The present study aimed to determine whether co-culture with bone marrow-derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of β-tubulin III-positive cells were detected by microscopy. The wingless-type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway was analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC-mediated NSC proliferation and differentiation. The results revealed that co-culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co-culture with EPCs markedly induced the expression levels of Wnt3a and β-catenin and inhibited the phosphorylation of glycogen synthase kinase 3β (GSK-3β). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC-mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC-mediated expression of β-catenin, and its phosphorylation and activation of GSK-3β. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC-mediated NSC proliferation and differentiation.
Spinal cord injury causes severe neurological dysfunction, which affects patients and their families as it requires substantial long-term healthcare expenditure and leads to permanent deprivation in quality of life (
There is increasing evidence that endothelial progenitor cells (EPCs) contribute to angiogenesis by promoting migration and proliferation (
The present study aimed to determine whether co-culture with bone marrow-derived EPCs affects spinal cord-derived NSC proliferation and differentiation. The data obtained in the present study is the first, to the best of our knowledge, to demonstrate that co-culture with bone marrow-derived EPCs promoted the proliferation and differentiation of spinal cord-derived NSC, at least in part, via modulation of the wingless-type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway. In conclusion, the results provided novel molecular insight into EPC-mediated neurogenesis during the repair of spinal cord injury.
All animal procedures were approved by the Animal Ethics Committee of the Institutional Animal Care and Use Committee of Anhui Medical University (Hefei, China), in accordance with the Guide for the Care and Use of Laboratory Animals in China (
Endothelial basal medium (EBM)-2 and EGM-2 Single Quots, containing 10 ml fetal bovine serum (FBS), 0.2 ml hydrocortisone, 2 ml human fibroblast growth factor (hFGF)-B, 0.5 ml vascular endothelial growth factor (VEGF), 0.5 ml R3-IGF-1, 0.5 ml ascorbic acid, 0.5 ml hEGF, 0.5 ml GA-1000 and 0.5 ml heparin, were purchased from Clonetics (San Diego, CA, USA). Dulbecco's modified Eagle's medium/F12 (DMEM/F12) medium, FBS, B27 supplement and L-glutamic acid (L-glutamine) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Basic (b) FGF and epidermal growth factor (EGF) were purchased from Peprotech, Inc. (Rocky Hill, NJ, USA). The rat bone marrow lymphocyte isolation kit was purchased from Tianjin Hao Yang Biological Products Technology Co., Ltd. (Tianjin, China). Rat fibronectin was purchased from Gene Operation, Inc. (Ann Arbor, MI, USA); DiI-labeled acetylated low-density lipoprotein (Di1-Ac-LDL) and Lipofectamine 2000 were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Fluorescein isothiocyanate UEA-1 (FITC-UEA-1), and poly-lysine were purchased from Sigma-Aldrich; Thermo Fisher Scientific, Inc. Antibodies against VEGF receptor (VEGFR)-2, β-tubulin III, β-actin and nestin were purchased from Santa Cruz Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against Wnt3a, phosphorylated (p)-glycogen synthase kinase 3β (GSK-3β), p-β-catenin, GSK-3β and β-catenin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody against CD133 was purchased from Biorbyt, Ltd. (San Fransisco, CA, USA). Rabbit anti-glial fibrillary acidic protein antibodies were purchased from Abcam (Cambridge, UK). The recombinant plasmid, pEGFP-short hairpin (sh) RNA-wnt3a, was purchased from Hefei Hao Xiang Biological Technology Co., Ltd (Anhui, China).
The SD rats (90–120 g) were sacrificed with an excess of 10% chloral hydrate anesthesia, following which both femurs and tibias were surgically dissected. The bone marrow mononuclear cell population was isolated using a commercially available kit (R&D systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol. The bone marrow mononuclear cells were then re-suspended in EBM-2 complete medium. To isolate the EPCs, the bone marrow mononuclear cells (5×105 cells/well) were plated on bovine fibronectin-coated 24-well plates. The plates were incubated in 5% CO2 at 37°C. The medium was replaced every 3 days until the first passage cells were ~70% confluent (14 days). The EPCs were identified by the expression of cell surface markers, CD133 and VEGFR-2, using fluorescence microscopy. In addition, the uptake of fluorescent Dil-ac-LDL was evaluated using confocal microscopy. The binding of UEA-1 was determined using FITC-conjugated UEA-1.
Newborn SD rats (5–7 days old) were sacrificed by cervical dislocation. The thoracolumbar spinal cord, stripped of soft meninges and blood vessels, were placed in ice-cold DMEM/F12 for further dissection. The spinal cord was cut it into sections measuring 1 mm3 with ophthalmic scissors, and filtrated through a 200 mesh cell sieve following repeated pipetting turbid suspension. The dissociated cell suspension was centrifuged at 800 x g for 5 min at room temperature, and the pellet was seeded (1×106 cells/ml) into flasks containing DMEM/F-12 with 2% B27, 20 ng/ml EGF, 10 ng/ml bFGF and 0.6 mg/ml L-glutamine. The flasks were incubated in 5% CO2 at 37°C. After 48 h, the medium was replaced the remove the non-adherent cells, and was replaced every 3 days thereafter. All the cells used in the experiments were obtained from passages 3–10. The NSCs were identified by positive staining for nestin under a light microscope (Carl Zeiss Inc., Jena, Germany), a molecular marker for multi-potent NSCs (
The EPCs were transfected with the mouse wnt3a pEGFP-shRNA plasmid using lipofectamine 2000 and Opti-MEM medium (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, the EPCs were plated on 6-well plates at 70–80% confluence 24 h prior to transfection. The Wnt3a pEGFP-shRNA plasmid (2
For
To determine the uptake of Dil-Ac-LDL and binding of FITC-UEA-1, the EPCs were incubated with Dil-Ac-LDL overnight at 37°C, and fixed with 4% paraformaldehyde for 20 min. The cells were then incubated with FITC-UEA-1 for 1 h at 37°C, and examined under a fluorescent microscope (Olympus Corporation).
To investigate the effect of EPC co-culture on the differentiation and proliferation of NSCs, an EPC/NSC co-culture assay was performed, as described in a previous report (
Following co-culture with EPCs for different periods of time, the viability of the NSCs was determined using an MTT Cell Proliferation and Cytotoxicity Assay kit (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. The cell viability was measured at 490 nm with a microplate reader (Tecan M200; Tecan Austria GmbH, Salzburg, Austria). NSC proliferation was measured using a BrdU cell proliferation kit (Roche, Mannheim, Germany), according to the manufacturer's protocol. The cell proliferation was measured at 450 nm with a microplate reader (Tecan M200; Tecan Austria GmbH).
In order to investigate the proliferation potential of the NSCs in the presence and absence of EPC co-culture, a neurosphere growth kinetics assay was performed, as described earlier. The NSC culture was passaged by gentle trituration, and the resulting single-cell suspension of NSCs was replated in a 12-well plate at a density of 5×104 cells/well for co-culture with or without EPCs. The number and diameter of the neurospheres were measured in all groups using an inverted phase-contrast microscope (Leica, Mannheim, Germany) and analyzed using Image J software (v1.50a, National Institutes of Health, Bethesda, MA, USA).
Total RNA was extracted from the differentiated NSCs using TRIzol (Takara Biotechnology Co., Ltd., Dalian, China). The total RNA was isolated and purified using an RNeasy minikit (Takara Biotechnology Co., Ltd.) with the addition of RNase-free DNase I (Takara Biotechnology Co., Ltd.). The total RNA (1
Western blot analysis was performed, as previously described (
The level of VEGF in the medium of the EPCs was measured using a commercially available ELISA kit (R&D Systems Europe, Ltd., Abingdon, UK), according to the manufacturer's protocol. After 1, 3, 5, 7 and 14 days of culture, the cell supernatants were collected to measure the levels of VEGF in the medium. Each assay was repeated at least three times.
The results of the experimental investigations are expressed as the mean ± standard error of the mean. Differences between the mean values of multiple groups were analyzed using one-way analysis of variance with Tukey's test for post-hoc comparisons. All data analysis was performed with the use of GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Bone marrow mononuclear cells were isolated by density gradient centrifugation; the isolated mononuclear cells were small and round in shape. After 24 h, a small number of adherent cells appeared. After 7 days, a proportion of the mononuclear cells had become spindle-shaped. On day 14, the nearby colonies had fused with each other, exhibiting a larger cell monolayer with a cobblestone-like morphology (
A specific characteristic of spinal cord-derived NSCs is the ability to self-renew. The ability to passage neurospheres clonally is an indicator of self-renewal. In the present study, only individual cells were observed 1 day post-seeding, with no cell spheres observed. The assembly of the neurospheres was slow and the spheres were relatively small. After 7–21 days of subculture, phase contrast microscopy indicated that neurosphere formation had occurred (
To assess the effects of co-culture with EPCs on the proliferation and viability of the NSCs, the NSCs were co-cultured with EPCs for different time periods and their viability was measured using an MTT assay. As shown in
Previous studies have identified that the Wnt/β-catenin pathway may be critical in the regulation of NSC proliferation and differentiation (
NSCs are functionally characterized as cells with the capacity to proliferate, self-renew and produce populous progeny, which can differentiate into neurons, astrocytes and oligodendrocytes (
To further assess the function of Wnt3a signaling, the NSCs were co-cultured with EPCs with Wnt3a knockdown. As shown in
The present study demonstrated that co-culture with EPCs promoted spinal cord-derived NSC proliferation and differentiation, and that these effects were not observed following Wnt3a knockdown in the EPCs. The data further demonstrated that Wnt3a was critical for EPC-mediated NSC proliferation and differentiation through modulation of the β-catenin and GSK-3β signaling pathway.
Spinal cord injury is a serious threat to human health and quality of life. One option for treating spinal tissue damage includes the replacement of lost neuronal cells, and NSC transplantation therapy offers the potential to promote the repair of neuronal loss and functional recovery of spinal cord-transected rats (
The activation of Wnt/β-catenin signaling is a key factor in initiating and promoting neurogenesis due to its ability to selectively trigger the expression of a panel of neuronal-associated genes for NSC proliferation and differentiation (
In conclusion, the results of the present study suggested that co-culture with EPCs promoted the proliferation and differentiation of NSCs through modulation of the Wnt3a/β-catenin and GSK-3β signaling pathway. The present study provided molecular insight into the EPC-mediated effects of neurogenesis during the process of repair following spinal cord injury.
This study was supported by grants from the National Natural Science Foundation of China (grant no 81171173) andt the Anhui Provincial Natural Science Foundation (grant no 11040606Q25).
Isolation and culture of bone marrow-derived EPCs and spinal cord-derived NSCs. Images show bone marrow-derived EPCs (A) 14 days following isolation and (B) following DAPI staining. The (C) expression of vascular endothelial growth factor-2 and (D) CD133, the (E) uptake of DiI-labeled acetylated low-density lipoprotein and (F) binding to fluorescein isothiocyanate UEA-1 were analyzed. (G) Spinal cord-derived NSCs 21 days following isolation, (H) expressing nestin and (I) following DAPI staining. Scale bar=50
Co-culture with EPCs promotes NSC proliferation and differentiation. (A) NSCs were seeded into 24-well plates and co-cultured with or without EPCs for the indicated time periods. (A) Cell viability of the NSCs was assessed using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Values are expressed as the mean ± standard error of the mean (n=3). (B) Images showing the number of neurospheres (magnification, ×100). (C) Cell proliferation of the NSCs was analyzed using a BrdU ELISA assay. (D) Images show NSC differentiation (β-tubulin III-positive cells). Values are expressed as the mean ± standard error of the mean (n=3). Scale bar=100
Co-culture with EPCs induces activation of the Wnt3a/β-catenin pathway in NSCs. Following co-culture for 7 days, reverse transcription-quantitative polymerase chain reaction analysis was used to analyze the mRNA levels of (A) β-catenin and (B) Wnt3a. GAPDH was used as the housekeeping gene. Western blot analysis was used to determine the protein levels of (C) β-catenin, (D) Wnt3a and (E) p-GSK3β. β-actin was used as a loading control. Values are expressed as the mean ± standard error of the mean (n=3). *P<0.05, vs. NSCs alone. EPCs, endothelial progenitor cells; NSCs, neural stem cells; Wnt3a, wingless-type MMTV integration site family, member 3a; p-GSK3β, phosphorylated glycogen synthase kinase 3β.
Wnt3a knockdown inhibits EPC co-culture-mediated NSC proliferation and differentiation. (A) Following treatment with a Wnt3a shRNA plasmid, western blot and densitometric analyses were performed to determine the level of Wnt3a, β-actin was used as loading control. (B) Following Wnt3a knockdown for the indicated time periods, the levels of VEGF released in the EPCs were determined. After 7 days co-culture with the EPCs, the (C) proliferation of the NSCs, the (D) number of neurospheres (magnification, ×100) and (E) differentiation of NSCs were determined. Scale bar=100
Wnt3a knockdown inhibits β-catenin signaling activation in the co-culture system. (A) Following co-culture with EPCs for 7 days, reverse transcription-quantitative polymerase chain reaction analysis was used to determine the mRNA levels of β-catenin. GAPDH was used as the housekeeping gene. Western blot analysis was used to determine the protein levels of (B) β-catenin, (C) p-β-catenin, (D) nuclear β-catenin and (E) p-GSK-3β, β-actin was used as a loading control. Values are expressed as the mean ± standard error of the mean (n=3). *P<0.05, vs. NSCs alone; #P<0.05, vs. EPC co-culture. EPCs, endothelial progenitor cells; NSCs, neural stem cells; GSK3β, glycogen synthase kinase 3β; p-, phosphorylated; shRNA, short hairpin RNA.