Research On The Treatment Of A Rat Model Of Epilepsy By Transplantation Of Hes1Silencing Bone Marrow Stromal Cells

Epilepsy is a common neurological disorder characterized by seizures, and3-5%of peoplehave the latent risk of suffering from epilepsy in their life time. However, traditionaltherapeutic methods still have some limitations, because the medication needs are basedon the individual seizure type and patient-specific factors, epilepsy surgery is used forpatients with focal seizures, and neuromodulation with vagus nerve stimulation and deepbrain stimulation still needs further evaluation of the sustained response. Therefore, it isnecessary to explore new anti-epilepsy strategies to improve the overall outcome. Amongthese, stem cell therapy has received much more attentions, because stem cells canproduce endogenous anticonvulsants, replace lost neurons or release disease modifyingsubstances for treatment of epilepsy. Previous studies demonstrated that gamma-aminobutyric acid (GABA)ergic cells play an important inhibitory role in regulation of cortexand hippocampus excitability, and loss of GABAergic neurons in brain can lead to variousdegrees of seizures. Given the importance of GABAergic system, medial ganglionic eminence-derived and embryonic stem cells-derived GABAergic neurons have beenattempted to treat epilepsy and made some successes. However, these efforts are limitedby lack of the stem cell resources.Since bone marrow stromal cells (BMSCs) can be easily obtained and thendifferentiate into various kinds of adult cells with minimal immunogenicity, it is morepractical to use BMSCs for both experimental and clinical applications. Hitherto, BMSCstransplantation has shown significant improvement in treatment of functional deficits,traumatic injury and autoimmune diseases. Recent studies have indicated that BMSCs notonly are capable of preventing the epileptic rat model from development of seizures,neuronal loss, and abnormal network in the acute phase, but also ameliorating learning andspatial memory impairments of the epileptic rats in chronic stage. However, promotingtransplanted BMSCs to differentiate into GABAergic neurons for treatment of epilepsyhas not been reported, and application of which may produce more therapeutic effectsgiven the importance of GABAergic system in epilepsy. Previous study reported thatBMSCs can differentiate into GABAergic neurons using potassium chloride as inducer.But, the inducing procedures referred to multiple factors (such as β-mercaptoethanol BME,retinoic acid RA and potassium chloride KCl), application of the multiple inducers in vivoprobably results in some unpredictable complications. Numerous data demonstrated that inthe absence of repressor-type basic helix–loop–helix (bHLH) genes (such as hairy andenhancer of split1Hes1, Hes3and Hes5), stem cells cannot proliferate sufficiently butprematurely differentiate into neurons, while the presence of repressor-type bHLH genescan antagonize the activator-type bHLH genes (such Mash1, Math and Neurogenin)effectively and maintain the stem cells to make cells not only in correct numbers but alsoin full diversity. Especially, the expression and activity of Hes1affects the GABAergicneurons differentiation of stem cells, and the neural phenotype differentiation of BMSCsis also associated with Hes1expression. Based on these results, the test was hypothesisthat inhibited Hes1gene of BMSCs maybe induce them to differentiate ino GABAergiccell. Our exerperiments were designed to inhibit the expression of Hes1gene throughsiRNA, and explored the differentiation capacity of Hes1silencing BMSCs(H-BMSCs) towards GABAergic neurons. Then the prepared rat model of epilepsy was transplanted byH-BMSCs, behavior and electroencephalography (EEG) of the recipient rats weremonitored in the following4weeks, followed by histological confirmation at last.1Hes1gene silencing improves GABAergic differentiation in the bone marrowstromal cellsObjective To study the influence of Hes1inhibition on the GABAergic differentiation ofBMSCs. Methods BMSCs were isolated from2-3weeks SD rats and purified in vitro,then characterized depending on immunocytochemistry of surface antigen andcorresponding antibodies, in addition to differentiated capacities. After that, Hes1gene ofBMSCs was inhibited by RNAi technology, then, BMSCs (control group) and H-BMSCs(experimental group) were induced by conditioned medium (1μmol of RA,1mmol N5,O2’-dibutyryl adenosine3’:5’-cyclic monophosphate,2%B27) respectively, finally,immunocytochemistry and immunoblotting were employed to analyze the neuraldifferentiation of BMSCs and H-BMSCs. Results Immunocytochemistry results showedthat the BMSCs had high expression of CD105(98.00±0.89)%, CD73(95.68±1.55)%and CD90(95.38±1.27)%, and almost no expression of CD34(1.62±0.44)%, CD11b(0.41±0.47)%. Flow cytometry results showed that the expression of CD105, CD73andCD90were99.6%,91.8%and91.4%respectively, the expression of CD11b and CD34were2.1%and0.6%respectively. Moreover, BMSCs were capable of differentiatinginto osteogenic, chondrogenic, and adipose cells cultured with conditioned medium. AfrerHes1siRNA was employed, immunoblotting results showed the BMSCs did not expressedHes1. When the experimental group and control group were induced by conditionedmedium for3-7days, the immunocytochemistry results showed that the differentiatedcells of experimental group expressed GAD67(32.8±2.2)%, NeuN (67.4±3.3)%, NF-H(71.9±4.6%) and Nestin (19.4±1.8)%, the GAD67, the number of NeuN, NF-H andNestin positive cells of control group were (22.4±4.4)%,(63.1±6.0)%,(67.1±6.3%)and30.5±2.2)%respectively after differentiation; immunoblotting results also showedthat the protein gray value ratio of GAD67or NeuN compare to the β-actin ofdifferentiated H-BMSCs was significantly higher than that of the control group through Bandscan software analysis. Conclusion Hes1gene silencing was capable of improvingGABAergic differentiation of BMSCs.2Research on the treatment of a rat model of epilepsy by transplantation of Hes1silencing bone marrow stromal cellsObjective To research the therapeutic effects of transplanted H-BMSCs in a rat model ofepilepsy. Methods Double-labeled H-BMSCs and BMSCs with5-bromo-2-deoxy uridineand ultra-small superparamagnetic iron oxide particles in vitro, lithium chloride-pilocarpine induced epileptic model were transplantated by saline(control group),BMSCs(experimental group I) and H-BMSCs(experimental group II) respectively, day3,day7, day14, day21and day28after transplantation, the experimental group and controlgroup were observed the mortality, SRS frequency and neurological deficit scores(NSS),then checked the different groups of electroencephalography(EEG) at different time points.Finally, histological study(immunehistochemistry and Nissl staining) was carried out toanalyze the histological difference among different group. Results Transplanted cells weretracked by MRI in vivo and positioned with3,3’-diaminobenzidine(DAB) staining, andfound that H-BMSCs and BMSCs could migrate to the hippocampus, the dentate gyrusand parahippocampal cortex, and the number of migrated cells in these places reachedpeak on day14. When the epileptic model were transplanted by saline, BMSCs orH-BMSCs, the mortality of control group, experimental group I and experimental groupII were41.7%,38.5%and21.4%respectively, the mortality was disappeared after1weekin all group; for the SRS frequency, the control group was0.27±0.25, experimental groupI was0.14±0.20, and the experimental group II wes0.09±0.14form day7to day28;NSS results showed that the score of experimental group II was significantly lower thanthe control group and the experimental group I at different time points; EEG resultsshowed that the frequency of epileptic waves of the experimental group II wassignificantly reduced than the experimental group I and the control group, particularly, thedifference was more significant than any other time points on day14;immunohistochemistry reaults showed that many glutamic acid decarboxylase(GAD)67-,neuron-specific enolase(NSE)-and a small mount of glial fibrillary acidic protein (GFAP)-positive cells co-localized with BrdU in the parahippocampal cortex of theexperimental group II, only a few GAD67-, NSE-or GAD67-positive cells co-localizedwith BrdU in the parahippocampal cortex of the experimental group I. But, the GAD67-,NSE-or GAD67-positive cells co-localized with BrdU in the hippocampus and dentategyrus were not found in all group; Nissl staining results were shown that the neuronaldensity of hippocampus in the experimental group I and experimental group II wasincreased significantly (P <0.01) compared with the control group, but there was notstatistically significant (P=0.137978698,>0.05) between the experimental group I andexperimental group II. Nevertheless, the neuronal density of parahippocampal cortex inthe experimental group II was significant higher than the experimental group I and thecontrol group. Conclusion Functional recovery and neuronal regeneration of a rat modelof epilepsy was improved by transplanted H-BMSCs.