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

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.

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Large- Scale Analysis of MicroRNA Expression in Motor Neuron- like Cells derived from Human Umbilical Cord Blood Mesenchymal Stem Cells

10.21203/rs.3.rs-16565/v1 ◽

2020 ◽

Author(s):

Davood Sanooghi ◽

Abolfazl Lotfi ◽

Faezeh Faghihi ◽

Afzal Karimi ◽

Zohreh Bagher ◽

...

Keyword(s):

Spinal Cord ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Motor Neuron ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Islet 1

Abstract Background Motor neuron- related disorders such as Spinal Cord Injuries and Amyotrophic Lateral Sclerosis are extremely common around the world. Many efforts have been made to use stem cells to modulate regeneration of spinal cord damages. Human umbilical cord blood mesenchymal stem cells (CB-MSCs) cover a class of cells with self-renewal feature and multilineage differentiation capacity. Retinoic acid(RA) and sonic hedgehog(Shh) are two morphogens responsible in motorneuron commitment during development. This study aims to explore the effect of Shh and RA on differentiation of CB-MSCs into motor neuron- like cells and to determine the related microRNA profile. To do that, human MSCs were isolated and then characterized using flowcytometry. The cells were induced using RA and Shh and the outcomes were assessed by immunocytochemistry, real-time- PCR, and flowcytometry. MicroRNA analysis was performed using Solexa system at three libraries, including Test 1 (with RA and Shh), Test 2 (After removing RA and Shh) and the Control. Results The isolated cells were spindle shape and could express MSC markers confirmed by flowcytometry. The cells could express motorneuron- related markers including Islet-1, Hb-9, SMI-32 and ChAT at the level of mRNA and protein, when treated with RA and Shh. Two weeks after induction, the expression of Neun and Islet-1 declined. The analysis of miRNA sequencing revealed a significant expression of mir-let-7b, mir-137 and mir-324-5p, which were responsible for neuron/motor neuron differentiation and suppression of neural progenitor cell proliferation. Moreover, some novel microRNAs involved in cholinergic, Jak- Stat, Hedgehog and Map kinase signaling pathways were revealed. Conclusion CB-MSC represents a type of cells with convenient accessibility, which can be differentiated into motor neuron- like cells in the presence of RA and Shh. We could also detect the expression of candid microRNAs responsible in motor neuron differentiation and some novel microRNAs involved in cholinergic, Jak- Stat, Hedgehog and Map kinase signaling pathways that must be functionally evaluated in further studies.

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