Contributed equally
Neural stem cells (NSCs) are characterized by the ability of self-renewal and capacity to proliferate and produce new nervous tissue. NSCs are capable of differentiating to three lineages of neural cells, including neurons, oligodendrocytes and astrocytes. Furthermore, hippocampal NSCs transplantation can improve the neurological deficits associated with expression of cytokines. Therefore, to compare the properties of NSCs of tree shrews and rats
Neural stem cells (NSCs) are a type of stem cell that possess self-renewal, self-replication and multi-differentiation properties. Under certain conditions, NSCs may be induced to differentiate into neurons, astrocytes and oligodendrocytes (
Several studies have demonstrated that NSCs are resident in various areas of the rat brain, including the hippocampus, cerebral hemisphere, hindbrain, spinal cord, cerebral ventricle area in the lateral ventricles, subventricle area and the cerebral cortex (
Tree shrews exhibit various characteristics that are similar to humans, including their biological features, metabolism, physiology, biochemistry and genome. Therefore, tree shrews are considered as a type of novel experimental animal model that may partially replace the primate models (
In the present study, the features of NSCs derived from rats and tree shrews were compared. Furthermore, the expression of certain growth factors was also compared, with the aim of increasing the understanding of the biological characteristics of tsNSCs and improving their application in research.
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 (
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 (
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
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
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 (
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 (
At 5 days following inoculation
At 5 days after induction by serum, certain NSCs exhibited radiate protuberance, and adherent and suspension culture was observed (
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;
Two primary conclusions were drawn based on the results of the present study. The first is that the ability of tsNSCs to differentiate into neurons was stronger compared with rNSCs. In addition, the level of NT3 expression in tsNSCs was significantly higher compared with rNSCs, and the level of BDNF expression was lower in tsNSCs.
In the current study, as revealed by
Using immunofluorescence staining, the present study demonstrated that tsNSCs and rNSCs, induced by serum, differentiated into neurons and astrocytes. This indicates that both types of NSCs exhibit multi-differentiation properties. However, tsNSCs exhibited a stronger ability to differentiate into neurons compared with rNSCs. Previously, certain studies reported that low concentrations of serum promoted the differentiation of NSCs derived from neonatal rats into neurons, while high concentrations facilitated the differentiation of NSCs into neural glial cells, such as GFAP positive cells (
Further immunofluorescence experiments demonstrated that tsNSCs expressed a lower level of BDNF and a higher level of NT3 compared with rNSCs, while no significant differences were observed for GDNF and TGFβ1 expression between the two groups. BDNF is widely distributed in various brain areas, including the hippocampus, thalamus, amygdala and cortical layer, and has essential roles in the survival, growth and development of neurons (
Concerning the application of tree shrews in research, researchers have reported that tree shrews were useful and easier to work with compared with rats in a hepatitis B virus injection study (
In conclusion, the results of the current study demonstrated that, compared with rNSCs, tsNSCs exhibited a weaker proliferative ability, however, their ability to differentiate into neurons was much stronger. These results provide valuable evidence for the increased use of tree shrews as models for CNS diseases in humans.
The present study was supported by the Program of Innovative Research Team In Science and Technology in University of Yunnan and the Program of Innovative Research Team In Science and Technology in Yunnan Province and supported by a grant from the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (grant no. 2014BAI01B10).
The authors declare that they have no competing interests.
Morphology of NSCs
Comparison of neurosphere number in tsNSCs and rNSCs at 5 days following inoculation. (A) Representative images demonstrating the formation of neurospheres at 5 days following inoculation in the rNSC and tsNSC groups. (B) Quantitative analysis of the average number of neurospheres in the rNSC and tsNSC groups. Data are presented as the mean + standard deviation. **P<0.01, as indicated. NSCs, neural stem cells; tsNSCs, tree shew NSCs; rNSCs, rat NSCs.
Comparison of the differentiation of tsNSCs and rNSCs into neurons and astrocytes. (A) Immunofluorescence staining of GFAP (red fluorescence) and nuclear staining with DAPI (blue fluorescence) in rNSCs and tsNSCs. Merged images are also presented. (B) Quantitative analysis of the proportion of GFAP positive cells. (C) Immunofluorescence staining of NeuN (red fluorescence) and nuclear staining with DAPI (blue fluorescence) in rNSCs and tsNSCs. Merged images are also presented. Examples of positive cells are indicated by white arrows. Scale bar, 50 µm. (D) Quantitative analysis of the proportion of GFAP positive cells. Data are presented as the mean + standard deviation. Scale bar=50 µm, applies to all images. **P<0.01, as indicated. NSCs, neural stem cells; tsNSCs, tree shrew NSCs; rNSCs, rat NSCs; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei protein.
Expression of neurotrophic factors in tsNSCs and rNSCs. (A) Immunofluorescence staining of NT3, BDNF, GDNF and TGFβ1 in rNSCs and tsNSCs. Additionally, the left images are for DAPI staining, the middle images are for staining with NT3/BDNF/GDNF/TGFβ1 and the right images are for merged DAPI and NT3/BDNF/GDNF/TGFβ1 staining. (B) Quantitative analysis of the proportion of NT3, BDNF, GDNF and TGFβ1 positive rNSCs and tsNSCs. (C) Negative control for immunofluorescence staining, where PBS was employed instead of a primary antibody. Data are presented as the mean + standard deviation. Scale bar=50 µm, applies to all images. *P<0.05 and #P<0.01, as indicated. NSCs, neural stem cells; tsNSCs, tree shrew NSCs; rNSCs, rat NSCs; NT3, neurotrophin 3; BDNF, brain-derived neurotrophic factor; GDNF, glial cell-derived neurotrophic factor; TGF, transforming growth factor.
Primary antibody details.
Primary antibody | Company | Concentration | Catalog number |
---|---|---|---|
BDNF | Boster | 1:50 | MGC34632 |
NT-3 | Abcam | 1:50 | ab65804 |
GDNF | ZhongShanJinQiao | 1:100 | EIA-1067 |
TGFβ1 | Abcam | 1:100 | ab92486 |
GFAP | ZhongShanJinQiao | 1:50 | ZA-0117 |
NeuN | Abcam | 1:100 | ab177487 |
Nestin | Abcam | 1:50 | ab92391 |
All primary antibodies were rabbit antibodies. BDNF, brain-derived neurotrophic factor; NT3, neurotrophin 3; GDNF, glial cell-derived neurotrophic factor; TGF, transforming growth factor; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei protein.