In Vitro Assessment of the Gene Expression Level of EZH-2 and P300 During Motor Neuron Differentiation of Human Cord Blood Mesenchymal Stem Cells

Abstract

Introduction: The dedication of stem cells for dissociation into a specific type of cell requires the over expression of genes related to a particular phenotype and suppression of the other genes. Through imposing corresponding alterations on the genome, the genome modulators such as transcription factors can be regulated by histone-modifying enzymes. Maintenance of the neurogenesis process depend on the function of some of these genes which can regulate shifting of cells from proliferation to differentiation such as Enhancer of zeste homolog 2 (EZH2) known as an evolutionarily conserved gene. Moreover, motor neurons (MN) in spinal cord can be regulated during neuronal differentiation via one of the histone acetyltransferase (P300). Up until now, the mechanism of epigenetic regulation and gene expression underlie transition process of human cord blood mesenchymal stem cells (hCB-MSCs) into MNs has not been clarified very well. Therefore, the aim of this study was to explore the quantitative expression of MN-related genes including ChAT, Islet-1, and Mnx-1 along with two epigenetic regulatory genes P300 and EZH2 involved in neurogenesis during differentiation of hCB-MSCs into MNs, using two morphogens including Sonic hedgehog (Shh) and Retinoic acid (RA) involved in the specification of MNs during the growth of nervous system. Methods: Flow cytometry was done to characterize the cells (hCB-MSCs). The cells were differentiated into MN-like cells according to our previous procedure using RA (0.01mM) and Shh (100ng/ ml) as the inducing morphogens. CB-MSCs with no treatment were assumed as control cells. RT-qPCR and Immunocytochemistry were performed to find the expression of interested genes in this study. Results: The expression of MN-related markers was confirmed at the level of mRNA and protein by induction of differentiation. The results was confirmed by immunocytochemistry showed that a number of cells about 55.33±15.885% and 49.67±13.796% could express Islet-1 and ChAT, respectively. The level of gene expression of Islet-1 and ChAT was significantly increased at the first and second week of exposure, respectively. After two weeks, expression level of P300 and EZH-2 genes was increased remarkably. No significant expression of Mnx-1 was detected when compared with the control sample. Conclusion: In this study MN-related markers, Islet-1 and ChAT, were detected in differentiated cells of hCB-MSCs supporting the potency of cord blood cells in regeneration of MN-related disorders. The over expression of Islet-1, as an early MN marker, in the presence of Shh and RA indicates the supportive effect of these morphogens for the onset of motor neuron generation. We could also detect significant expression of two potent epigenetic regulatory genes involved in neurogenesis, P300 and EZH2 accompanied by ChAT as the mature motor neuron marker at the second week of exposure due to the elimination of Shh and RA at later time of differentiation. To our knowledge, the evaluation of P300 and EZH2 during differentiation of hCB-MSCs into MN-like cells was performed in this study for the first time. However, the assessment of these epigenetic regulatory genes at the level of protein can be suggested to confirm their functional epigenetic modifying effects during motor neuron differentiation.