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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

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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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Authors and Affiliations

  1. Department of Biology, University of Sistan and Baluchestan, P.O. Box 98155-411, Zahedan, Iran

    Reihane Ebadi & Dor Mohammad Kordi-Tamandani

  2. Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

    Kamran Ghaedi

  3. Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, P.O. Box 816513-1378, Isfahan, Iran

    Kamran Ghaedi & Mohammad Hossein Nasr-Esfahani

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  1. Reihane Ebadi
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  2. Dor Mohammad Kordi-Tamandani
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  3. Kamran Ghaedi
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  4. Mohammad Hossein Nasr-Esfahani
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Contributions

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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Correspondence to Dor Mohammad Kordi-Tamandani, Kamran Ghaedi or Mohammad Hossein Nasr-Esfahani.

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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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