Pregnant (E16) Sprague-Dawley rats (4 months old, n=3) and pregnant (E38) tree shrews (8 months old, n=3) of clean grade, weighing 170 g, were used in the present study. All animals were provided by the Animal Experimental Center of Kunming Medical University (Kunming, China). Animal care and all experimental protocols were approved by the guidelines of the Institutional Medical Experimental Animal Care Committee of Sichuan University, West China Hospital, (Chengdu, China). Guidelines for Laboratory Animal Care and Safety from the National Institutes of Health were also followed (24). The animals were bred in separate cages in a temperature (20±5°C), CO₂ (0.03%) and humidity (40–60%)-controlled room with a 12 h light/dark cycle and free access to pellet chow and water.

Pregnant (E16) Sprague-Dawley rats and pregnant tree shrews (E38) were sacrificed after being anesthetized by intraperitoneal injection of 3.6% chloral hydrate (1 ml/100 g). Following rinsing in 75% ethanol for 3 min, the embryonic rat and tree shrews were removed under sterile conditions and kept in a culture dish containing Hank's balanced salt solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on ice. The skulls were dissected and the brain hemisphere was removed, subsequently, the brain tissues were placed into pre-cooled PBS. Under an anatomic microscope, the meninges, olfactory bulb, cerebellum and brain stem were attentively removed and the hippocampal tissues were exposed and harvested. The samples were washed twice with pre-cooled PBS and placed into centrifuge tubes (25).

Hippocampal tissues were sheared into 1 mm³ sized tissue blocks. Trypsin (0.25%; 1:250; EMD Millipore, Billerica, MA, USA) was used to digest the tissue block at room temperature for 20 min. The digested tissue solution was collected and placed in a 15-ml centrifuge tube and Dulbecco's modified Eagle's medium/F12 (DMEM/F12; 1:1; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (Thermo Fisher Scientific, Inc.) was added to stop the digestion. Centrifugation was subsequently performed at 560 × g (4°C) for 5 min. The supernatant was discarded and the cell suspension was harvested using DMEM/F12 culture media (1:1; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2% B-27 (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, Inc., Minneapolis, MN, USA), 20 ng/ml epidermal growth factor (EGF; R&D Systems, Inc.), 2 mmol/l glutamine (Gibco; Thermo Fisher Scientific, Inc.), 10,000 U/l penicillin and 10 mg/l streptomycin.

Cell density was determined in the cell suspension and the density was adjusted to 5×10⁵/ml. The cells were inoculated onto the culture plates or bottles and kept in an incubator containing 5% CO₂ at 37°C. Half of the culture medium was replaced every other day.

At 7 days post-culture, the diameter of neurospheres was commonly ~100 µm and subculturing was performed. In detail, NSCs were digested using 0.25% trypsin (1:250, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 10 min and DMEM/F12 (1:1; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing serum was used to stop the digestion. NSC suspension was collected into a 15 ml centrifuge tube. Subsequently, centrifugation at 560 × g (4°C) was performed for 5 min. The supernatant was discarded. The cell suspension was resuspended in DMEM/F12 (1:1; Gibco; Thermo Fisher Scientific, Inc.) containing 2% B-27 (Gibco; Thermo Fisher Scientific, Inc.), 20 ng/ml bFGF (R&D Systems, Inc.), 20 ng/ml EGF (R&D Systems, Inc.), 2 mmol/l glutamine (Gibco; Thermo Fisher Scientific, Inc.), 10,000 U/l penicillin and 10 mg/l streptomycin. The cellular density was adjusted to 1.5–2.5×10⁶/ml and inoculated into a culture bottle (25 ml in volume), which was gently swayed for even distribution. The cells were incubated in an incubator at 37°C.

During the primary and secondary culture of cells derived from the hippocampus of rats and tree shrews, inverted phase contrast microscopy (Leica Microsystems GmbH, Wetzlar, Germany) was employed to observe and record the morphology and growth of NSCs. Prior to observation, cells were fixed with 4% paraformaldehyde for 20 min at room temperature.

In order to confirm the cultured NSCs and detect the expression of neurotrophin 3 (NT3), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) and transforming growth factor (TGF) β1, NSCs were cultured to the third generation, then immunofluorescence staining of nestin (a maker of NSCs) and neurotrophic factors (NT3, BDNF, GDNF and TGFβ1) was performed. For detecting the differentiation of NSCs, immunofluorescence staining of neuronal nuclei protein (NeuN; a neuronal marker) and glial fibrillary acidic protein (GFAP; an astrocyte marker) was performed to identify the characteristics of NSCs following serum induction (DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.)) for 48 h. The antibodies used were showed in Table I. Briefly, after discarding the culture medium of the third passage of NSCs, 0.25% trypsin (1:250, Sigma-Aldrich; Merck KGaA) was added. Inverted phase contrast microscopy (Leica Microsystems GmbH, Wetzlar, Germany) was employed to observe the morphology of the NSCs. The majority of the cells were round in shape with cytoplasmic retraction and were loosened and floating. An appropriate amount of culture medium containing 10% fetal bovine serum was added to stop the digestion. The cell suspension was gently blended for even distribution using pap dropper. Subsequently, the cell suspension was transferred to 6-well plates and dropped onto sterile cover slips. The 6-well plates containing 1.2×10⁶ per well NSCs were incubated in an incubator at 37°C for 4 h. After 5 days of culture, tsNSCs and rNSCs were fixed with 4% paraformaldehyde for 30 min at room temperature, rinsed with 0.01 M PBS and incubated with 3% goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37°C to quench non-specific binding. Subsequently, cells were incubated overnight at 4°C with primary antibodies (Table I). As for the control group, the primary antibody was substituted with 0.01 M PBS. The glass slides were washed with 0.01 M PBS three times, each for 2 min, which was followed by incubation with cyanine 3-labeled anti-rabbit secondary antibody (1:200; cat. no. 111-165-003; Jackson Laboratory, Ben Harbor, ME, USA) at 37°C for 30 min. Sections were observed under a fluorescent microscope. DAPI was used to counterstain the nuclei and the images were acquired using a Leica AF6000 cell station (Leica Microsystems GmbH). Finally, the proportion of positive cells was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Each detection involved the preparation of six plates (6-well plates) of cells and each well was put into one sterile cover slip. Three random fields were selected per slide and evaluated by three investigators blinded to the experimental information, and the mean proportion of positive cells for each detection was calculated.

To investigate the expansion rates, tsNSCs and rNSCs were seeded in 6-well plates (1.2 ×10⁶ per well) and cultured at 37°C for up to 72 h. Neurospheres formed within 2–3 days in vitro. The aforementioned culture medium: DMEM/F12 supplemented with 2% B-27, 20 ng/ml bFGF, 20 ng/ml EGF;, 2 mmol/l glutamin, 10,000 U/l penicillin and 10 mg/l streptomycin, was changed every 2 days. Numbers of neurospheres of tsNSCs and rNSCs were counted after culturing for 72 h. The images were captured with a Leica AF6000 cell station (Leica Microsystems GmbH). The number of neurospheres was quantified by using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). Five fields of vision were randomly selected per well and evaluated by three blinded investigators, and the mean number of neurospheres per well was calculated.

SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Experimental data are presented as the mean + standard deviation and were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Following the inoculation of tsNSCs and rNSCs, cells were well distributed under the microscope, cells commonly exhibited a round shape with a transparent cytoplasm. At 1 day after inoculation, the majority of cells were single celled and only a few exhibited a proliferative growth style. At this time, 2 or 4 cells connecting together was observed, and the cells were in a good growth state with good refraction and a transparent cytoplasm (Fig. 1). At day 3 of culture, the number of cell spheres increased and the size of cell spheres was uneven. Certain cells proliferated and formed embryonic spheres consisting of tens to several tens of cells. The cytoplasm of all the cell spheres was transparent. Occasionally, individual cells exhibited an adherent growth style with outgrowing processes (Fig. 1). At day 5 of culture, the volume of the cell spheres was enlarged in addition to the number of cell spheres. At this time, the majority of cells exhibited a suspended growth style, with regular morphology and strong refraction, without process outgrowth (Fig. 1).

As revealed by immunofluorescence staining of nestin, primary cultured neurospheres from tree shrews and rats exhibited positive nestin staining. The cytoplasm of neurospheres exhibited a clear red color (Fig. 2), indicating strong positive nestin expression. DAPI staining stained the nuclei of NSCs blue (Fig. 2). Merged synthetic images demonstrated that nestin (red) and DAPI (blue) were co-localized in the cultured cells (Fig. 2). These results indicate that the separation and culture tsNSCs and rNSCs was successful in the present study, which allowed subsequent experiments to be performed.

At 5 days following inoculation in vitro, tsNSCs and rNSCs were present as cell colonies with round shapes (Fig. 3A). In order to detect the proliferation ability of rNSCs and tsNSCs, quantitative analysis of the average number of neurospheres demonstrated that the number of neurospheres in the cultured tsNSCs was significantly decreased compared with rNSCs (P=0.0031; Fig. 3B).

At 5 days after induction by serum, certain NSCs exhibited radiate protuberance, and adherent and suspension culture was observed (Fig. 4A). Following immunofluorescence staining, compared with rNSCs, tsNSCs exhibited stronger GFAP positive immunoreactivity, however, the proportion of GFAP positive tsNSCs was not significantly different compared with rNSCs (P=0.665; Fig. 4A and B). NeuN immunofluorescence staining demonstrated that tsNSCs exhibited stronger NeuN positive reactivity compared with rNSCs (Fig. 4C). In addition, quantitative analysis demonstrated that the proportion of NeuN tsNSCs was markedly higher compared with rNSCs (P=0.0002; Fig. 4D).

Immunofluorescence staining was also performed to detect the expression of NT3, BDNF, GDNF and TGFβ1 in rNSCs and tsNSCs. Compared with rNSCs, tsNSCs expressed stronger NT3 positive immunoreactivity, and the proportion of NT3 positive tsNSCs was markedly higher compared with rNSCs (P<0.01; Fig. 5A and B). In addition, the BDNF positive immunoreactivity in rNSCs was stronger compared with tsNSCs, and quantitative analysis showed that the proportion of BDNF positive tsNSCs was markedly lower compared with rNSCs (P=0.045; Fig. 5A and B). Compared with rNSCs, GDNF positive immunoreactivity in tsNSCs was higher. However, quantitative analysis of the proportion of GDNF positive cells indicated no significant difference between the two groups (P=0.173; Fig. 5A and B). Furthermore, according to immunofluorescence staining analysis, the proportion of TGFβ1 tsNSCs was not significantly different compared with rNSCs (P=0.26; Fig. 5A and B). The negative control exhibited no positive immunoreactivity (Fig. 5C).