ICA (purity >98%, high-performance liquid chromatography grade) with the molecular formula C₃₃H₄₂O₁₅ and a molecular weight of 676.65 was purchased from Shaanxi Scidoor Hi-tech Biology Co., Ltd. (Xi'an, China). Hank's Balanced Salt Solution (HBSS), Dulbecco's modified Eagle's medium (DMEM)/F12 medium, fetal bovine serum (FBS), B27, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), Glutmax and poly-L-lysine-coated plates were purchased from Thermo Fisher Scientific, Inc. (Beijing, China). Mouse anti-nestin was obtained from BD Pharmingen (cat. no. 560341; San Jose, CA, USA), mouse anti-β-III-tubulin (cat. no. T8578), mouse anti-glial fibrillary acidic protein (GFAP; cat. no. G3893) and rabbit anti-galactocerebroside (GalC; cat. no. G9152) were obtained from Sigma Aldrich (Shanghai) Trading. Co., Ltd.(Shanghai, China). Cy2- or Cy3-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). RNeasy kit and AMV reverse transcriptase were obtained from Qiagen, Inc. (Valencia, CA, USA). Cy3-dCTP and Cy5-dCTP were purchased from CapitalBio, Inc. (Beijing, China). SYBR Premix Ex Taq for quantitative polymerase chain reaction (qPCR) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China).

The use of human fetuses obtained following spontaneous abortion was approved by the Ethic Committee of Xuanwu Hospital of Capital Medical University (Beijing, China), and written informed consent was obtained from the study participants. The corpus striatum of 16–20-week fetuses were obtained from 5 fetuses following spontaneous abortion, and were dissected and chopped in cooled HBSS using a tissue chopper. The tissue was further triturated by pipetting up and down 10 times to dissociate cells. After filtering through a 300 micron stainless steel sieve, in order to remove the large pieces of tissue, the dissociated cells were pelleted by centrifugation at 200 × g for 5 min at room temperature, washed once with HBSS, and were re-suspended at a density of 2×10⁶ cells/ml in DMEM/F12 medium containing 2% B27, 20 ng/ml EGF, 20 ng/ml bFGF and 1% Glutmax (DMEM/F12 complete medium). Cells were seeded in 100 mm dishes and cultured at 37°C in a humidified atmosphere containing 5% CO₂. Cell medium was changed every 3 days. After an initial culture for 7–10 days typical NSC clusters appeared, which were pelleted by centrifugation at 200 × g for 5 min at room temperature and were treated with accutase. Accutase-digested cells were washed once with HBSS, re-suspended in DMEM/F12 complete medium and used for all subsequent assays. For NSC characterization, the cells were seeded onto poly-L-lysine-coated chamber slides (1×10⁴/cm²). Following a 12 h culture, the cells were subjected to immunostaining for the analysis of nestin expression.

Growth factor-free DMEM/F12 medium supplemented with 1% FBS was used to induce cell differentiation. Cell clusters were digested with accutase as aforementioned, were washed once with HBSS, re-suspended in differentiation medium, and were seeded onto poly-L-lysine-coated chamber slides (1×10⁴/cm²). Cells were cultured for 7 days, and were subsequently subjected to fixation for immunostaining, in order to determine the expression of β-III-tubulin, GFAP and GalC.

Cells were fixed in ice-cold 4% paraformaldehyde for 15 min, followed by three washes with phosphate-buffered saline (PBS; pH 7.4). After blocking with 10% goat serum (Jackson ImmunoResearch, Inc., West Grove, PA, USA) in PBS containing 0.3% Triton X-100 at 37°C for 1 h, the cells were incubated with various primary antibodies at 4°C overnight. The antibodies used were as follows: Mouse anti-nestin (1:1,000), mouse anti-β-III-tubulin (1:1,000), mouse anti-GFAP (1:500) and rabbit anti-GalC (1:100). After three washes with PBS, Cy2- or Cy3-conjugated secondary antibodies (1:500) were added to the cells and were incubated at 37°C for 30 min, followed by three washes with PBS. Cells were then mounted with VectaShield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and 4′,6-diamidino-2-phenylindole was used for nuclear counter-staining. Fluorescent signals were visualized using a confocal laser microscope system (MRC1024; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

To study the effects of ICA on NSC proliferation, accutase-dissociated single cells were suspended in DMEM/F12 complete medium at a density of 2×10⁵ cells/ml. A 100 µl cell suspension was added into each well of a 96-well plate, which had previously been coated with poly-L-lysine. Following a 24 h culture, the medium was replaced with serum- and growth factor-free medium containing various concentrations of ICA (final concentrations, 0.1, 1 and 10 µM). After a further 24 h of culture, the medium was removed and serum- and growth factor-free medium (without phenol red) containing 10% Cell Counting kit (CCK)-8 reagent was added (Dojundo Molecular Technologies, Inc., Kumamoto, Japan). The cells were cultured for 4 h at 37°C, and the optical density (OD) of each well was measured at 450 nm (OD450) using a microplate reader. Cells cultured in serum- and growth factor-free medium served as a control. In a separate experiment, individual NSCs in DMEM/F12 complete medium were seeded into each well of a 24-well plate (10⁴ cells/cm²) coated with poly-L-lysine. Following a 24 h culture, the medium was replaced with serum- and growth factor-free medium containing 10 µM ICA (final concentration). Cells were maintained in culture for 7 days, and the formation of neurospheres was observed by microscopy. The diameters of 15 randomly chosen neurospheres from each well were measured using a micrometer installed in the microscope (IX70; Olympus Corporation, Tokyo, Japan). The results from three wells of each treatment group were averaged and compared.

cDNA microarray analysis was performed for gene expression profiling, and the results were compared between the cells treated with ICA and the untreated control cells. Cell treatment with 10 µM ICA was conducted, as aforementioned. NSCs cultured in serum- and growth factor-free medium without ICA served as a control. RNA was extracted from the cells using the RNeasy kit, according to manufacturer's protocol, and was quantified using the spectrophotometric method. Total RNA (2 µg) was used for first-strand cDNA synthesis using AMV reverse transcriptase, according to the manufacturer's protocol. Cy3-dCTP was added to the cDNA of the control cells, whereas Cy5-dCTP was added to the cDNA of ICA-treated cells. Microarray analysis was performed using the 22 K Human Genome Array (CapitalBio, Inc.). The slide contained gene-specific 70-mer oligonucleotides representing 21,329 human genes, including four human housekeeping genes as positive controls, and 12 negative controls, which were designed to have no significant homology with known human gene sequences. The labeled samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed into 80 µl hybridization solution [3X saline-sodium citrate (SSC), 0.2% sodium dodecyl sulfate (SDS), 25% formamide and 5X Denhardt's). cDNA in hybridization solution was denatured at 95°C for 3 min prior to loading onto the microarray. The array was hybridized at 42°C overnight, and washed once with washing solution (0.2% SDS and 2X SSC) at 42°C for 5 min, followed by one wash with 0.2% SSC at room temperature for 5 min. Finally, the array was scanned using a confocal LuxScan 10 KA scanner (CapitalBio, Inc.). The data of the obtained images were extracted using LuxScan 3.0 software (CapitalBio, Inc.). Genes with a signal intensity >800 (Cy3 or Cy5) were considered to be expressed. In every two channel slides, the intensity ratio of Cy5 to Cy3 of each spot was calculated following normalization with LOWESS regression. Statistical data and differential analysis files were generated using SAM software 3.0 (Stanford University, Stanford, CA, USA). Significantly altered genes were selected based on a P<0.05 and >2 fold change. All differentially expressed genes were analyzed using the free web-based Molecular Annotation System 2.0 (MAS 2.0; http://bioinfo.capitalbio.com/mas3/).

qPCR was performed to verify the array results. Primers (Table I) were designed based on published gene sequences using the Oligo 5.0 program (Molecular Biology Insights, Inc., Colorado Springs, CO, USA), and were synthesized by Sangon Biotech Co., Ltd. The PCR mix (20 µl) contained 10 µl 2X SYBR Taq Premix buffer, 1 µl cDNA, 200 nM of each primer (final concentration) and 9.0 µl nuclease-free water. The reaction mixture was incubated at 95°C for 10 sec, followed by 40 amplification cycles at 95°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. A melting curve analysis was run to determine the specificity of amplification following each qPCR analysis. Relative mRNA expression levels were calculated using the 2^−∆∆Cq method (14), following normalization against glyceraldehyde 3-phosphate dehyrdrogenase levels.

All results are expressed as the mean ± standard error of the mean, and were analyzed using one-way analysis of variance followed by Tukey's post-hoc test using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

NSCs were isolated from the corpus striatum of 16–20-week human fetuses obtained following spontaneous abortion. After culturing in DMEM/F12 complete medium for 7–10 days, typical NSC clusters appeared in the culture. Immunostaining analysis revealed that neurosphere-forming cells expressed nestin, a marker of NSCs (Fig. 1A). Cell differentiation assay demonstrated that NSCs were able to differentiate into different cell types of the nervous system, as indicated by the expression of β-III-tubulin, a specific neuronal marker (Fig. 1B); and GFAP, an astrocyte marker (Fig. 1C). Few cells expressed GalC, an oligodendrocyte marker (data not shown).

Using a CCK-8 cell proliferation assay kit, the effects of ICA on NSC proliferation were determined. As shown in Fig. 2, NSCs were incubated with various concentrations of ICA. In the presence of 0.1 or 1 µM ICA, cells did not exhibit markedly increased proliferation compared with the control cells (Fig. 2A). Conversely, cells incubated with 10 µM ICA exhibited a significantly increased proliferation rate compared with the untreated cells (n=4, P<0.05; Fig. 2A). When ICA concentration increased to 50 µM, cell toxicity was observed (data not shown). In a separate assay, the formation of neurospheres under the influence of ICA was examined. Neurospheres formed in ICA-treated cells appeared larger compared with those in the control group (Fig. 2B and C). By measuring neurosphere diameter, it was demonstrated that the diameter of neurospheres formed in the ICA-treated group was significantly greater than that of the neurospheres in the control group (n=5, P<0.05; Fig. 2D).

Gene expression in 10 µM ICA-treated cells and control cells was profiled by cDNA microarray, and the results were compared. A total of 779 genes were differentially expressed between the ICA-treated group and the control group, based on P<0.05 and >2 fold change criteria (data not shown). Of the altered genes, those involved in the top 10 cell signaling pathways analyzed using MAS software were listed in Table II. Since the Wnt signaling pathway and the bFGF pathway have important roles in NSC proliferation and neurogenesis, several differentially expressed genes important in the Wnt signaling pathway were selected (Table III). Fibroblast growth factor receptor 1 was also further analyzed by qPCR to validate the array results.

The mRNA expression levels of fibroblast growth factor receptor 1 (FGFR1), and four genes of the Wnt signaling pathway: Frizzled class receptor 7 (FZD7), dishevelled segment polarity protein 3 (DVL3), catenin beta-1 (CTNNB1) and glycogen synthase kinase-3β (GSK-3β), were further determined by qPCR. The results indicated that the mRNA expression levels of FZD7 (Fig. 3A) and DVL3 (Fig. 3B) were significantly elevated in the ICA-treated group compared with the control group (P<0.01 and P<0.05, respectively); CTNNB1 expression was increased in ICA-treated cells; however, the increase was not significantly different compared with the control cells, as determined by statistical analysis (Fig. 3C). The mRNA expression levels of GSK-3β were markedly reduced in the ICA-treated cells (n=6, P<0.01; Fig. 3D). In addition, FGFR1 expression was significantly upregulated by ICA (P<0.01; Fig. 3E).