NRSF Involves In The Neuronal And Pancreatic Differentiation Of MSCs

More and more people suffer from diseases of central neural system and diabetes. To many patients suffering from these diseases, cell transplantation represents an ideal method to cure them. But the source of cells such as neurons and islet are limited. Because of their self-renewal and pluripotency, stem cells represent candidate of cell transplantation. Mesenchymal stem cells (MSCs) derived from bone marrow are multipotent cells and have many good qualities, such as easy acquisition, multipotency, no ethical debate and low immunological rejection, so they are considered as the ideal"seed cells"of cell transplantation. But there are still many problems in induction MSCs into neurons or pancreatic cells, the induction efficiency is low, and the mechanisms responsible for MSCs'differentiation are still unknown. Our aim is to improve the induction efficiency and make the mechanisms responsible for MSCs'neuronal and pancreatic differentiation more clear. To our knowledge, NRSF plays an important role both in neuronal differentiation and pancreatic differentiation.Neuronal restrictive silencing factor (NRSF), also known as repressor element silencing transcription factor (REST), is a member of the Kruppel family of zinc-finger transcription factors. The estimated 116kD molecular protein contains a DNA-binding domain with eight zinc-finger regions and two independent inhibitory domains in the two ends of NRSF protein. The 6~8 zinc-fingers bind target DNA sequence, and the 2~5 zinc fingers'function is nucleic localization. NRSF binds sequence named NRSE (neuronal restrictive silencer element), and then recruits histone deacetylases (HDACs) which can deacetylate histones and repress the expression of target genes. NRSF repress a battery of neuron specific genes expression, including ion channels, neurotransmitter receptors, growth factors, synaptic vesicle protein, and neuronal trophic factors. NRSF/REST has been found to be expressed in embryonic stem cells and most adult cells. There is no NRSF expression only in mature neurons and pancreatic cells.It has been reported that NRSF plays an important role in the self-renewal and differentiation of stem cells. In neural stem cells, the repression of endogenous REST/NRSF can activate multiple neuronal genes which are specific markers for neuronal differentiation. Knock-down of NRSF expression by RNA interference technology in mouse neuroblastoma cell line N18 induces a clear increase in neurite length. Recently it is reported that NRSF could maintain the self-renewal and pluripotency of mouse embryonic stem cells (mESCs) by repressing miRNA-21 which specifically suppresses the self-renewal of mESCs. Down-regulation of NRSF during neurogenesis is necessary for proper neuronal differentiation, and over-expression of REST/NRSF in differentiating neurons disrupts neuronal gene expression and causes axon guidance errors.We find NRSF is highly expressed in mesenchymal stem cells (MSCs), indicating that NRSF may play an important role in the stemness maintenance of MSCs. We down-regulate the expression of NRSF by RNA interference and then analyze the potential of NRSF silencing MSCs differentiate into neurons and pancreatic cells. First we constructed a lentiviral-mediated RNA interference vector that could stably down-regulate the expression of NRSF. We found that the lentiviral-mediated RNA interference vector could stably down-regulate the mRNA and protein expression of NRSF in HeLa cells. NRSF silencing could initiate the expression of NRSF target genes such as superior cervical ganglion 10 (SCG10), brain-derived neurotrophic factor (BDNF), paired box gene 4 (pax4) and insulin. A luciferase report system which includes human insulin core promoter and luciferase CDS was also constructed. By this luciferase report system, we found NRSF silencing increased the insulin promoter activity.Similarly, the lentiviral-mediated RNA interference vector could also stably down-regulate the mRNA and protein expression of NRSF in MSCs without off-target effects. MSCs infected with lentivirus contained si-NRSF (MSCs-siNRSF) had a lower growth rate than MSCs infected with lentivirus contained scrambled sequence (MSCs-siControl). When cultured under normal culture conditions (LDMEM plus 10% fetal bovine serum), some of the MSCs-siNRSF stretched out short neurites-like structures 7 days later. Fourteen days after infection, most of the MSCs-siNRSF exhibited a neuron-like morphological change, with long neurites-like structures. No obvious morphological changes were observed in MSCs-siControl under these same culture conditions, all the MSCs-siControl were as long fusiform as before. RT-PCR revealed that the differentiated MSCs-siNRSF had mRNA expression of SCG10, BDNF, synaptophysin (SYP), neuron specific enolase (NSE), neurogenin (NGN3), but without glial fibrillary acidic protein (GFAP). Immunofluorescence staining was also performed to confirm the expression of the neuronal marker proteins. The results showed that the MSCs-siNRSF cultured for 14 days express mature neuronal markers such asβ-tubulinⅢ, neurofilament (NF-200), microtubule-associated protein 2 (MAP-2) and NSE both in the cell bodies and in their neurites. No positive staining signal for GFAP and oligodendrocyte transcription factor 2 (Olig2) in these differentiated cells was detected. These results showed that no glial differentiation occurred in these cells. To evaluate expression of functional voltage-gated ion channels, we assessed the voltage-gated fast sodium currents by whole cell patch-recording in differentiated MSCs-siNRSF. Recordings in voltage-clamp mode displayed typical Na+ channel currents in the 14 days MSCs-siNRSF, and the Na+ currents could be blocked by tetrodotoxin (TTX). This result suggests that the induced cells are not only morphological similar to the neurons, but also possess the electrical excitability associated with the function of neurons.NRSF also plays an important role in pancreatic development. The over-expression of NRSF in mouse pancreaticβTC3-cell line will impair the capacity to secrete insulin in response to mitochondrial fuels, a particularity of matureβ-cells. Transgenic mice featuring a beta cell-targeted expression of REST exhibited glucose intolerance and reduced beta cell mass. In our experiment, NRSF silencing is good to MSCs'differentiation into pancreatic cell-like cells in the presence of glucagon-like peptide 1 (GLP-1) and basic fibroblast growth factor (bFGF). These differentiated cells expressed insulin and were positive for dithizone (DTZ) staining. Further experiments are needed to determine the functions of these cells.Overall, NRSF silencing induces neuronal differentiation of human mesenchymal stem cells; the MSCs-siNRSF also can be induced into insulin-producing cells under certain conditions. Our results provide a novel method to obtain neurons from MSCs without the contamination of glial cells, and will be helpful to clarify the role of NRSF in the neuronal and pancreatic development.