 μm. (G) Histogram indicating the number of cells passing through the filter, showing that no statistical differences were observed in all treatments. P b 0.05 (*) compared to untreated controls.
FIG. 5. HS treatment enhanced p65 protein nuclear translocation in a dose-dependent manner. P b 0.05 (*) compared to untreated controls.
cultures (** P b 0.01). This concludes that HS acted synergistically with FGF-2 and enhanced FGF-2 effect on neurosphere formation. However, a detailed structure of HS and its interaction with FGF-2 remain to be investigated. In addition, we attempted to explain the motility of NSPCs in HS culture as chemotactic migration using the classic transwell chamber assay. But, we were unable to detect any correlation between our observation and this particular property. In fact, it has been reported that rapid re-aggregation will interfere with migration assays; migration of NSPCs as induced by cytokines and growth factors (such as SCF) has only been observed 3 h after plating, longer than our observed aggregation (30). However, it has been reported that cell aggregation was disadvantageous for the proliferation of NSPCs because of restricted supplies of nutrients and heterogeneous populations within neurospheres (1,7). We here believe that cell aggregates formed at the early culture stage might provide a favorable environment for the proliferation of NSPCs. Thus, we propose a new model for the proliferation of NSPCs induced by HS. Firstly, several NSPCs dispersed on the plate will begin to aggregate in response to a signal from HS
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
485
FIG. 6. Multi-lineage potential of NSPCs from HS-stimulated formation of neurospheres. HS-treated neurospheres (A) were collected at 72 h, fixed and stained for Nestin (B) as described in Materials and Methods. Part of HS-treated neurospheres were dissociated, plated on coverslips and cultured in the medium containing 1% FBS for 5 days. The cells were fixed, stained for Hoechst 33342 and three lineage marker, O4 positive oligodendrocytes (red, C), GFAP positive astrocytes (green, D), MAP2 positive neuron (green, E) and Hoechstlabeled nuclei (blue). Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and/or other cell–cell interaction signals, and then 3–5 cells will aggregate to form neurosphere units. Subsequently, the cells within the neurosphere units will begin to divide, each neurosphere will become larger through the cell division and further aggregation will occur with single cells and/or neurosphere units. HS exposure was maintained throughout the process of formation of neurospheres. Thus, the proliferation and aggregation of NSPCs leading to the formation of neurospheres was induced by HS. Furthermore, the faster formation of neurospheres induced by HS was closely related to the activation of the NF-κB signaling pathway. In conclusion, HS can promote neurosphere formation, and act synergistically with FGF-2. The effect of HS on NSPCs was related to the activation of the transcription factor NF-κB. These results suggest that HS would be a useful adjuvant for promoting the proliferation of NSPCs, a feature that might be useful for clinical applications. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (30873198) and the Jointly-Funded Universities Projects of Weihai (0000413420614). REFERENCES 1. Ahmed, S.: The culture of neural stem cells, J. Cell. Biochem., 106, 1–6 (2009). 2. Pitman, M., Emery, B., Binder, M., Wang, S., Butzkueven, H., and Kilpatrick, T. J.: LIF receptor signaling modulates neural stem cell renewal, Mol. Cell. Neurosci., 27, 255–266 (2004). 3. Kokuzawa, J., Yoshimura, S., Kitajima, H., Shinoda, J., Kaku, Y., Iwama, T., Morishita, R., Shimazaki, T., Okano, H., Kunisada, T., and Sakai, N.: Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos, Mol. Cell. Neurosci., 24, 190–197 (2003). 4. Ida, M., Shuo, T., Hirano, K., Tokita, Y., Nakanishi, K., Matsui, F., Aono, S., Fujita, H., Fujiwara, Y., Kaji, T., and Oohira, A.: Identification and functions of chondroitin sulfate in the milieu of neural stem cells, J. Biol. Chem., 281, 5982–5991 (2006). 5. Sirko, S., von Holst, A., Wizenmann, A., Götz, M., and Faissner, A.: Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells, Development, 134, 2727–2738 (2007). 6. Caldwell, M. A. and Svendsen, C. N.: Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells, Exp. Neurol., 152, 1–10 (1998).
7. Mori, H., Ninomiya, K., Kino-oka, M., Shofuda, T., Islam, M. O., Yamasaki, M., Okano, H., Taya, M., and Kanemura, Y.: Effect of neurosphere size on the growth rate of human neural stem/progenitor cells, J. Neurosci. Res., 84, 1682–1691 (2006). 8. Widera, D., Mikenberg, I., Kaus, A., Kaltschmidt, C., and Kaltschmidt, B.: Nuclear Factor-κB controls the reaggregation of 3D neurosphere cultures in vitro, Eur. Cell Mater., 11, 76–84 (2006). 9. Widera, D., Mikenberg, I., Elvers, M., Kaltschmidt, C., and Kaltschmidt, B.: Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-κB signaling, BMC Neurosci., 7, 64–82 (2006). 10. Cyert, M. S.: Regulation of nuclear localization during signaling, J. Biol. Chem., 276, 20805–20808 (2001). 11. Karin, M. and Lin, A.: NF-κΒ at the crossroads of life and death, Nat. Immunol., 3, 221–227 (2002). 12. Goldschmidt, B., Wider, D., and Goldschmidt, C.: Signaling via NF-κB in the nervous system, Biochim. Biophys. Acta, 1745, 287–299 (2005). 13. Zhang, Y. J., Song, S. L., Song, D., Liang, H., Wang, W. L., and Ji, A. G.: Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus, J. Biosci. Bioeng., 109, 67–72 (2010). 14. Kosaka, N., Kodama, M., Sasaki, H., Yamamoto, Y., Takeshita, F., Takahama, Y., Sakamoto, H., Kato, T., Terada, M., and Ochiya, T.: FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation, FASEB J., 20, 1484–1485 (2006). 15. Lum, M., Croze, E., Wagner, C., McLenachan, S., Mitrovic, B., and Turnley, A. M.: Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation, J. Neuroimmunol., 206, 32–38 (2009). 16. Kimura, A., Ohmori, T., Ohkawa, R., Madoiwa, S., Mimuro, J., Murakami, T., Kobayashi, E., Hoshino, Y., Yatomi, Y., and Sakata, Y.: Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury, Stem Cells, 25, 115–124 (2007). 17. Mo, S. J., Son, E. W., Lee, S. R., Lee, S. M., Shin, D. H., and Pyo, S.: CML-1 inhibits TNF-α induced NF-κB activation and adhesion molecule expression in endothelial cells through inhibition of IkB-α kinase, J. Ethnopharmacol., 109, 78–86 (2007). 18. Wang, T. Y., Sen, A., Behie, L. A., and Kallos, M. S.: Dynamic behavior of cells within neurospheres in expanding populations of neural precursors, Brain Res., 1107, 82–96 (2006). 19. Nikitovic, D., Assouti, M., Sifaki, M., Katonis, P., Krasagakis, K., Karamanos, N. K., and Tzanakakis, G. N.: Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factormediated proliferation in human metastatic melanoma cell lines, Int. J. Biochem. Cell Biol., 40, 72–83 (2008). 20. Sweeney, E. A., Priestley, G. V., Nakamoto, B., Collins, R. G., Beaudet, A. L., and Papayannopoulou, T.: Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence, Proc. Natl. Acad. Sci. USA, 97, 6544–6549 (2000). 21. Li, F., Shetty, A. K., and Sugahara, K.: Neuritogenic activity of chondroitin/ dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from
486
22.
23. 24.
25.
ZHANG ET AL.
shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways, J. Biol. Chem., 282, 2956-2066 (2007). Akita, K., von Holst, A., Furukawa, Y., Mikami, T., Sugahara, K., and Faissner, A.: Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult central nervous system implies that complex chondroitin sulfates have a role in neural stem cell maintenance, Stem Cells, 3, 798–809 (2008). Sugahara, K. and Mikami, T.: Chondroitin/dermatan sulfate in the central nervous system, Curr. Opin. Struct. Biol., 17, 536–545 (2007). Berteau, O. and Mulloy, B.: Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide, Glycobiology, 13, 29R–40R (2003). Pereira, M. S., Mulloy, B., and Mourao, P. A. S.: Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae, J. Biol. Chem., 274, 7656–7667 (1999).
J. BIOSCI. BIOENG., 26. Mori, H., Kanemura, Y., Onaya, J., Hara, M., Miyake, J., Yamasaki, M., and Kariya, Y.: Effects of heparin and its 6-O- and 2-O-desulfated derivatives with low anticoagulant activity on proliferation of human neural stem/progenitor cells, J. Biosci. Bioeng., 100, 54–61 (2005). 27. Reynolds, B. A. and Weiss, S.: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science, 255, 1707–1710 (1992). 28. Raballo, R., Rhee, J., Lyn-Cook, R., Leckman, J. F., Schwartz, M. L., and Vaccarino, F. M.: Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex, J. Neurosci., 20, 5012–5023 (2000). 29. Qian, X., Davis, A. A., Goderie, S. K., and Temple, S.: FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells, Neuron, 18, 81–93 (1997). 30. Sun, L., Lee, J., and Fine, H. A.: Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury, J. Clin. Invest., 113, 1364–1374 (2004).
Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro Yuejie Zhang,1,3 Shuliang Song,2 Hao Liang,2 Yunshan Wang,2 Weili Wang,2 and Aiguo Ji1,2,⁎ School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China 1 International Biotechnology Research and Development Centre, Shandong University at Weihai, Weihai 264209, China 2 and School of Life Sciences, Shandong University of Technology, Zibo 250049, China 3 Received 15 April 2010; accepted 13 May 2010 Available online 11 June 2010
Neural stem/progenitor cells (NSPCs) exhibit therapeutic potential in neuronal diseases. Previously, we reported that a sulfated polysaccharide (HS) from the sea cucumber Stichopus japonicus increased the proliferation of NSPCs. Since the formation of neurospheres is related with NSPCs proliferation, we investigated the mechanism leading to neurosphere formation with and without HS. The results showed that HS significantly promoted neurosphere formation in a dosedependent manner at concentrations between 2 and 8 μg/ml. Cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. On the other hand, we observed a significantly rapid aggregation of NSPCs, resulting in formation of neurospheres as early as 2 h after HS treatment. However, the aggregation was not caused by chemotactic migration of NSPCs, as evidenced by the transwell chamber assay. Furthermore, the effect of HS on NSPCs was similar to the tumor necrosis factor-α (TNF-α) that activated nuclear factor NF-κB. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Neurosphere formation; Neural stem/progenitor cells; Sulfated polysaccharide; Nuclear factor NF-κB]
Neural stem/progenitor cells (NSPCs) are defined as undifferentiated neural cells that are endowed with a high potential for proliferation and the capacity for generating all the major lineages of the central nervous system (CNS). Currently, neurosphere culture system is considered to be the most widely used technique for expansion of NSPCs in vitro, which seems to be a less time-consuming, and more economical and accurate method. The formation of neurospheres is considered to be an efficient indicator for proliferation of NSPCs (1). Neurosphere formation is associated with (a) neurotrophic factors, (b) cell contacts, and (c) cytokines. Studies show that neurotrophic factors and glycosaminoglycans within the extracellular matrix milieu of CNS provide signals for cell proliferation, differentiation, adhesion and migration (2–6). It is known that cell contacts are important to cell survival and growth (especially in regard to primary culture of NSPCs), and aggregation of NSPCs could contribute to their own proliferation through the formation of neurospheres (7). Tumor necrosis factor-α (TNF-α) increases aggregation and promotes proliferation of isolated NSPCs by activating the upstream nuclear factor kappa-B (NF-κB) pathway (8,9). NF-κB is ubiquitously expressed throughout the nervous system, and the pathway is
⁎ Corresponding author. School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. Tel./fax: + 86 631 5685049. E-mail addresses: [email protected] (Y. Zhang), [email protected] (A. Ji).
activated by cell surface receptors that signal to degrade its inhibitor IκB, leading to NF-κB nuclear translocation (10). The NF-κB signaling pathway plays a central role in neuronal integrity, synaptic plasticity, neuroprotection and neurogenesis (11,12). Recently, we demonstrated that a sulfated polysaccharide (HS) extracted from the sea cucumber Stichopus japonicus, was able to promote NSPC proliferation in a dose-dependent manner, and it synergized with fibroblast growth factor, FGF-2 (13). Since the formation of neurospheres is important for NSPCs, the studies of optimization of culture systems and exploration on mechanism of neurosphere formation will provide a further understanding of neural development and their potential clinical use. The aim of the present study was to test the effect of HS on formation of neurospheres, and to define its underlying molecular mechanism. MATERIALS AND METHODS Preparation of sample Sulfated polysaccharide (HS) was extracted from the sea cucumber Stichopus japonicus by procedures described previously (13). The glycosaminoglycan consists of a homogenous fraction with an average molecular weight of 4.23 × 105 Da, as determined by analytical high-performance liquid chromatography. Its chemical composition is 38.12% fucose, 16.52% uronic acid, 32.64% sulfate group and small amounts of galactose. HS was dissolved in cell culture medium as described below. Primary culture of NSPCs and treatment Animal experiments were approved by the Ethics Committee of Shandong University. Isolation of rat NSPCs was accomplished by previously described methods (14) with minor modifications. Briefly, Wistar rats were sacrificed by cervical dislocation under anesthesia. Embryos were harvested at day 14 (E14), and the brains were removed using a dissection microscope.
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.05.009
480
ZHANG ET AL.
J. BIOSCI. BIOENG.,
Cerebral cortex tissues were carefully isolated by syringes and collected in cold serumfree medium consisting of DMEM/F-12 nutrient (1:1; Invitrogen, Carlsbad, CA, USA). The tissue was mechanically and enzymatically dissociated into cell suspensions, which were plated in 96-well plates or T75 flasks at the density of 2 × 105 cells/ml. To examine effects of HS on the formation of neurospheres, NSPCs were treated with different concentrations of HS (1, 2 , 4 , and 8 μg/ml) and were incubated for 96 h (h). After 48 h, half of the media was exchanged for fresh media, and the additives were supplemented accordingly. FGF-2 (20 ng/ml, Invitrogen) was used as the positive control. At the end of the incubation period, the number of neurospheres (with a diameter over 30 μm) was counted in five random microscopic areas at the center of each well under the microscope for use in further quantitative analysis.
Sigma) for 10 min at RT. The images were acquired using a fluorescence microscope with a high-resolution digital camera (DMRA, Q-Fish system, Leica, Germany). Statistical analysis Experiments were performed in triplicate. Results are shown as the mean ± standard deviations. Statistical differences between the control and experimental group were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test. P value less than 0.05 was considered statistically significant.
Cell cycle analysis NSPCs were plated in T75 flasks at a concentration of 3 × 105 cells/ml. The additives were added into the media 6 h after seeding the cells and supplemented every second day, thereafter. After incubation for 72 h, neurospheres were harvested and dissociated into single cells by incubation in 0.125% trypsin (Gibco, Grand Island, NY, USA). The enzymatic reaction was blocked with trypsin inhibitor (0.125%, Invitrogen) to inactivate the trypsin. Subsequently, cells were washed with ice-cold phosphate buffered saline (PBS), fixed by ice-cold 100% ethanol and resuspended in ice-cold PBS containing 2% fetal calf serum, RNase (100 μg/ml, Invitrogen), and propidium iodide (50 μg/ml) for 15 min. DNA analysis was then performed using FACS Calibur instrument (BD Biosciences). Dead cells and cell aggregates were eliminated from the analysis by gating. The cell cycle was evaluated by measuring the percentage of remaining cells at G0/G1-, S-, and G2/M phase cells by side scatter analysis (15).
Effects of sulfated polysaccharide (HS) on neurosphere formation The sulfated polysaccharide HS, extracted from sea cucumber Stichopus japonicus, has been shown to increase NSPC viability in a dose-dependent manner, and does not induce apoptosis of NSPCs (13). To examine the effects of HS on the formation of neurospheres, we counted the number of neurospheres present on day 4 so to avoid the interference of the coalescence of neurospheres in later culture (18). As shown in Fig. 1, NSPCs were inclined to differentiate and display some neurite outgrowth in the absence of HS or FGF-2 (Fig. 1A). HS alone promoted formation of neurospheres in a dose-dependent manner, and the effect peaked at 8 μg/ml, a level which was equivalent to cultures treated with 20 ng/ml FGF-2 (Fig. 1E, * P b 0.05). Furthermore, HS acted synergistically with FGF-2 to promote the formation of neurospheres (Fig. 1E, * P b 0.05). These data suggested that HS significantly increased neurosphere formation and synergistically promoted proliferation of NSPCs with FGF-2. Mechanism of neurosphere formation induced by sulfated polysaccharide (HS) Cell cycle analysis revealed that HS induced an increase in the percentage of cells in S phase and decreased those in G1 phase at 72 h (Fig. 2, * P b 0.05). There was an approximately 2.8-fold increase in the S phase cells in the culture incubated with HS (4 μg/ml), as compared to the untreated control cells. NSPCs treated with HS and FGF-2 (20 ng/ml) displayed an increase in the percentage of S phase cells higher than that obtained with FGF-2, suggesting that HS acted synergistically with FGF-2 to promote cell division. These findings suggest that the increased amount of cells in the S phase, promoted by HS, may attribute to formation of neurospheres and maintaining an undifferentiated potential in vitro. The movement of neural cells is important in the development of neurospheres and neurosphere properties involving cell–cell or cell– environment interactions (18). We used a time-lapse observation technique to investigate the behavior of cells from a single cell suspension (Fig. 3). As seen in Fig. 3, the cells began to aggregate as early as half an hour after HS treatment, and the aggregate of 4–6 cell clusters was observed at 2 h after HS treatment. The cells in the control also displayed some random motility, but did not aggregate into cell clusters as seen in the HS treatment group. Compared to a control culture, the aggregation level showed a 2.6-fold increase after HS treatment (Fig. 3, ** P b 0.01). Thus, HS treatment activated a faster motility and aggregation of NSPCs, resulting in a faster neurosphere formation. To verify the potential mechanism underlying the aggregation effects induced by HS, we examined the chemotactic migration of NSPCs using the transwell chamber assay. The results showed that the treatment with different concentrations of HS did not display any differences in the number of cells able to pass through the filter (Fig. 4, * P b 0.05). The positive control treatment, FGF-2 group, induced a weak but non-statistically significant increase in migratory cells compared with the control. These results suggested that NSPCs chemotactic migration properties were not stimulated by HS. Since NF-κB is known to control the proliferation and aggregation of NSPCs, we examined interaction between the effects of HS on NSPCs and the activation of nuclear factor NF-κB. We used an ELISA kit to measure the amount of p65 in the nucleus of NSPCs that indicated the extent of activation of NF-κB. As shown in Fig. 5, HS significantly
Cell aggregation assay Aggregation behavior of NSPCs, from the single cells in suspension culture, is likely to involve the regeneration and self-renewal of neurospheres (8). In order to investigate further the functions of HS, we tested the influence of HS on motility and/or movement behavior of NSPCs. NSPCs were plated in 96-well plates and stimulated with 50 μg/ml HS in the growth medium. Aggregation was recorded for up to 2 h after HS stimulation and analyzed using Image J software (National Institute of Health, USA; http://rsb.info.nih.gov/ij). Aggregation level was determined as the size of free space between cell aggregations, that is, the bigger the size of free space between cell aggregations was, the higher the aggregation level was. Maximal aggregation level was set as 100%. Cell migration assay NSPC migration was assessed by a modified Boyden's chamber assay in 24-well transwell chamber (Corning, NY, USA) (16). The upper surface was composed of polycarbonate filters with 8 μm pores. A suspension of cells (3000 cells/100 μl resuspended in serum-free DMEM/F-12 medium) was placed in the upper chambers. The lower chambers were filled with 600 μl of different concentrations of HS or growth factors. The NSPCs were allowed to migrate toward the indicated chemoattractant in the lower chamber. After 24 h of incubation at 37 °C under optimal conditions, the filters were fixed with 10% buffered formalin and stained with hematoxylin. After removal of the non-migrated cells by wiping with cotton swabs, the number of cells was counted in five random microscopic fields per filter at a magnification of 200×. NF-κB activation assay Nuclear extracts were prepared as described previously (17) with minor modifications. Neurospheres were stimulated with different concentrations of HS (1, 2, 4, 5, 30, and 50 μg/ml) for 45 min, then the cells were harvested and nuclear protein extracted to detect NF-κB p65 proteins. In brief, the cells were washed with ice-cold PBS and suspended in 800 ul of a lysis buffer and incubated on ice for 5 min. Cells were then lysed by adding 0.1% Nonidet P-40 and vortexed vigorously for 20 s. After incubation for 5 min at 4 °C, the samples were subjected to centrifugation at 2000 g for 10 min at 4 °C. The precipitate was resuspended in 100 μl of ice-cold nuclear extraction buffer and vortexed vigorously for 20 s. After incubation for 30 min at 4 °C, the samples were subjected to centrifugation at 12000 g for 2 min at 4 °C, and the resulting nuclear extract supernatant was collected in a chilled Eppendorf tube. An aliquot was taken and its protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). NF-κB p65 protein content was quantified using a commercial ELISA kit from R & D system (Minneapolis, MN, USA). All procedures were performed according to the manufacturer's instructions. Immunochemistry assay After incubation with HS (4 μg/ml) for 72 h in culture, neurospheres were harvested for immunostaining. For differentiation assay of the neurospheres stimulated by HS, dissociated cells were plated onto poly-D-lysine (0.1 mg/ml, Sigma) coated coverslips in 24-well plates in the medium containing 1% fetal bovine serum (FBS, Invitrogen) at the density of 1 × 105 cells/ml. The cultures were allowed to differentiate for 5 days in vitro before being fixed for immunostaining. Both neurospheres and cells were fixed in 4% paraformaldehyde for 20 min at room temperature (RT), then washed and blocked in 0.3% Triton X-100-containing 10% goat serum in PBS for 30 min at RT, and then incubated with the following primary antibodies overnight at 4 °C: the monoclonal mouse antibodies IgG against Nestin (1:800, Chemicon) for NSPCs, the monoclonal mouse antibodies IgM against O4 (1:600, R& D) for oligodendrocytes, glial fibrillary acidic protein (GFAP, 1:200, Sigma) for astrocytes and microtubule-associated protein 2 (MAP2, 1:200, Sigma) for neurons. After being washed with PBS, neurospheres or cells were incubated for 60 min at 37 °C with the appropriate secondary antibody: fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (1:100, Sigma), tetramethyl rhodamine iso-thiocyanate (TRITC)conjugated goat anti-mouse IgG (1:100, Sigma). After being washed with PBS, the coverslips were incubated with a fluorescent nuclear dye, Hoechst 33342 (10 μg/ml,
RESULTS
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
481
FIG. 1. HS promoted proliferation of neurospheres. Phase-contrast images of neurospheres in untreated controls (A), HS (4 μg/ml, B), FGF-2 (20 ng/ml, C), and FGF-2 (20 ng/ml) + HS (4 μg/ml, D). Scale bar = 100 μm. Histogram indicating dose-independent response of HS-treated neurospheres compared to FGF-2 used as a positive control (E). P b 0.05 (*) compared to positive control.
activated the translocation of NF-κB and the effects were dosedependent. HS (50 μg/ml) increased NF-κB nuclear translocation nearly 1.5-fold greater than the controls (Fig. 5, *P b 0.05). These findings suggest that HS stimulation significantly enhanced NF-κB activities.
Hoechst (blue). These findings suggest that HS stimulation might not influence stemness of neurospheres and their multi-lineage potential.
Multi-lineage potential of NSPCs HS-stimulated formation of neurospheres did not alter the lineage of after differentiation. Neurospheres were remained positive for intermediate filament protein (Nestin), a NSC marker, and the expression of the marker did not change after HS treatment (Fig. 6A and B). As expected, neurospheres were negative for the neuronal marker MAP2 and the astrocytes marker GFAP (date not shown). To elucidate whether neurosphere formed with HS had the ability of multi-lineage potential, the markers of three neural lineages were investigated. The neurospheres, which were cultured in the medium containing 4 μg/ml HS for 3 days, were dissociated and plated on coverslips with 1% FBS for 5 days. The cells were immunostained with oligodendrocytes marker, O4 (red) (Fig. 6C), astrocytes marker, GFAP (green) (Fig. 6D), neuronal marker, MAP2 (green) (Fig. 6E), and nuclear stain,
In the present study, we demonstrated that HS alone was able to promote the formation of neurospheres in a dose-dependent manner, and acted synergistically with FGF-2. Its proliferative effect was due to an increased number of cells existing in S phase. On the other hand, HS modulated the motility behavior of NSPCs and resulted in a rapid neurosphere formation. However, these effects were not due to chemotactic migration. The mechanism was related to the activation of NF-κB signaling pathway. It has been reported that some polysaccharides act as accessory molecules that regulate growth factors binding to their receptors and the activation of the cognate signaling pathways (19). A polysaccharide, fucoidan, extracted from brown seaweed, was shown to increase the presence of circulating mature white blood cells and mobilize hematopoietic progenitor/stem cells in a selectin-independent
DISCUSSION
482
ZHANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. HS promoted cell division and increased the number of the cells in the S phase. Images of flow cytometric analysis in untreated controls (A), HS (4 μg/ml, B), FGF-2 (20 ng/ml, C), and FGF-2 + HS (D). Histogram indicating an increase in the S phase and less in G1 phase in HS-treated culture and the synergism with FGF-2 to promote cell division (E). P b 0.05 (*) compared to untreated controls.
manner in mice and non-human primates (20). Other polysaccharides also have been shown to serve as signaling molecules in stem cell proliferation, homing, neurogenesis and neural network formation (21,22). It is possible that exogenous polysaccharides may be able to recruit minute amounts of endogenous growth factors. The application of polysaccharides, in lieu of growth factors, has been suggested as an alternative therapeutic strategy (23). Fucoidan composed of L-fucose and ester sulfate, is a good alternative to heparin due to its antithrombotic activity and its relative safety: being of invertebrate origin fucoidan is less likely to contain infectious agents, such as viruses or prions (24). The anticoagulant activity of fucoidan is lower than heparin (25), so that the hemorrhage risk is lowered for clinical use of transplanting NSPCs into the CNS if contaminated with fucoidan (26). In the present study, we found that HS promoted the formation
of neurospheres at the early culture stage and acted synergistically with FGF-2 in both neurosphere formation assay and cell cycle analysis. Moreover, HS-stimulated formation of neurospheres did not alter the neuronal lineage of NSPCs after differentiation. Thus, our finding indicated that HS could be a more effective and lower-risk growth co-adjuvant for clinical use in culturing NSPCs. We have shown that the most effective dose of HS to promote the formation of neurospheres was between 4 and 8 μg/ml. Thus, the concentration of 4 μg/ml HS was applied in the subsequent experiments, including cell cycle analysis and immunochemistry assay. In aggregation assay of NSPCs, if the concentration of HS in the medium was 5 μg/ml, the record was up to 24 h for appearance of 4–6 cell clusters (date not shown). Therefore, we carried out HS at a higher concentration 50 μg/ml in order to facilitate quantitative analysis of
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
483
FIG. 3. HS induced enhanced aggregation of NSPCs. NSPCs were stimulated with 50 μg/ml HS (upper panel) in growth medium; the lower panel corresponds to the control. (A) 30 min after HS treatment. (B) 120 min after HS treatment. (C) Control 30 min. (D) Control 120 min. Scale bar = 100 μm. Histogram indicating the quantification of aggregation level, and suggested that HS induced a fast aggregation of NSPCs and a rapid neurosphere formation at the early culture stage. P b 0.01 (**) compared to untreated controls.
aggregation level in this assay. In cell migration assay, we did not find a significant difference in migratory cells in all treatment, even though at a broad concentration ranging from 50 to 5000 ng/ml. Although we did not find a significant difference at a concentration ranging from 1 to 4 μg/ml in NF-κB activation assay, there was a good linear relationship at a higher concentration ranging from 5 to 50 μg/ml. Therefore, we carried out HS at a higher concentration in this assay. Isolation of NSPCs from the CNS using the neurosphere formation assay was first described in 1992 (27). Neurosphere culture system using FGF-2 and/or EGF as the mitogen is considered to be the most widely used technique for the in vitro expansion of NSPCs. However, the mechanism leading to neurosphere formation remains largely unclear. It is reported that FGF-2 not only promotes cell proliferation of embryonic cortical stem cells (28), but also controls their differentiation into neuronal and glial lineages under differentiation conditions (29). Thus, we think that FGF-2 is essential for proliferation of NSPCs in process of neurosphere formation. In general, neurospheres can form from dissociated cells after only 3 days of culture. Since NSPCs double approximately every 20 h, after
3 days neurospheres contain, on the average, eight cells, and are identified as neurosphere forming units (1). In the present study, we observed a significantly faster formation of neurosphere units on the same day of plating. Meanwhile, a larger free space between cells and/or neurosphere units appeared in HS cultures, as compared to the control group. Thus, we attempted to explore the mechanisms of the faster formation of neurosphere units from these two aspects: aggregation and proliferation of NSPCs. The results showed that HS induced an increase in the percentage of cells in S phase and promoted the proliferation of NSPCs, similar to reports of heparin and chondroitin sulfate glycosaminoglycans. On the other hand, a rapid aggregation occurred as early as 30 min after HS treatment, resulting in the formation of neurosphere units of 3–5 cells after 2 h treatment. Although HS had a less effect on proliferation than FGF-2 as indicated in neurosphere formation assay and cell cycle analysis, HS treatment activated a faster cell aggregates formed at the early culture stage. The cell aggregates might provide a favorable environment for the proliferation of NSPCs. Therefore, there was more neurospheres appeared in FGF-2 cultures with HS treatment than that of FGF-2
484
ZHANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 4. Chemotactic migration of NSPCs of HS-enhanced aggregation of NSPCs by a modified transwell chamber assay. (A) no additive control. (B) 50 ng/ml HS. (C) 500 ng/ml HS. (D) 5000 ng/ml HS. (E) 20 ng/ml FGF-2. (F) 20 ng/ml FGF-2 + HS 500 ng/ml. Scale bar = 100 μm. (G) Histogram indicating the number of cells passing through the filter, showing that no statistical differences were observed in all treatments. P b 0.05 (*) compared to untreated controls.
FIG. 5. HS treatment enhanced p65 protein nuclear translocation in a dose-dependent manner. P b 0.05 (*) compared to untreated controls.
cultures (** P b 0.01). This concludes that HS acted synergistically with FGF-2 and enhanced FGF-2 effect on neurosphere formation. However, a detailed structure of HS and its interaction with FGF-2 remain to be investigated. In addition, we attempted to explain the motility of NSPCs in HS culture as chemotactic migration using the classic transwell chamber assay. But, we were unable to detect any correlation between our observation and this particular property. In fact, it has been reported that rapid re-aggregation will interfere with migration assays; migration of NSPCs as induced by cytokines and growth factors (such as SCF) has only been observed 3 h after plating, longer than our observed aggregation (30). However, it has been reported that cell aggregation was disadvantageous for the proliferation of NSPCs because of restricted supplies of nutrients and heterogeneous populations within neurospheres (1,7). We here believe that cell aggregates formed at the early culture stage might provide a favorable environment for the proliferation of NSPCs. Thus, we propose a new model for the proliferation of NSPCs induced by HS. Firstly, several NSPCs dispersed on the plate will begin to aggregate in response to a signal from HS
VOL. 110, 2010
A SULFATED POLYSACCHARIDE FOR NEUROSPHERE FORMATION
485
FIG. 6. Multi-lineage potential of NSPCs from HS-stimulated formation of neurospheres. HS-treated neurospheres (A) were collected at 72 h, fixed and stained for Nestin (B) as described in Materials and Methods. Part of HS-treated neurospheres were dissociated, plated on coverslips and cultured in the medium containing 1% FBS for 5 days. The cells were fixed, stained for Hoechst 33342 and three lineage marker, O4 positive oligodendrocytes (red, C), GFAP positive astrocytes (green, D), MAP2 positive neuron (green, E) and Hoechstlabeled nuclei (blue). Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
and/or other cell–cell interaction signals, and then 3–5 cells will aggregate to form neurosphere units. Subsequently, the cells within the neurosphere units will begin to divide, each neurosphere will become larger through the cell division and further aggregation will occur with single cells and/or neurosphere units. HS exposure was maintained throughout the process of formation of neurospheres. Thus, the proliferation and aggregation of NSPCs leading to the formation of neurospheres was induced by HS. Furthermore, the faster formation of neurospheres induced by HS was closely related to the activation of the NF-κB signaling pathway. In conclusion, HS can promote neurosphere formation, and act synergistically with FGF-2. The effect of HS on NSPCs was related to the activation of the transcription factor NF-κB. These results suggest that HS would be a useful adjuvant for promoting the proliferation of NSPCs, a feature that might be useful for clinical applications. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (30873198) and the Jointly-Funded Universities Projects of Weihai (0000413420614). REFERENCES 1. Ahmed, S.: The culture of neural stem cells, J. Cell. Biochem., 106, 1–6 (2009). 2. Pitman, M., Emery, B., Binder, M., Wang, S., Butzkueven, H., and Kilpatrick, T. J.: LIF receptor signaling modulates neural stem cell renewal, Mol. Cell. Neurosci., 27, 255–266 (2004). 3. Kokuzawa, J., Yoshimura, S., Kitajima, H., Shinoda, J., Kaku, Y., Iwama, T., Morishita, R., Shimazaki, T., Okano, H., Kunisada, T., and Sakai, N.: Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos, Mol. Cell. Neurosci., 24, 190–197 (2003). 4. Ida, M., Shuo, T., Hirano, K., Tokita, Y., Nakanishi, K., Matsui, F., Aono, S., Fujita, H., Fujiwara, Y., Kaji, T., and Oohira, A.: Identification and functions of chondroitin sulfate in the milieu of neural stem cells, J. Biol. Chem., 281, 5982–5991 (2006). 5. Sirko, S., von Holst, A., Wizenmann, A., Götz, M., and Faissner, A.: Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells, Development, 134, 2727–2738 (2007). 6. Caldwell, M. A. and Svendsen, C. N.: Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells, Exp. Neurol., 152, 1–10 (1998).
7. Mori, H., Ninomiya, K., Kino-oka, M., Shofuda, T., Islam, M. O., Yamasaki, M., Okano, H., Taya, M., and Kanemura, Y.: Effect of neurosphere size on the growth rate of human neural stem/progenitor cells, J. Neurosci. Res., 84, 1682–1691 (2006). 8. Widera, D., Mikenberg, I., Kaus, A., Kaltschmidt, C., and Kaltschmidt, B.: Nuclear Factor-κB controls the reaggregation of 3D neurosphere cultures in vitro, Eur. Cell Mater., 11, 76–84 (2006). 9. Widera, D., Mikenberg, I., Elvers, M., Kaltschmidt, C., and Kaltschmidt, B.: Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-κB signaling, BMC Neurosci., 7, 64–82 (2006). 10. Cyert, M. S.: Regulation of nuclear localization during signaling, J. Biol. Chem., 276, 20805–20808 (2001). 11. Karin, M. and Lin, A.: NF-κΒ at the crossroads of life and death, Nat. Immunol., 3, 221–227 (2002). 12. Goldschmidt, B., Wider, D., and Goldschmidt, C.: Signaling via NF-κB in the nervous system, Biochim. Biophys. Acta, 1745, 287–299 (2005). 13. Zhang, Y. J., Song, S. L., Song, D., Liang, H., Wang, W. L., and Ji, A. G.: Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus, J. Biosci. Bioeng., 109, 67–72 (2010). 14. Kosaka, N., Kodama, M., Sasaki, H., Yamamoto, Y., Takeshita, F., Takahama, Y., Sakamoto, H., Kato, T., Terada, M., and Ochiya, T.: FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation, FASEB J., 20, 1484–1485 (2006). 15. Lum, M., Croze, E., Wagner, C., McLenachan, S., Mitrovic, B., and Turnley, A. M.: Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation, J. Neuroimmunol., 206, 32–38 (2009). 16. Kimura, A., Ohmori, T., Ohkawa, R., Madoiwa, S., Mimuro, J., Murakami, T., Kobayashi, E., Hoshino, Y., Yatomi, Y., and Sakata, Y.: Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury, Stem Cells, 25, 115–124 (2007). 17. Mo, S. J., Son, E. W., Lee, S. R., Lee, S. M., Shin, D. H., and Pyo, S.: CML-1 inhibits TNF-α induced NF-κB activation and adhesion molecule expression in endothelial cells through inhibition of IkB-α kinase, J. Ethnopharmacol., 109, 78–86 (2007). 18. Wang, T. Y., Sen, A., Behie, L. A., and Kallos, M. S.: Dynamic behavior of cells within neurospheres in expanding populations of neural precursors, Brain Res., 1107, 82–96 (2006). 19. Nikitovic, D., Assouti, M., Sifaki, M., Katonis, P., Krasagakis, K., Karamanos, N. K., and Tzanakakis, G. N.: Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factormediated proliferation in human metastatic melanoma cell lines, Int. J. Biochem. Cell Biol., 40, 72–83 (2008). 20. Sweeney, E. A., Priestley, G. V., Nakamoto, B., Collins, R. G., Beaudet, A. L., and Papayannopoulou, T.: Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence, Proc. Natl. Acad. Sci. USA, 97, 6544–6549 (2000). 21. Li, F., Shetty, A. K., and Sugahara, K.: Neuritogenic activity of chondroitin/ dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from
486
22.
23. 24.
25.
ZHANG ET AL.
shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways, J. Biol. Chem., 282, 2956-2066 (2007). Akita, K., von Holst, A., Furukawa, Y., Mikami, T., Sugahara, K., and Faissner, A.: Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult central nervous system implies that complex chondroitin sulfates have a role in neural stem cell maintenance, Stem Cells, 3, 798–809 (2008). Sugahara, K. and Mikami, T.: Chondroitin/dermatan sulfate in the central nervous system, Curr. Opin. Struct. Biol., 17, 536–545 (2007). Berteau, O. and Mulloy, B.: Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide, Glycobiology, 13, 29R–40R (2003). Pereira, M. S., Mulloy, B., and Mourao, P. A. S.: Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae, J. Biol. Chem., 274, 7656–7667 (1999).
J. BIOSCI. BIOENG., 26. Mori, H., Kanemura, Y., Onaya, J., Hara, M., Miyake, J., Yamasaki, M., and Kariya, Y.: Effects of heparin and its 6-O- and 2-O-desulfated derivatives with low anticoagulant activity on proliferation of human neural stem/progenitor cells, J. Biosci. Bioeng., 100, 54–61 (2005). 27. Reynolds, B. A. and Weiss, S.: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science, 255, 1707–1710 (1992). 28. Raballo, R., Rhee, J., Lyn-Cook, R., Leckman, J. F., Schwartz, M. L., and Vaccarino, F. M.: Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex, J. Neurosci., 20, 5012–5023 (2000). 29. Qian, X., Davis, A. A., Goderie, S. K., and Temple, S.: FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells, Neuron, 18, 81–93 (1997). 30. Sun, L., Lee, J., and Fine, H. A.: Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury, J. Clin. Invest., 113, 1364–1374 (2004).