Facilitated Neural Differentiation Of Adipose Tissue-derived Stem Cells By Electrical Stimulation And Nurr-1Gene Transfection

Spinal injury are characterized by loss of neuronal cell populations. Thus far,extensive efforts, such as medication, surgery and gene delivery, have been tried tominimize the loss of neuronal cells, and relieve the symptoms. However, restoring spinaltissue and its function remains to be unsolved after nerve lesions. Progress in stem cellresearches offers new avenues for stem cell therapy in the treatment of lesions in spinalinjury. Of various stem cells, ADSCs attracted lots of attention because of their abundance,easy harvesting and excellent pluripotency potential. Thus far, many strategies have beenproposed, including neurotrophic factor, gene transfection, preinducer treatment andsub-sonic vibration treatment, to promote neuronal differentiation of ADSCs. However,the differentiation rate of ADSCs into neuronal cells remains unsatisfactory, whichsignificantly limits their application in the treatment of spinal injury lesions.Nurr-1is a transcription factor, which is essential in the generation, maturation,migration, as well as differentiation of midbrain dopaminergic cells. It has been reportedthat Nurr-1plays a critical role in mesencephalic dopaminergic neuronal development inassociation with dopamine D2receptor (D2R) through extracellular signal-regulated kinase (ERK) signaling pathway. In addition, over-expressing Nurr-1gene in embryoniccortical precursor cells has been found to be able to promote neuron differentiation andinhibit astrocyte formation. In a recent study, Nurr-1gene over-expression has been shownto be capable of initiating neural differentiation of bone marrow mesenchymal stromalcells (BMMSCs). All these evidences indicate the essential role of Nurr-1gene ininitiation of neuronal differentiation in embryonic cortical precursor cells and BMMSCs.However, whether Nurr-1is capable of initiating neuronal differentiation in ADSCsremains unclear.Neurons are excitable cells, which can respond quickly to ES at higher frequencies(>50Hz) by instant depolarization. It has been reported that ES is able to promote neuriteoutgrowth, which is an early and essential process during neuronal differentiation. Allthese evidences drive researches to investigate the application of ES in facilitatingneuronal differentiation in stem cells. It has been reported that mild ES (10V) is capableof inducing embryonic stem cells into neuronal fate. In addition, ES has also been shownto promote neuronal differentiation of BMMSCs. All these findings suggest a beneficialrole of ES in neuronal differentiation of stem cells. A most recent study has shown thatADSCs are able to respond to low-frequency alternating current electric fields byelevating cytoplasmic calcium, highlighting the possibility of using ES to promoteneuronal differentiation of ADSCs. Thus far, the possible role of ES in neuronaldifferentiation of ADSCs has not been investigated. In addition, Nurr-1gene has beenshown to be able to initiate neuronal differentiation of many stem cells, but not reported inADSCs. Whether Nurr-1gene over-expression is able to initiate neuronal differentiation ofADSCs and whether it is possible for ES to further promote neuronal differentiation ofADSCs which has been initiated by Nurr-1gene over expression, are all interesting issueswhich needs to be clarified. The present study was designed to investigate the role of ESand Nurr-1gene over-expression in neuronal differentiation of ADSCs.Part1Enhanced neural differentiation of adipose tissue-derived stem cells by Nurr-1gene transfectionObjectiveTo explore the role of Nurr-1gene over-expression in neuronal differentiation ofadipose tissue-derived stem cells (ADSCs).MethodThe characterization of ADSCs was used flow cytometric technology, adipogenicinduction and osteogenic induction. The differentiation of ADSCs into neuron-like cellswas examined by immunofluorescence staining and RT-PCR. The gene expression ofmicrotubule-associated protein2(MAP-2), β-tubulin and neurofilaments200(NF-200),was examined by RT-PCR. The proliferation of cells after Nurr-1gene transfection wascharacterized using a CCK-8assay.ResultsFlow cytometric analysis demonstrated that cells were positive for CD90and CD29,but only0.3%of the cells were positive for CD31and1.2%of the cells were positive forCD45. After differentiation into adipocytes, the cells were stained with Oil Red O, and redcolored lipid vacuoles were visualized. After osteogenic differentiation, the cells werestained with Alizarin-Red-S, and an immense production of mineral deposits wasdisplayed. The MAP-2and β-tubulin were positive in Nurr-1group afterimmunofluorescence staining. The gene and protein expression of MAP-2, β-tubulin andwas significantly enhanced compared with control group.ConclusionNurr-1gene transfection is able to enhance the neuronal differentiation of ADSCs. Part2The optimal intensity of electrical stimulation promotes theneural differentiation of adipose tissue-derived stem cellsObjectiveTo explore the role of electrical stimulation (ES) in neuronal differentiation ofadipose tissue-derived stem cells (ADSCs) and select the optimal intensity of ES.MethodThe proliferation of cells was characterized using a CCK-8assay. The differentiationof ADSCs into neuron-like cells was examined by immunofluorescence staining, RT-PCRand Western blotting. The gene expression of microtubule-associated protein2(MAP-2),β-tubulin, and neurofilaments200(NF-200) was examined by RT-PCR.ResultsThe CCK-8assay showed1V/cm was beneficial for the proliferation of ADSCs. TheMAP-2and β-tubulin were positive in1V/cm group after immunofluorescence staining.The gene and protein expression of MAP-2, β-tubulin and was significantly enhancedcompared with control and0.5V/cm group.ConclusionThe optimal intensity of ES is able to promote the neuronal differentiation of ADSCsand is beneficial for the proliferation of ADSCs, which highlight the potential applicationin the treatment of spinal injury.Part3The optimal intensity of electrical stimulation promotes theneural differentiation of adipose tissue-derived stem cellsObjectiveIn the present study, we tested the possibility of using a combination of electrical stimulation (ES) with Nurr-1gene transfection to promote neuronal differentiation ofADSCs.MethodThe morphology of cells was examined by scanning electron microscopy (SEM). Thedifferentiation of ADSCs into neuron-like cells was examined by immunofluorescencestaining, RT-PCR and Western blotting. The gene expression of microtubule-associatedprotein2(MAP-2), β-tubulin, neurofilaments200(NF-200), octamer binding transcriptionfactor4(OCT-4), and glial fibrillary acidic protein (GFAP) after stimulation wasexamined by RT-PCR.ResultsThe percentage of MAP-2positive cells was52.13±3.12%(p <0.01) in the ES+Nurr-1group, which was significantly higher than that in the control group (2.11±1.21%),Nurr-1group (32.31±2.46%), and ES group (22.10±2.01%). The percentage ofβ-tubulin positive cells was66.12±5.03%(p <0.01) in the ES+Nurr-1group, which wassignificantly higher than that in the control cells (4.20±2.13%), Nurr-1group (39.72±4.03%), and ES group (26.82±3.02%).Western blotting analysis showed that the protein levels of MAP-2and β-tubulin washighest in the ES+Nurr-1group, followed by Nurr-1group and ES group.RT-PCRanalysis showed that the gene expression of MAP-2, β-tubulin and NF-200were promotedin ES+Nurr-1group, but the gene expression of OCT-4and GFAP was decreased in ES+Nurr-1group.The length of neurite-like process in β-tubulin positive cells was174.2±8.7(p <0.01) μm in the ES+Nurr-1group,74.1±7.8(p <0.05) μm in the ES group,61.2±7.1(p <0.05) μm in the Nurr-1group and11.8±5.3μm in the control group. Themorphological appearances of cells in each group were evaluated by. As shown in theSEM, the cells in the control group attached, expanded, and showed polygonal appearancewith multiple processes. When ADSCs were subjected to ES (1V/cm), the cells becamespindle-shaped with bipolarly extended processes. After Nurr-1gene transfection, the cells showed spherical-shaped and refractile appearances with multiple processes or bipolarprocesses. When the Nurr-1transfected cells were subjected to ES, the cells becamespindle or triangle shaped, with long extended processes, showing a neuron-likeappearance.ConclusionThe combination of ES with Nurr-1gene transfection could facilitate neuronaldifferentiation of ADSCs, which raises the possibility of its application in the treatment ofspinal injury.