Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Esteban Jim&#233;nez-Villalba; Laura L&#225;zaro-Carot; Carmen M. Mateos-Mart&#237;nez; Jennifer D&#237;az-Moncho; Daniel Samper-Llavador; Marta Igual-L&#243;pez; Jordi Planells; Sacri  R. Ferr&#243;n

doi:10.3791/66651

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

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09:19 min

June 14, 2024

DOI: 10.3791/66651-v

Esteban Jiménez-Villalba*¹, Laura Lázaro-Carot*¹, Carmen M. Mateos-Martínez¹, Jennifer Díaz-Moncho¹, Daniel Samper-Llavador¹, Marta Igual-López¹, Jordi Planells¹, Sacri R. Ferrón¹

¹Instituto de Biotecnología y Biomedicina (BIOTECMED), Departamento de Biología Celular, Biología Funcional y Antropología Física,Universitat de València

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Overview

This study presents a novel nucleofection system aimed at enhancing gene delivery efficiency in neural stem cells (NSCs) derived from the adult murine subventricular zone. The findings demonstrate significant improvements in gene perturbation and cell survival rates compared to traditional transfection methods.

Key Study Components

Area of Science

Neuroscience
Gene Delivery
Stem Cell Biology

Background

Neural stem cells (NSCs) in the adult brain are crucial for neurogenesis.
Current transfection methods can affect cell viability and functionality.
Efficient methods for manipulating gene expression in NSCs are needed.
This study examines a new protocol to improve gene delivery in NSCs.

Purpose of Study

To develop a refined method for gene delivery in NSCs.
To enhance the survival rate of NSCs during gene perturbation.
To provide a reproducible approach for future research and therapeutic applications.

Methods Used

Cell culture techniques were employed using NSCs isolated from the murine subventricular zone.
The nucleofection system was applied for gene delivery.
Traditional gene perturbation methods were compared to the new nucleofection protocol.
Key steps included the preparation of the murine brain and isolation of NSCs from the subventricular zone.
Survival rates and gene delivery efficiency were assessed post-nucleofection.

Main Results

The nucleofection system yielded higher gene delivery efficiency in NSCs.
Survival rates of Neural stem cells exceeded 80% after nucleofection.
These improvements surpass those achieved with conventional transfection methods.
Significant advancements in gene perturbation effectiveness were demonstrated.

Conclusions

This study establishes an improved protocol for gene delivery in NSCs.
The findings have strong implications for advancing research in neural stem cell biology and potential therapeutic applications.
Enhanced survival and perturbation efficiency can facilitate future studies on NSC dynamics and their roles in neurogenesis.

Frequently Asked Questions

What are the advantages of the nucleofection system?

The nucleofection system offers higher efficiency in gene delivery and improved survival rates compared to traditional methods, making it a valuable tool for NSC research.

How are neural stem cells isolated in this study?

Neural stem cells are isolated from the murine subventricular zone after careful brain dissection to retain the integrity of the tissue.

What types of outcomes can be obtained using this method?

The method allows for effective gene perturbation in NSCs while maintaining high viability, useful for studying gene function and cellular behavior.

Can this method be applied to other cell types?

While this study focuses on NSCs, the nucleofection protocol may be adaptable for other cell types in need of efficient gene delivery.

What are the key limitations of this nucleofection approach?

Potential limitations include the need for precise implementation and monitoring to ensure optimal gene delivery efficiency and cell viability.

How does this study contribute to stem cell research?

It provides a refined protocol that enhances gene delivery efficiency, thus enabling more thorough investigations into NSC biology and therapeutic potential.

ここでは、成体マウス脳室下ゾーンから単離された拡大神経幹細胞(NSC)における遺伝子導入効率を高めるように設計されたヌクレオフェクションシステムについて説明します。この知見は、この方法が神経幹細胞における遺伝子摂動を大幅に改善し、従来のトランスフェクションプロトコルの有効性を上回り、細胞の生存率を向上させることを示しています。

私たちの研究は、成体哺乳類の脳内で神経幹細胞集団がどのように制御されているかを研究することに焦点を当てています。神経原性ニッチ内の神経幹細胞を調節する内因性および外因性の分子メカニズムを理解することは、生物学を理解し、将来の潜在的な治療応用を開発するために重要です。神経幹細胞の挙動を研究するために、in vivoおよびin vitroの両方のアプローチが広く使用されています。

In vitro神経幹細胞培養は、制御された環境を提供し、この細胞集団を容易に操作および監視する可能性を提供します。in vitroでの遺伝子発現保存技術は、細胞生物学を支配する分子メカニズムを研究するための汎用性の高いアプローチです。しかし、神経幹細胞の候補遺伝子を過剰発現させ、ノックダウンするための効率的で再現性の高い方法は、この分野ではまだ課題となっています。

遺伝子導入のための従来のトランスフェクション法は、中枢神経系細胞で有効であることが証明されています。さらに、これらの方法は細胞の生存率と機能に影響を与えます。したがって、神経幹細胞の遺伝子発現を操作するためには、代替アプローチの改良が重要です。

このプロトコルにより、成体マウス脳室下ゾーンからの神経幹細胞培養における遺伝子導入の高効率と、80%を超える生存率を達成する改良されたヌクレオフェクションシステムを紹介します

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