Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen Neu N, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ? anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.     

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.     

Neuronal HIF-1 alpha protein and VEGFR-2 immunoreactivity in functionally related motor areas following a focal M1 infarct.

Clinical and experimental data support a role for the intact cortex in recovery of function after stroke, particularly ipsilesional areas interconnected to the infarct. There is, however, little understanding of molecular events in the intact cortex, as most studies focus on the infarct and peri-infarct regions. This study investigated neuronal immunoreactivity for hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) in remote cortical areas 3 days after a focal ischemic infarct, as both HIF-1alpha and VEGFR-2 have been implicated in peri-infarct neuroprotection. For this study, intracortical microstimulation techniques defined primary motor (M1) and premotor areas in squirrel monkeys (genus Saimiri). An infarct was induced in the M1 hand representation, and immunohistochemical techniques identified neurons, HIF-1alpha and VEGFR-2. Stereologic techniques quantified the total neuronal populations and the neurons immunoreactive for HIF-1alpha or VEGFR-2. The results indicate that HIF-1alpha upregulation is confined to the infarct and peri-infarct regions. Increases in VEGFR-2 immunoreactivity occurred; however, in two remote regions: the ventral premotor hand representation and the M1 hindlimb representation. Neurons in these representations were previously shown to undergo significant increases in VEGF protein immunoreactivity, and comparison of the two data sets showed a significant correlation between levels of VEGF and VEGFR-2 immunoreactivity. Thus, while remote areas undergo a molecular response to the infarct, we hypothesize that there is a delay in the initiation of the response, which ultimately may increase the 'window of opportunity' for neuroprotective interventions in the intact cortex.     

Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

Cytoskeletal proteins are involved in neuronal survival.Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury.However,the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear.To examine this,we established a rat model of traumatic brain injury by controlled cortical impact.At 72 hours after injury,2 × 10~7 cells/m L neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells(3 m L) were injected into the injured cortex.At 1–3 weeks after transplantation,expression of neurofilament 200,microtubule-associated protein 2,actin,calmodulin,and beta-catenin were remarkably increased in the injury sites.These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival,growth,and differentiation in the injury sites.The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.     

Regulation of nestin expression after cortical ablation in adult rat brain

During embryogenesis, transient expression of nestin in proliferating neuroepithelial stem cells signals the commitment of progenitor cells to differentiate. Although adult mammalian brain contains very little nestin, significant upregulation of nestin has been reported following cerebral injury, leading to speculation that nestin may be involved in brain repair. In this study, we assessed the temporal profile of nestin expression following ablation injury of the sensory barrel cortex and investigated the influence of contralateral whisker stimulation on nestin expression. Since the adult mammalian brain contains proliferating neuronal progenitor cells that can be labeled with bromodeoxyuridine (BrdU), we also determined the association of nestin reexpression with BrdU-labeled cells. Nestin reexpression was detected predominantly in the ipsilateral cortex 3 days post-ablation. There was no significant nestin upregulation in the subcortical region. Nestin reexpression was most marked surrounding the lesion, but also extended throughout the entire lateral cortex. Nestin in the ipsilateral cortex subsided by day 7, although perilesional nestin expression was still apparent 28 days post-injury. Western blot analysis of nestin expression 3 days post-ablation confirmed a significant two-fold increase in nestin expression (p<0.05). Double immunofluorescence labeling demonstrated that the majority of nestin expression occurred in astrocytes. We were unable to detect any colocalization with neuronal makers. However, BrdU-labeled cells, which were readily detected in the subventricular zone prior to injury, were readily detected in the perilesional area 3 days post-ablation, concomitant with nestin in this area. Confocal microscopy detected several BrdU-positive cells expressing nestin. Taken together, the data support a potential role for nestin reexpression in brain repair.     

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor(100 μg/L) to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.     

Mild hypothermia combined with neural stem cell transplantation for hypoxic-ischemic encephalopathy： neuroprotective effects of combined therapy

Neural stem cell transplantation is a useful treatment for ischemic stroke, but apoptosis often occurs in the hypoxic-ischemic environment of the brain after cell transplantation. In this study, we determined if mild hypothermia(27–28°C) can increase the survival rate of neural stem cells(1.0 × 105 /μL) transplanted into neonatal mice with hypoxic-ischemic encephalopathy. Long-term effects on neurological functioning of the mice were also examined. After mild hypothermia combined with neural stem cell transplantation, we observed decreased expression levels of inflammatory factor nuclear factor-kappa B and apoptotic factor caspase-3, reduced cerebral infarct volumes, increased survival rate of transplanted cells, and marked improvements in neurological function. Thus, the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation are superior to those of monotherapy. Moreover, our findings suggest that the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation on hypoxic-ischemic encephalopathy are achieved by anti-inflammatory and anti-apoptotic mechanisms.     

Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex-derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin-positive cells in the post-stroke cerebral cortex, but not in the non-ischemic cortex. Cells obtained from the ischemic core of the post-stroke cerebral cortex formed neurosphere-like cell clusters expressing nestin; such cells had the capacity for self-renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin-producing oligodendrocytes. Nestin-positive cells from the stroke-affected cortex migrated into the peri-infarct area and differentiated into glial cells in vivo . Although we could not detect differentiation of nestin-positive cells into neurons in vivo , our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post-stroke cerebral cortex.     

Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

Transplantation of Flk-1+ human bone marrow-derived mesenchymal stem cells promotes angiogenesis and neurogenesis after cerebral ischemia in rats

Transplantation of bone marrow‐derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. Although BMSCs‐induced angiogenesis is considered important for neurological functional recovery, the neurorestorative mechanisms are not fully understood. We examined whether BMSCs‐induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. Adult female rats subjected to 2 h middle cerebral artery occlusion (MCAO) were transplanted with a subpopulation of human BMSCs from male donors (Flk‐1+ hBMSCs) or saline into the ipsilateral brain parenchymal at 3 days after MCAO. Flk‐1+ hBMSCs‐treated rats exhibited significant behavioral recovery, beginning at 2 weeks after cerebral ischemia compared with controls. Moreover, rats treated with Flk‐1+ hBMSCs showed increased glucose metabolic activity and reduced infarct volume. Flk‐1+ hBMSCs treatment significantly increased the expression of vascular endothelial growth factor and brain‐derived neurotrophic factor, promoted angiogenesis, and facilitated cerebral blood flow in the ischemic boundary zone. Further, Flk‐1+ hBMSCs treatment enhanced proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone and subgranular zone of the hippocampus. Finally, more NSPCs migrated toward the ischemic lesion and differentiated to mature neurons or glial cells with less apoptosis in Flk‐1+ hBMSCs‐treated rats. These data indicate that angiogenesis induced by Flk‐1+ hBMSCs promotes endogenous neurogenesis, which may cause functional recovery after cerebral ischemia.     

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.     

Ischemia induced neural stem cell proliferation and differentiation in neonatal rat involved vascular endothelial growth factor and transforming growth factor-beta pathways

Brain ischemia is a leading cause of mortality and morbidity in premature infants. Knowing the fate of neural stem cells in the subventricular zone (SVZ) after ischemia and the mechanisms that determine this fate would be useful in manipulating neural stem cell proliferation and differentiation and possibly in reversing ischemic damage. We sought to identify the genes involved in the proliferation and differentiation of neural stem cells after exposure to ischemia in a 3-day-old rat model that approximates ischemia in premature infants. Proliferating cells were labeled by bromodeoxyuridine (BrdU) through intraperitoneal injection. Using immunfluorescence assays, we observed the proliferation and differentiation of neural stem cells. Genes were identified with GeneChip and real-time quantitative polymerase chain reaction analysis. Ischemic rats had more BrdU-positive cells in the SVZ at all four time points and more neural stem cells differentiation into neurons, astrocytes, and oligodendrocytes. GeneChip analysis showed a 3- to 10-fold increase in the mRNA expression of vascular endothelial growth factor, transforming growth factor-beta, and their receptors in the SVZ. PCR assays and Western blot analyses confirmed these results, indicating that vascular endothelial growth factor and transforming growth factor-beta might be two of the factors that involve post-ischemic neural stem cell proliferation and differentiation.     

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.     

Effects of cytokines and chemokines on migration of mesenchymal stem cells following spinal cord injury

We investigated the effects of cytokines and chemokines and their associated signaling pathways on mesenchymal stem cell migration after spinal cord injury, to determine their roles in the curative effects of mesenchymal stem cells. This study reviewed the effects of tumor necrosis factor-α, vascular endothelial growth factor, hepatocyte growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin like growth factor-1, stromal cell-derived factor and monocyte chemoattractant protein-1, 3 during mesenchymal stem cell migration to damaged sites, and analyzed the signal transduction pathways involved in their effects on mesenchymal stem cell migration. The results confirmed that phosphatidylinositol 3-kinase/serine/threonine protein kinases and nuclear factor-κB play crucial roles in the migration of mesenchymal stem cells induced by cytokines and chemokines.     

Changes of hypoxia-inducible factor-1 signaling and the effect of cilostazol in chronic cerebral ischemia

Hypoxiainducible factor1 and its specific target gene heme oxygenase1, are involved in acute cerebral ischemia. However, very few studies have examined in detail the changes in the hy poxiainducible factor1/heme oxygenase1 signaling pathway in chronic cerebral ischemia. In this study, a rat model of chronic cerebral ischemia was established by permanent bilateral common carotid artery occlusion, and these rats were treated with intragastric cilostazol （30 mg/kg） for 9 weeks. Morris water maze results showed that cognitive impairment gradually worsened as the cerebral ischemia proceeded. Immunohistochemistry, semiquantitative PCR and western blot analysis showed that hypoxiainducible factorla and heme oxygenase1 expression levels in creased after chronic cerebral ischemia, with hypoxiainducible factorla expression peaking at 3 weeks and heme oxygenase1 expression peaking at 6 weeks. These results suggest that the elevated levels of hypoxiainducible factorla may upregulate heine oxygenase1 expression fol lowing chronic cerebral ischemia and that the hypoxiainducible factor1/heme oxygenase1 sig naling pathway is involved in the development of cognitive impairment induced by chronic cerebral ischemia. Cilostazol treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, decreased hypoxiainducible factorla and heme oxygenase1 expression levels, and reduced apoptosis in the frontal cortex. These findings demonstrate that cilostazol can protect against cognitive impairment induced by chronic cerebral ischemic injury through an antiapoptotic mechanism.     

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor(β-NGF) on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS(β-NGF + PBS) into the right-hand side defect, and PBS into the left(control) defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF + PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF + PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.     

Restorative effect and mechanism of mecobalamin on sciatic nerve crush injury in mice

Mecobalamin,a form of vitamin B12 containing a central metal element(cobalt),is one of the most important mediators of nervous system function.In the clinic,it is often used to accelerate recovery of peripheral nerves,but its molecular mechanism remains unclear.In the present study,we performed sciatic nerve crush injury in mice,followed by daily intraperitoneal administration of mecobalamin(65 μg/kg or 130 μg/kg) or saline(negative control).Walking track analysis,histomorphological examination,and quantitative real-time PCR showed that mecobalamin significantly improved functional recovery of the sciatic nerve,thickened the myelin sheath in myelinated nerve fibers,and increased the cross-sectional area of target muscle cells.Furthermore,mecobalamin upregulated m RNA expression of growth associated protein 43 in nerve tissue ipsilateral to the injury,and of neurotrophic factors(nerve growth factor,brain-derived nerve growth factor and ciliary neurotrophic factor) in the L4–6 dorsal root ganglia.Our findings indicate that the molecular mechanism underlying the therapeutic effect of mecobalamin after sciatic nerve injury involves the upregulation of multiple neurotrophic factor genes.     

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.     

Secretion of nerve growth factor,brain-derived neurotrophic factor,and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7-10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.