Objective To establish a better method of isolating andculturing ofneural stem cells(NSCs) in neonatal rat brain. Methods Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used toculture NSCs of neonatal rat. The culture concentration used were 1×10 4, 1×105, 1×106and 1×107/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate asto be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. Results The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining longterm survival in vitro. The cells could be differentiated into neurons and glia.It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS)into the medium at the beginning of the culturing. When 10% FBS was added intothe medium, the neurospheres differentiated quickly. When concentration 1×106/ ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. Conclusion A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.
To summarize Notch, basic hel ix-loop-hel ix (bHLH) and Wnt gene signal transduction pathways in the process of differentiation and development of neural stem cells. Methods The l iterature on the gene signal transduction pathway in the process of differentiation and development of neural stem cells was searched and then summarized and analyzed. Results The formation of Nervous System resulted from common actions of multi-signal transduction pathways. There may exist a fixed threshold in the compl icated selective system among Notch, bHLH and Wnt gene signal transduction pathways. Conclusion At present, the specific gene signal transduction pathway of multi pl ication and differentiation of neural stem cells is still unclear.
Objective To observe the biocompatibil ity of self-assembled FGL peptide nano-fibers scaffold with neural stem cells (NSCs). Methods FGL peptide-amphiphile (FGL-PA) was synthesized by sol id-phase peptide synthesistechnique and thereafter It was analyzed and determined by high-performance l iquid chromatography (HPLC) and massspectrometry (MS). The diluted hydrochloric acid was added into FGL-PA solution to reduce the pH value and accordinglyinduce self-assembly. The morphological features of the assembled material were studied by transmission electron microscope (TEM). NSCs were cultured and different concentrations of FGL-PA assembled material were added with the terminal concentrations of 0, 50, 100, 200, 400 mg/L, respectively. CCK-8 kit was used to test the effect of FGL assembled material on prol iferation of NSCs. NSCs were added into differentiation mediums (control group: DMEM/F12 medium containing 2% B27 supplement and 10% FBS; experimental group: DMEM/F12 medium containing 2% B27 supplement, 10% FBS and 100 mg/L FGL-PA, respectively). Immunofluorescence was appl ied to test the effect of FGL-PA assembled material on differentiation of NSCs. Results FGL-PA could be self-assembled to form a gel. TEM showed the self-assembled gel was nano-fibers with diameter of 10-20 nm and length of hundreds nanometers. After NSCs were incubated for 48 hours with different concentrations of FGL-PA assembled material, the result of CCK-8 assay showed that FGL-PA with concentrations of 50, 100 or 200 mg/L could promote the prol iferation of NSCs and absorbance of them was increased (P lt; 0.05). Immunofluorescence analysis notified that the differentiation ratio of neurons from NSCs in control group and experimental group were 46.35% ± 1.27% and 72.85% ± 1.35%, respectively, when NSCs were induced to differentiation for 14 days, showing significant difference between 2 groups (P lt; 0.05). Conclusion FGL-PA can self-assemble to nano-fiber gel, which has good biocompatibil ity and neural bioactivity.
Objective To investigate the memory amelioration of the Alzheimer disease (AD)model rat after being transplanted the single neural stem cells(NSC) and NSC modified with human brain-derived neurotrophic factor（hBDNF） gene. Methods Forty SD rats were divided evenly into 4 groups randomly. The AD model rats were made by cutting unilaterallythe fibria fornix of male rats. Ten to twelve days after surgery, the genetically modified and unmodified NSC were implanted into the lateral cerebral ventricle of group Ⅲ and group Ⅳ respectively. Two weeks after transplantation, theamelioration of memory impairment of the rats was detected by Morris water maze. Results The average escaping latency of the group Ⅲ and group Ⅳ (41.84±21.76 s,25.23±17.06 s respectively) was shorter than that of the group Ⅱ(70.91±23.67 s) (Plt;0.01). The percentage of swimming distance inthe platform quadrant in group Ⅲ (36.9%) and in group Ⅳ(42.0%) was higherthan that in the group Ⅱ(26.0%) (Plt;0.01). More marginal and random strategies were used in group Ⅱ.The percentage of swimming distance in the platform quadrant in group Ⅳ was also greater than that in group Ⅲ(Plt;0.05). There were no significant differences in the average escaping latency, the percentage of swimming distance in the platform quadrant and the probe strategy between group Ⅳ and group Ⅰ(Pgt;0.05).More lineal and oriented strategies were used in group Ⅳ. Conclusion The behavioral amelioration of AD model rat was obtained by transplanting single NSC and hBDNF-gene-modified NSC. The effect of the NSC group modified with hBDNF gene is better than that of the groupⅢ.
ObjectiveTo observe the effect of transplantation of neural stem cells (NSCs) induced by all-trans-retinoic acid (ATRA) combined with glial cell line derived neurotrophic factor (GDNF) and chondroitinase ABC (ChABC) on the neurological functional recovery of injured spinal cord in Sprague Dawley (SD) rats. MethodsSixty adult SD female rats, weighing 200-250 g, were randomly divided into 5 groups (n=12): sham operation group (group A), SCI model group (group B), NSCs+GDNF treatment group (group C), NSCs+ChABC treatment group (group D), and NSCs+GDNF+ChABC treatment group (group E). T10 segmental transversal injury model of the spinal cord was established except group A. NSCs induced by ATRA and marked with BrdU were injected into the site of injury at 8 days after operation in groups C-E. Groups C-E were treated with GDNF, ChABC, and GDNF+ChABC respectively at 8-14 days after operation;and group A and B were treated with the same amount of saline solution. Basso Beattie Bresnahan (BBB) score and somatosensory evoked potentials (SEP) test were used to study the functional improvement at 1 day before remodeling, 7 days after remodeling, and at 1, 2, 5, and 8 weeks after transplantation. Immunofluorescence staining and HE staining were performed to observe the cells survival and differentiation in the spinal cord. ResultsFive mouse died but another rats were added. At each time point after modeling, BBB score of groups B, C, D, and E was significantly lower than that of group A, and SEP latent period was significantly longer than that of group A (P<0.05), but no difference was found among groups B, C, D, and E at 7 days after remodeling and 1 week after transplantation (P>0.05). BBB score of groups C, D, and E was significantly higher than that of group B, and SEP latent period was significantly shorter than that of group B at 2, 5, and 8 weeks after transplantation (P<0.05);group E had higher BBB score and shorter SEP latent period than groups C and D at 5 and 8 weeks, showing significant difference (P<0.05). HE staining showed that there was a clear boundary between gray and white matter of spinal cord and regular arrangement of cells in group A;there were incomplete vascular morphology, irregular arrangement of cells, scar, and cysts in group B;there were obvious cell hyperplasia and smaller cysts in groups C, D, and E. BrdU positive cells were not observed in groups A and B, but could be found in groups C, D and E. Group E had more positive cells than groups C and D, and difference was significant (P<0.05). The number of glial fibrillary acidic protein positive cells of groups C, D, and E was significantly less than that of groups A and B, and it was significantly less in group E than groups C and D (P<0.05). The number of microtubule-associated protein 2 positive cells of groups C, D, and E was significantly more than that of groups A and B, and it was significantly more in group E than groups C and D (P<0.05). ConclusionThe NSCs transplantation combined with GDNF and ChABC could significantly promote the functional recovery of spinal cord injury, suggesting that GDNF and ChABC have a synergistic effect in the treatment of spinal cord injury.
Objective To investigate the method of cultivation and the feature of differentiation of spinal cordderived stem cells in vitro.Methods The neural stemcells from spinal cord of 15 days fetal rats were harvested and cultivated in aserumfree limited medium. The stem cells were induced to differentiate and theresults were identified by cellular immunohistochemistry. Results Lots of stem cells were obtained from the spinal cord of fetal rats and the sphere of stemcells was formed about 10 days. Neural stem cells can give rise to mature neurons and astrocytes.Conclusion Epidermal growth factor/basic fibroblast growth factor serum-free limited medium can promote the proliferation activity ofthe stem cells. Spinal cord-derived stem cells can differentiate into glial cells and neurons.
Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups (n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSCsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectivel. The generalcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus tricepssurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps surae was (0.849 ± 0.064) g and (0.596 ± 0.047) g in the experimental group, respectively, and was (0.651 ± 0.040) g and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P lt; 0.05). The fiber section area of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ± 4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P lt; 0.05). Mallory trichrome staining of muscle notified that there was more collagen fiber hyperplasia of denervated gastrocnemius in the control group than that in the experimental group at 4 and 12 weeks postoperatively. After 12 weeks of operation, the area of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as (61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant differences (P lt; 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new approach to prevent and treat the denervated muscle atrophy cl inically.
ObjectiveTo study the possibility of the C17.2 neural stem cells (NSCs) differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells (OECs) and to detect the cell viability of the differentiated cells. MethodsOECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining[microtubule-associated protein 2 (MAP-2) and β-tubulin-Ⅲ] and Western blot (Nestin, β-tubulin-Ⅲ, and MAP-2) were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase (LDH) release in the medium. ResultsIn the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that β-tubulin-Ⅲ and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of β-tubulin-Ⅲ and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group (P<0.05). The LDH release and the cell viability were 130.60%±6.86% and 62.20%±3.82% in the experimental group, and were 178.20%±5.44% and 18.00%±3.83% in the control group respectively, showing significant differences between 2 groups (P<0.05). The LDH release and the cell viability of experimental group and control group were significantly lower than those of blank control group (100%) (P<0.05). ConclusionNeurotrophic factors from OECs play an important role in inducing C17.2 NSCs differentiation into neural cells and keeping the viability of differentiated cells after induction.
ObjectiveTo observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis, and the therapeutic effects of glial cell derived neurotrophic factor (GDNF) and/or ciliary neurotrophic factor (CNTF) based on neural stem cells (NSC) on mouse photoreceptor cells. MethodsA total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group, with 80 and 20 mice respectively. Twenty C57BL/6J mice aged 14 days were assigned as wild-type group (WT group). Mice in control group and WT group did not receive any interventions. At 2, 4, and 6 months of age, immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones, rod-bipolar cells, and cone-bipolar cells within the retinal of mice while electroretinography (ERG) examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes. At 14 days of age, the mice in the experimental group were intravitreally injected with 2 μl of CNTF-NSC, GDNF-NSC, and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC (GDNF/CNTF-NSC). Those mice were then subdivided into the CNTF-NSC group, the GDNF-NSC group, and the GDNF/CNTF-NSC group accordingly. The contralateral eyes of the mice were injected with 2 μl of control NSC without neurotrophic factor (NTF) as their own control group. At 2 and 4 months of age, the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes. At 4 months of age, the differentiation of grafted NSC and the expression of NTF were observed. Statistical comparisons between the groups were performed using a two-way ANOVA. ResultsCompared with WT group, the density of cones in the peripheral region of the control group at 2, 4 and 6 months of age (F=285.10), rod-bipolar cell density in central and peripheral retina (F=823.20, 346.20), cone-bipolar cell density (F=356.30, 210.60) and the scotopic amplitude of a and b waves (F=1 911.00, 387.10) in central and peripheral retina were significantly decreased, with statistical significance (P＜0.05). At the age of 4 and 6 months, the density of retinal cone cells (F=127.30) and b-wave photopic amplitude (F=51.13) in the control group were significantly decreased, and the difference was statistically significant (P＜0.05). Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes, and stably expressed CNTF and GDNF at high levels. Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages: CNTF-NSC group, at 2 months of age: the whole, central and peripheral regions were significantly different (F=31.73, 75.06, 75.06; P＜0.05); 4 months of age: The difference between the whole area and the peripheral region was statistically significant (F=12.27, 12.27; P＜0.05). GDNF/CNTF-NSC group, 2 and 4 months of age: the whole (F=27.26, 27.26) and the peripheral area (F=16.01, 13.55) were significantly different (P＜0.05). In GDNF-NSC group, there was no statistical significance at all in the whole, central and peripheral areas at different months of age (F=0.00, 0.01, 0.02; P＞0.05). ConclusionsCLN7 neuronal ceroid-lipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing, aligning with both morphological and functional observations. Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells. Nevertheless, the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect. GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.
Objective To compare single cell suspension of neural stem cells (NSCs) with neurospheres transplantation for spinal cord injury (SCI) so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley (SD) rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and gl ial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats (weighing 230-250 g) were made the SCI models at T10 level with modified Allen method and randomlydivided into 3 groups (20 rats in each). The injury sites were treated by injecting 5 μL sal ine (group A), 5 μL single cellssuspensions of NSCs at passage 3 (group B), and 5 μL neurospheres cell suspensions at passage 3 (group C). At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 (MAP-2). Results The cells cultured were NSCs by morphological observation and immunofluorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days (P gt; 0.05). BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences (P lt; 0.05). HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days (P gt; 0.05). At 14, 21, and 28 days, the number of MAP-2 positive cells of groups B and C was significantly more than that of group A, and group C was more than group B, showing significant differences (P lt; 0.05). Conclusion Transplantation of neurospheres suspension compared with single cell can significantly promote NSCsto differentiate into neurons and is conducive to recover the lower extremity function after SCI.