Volume 27, Issue 1 (March 2023)
Physiol Pharmacol 2023, 27(1): 80-91 |
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Khayamabed R, Rezaie N, Poorgolizadeh E, Homayouni Moghadam F, Dorminani K, Nasr-Esfahani M H. Efficient Modified-mRNA Transfection in Neural Stem Cells. Physiol Pharmacol 2023; 27 (1) :80-91
URL: http://ppj.phypha.ir/article-1-1847-en.html
URL: http://ppj.phypha.ir/article-1-1847-en.html
Reyhaneh Khayamabed 
, Naeimeh Rezaie , Elnaz Poorgolizadeh , Farshad Homayouni Moghadam 
, Kianoush Dorminani , Mohammad Hossein Nasr-Esfahani
, Naeimeh Rezaie , Elnaz Poorgolizadeh , Farshad Homayouni Moghadam , Kianoush Dorminani , Mohammad Hossein Nasr-Esfahani
Abstract: (1904 Views)
Introduction: Neural stem cells (NSCs) are multipotent stem cells residing in the central nervous system that is capable of self-renewal to support ongoing requirements for neurogenesis in the adult brain. Since NSCs are considered potential candidate cells for neuro-regenerative medicine, applying safe induction methods for them is very important. Synthetic modified-mRNA (mmRNA) as an alternative to traditional DNA- or protein-based methods, is regarded as a powerful tool for inducing short-term gene expression in cells with no genetic manipulation.
Methods: Here, we aimed to develop an optimized condition for mmRNA transfection in primary NSCs. In vitro-transcribed EGFP mmRNA (mmRNAEGFP) was delivered to human embryonic kidney cells (HEK293T) and mouse NSCs by using two commercial agents, Lipofectamine-2000 (LF2000) and TransIT. Also, a plasmid DNA was used to transfect cells considered EGFP-expressing positive control. In addition, the poly(A) tail (poly adenosine tail) elongation and chloroquine (CQ) treatment were performed to improve transfection efficiency. Finally, flow cytometry, fluorescence microscopy, and MTT assays were performed to assess the cells.
Results: In comparison with HEK293T, NSCs were very sensitive to transfection, the efficacy of transfection using DNA/LF2000 was higher in HEK293T cells, but mmRNAEGFP/ TransIT showed better transfection efficacy in NSCs. Poly(A) tail elongation; also, treating the cells with CQ before transfection significantly improved its efficacy.
Conclusion: The mmRNA poly(A) tail elongation and the use of specific transfection agents in combination with TLR inhibitors can lead to a more effective transfection in NSCs.
Methods: Here, we aimed to develop an optimized condition for mmRNA transfection in primary NSCs. In vitro-transcribed EGFP mmRNA (mmRNAEGFP) was delivered to human embryonic kidney cells (HEK293T) and mouse NSCs by using two commercial agents, Lipofectamine-2000 (LF2000) and TransIT. Also, a plasmid DNA was used to transfect cells considered EGFP-expressing positive control. In addition, the poly(A) tail (poly adenosine tail) elongation and chloroquine (CQ) treatment were performed to improve transfection efficiency. Finally, flow cytometry, fluorescence microscopy, and MTT assays were performed to assess the cells.
Results: In comparison with HEK293T, NSCs were very sensitive to transfection, the efficacy of transfection using DNA/LF2000 was higher in HEK293T cells, but mmRNAEGFP/ TransIT showed better transfection efficacy in NSCs. Poly(A) tail elongation; also, treating the cells with CQ before transfection significantly improved its efficacy.
Conclusion: The mmRNA poly(A) tail elongation and the use of specific transfection agents in combination with TLR inhibitors can lead to a more effective transfection in NSCs.
Keywords: Modified-mRNA, In vitro transcription, Neural stem cell, Chloroquine, Transfection, Cell culture.
Type of Manuscript: Experimental research article |
Subject:
Cell, Stem Cell and Cancer
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