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

Introduction: Maintenance of neurogenesis depends on the function of some histone-modifying enzymes; including Enhancer of zeste homolog 2 (EZH2) and histone acetyltransferases (P300). The mechanism of epigenetic regulation and gene expression underlying the transition of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) into MNs has not been fully clarified.  Methods: Two morphogens; sonic hedgehog (Shh: 100 ng/mL) and retinoic acid (RA: 0.01 mM) were involved in the specification of hUCB-MSCs into MNs after MSC characterization using Flow cytometry. Real time-quantitative PCR and immunocytochemistry were performed to find the expression of the genes at the level of mRNA and protein.  Results: The expression of MN-related markers was confirmed at the level of mRNA and protein by induction of differentiation. The results were confirmed by immunocytochemistry and showed those mean cell percentages of 55.33%±15.885% and 49.67%±13.796% could express Islet-1 and ChAT, respectively. The gene expression level of Islet-1 and ChAT was significantly increased in the first and second week of exposure, respectively. After two weeks, the expression level of P300 and EZH-2 genes increased remarkably. No significant expression of Mnx-1 was detected when compared to the control sample. Conclusion: MN-related markers, Islet-1 and ChAT, were detected in differentiated cells of hUCB-MSCs, supporting the potency of cord blood cells in the regeneration of MN-related disorders. Assessing these epigenetic regulatory genes at the protein level can be suggested to confirm their functional epigenetic modifying effects during motor neuron differentiation.