Establishment Of GAD₆₅-engeneered Neural Stem Cells And A Pilot Study Of Its Application In Treating Epilepsy

Epilepsy is a common disease in functional neurosurgery. It is characterized by a series of repeated bursts of ultra-synchronized discharges by neurons. It may caused by a lot of pathogenic factors. In clinic practice, about 20%-30% epileptic patients fail to respond to a well-disciplined chemotherapy, which is named refractory epilepsy. Though surgery may help some patients suffering from refractory epilepsy, the long-term therapeutic effectiveness is only 60%, leaving much room to improve. Moreover, many patients are unsuitable for an operation, since the epileptic focuses are diffused or unidentified. Thus, new therapy should be developed now.The core of the pathogenesis of epilepsy is disequilibrium between exciting and inhibiting amino acid neurotransmitters. The morphologic study revealed that inhibiting intermediate neurons as well as inhibiting synapses were exclusively damaged in the epileptic focus. Meanwhile, hyperplasia of astrocytes and atrophy of normal neuronal tissue were common pathological changes. As the concept of "cellular and molecular neurosurgery" is widely accepted nowadays, scientists have tried to supply GABA-producing cells and rebuild the balance between exciting and inhibiting amino acid neurotransmitters by transplantation. Allograft of fetal hippocampal neurons or GAD-engineered immortalized neurons has been proven effective. However, fetal neuronal tissue is unavailable in clinical practice and unacceptable ethically. Genetically engineered immortalized neurons is not a good alternatives either, since the potential threat of carcinogenesis of immortalized cells is considerable. Wesuppose neural stem cell might be a better candidate for cell therapy of epilepsy. Neural stem cells are capable of self-renewal and multi-linage differentiation. If transplanted into hippocampus, they tend to differentiate into neurons rather than glias. Neural stem cells are capable of long-distance migration towards the focus, so they are suitable for tracing extensive or diffused lesions in central nervous system. Neural stem cells could integrated into the host cerebral tissues. Because of these advantages, we try to establish GAD₆5-engeneered neural stem cells and transplant them into epileptic rat brains, so as to study their effectiveness in anti-epilepsy cell therapy.Since traditional method for neural stem cell cannot prevent cell clusters from attachment and differentiation, we first established a novel anti-attachment culture system for long-term large-quantity neural stem cell culture. Then we cloned the cDNA of rat GAD65 gene and construct a recombinant plasmid which expresses GAD65 in eukaryotic cells. Neural stem cells were transfected with this recombinant plasmid and then transplanted into the rat brains of kainic acid-inducing epilepsy model. Expression of GAD65 in vivo was analyzed, so were the survival and therapeutic effect of donor neural stem cells.Materials and Methods1. Neural stem cell culture: Hippocampal tissue was extracted from newborn Sprague-Dawley rats and digested into single-cell suspension. Cells were seeded in flasks at a density of 5X 105/ml. The medium is DMEM/F12 supplemented with 20ng/ml EGF, 20ng/ml bFGF and 2%B27. Neural stem cells were passaged every 7 days.2. Analysis of the suspending stability of neurosphere: Suspending stability of small particles in liquid is reflected by the settling velocity (v). In the viscous medium, forces impressed on a floating neurosphere includes gravity (G), flotage (f) and viscosity resistance force (F). If the density (P 0, viscosity coefficient (1) of the medium and the density (Po)? radius (r) of neurosphere were determined, the settling velocity can be calculated with theequation: v = 0.222gr2 (/??-/?,)/ 77 Therefore the main factors that influence theneurosphere's suspending stability could by determined.3. Design of novel anti-attachment culture system: Based on the study of suspending stability of neurosphere, we try to establish a novel anti-attachment culture system for long-term neural stem cell cultivation. The study include 3 groups: medium-modified group, flask-modified group and control. The medium-modified group used medium supplemented with 5 % mannitol so as to increase its density and velocity. The flask-modified group used flasks coated with a thin slice of 1.5% agarose gel. In the process of long-term culture, the proliferation cell vigor and differentiation of stem cells were analyzed.4. Cloning of cDNA of GAD65 gene: According to the sequence data in GenBank, we designed and propared the GAD65 primers as following: forward primer: 5 ' -AGTGAATTCAGAACCCATGGCATCTCC-3;backward primer: 5 -CGTCCAGCTCGAGCAAAGTGATTACAA-3. Total RNA was extracted from Sprague-Dawley rat brain tissue. Using Promega Access RT-PCR System, reverse transcript reaction was performed with Oligo(dT) primer and RNA. Then polymerase chain reaction was carried out to expand the cDNA of GAD65 gene, with the GAD65 primers and reverse transcript reaction product as template.5. Constructing recombinant eukaryonic-expression plasmid pcDNA3-GAD65: PCR product was extracted, purified and linked with Pucm-T vector by T4 ligase. Pucm-GAD65 was sequenced to authenticate the cDNA. Afterwards, GAD65 fragment was released by double-digestion with EcoR I and Xho I. The fragment was purified and inserted into multiple cloning site of pcDNA3.1(+) plasmid. This recombinant plasmid pcDNA3-GAD65 was expanded in E. coli and extracted with Invitrogen Hipure Plasmid Midi-prepare Kit.6. Transfection and expression of GAD65 in neural stem cells: Mediated by Qiagen Effectene Transfection Reagent, neural stem cells were transfected by purified pcDNA3-GAD65. The neural stem cells not transfected was set as control. The expression of GAD6s was detected by RT-PCR,Western blotting and immunocytochemistry 48 hours later. The medium GABA concentrations of transfected cells and control were detected by high-performance liquid chromatography.7. Epilepsy model and transplantation: The day before transplantation, neural stem cells were labeled with BrdU. Sixty adult Sprague-Dawley rats were subjected to lcainic acid-inducing epilepsy, and assigned randomly to 3 groups: ?GAD-NSC transplantation group (n=20): epilepsy model, transplanted with GAD65-engineered neural stem cells. ?NSC transplantation group (n=20): epilepsy model, transplanted with neural stem cells. ?Control group (n=20): epilepsy model, same amount of Saline instead of stem cells.8. Observation of acute seizure: Between 4-5 hours after modeling and transplantation, seizure frequencies were recorded. At the same time and even 24 hours later, electroencephalogram was performed. In the electroencephalogram, sharp wave, spike wave and their combination with slow wave were considered as seizure discharge. Seizure discharge frequencies were recorded, too.9. Small-dose pentetrazole inducing test: At different stage (Id, 2d, 3d, lw, 2w, 4w, 8w after modeling), small-dose pentetrazole (20mg/kg) inducing tests were performed to detect the epileptic sensitivity according to Racine grading standard.10. Detection of in vivo GAD6s expression: One week after modeling, 5 rats of each group were sacrificed. GAD65 expression in the hippocampus was detected with RT-PCR and Western blotting.11. Pathological examination: Two months after modeling, all animals were sacrificed. The brains was sliced and stained immunohistochemically targeting at BrdU and GAD65. HE staining was also performed. Pathological examination revealed the survival and expression of GAD65 of donor neural stem cells.12. Statistical analysis: AH data were expressed as mean±standard deviation (X ±s). We used SPSS 10.0 software in statistical analysis. Analysis of variance, student t test and^2 test were applied to analyze differences between groups. Statistical significance was assigned to Pvalue of <0.05.ResultsNeural stem cells were cultured well in vitro. They expanded rapidly and tended to attach to the flask floor. All neurospheres attached 6 days after passage. The density and viscosity coefficient of the medium and the density of clusters did not differ significantly. As the volume of neurosphere expanded, the settling velocity increased dramatically, so the volume of neurosphere is the major factor contributed to the change of suspending stability. When the medium was modified, neurospheres kept floating, however, S-phase labeling index (LI) decreased and cells died gradually. When the flask bottom were covered with agarose gel, neural stem cells grew rapidly without attachment. Even cultured for 3 months, only 0.64% cells had differentiated, while most cells still maintained their multipotency.Molecular cloning: Total RNA of rat brain tissue was extracted successfully. Electrophoresis and OD value (A260/280=1.72) demonstrated the integrity of RNA. A DNA fragment about 1.7kb was expanded in reverse-transcript polymerase chain reaction. The fragment was linked to Pumc-T vector and comfirmed to be the cDNA of GAD65 gene by sequencing. Then a recombinant eukaryonic expression plasmid was constructed successfully, namely pcDNA3-GAD65. This plasmid was expanded and purified, with a concentration of 0.8mg/ml.Transfection and expression: RT-PCR and Western blotting demonstrated high expression of GAD65 in neural stem cells 48 hours after transfection while in the control group no similar band found. Immunocytochemistry revealed that 27.4% cells of the transfection group were GAD65 positive, and only 0.3% cells of the control were positive (P<0.01). Medium GABA concentration of the transfection group was 61072 ± 826 nmol / L, and that of the control group was 3540 ± 166 nmol/L(P<0.01).Animal experiment: All animals presented typical seizures. Four hours after modeling, theseizure frequency of GAD-NSC transplantation group was 87.2+9.5/h (P>0.05), that of NSC transplantation group was 85.6+12.4/h (P>0.05), and that of control was 87.6+11.9/h. At the same time, electroencephalogram recorded crowded spike waves and sharp waves. Seizure discharges were synchronized strictly with convulsion. One day after modeling, electroencephalogram recorded many 0 > 5 waves, mixed with some sharp waves, the seizure discharge frequency of GAD-NSC transplantation group was 7.0+0.4/h (P<0.01), that of NSC transplantation group was 60.3+10.2/h (P>0.05), and that of control was 58.1+9.5/h.. Small-dose pentetrazole inducing test revealed that the epileptic sensitivity of GAD-NSC transplantation group decreased as early as 2d after modeling. But that of the NSC transplantation group decreased only after 2 weeks and still much higher than GAD-NSC transplantation group. RT-RCR and Western blotting demonstrate high-level GAD65 expression in vivo. Pathological changes of the control group included severe hippocampal neuron damage and depletion, massive hyperplasia of glias and formation of glial scar, mossy fiber sprouting, and et al. Changes of the GAD-NSC and NSC transplantation groups were minor. In the hippocampal tissues of GAD-NSC transplantation group, percentage of BrdU (+) cells was 0.183+0.042, while that of the NSC transplantation group was 0.054+0.010 (P<0.01). The percentage of GAD65 (+) cells of GAD-NSC transplantation group was 0.265+0.085, while that of the NSC transplantation group was 0.072+0.009 (P<0.01).Conclusions1. It is vital to keep neural stem cells in a undifferentiated state in many studies. But traditional culture method can not prevent neurosphere from attachment and differentiation, mainly because of the decreasing suspending stability as the neurosphere expanded. If the culture flask was coated with 1.5% agarose gel, the neurosphere would be kept floating without influence on the proliferating rate. Therefore, we have established a novel anti-attachment culture system for long-term neural stem cell cultivation.2. Rat GAD65 gene was cloned successfully. Its sequence was completely consistent with that listed in GenBank database. Then we constructed a recombinant eukaryonic expression plasmid pcDNA3-GAD65. After transfected with this plasmid, neural stem cells expressed GAD65 in a high-level, as demonstrated by RT-PCR, Western blotting and immunocytochemistry. Moreover, transfected neural stem cells secrete large quantity of GABA.3. Animal model of kainic acid induing epilepsy was set up successfully. GAD65-engineered neural stem cells transplanted in the hippocampus of epileptic rat survived as long as 2 months. They expressed GAD65 in a high-level in vivo. Only one day after transplantation, the engineered stem cells began to present their therapeutic effect of inhibiting seizures, protecting host hippocampal neurons and improving the pathological change.