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

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.