Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functional
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ORIGINAL ARTICLE
Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functionality Genes Wissam Khalil 1 & Taki Tiraihi 1 & Masoud Soleimani 2 & Nafiseh Baheiraei 2 & Kazem Zibara 3 Received: 17 January 2020 / Revised: 1 June 2020 / Accepted: 17 June 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Conversion of mesenchymal stem cells (MSC) into neuron-like cells (NLC) is a feasible cell therapy strategy for replacing lost neurons in neuronal disorders. In this study, adipose-derived MSC (ADMSC) were converted into neural stem cells (NSC) via neurosphere. The resulting NSC were then differentiated into NLC by transduction with microRNA-218, using a lentiviral vector. ADMSC, NSC, and NLC were first characterized by flow cytometry, RT-PCR, and immunocytochemistry. The functionality of the NLC was evaluated by qRT-PCR and patch clamp recording. Immunophenotyping of ADMSC showed their immunoreactivity to MSC markers CD90, CD73, CD105, and CD49d, but not to CD31 and CD45. RT-PCR results demonstrated the expression of nestin, neurogenin, neurod1, neurofilament light, and GAP43 genes in NSC while NLC expressed synaptophysin, neurofilament heavy, and GAP43. In addition, NSC morphology changed into multipolar with long processes after transduction with miR-218. Moreover, using qRT-PCR, the expression levels of miR-218 and functionality genes CACNA1C, SNAP25, KCNH1, KCNMA1, and SCN9A were significantly increased in NLC, compared with NSC, and ADMSC at 3 weeks and 5 months post-transduction. Furthermore, the generated NLC expressed significantly higher protein levels of neurofilament heavy polypeptide (NFh) and enolase 2 (Eno2) neuronal markers, compared with ADMSC and NSC. Finally, action potentials were successfully recorded by the generated NLC, using patch clamp. In summary, ADMSC-derived NSC differentiated into functional NLC by transduction with miR-218. The generated NLC expressed functional SNAP25, CACNA1C, KCNH1, KCNMA1, and SCN9A and produced an action potential, which provides useful insights into the generation of functional neuronal cells. Keywords ADMSC . Genetic transduction . miR-218 . Neuron-like cell . Functionality genes . Patch clamp
Introduction The generation of functional neuron-like cells (NLC) from NSC in vitro remains a challenge since the optimization of a safe conversion protocol is still needed (Dharmadasa et al.
2016). Hence, microRNAs (miRNA) could provide such a secure and effective induction tool to produce and reprogram cells for translational regenerative medicine (Moradi et al. 2014). Neural stem cells (NSC) are considered a promising option for treating injuries of the central nervous system
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12640-020-00244-7) contains supplementary material, which is available to authorized users. * Taki Tiraihi [email protected]; [email protected]
Kazem Zibara [email protected]
Wissam Khalil vm.khalil@modare
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