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.
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Abbreviations
- ADMSC:
-
adipose-derived mesenchymal stem cells
- NS:
-
neurospheres
- NSC:
-
neural stem cells
- NLC :
-
neural-like cells
- Mir-218:
-
microRNA-218
- CD90:
-
thy-1 cell surface antigen
- CD73:
-
5′ecto-nucleotidase
- CD49d:
-
integrin subunit alpha 4
- CD105:
-
endoglin
- CD31:
-
platelet and endothelial cell adhesion molecule 1
- CD45:
-
protein tyrosine phosphatase receptor type C
- Syp:
-
synaptophysin
- NFl:
-
neurofilament light
- NFh:
-
neurofilament heavy
- Nes:
-
nestin
- Sox2:
-
SRY-box transcription factor 2
- Oct4:
-
POU class 5 homeobox 1
- Neurog1:
-
neurogenin
- Neurod1:
-
neuronal differentiation 1
- GAP43:
-
growth-associated protein 43
- SNAP25:
-
synaptosome-associated protein 25
- CACNA1C:
-
calcium voltage-gated channel subunit alpha1 C
- KCNH1:
-
potassium voltage-gated channel subfamily H member 1
- KCNMA1:
-
potassium calcium-activated channel subfamily M alpha 1
- SCN9A:
-
sodium voltage-gated channel alpha subunit 9
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Acknowledgments
The authors are thankful for the help and advice of the laboratory members at Stem Cell Technology Research Center, Tehran, Iran.
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The project was funded by Tarbiat Modares University.
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Conceptualization: Taki Tiraihi; formal analysis: Wissam Khalil; investigation: Wissam Khalil; methodology: Wissam Khalil; project administration: Taki Tiraihi; resources: Masoud Soleimani and Nafiseh Baheiraei; supervision: Taki Tiraihi; validation: Taki Tiraihi, Masoud Soleimani, and Kazem Zibara; visualization: Wissam Khalil; writing—original draft: Wissam Khalil; writing—review and editing: Taki Tiraihi, Kazem Zibara, and Nafiseh Baheirae.
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All the applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocols performed in this study were approved by the ethical committee of the Faculty of Medical Sciences at Tarbiat Modares University (IRB NO. IR.TMU.REC.1394.279), and the experiments were done according to the principles expressed in the declaration of Helsinki.
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Khalil, W., Tiraihi, T., Soleimani, M. et al. Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functionality Genes. Neurotox Res 38, 707–722 (2020). https://doi.org/10.1007/s12640-020-00244-7
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DOI: https://doi.org/10.1007/s12640-020-00244-7