Abstract
Neural cells derived from embryonic stem cells (ESCs) have potential usefulness for the treatment of neurodegenerative disorders. Modulation of intrinsic growth factors expression such as neurotrophins and their respective receptors by these cells is necessary to obtain functional neural cells for transplantation. In present study, we compared neural differentiation potential of two different media, NB + 5%ES-FBS + N2B27 and Ko-DMEM + 5%ES-FBS for conversion of mESC derived neural progenitors (NPs) into mature neural cells with emphasis on effect of the these two media on neurotrophins and their respective receptors expression. Immunofluorescence staining, RT-qPCR and western blot analysis showed that the expression of neuronal specific markers, MAP2 and Tuj-1, in NB + 5%ES-FBS + N2B27 medium was significantly higher than the other medium. Western blot assay revealed that the expression of BDNF and NGF increased significantly in mature neural cells obtained from NB + 5%ES-FBS + N2B27 medium but decreased in neural cells from Ko-DMEM + 5%ES-FBS medium compared to mESCs. TrkB protein was not detectable in mESCs but its expression increased in neural cells obtained from both media although its expression in NB + 5%ES-FBS + N2B27 medium was significantly higher than the other medium. In contrast to TrkB, p75NTR protein was detectable in mESCs and is remained constant in neural cells cultured in NB + 5%ES-FBS + N2B27 medium but decreased significantly in the other medium. In conclusion, our results indicated that NB + 5%ES-FBS + N2B27 medium promoted neural differentiation process of mESCs and caused enhancement of neurotrophins protein expression in addition to their cognate receptors.
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Abbreviations
- ANOVA:
-
Two-way analysis of variance
- BDNF:
-
Brain-derived neurotrophic factor
- bHLH:
-
Basic helix–loop–helix
- BSA:
-
Bovine serum albumin
- CNS:
-
Central nervous system
- CTCF:
-
Corrected total cell fluorescence
- DAPI:
-
4,6-Diamidino-2-phenylindole
- DMEM/F12:
-
Dulbecco’s modified Eagle’s medium/Hams F12 medium
- EB:
-
Embryoid body
- ECL:
-
Enhanced chemiluminescence
- ERK:
-
Extracellular signal-regulated kinase
- ESC:
-
Embryonic stem cell
- ES-FBS:
-
Embryonic stem cell qualified fetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- GFAP:
-
Glial fibrillary acidic protein
- HRP:
-
Horse radish peroxidase
- JNK:
-
Jun N-terminal kinase
- Ko-DMEM:
-
Knock-out DMEM
- LIF:
-
Leukemia inhibitory factor
- MAP2:
-
Microtubule associated protein 2
- mESC:
-
Mouse embryonic stem cell
- miRNA:
-
MicroRNAs
- NB:
-
Neurobasal
- NC:
-
Neural cell
- NGF:
-
Nerve growth factor
- NP:
-
Neural progenitor
- NSC:
-
Neural stem cell
- NT:
-
Neurotrophin
- NT-3:
-
Neurotrophin-3
- NT-4:
-
Neurotrophin-4
- p75NTR:
-
p75 neurotrophin receptor
- PBS:
-
Phosphate buffered saline
- PI3:
-
Phosphatidylinositol-3-kinase
- PVDF:
-
Polyvinylidene difluoride
- RA:
-
Retinoic acid
- RT:
-
Room temprature
- RT-qPCR:
-
Real-time quantitative polymerase chain reaction
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SEM:
-
Standard error of the mean
- TRI:
-
Total RNA isolation
- Trk:
-
Tropomyosin related kinase
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Acknowledgements
Authors would like to thank University of Sistan and Baluchestan and Royan Institute for the financial support of this project. The authors are thankful to other members of Royan Institute for their excellent technical assistance and advice.
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RE experimental design, data collection, data analysis, data interpretation and manuscript writing. DMKT experimental design, financial support, data analysis, data interpretation, and final approval of the manuscript. KG experimental design, data analysis, data interpretation, manuscript writing, and final approval of the manuscript. MHNE experimental design, financial support, data analysis, data interpretation, and final approval of manuscript.
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Ebadi, R., Kordi-Tamandani, D.M., Ghaedi, K. et al. Comparison of two different media for maturation rate of neural progenitor cells to neuronal and glial cells emphasizing on expression of neurotrophins and their respective receptors. Mol Biol Rep 45, 2377–2391 (2018). https://doi.org/10.1007/s11033-018-4404-4
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DOI: https://doi.org/10.1007/s11033-018-4404-4