Human umbilical cord blood-derived mesenchymal stem cells differentiate into neuron-like cells induced by combination of mouse nerve growth factor and brain-derived neurotrophic factor / 中国组织工程研究

BACKGROUND: We have found that mouse nerve growth factor has the ability to induce differentiation of umbilical cord blood mesenchymal stem cells (UCB-MSCs) into neurons in vitro. In order to further explore the method of improving the induction efficiency of nerve cells, we attempt to combine a variety of cell growth factors for cell induction. OBJECTIVE: To explore the effect of mouse nerve growth factor combined with brain-derived neurotrophic factor on the differentiation of UCB-MSCs into neuron-like cells in vitro . METHODS: After the donated primary UCB-MSCs were resuscitated and cultured, the passage 5 UCB-MSCs were divided into five groups. The first four groups served as pre-induced groups, and fibroblast growth factor and epidermal growth factor were added to pre-induce cells for 24 hours, and mouse nerve growth factor and brain-derived neurotrophic factor, alone or in combination, were used thereafter to induce UCB-MSCs, while in control group, only the same amount of cell medium was added. The last group was non-pre-induced group, in which the cells were cultured in the cell culture medium for 24 hours, and then mouse nerve growth factor and brain-derived neurotrophic factor were both added to induce UCB-MSCs. The morphological changes of cells were observed under inverted microscope. The expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemistry technique. Real-time qPCR was used to detect the relative expression of NSE and GFAP at mRNA level. RESULTS AND CONCLUSION: (1) The cell morphology of UCB-MSCs was in long shuttle shape and spindle shape with unequal size. After induction, the cell bodies gradually retracted and became rounded, and the projections extended to one-side or multi-sides, presenting with the neuron-like changes. (2) Immunocytochemistry and real-time qPCR results showed that NSE and GFAP were positive in each experimental group, and the positive rate and mRNA expression of NSE and GFAP in the combined induction group were higher than those in the other groups. (3) Either mouse nerve growth factor or brain-derived neurotrophic factor could induce UCB-MSCs to differentiate into neuron-like cells. Moreover, there was a cumulative effect between the two cytokines, and their combined use could effectively improve the efficiency of induction.

Human umbilical cord blood mesenchymal stem cells differentiate into neuron-like cells:in vitro induction by mouse nerve growth factor / 中国组织工程研究

BACKGROUND: Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are a kind of adult stem cells in the human umbilical cord blood, which have the potential to differentiate into neuron-like cells and can be used for the treatment of a variety of nervous system diseases. How to effectively induce hUCB-MSCs differentiation into neuron-like cells is always a hotspot in the stem cell research, which is of high scientific research value. OBJECTIVE: To explore the induction effect of mouse nerve growth factor (mNGF) on the differentiation of human umbilical cord blood-derived mesenchymal stem cells into neuron-like cells in vitro. METHODS: The donated hUCB-MSCs were resuscitated and the cell morphology after culture was observed to draw a cell growth curve. Passage 5 cells were cultured in the culture medium containing 0 (blank control), 50, 100, 150, 200 μg/L mNGF, and the cell morphology was observed and recorded daily under inverted phase contrast microscope. Immunocytochemistry detection was used to examine the expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) at 7 days of induction, and then the NSE and GFAP positive expression was calculated by Image-Pro Plus 6.0 software. RESULTS AND CONCLUSION: The viability of resuscitated hUCB-MSCs was up to over 90%. The cell morphology was in long shuttle shape and spindle shape with unequal size, and the cells presented with "S"-shaped growth curve and entered the logarithmic growth phase at 3-6 days. Typical neuron-like changes were observed after induction by mNGF; however, there was no change in the cell morphology in the control group. Immunocytochemical staining showed that the induced cells were positive for both NSE and GFAP, and the highest positive rates of NSE and GFAP were observed after induction by 100 μg/L mNGF (P < 0.05). In the control group, there was no positive expression of NSE and GFAP.To conclude,mNGF can induce the in vitro differentiation of hUCB-MSCs into neuron-like cells,and 100 μg/L mNGF can achieve the best induction effect.

Brain-derived neurotrophic factor and neural stem cells transplantation in treatment of hypoxic-ischemic brain injury in rats / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the effects of brain-derived neurotrophic factor (BDNF) on survival, migration and differentiation of neural stem cells (NSCs) transplanted into the brain of newborn rats with hypoxic-ischemic brain damage and the recovery of nervous functions.</p><p><b>METHODS</b>The NSCs were separated from hippocampus of neonatal Wistar rats within 24 h after birth. Brdu, NSE and GFAP were used as markers of differentiation and proliferation of NSCs. The newborn rats were subjected to hypoxic-ischemic condition to induce brain damage. Seven days later, NSCs transplantation was performed for the animals. The rats were divided into normal control group, HIBD group, PBS group, NSCs transplantation group, BDNF group and BDNF + NSCs transplantation group randomly. At 4 weeks after transplantation the nervous function of rats was observed by Y-maze and nerve behavior test. After they were sacrificed, the rat brains were examined by immunocytochemistry for Brdu and by immunofluorescence for NSE/Brdu.</p><p><b>RESULTS</b>The hippocampus NSCs of newborn rat could be well cultured and they expressed nestin and they could differentiate into NSE, GFAP. Most of NSCs survived in cerebral ventricle 4 weeks after transplantation in brain through Brdu immunocytochemistry and they migrated into regions of brain extensively, especially to the injured side of cortex and hippocampus. The number of living NSCs in the injured side of cortex and hippocampus of BDNF + NSCs transplantation group increased evidently and the percentage of NSCs differentiated into NSE was higher than that in the NSCs transplantation group (P < 0.05). The nerve function recovery of the rats in BDNF and NSCs treated group was significantly better than that in the other groups (P < 0.05). The NSCs group had no prominent changes as compared with the model groups (P > 0.05).</p><p><b>CONCLUSIONS</b>NSCs can be isolated from newborn rats hippocampus and cultured in vivo. NSCs can survive, migrate and differentiate into neurons through cerebral ventricle. BDNF could significantly accelerate proliferation and differentiation of NSCs transplanted into the brain of rats with HIBD. The nervous function recovery was improved prominently by transplantation of NSCs with BDNF application, which may become a potentially effective method to treat HIBD.</p>

Combined transplantation of neurotrophin-3 and neural stem cells in treatment of hypoxic-ischemic brain injury in rats / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of combined transplantation of neurotrophin-3 and neural stem cells in treatment of hypoxic-ischemic brain injury in rats, and study the possible mechanism.</p><p><b>METHODS</b>Neural stem cells (NSCs) isolated from hippocampi of newborn wistar rats were cultured and identified. The hypoxic-ischemic brain injury models established with 7-day-old wistar rats, and neural stem cells or others were transplantated into the ipsilateral ventricles 7 days later. The 7-day-old rats were randomized into 5 groups: normal group, model group, sham-transplantation group, NSCs transplantation group, and NT-3 combined with NSCs transplantation group. There were 12 rats per group. The functional test and immunohistochemistry were examined 4 weeks later.</p><p><b>RESULTS</b>The neural stem cells from new-born rats' hippocampi were successfully cultured. It was found that they formed typical neurospheres in suspension, and the majorities of cells expressed nestin, which was the marker for neural stem cells. The rats from combined transplantation group performed significantly better in the ability to study and in memory and the limb function than the rats from NSCs transplantation group (P < 0.05). The rate of neural stem cells differentiating into neurons from combined transplantation group was higher, too (50% vs. 30%).</p><p><b>CONCLUSION</b>Combined NT-3 and NSCs transplantation could improve the ability to study, memory and the limb function of rats after hypoxic-ischemic brain injury, and improve the rate of NSCs differentiating neurons. Combined NT-3 and NSCs transplantation had better effects on hypoxic-ischemic rats than transplantation of NSCs alone.</p>

Nerve Stem Cells Orientation Differentiation in Neonatal Rat Hippocampus Induced by Brain-Derived Neurotrophic Factor in Vitro / 实用儿科临床杂志

Objective To explore the influence of brain-derived neurotrophic factor (BDNF) on the differentiation of nerve stem cells (NSCs) from neonatal rat hippocampus in vitro and to find new revulsant of NSCs,which can improve the percentage of NSCs differentiating into neurons.Methods Twenty-four hours neonatal rats were selected to obtain hippocampus tissue to culture NSCs in serum-free culture medium by suspending culture.The high pure NSCs were obtained after passing 2 generations.The culture cells were identified as NSCs by staining of nestin,which was NSCs special marker.After passaged three generations,the NSCs were randomly classified into 2 groups:test group and control group.There were 15 pieces per group.There was 2 mL per piece,which contains 1?105 cells.50 g/L fetal bovine serum(FBS) and 20 ?g/L BDNF were added into foundational culture medium in test group;only 50 g/L FBS was added into foundational culture medium in control group.The neurons and their percentage were tested using the immunofluorescence labeling and flow cytometer after 7 days of differentiated cultivation.Results The hippocampus tissue cells grew in globular in serum-free culture medium by suspending culture,which expressed highly positive by nestin immunofluorescence staining.Its purity was above 90%.The percentage of neurone specific enolase(NSE)-positive cells in test group was 60.45%,which was obviously higher than that of control group (23.67%).The difference was significant between 2 groups(?2=27.75 P

Serum-Free Culture and Identification of Neural Stem Cells from Hippocampus of Neonatal Rats / 实用儿科临床杂志

Objective To explore the best serum-free cultural way in neural stem cells from the hippocampus of the newborn rats and observe the characteristics of growth,proliferation,and induced differentiation of neural stem cells.Methods The neonatal rats′ hippocampus tissues were dissociated mechanically.The cells were cultured in the serum-free cultural medium which were added separately basic fibroblast growth factor(bFGF),epithelium growth factor(EGF),bFGF+EGF by suspended cultural way.The neural stem cells were identified by nestin immunofluorescence.After induced differentiation of embryonic cow serum,the differentiated cells were identified.Results The serum-free medium added bFGF and EGF could perfectly induce neural stem cells to proliferate and the differentiated cells expressed the specific antigen of neurons,astrocytes and oligodendrocytes.Conclusion The serum-free medium with bFGF and EGF can be used to culture neural stem cells from hippocampus tissue of rats in vitro.