en Cell, Stem Cell and Cancer Cell, Stem Cell and Cancer Experimental research article Experimental research article <div style="text-align: justify;"><strong>Introduction:</strong> 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.<br /> <strong>Methods:</strong> 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.<br /> <strong>Results: </strong>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.<br /> <strong>Conclusion:</strong> 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.</div> Modified-mRNA, In vitro transcription, Neural stem cell, Chloroquine, Transfection, Cell culture. 80 91 http://ppj.phypha.ir/browse.php?a_code=A-10-146-2&slc_lang=en&sid=1 Reyhaneh Khayamabed 3200319475328460032151 3200319475328460032151 No Department of Biology, ACECR Institute of Higher Education, Isfahan, Iran Naeimeh Rezaie 3200319475328460032152 3200319475328460032152 No Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran Elnaz Poorgolizadeh 3200319475328460032153 3200319475328460032153 No Department of Biology, ACECR Institute of Higher Education, Isfahan, Iran Farshad Homayouni Moghadam homayouni@royan-rc.ac.ir 3200319475328460032154 3200319475328460032154 Yes Department of Biology, ACECR Institute of Higher Education, Isfahan, Iran Kianoush Dorminani 3200319475328460032155 3200319475328460032155 No Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran Mohammad Hossein Nasr-Esfahani 3200319475328460032156 3200319475328460032156 No Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran