Isolation and Culture of Embryonic Mouse Neural Stem Cells

Farshad Homayouni Moghadam; Maryam Sadeghi-Zadeh; Bahareh Alizadeh-Shoorjestan; Reza Dehghani-Varnamkhasti; Sepideh Narimani; Leila Darabi; Abbas Kiani Esfahani; Mohammad Hossein Nasr Esfahani

doi:10.3791/58874

Isolamento e cultura de células-tronco neurais embrionárias de rato

JoVE Journal
Neuroscience
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JoVE Journal Neuroscience

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Isolamento e cultura de células-tronco neurais embrionárias de rato

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09:04 min

November 11, 2018

DOI: 10.3791/58874-v

Farshad Homayouni Moghadam¹, Maryam Sadeghi-Zadeh^1,3, Bahareh Alizadeh-Shoorjestan^1,3, Reza Dehghani-Varnamkhasti^1,3, Sepideh Narimani^1,2, Leila Darabi^1,3, Abbas Kiani Esfahani¹, Mohammad Hossein Nasr Esfahani¹

¹Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology,ACECR, ²Department of Biology, Faculty of Basic Sciences,Islamic Azad University, ³Department of Biology,ACECR Institute of Higher Education

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Overview

This study introduces a novel microsurgical technique for isolating neural stem cells from the ganglionic eminence of E 13 mouse embryos. The aim is to provide a reliable and efficient protocol for the extraction and culture of these cells, which are essential for understanding neural development and stem cell biology.

Key Study Components

Area of Science

Cellular and Molecular Biology
Neuroscience
Stem Cell Research

Background

Neural stem cells play a critical role in brain development.
The ganglionic eminence is a key region for neurogenesis.
Current methods for isolating these cells can be cumbersome and inefficient.
This technique aims to streamline the process and improve cell yield.

Purpose of Study

To establish a reliable method for the isolation of neural stem cells.
To enhance the understanding of neurogenesis in early embryos.
To provide a protocol that can be utilized in various research settings.

Methods Used

The technique involves microsurgical dissection of E 13 mouse embryos.
It includes specific steps for tissue dissociation and cell plating in culture.
Important steps include the use of HEPES MEM buffer and careful microsurgery for brain extraction.
Results are obtained after culturing the cells, allowing for the observation of neurosphere formation.

Main Results

The protocol successfully yields a high percentage of viable neural stem cells.
Neurospheres form within 3 days, indicating successful stem cell proliferation.
Flow cytometry reveals that approximately 95% of cells are Nestin positive, confirming their identity as neural stem cells.
Cells show differentiation potential, with 94% exhibiting neuronal characteristics after further culture.

Conclusions

This study demonstrates a practical approach to isolate neural stem cells from mouse embryos.
The method lays the groundwork for future research into neural development and disease models.
It has potential implications for stem cell therapy and regenerative medicine.

Frequently Asked Questions

What are the advantages of the microsurgical technique?

This technique is efficient, provides high yields of viable neural stem cells, and streamlines the isolation process compared to traditional methods.

How are the embryos harvested for the protocol?

Embryos are harvested from the uterus and subjected to micro-dissection to isolate the ganglionic eminence for neural stem cell extraction.

What types of cells are generated through this method?

The method primarily yields neural stem cells, which can differentiate into neurons and glia under appropriate culture conditions.

How can this technique be applied in research?

The isolated neural stem cells can be used for studies on neurogenesis, drug screening, and regenerative therapies, contributing to a better understanding of neural development.

What critical steps are involved in the protocol?

Key steps include careful microsurgery, tissue dissociation in neural media, and cell culture for observing neurosphere formation.

Are there any limitations to this method?

The technique requires precision in microsurgery and may necessitate advanced skills, which could limit accessibility to some researchers.

Aqui, apresentamos uma nova técnica microcirúrgica para o isolamento de células-tronco neurais da Eminência ganglionar embrião de rato E¹³.

O objetivo deste protocolo de vídeo é estabelecer um protocolo de origem e eficiente para o isolamento e cultura das células-tronco neurais do rato embrionário. Olá, sou Maryam Sadeghi-Zadeh. Sou um estudante de mestrado em Biologia Celular e Molecular no Instituto Royan.

Hoje eu e meu colega queremos mostrar como colher células-tronco neurais de 13 eminência gangliônica de camundongos embriões. Olá, eu sou Bahareh Alizadeh-Shoorjestan. Sou um estudante de mestrado em Biologia Celular e Molecular no Departamento de Células-Tronco do Instituto Royan de Biotecnologia.

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