Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Yongchao Mou; Sukhada Mukte; Eric Chai; Joshua Dein; Xue-Jun Li

doi:10.3791/60548

遺伝性痙性対麻痺におけるヒト人工多能性幹細胞由来ニューロンにおけるミトコンドリア輸送と形態学の解析

JoVE Journal
Neuroscience

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

遺伝性痙性対麻痺におけるヒト人工多能性幹細胞由来ニューロンにおけるミトコンドリア輸送と形態学の解析

Full Text

8,313 Views

07:32 min

February 9, 2020

DOI: 10.3791/60548-v

Yongchao Mou^1,2, Sukhada Mukte¹, Eric Chai¹, Joshua Dein³, Xue-Jun Li^1,2

¹Department of Biomedical Sciences,University of Illinois College of Medicine Rockford, ²Department of Bioengineering,University of Illinois at Chicago, ³MD Program,University of Illinois College of Medicine Rockford

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study investigates mitochondrial transport and morphology using induced pluripotent stem cell-derived forebrain neurons in the context of hereditary spastic paraplegia. The protocol allows for detailed assessment of mitochondrial dynamics along axons, contributing to the understanding of neurodegenerative diseases.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Neurodegenerative Diseases

Background

Mitochondrial dysfunction is a key factor in various neurodegenerative diseases.
Impaired mitochondrial transport and morphology have been linked to axonal degeneration.
The use of induced pluripotent stem cells provides a relevant model for studying human neural processes.

Purpose of Study

To develop a protocol for examining mitochondrial behavior in axons.
To elucidate the relationship between mitochondrial dynamics and neurodegenerative disease mechanisms.
To identify potential therapeutic targets for conditions like hereditary spastic paraplegia.

Methods Used

Cell culture of induced pluripotent stem cell-derived forebrain neurons.
Live cell imaging combined with mitochondrial labeling to assess mitochondrial tracking.
Important steps include dissociation of neurospheres and proper staining with fluorescent dyes.
Image analysis performed using ImageJ, including the generation of kymographs.

Main Results

Characterization of mitochondrial transport revealed significant differences in mitochondrial dynamics.
Quantitation of mitochondrial length and movement showed reduced motility in neurons derived from hereditary spastic paraplegia models.
Findings underscore the importance of mitochondrial function in neural health and disease.

Conclusions

This study provides a vital experimental approach to analyze mitochondrial dynamics in the context of neurodegeneration.
The insights gained can inform future therapeutic strategies targeting mitochondrial dysfunction.
The methodology may enhance our understanding of neuronal mechanisms and disease progression.

Frequently Asked Questions

What are the advantages of using induced pluripotent stem cells for this study?

Induced pluripotent stem cells offer a human-relevant model to explore mitochondrial function and dynamics, allowing for insights directly applicable to human disease.

How is mitochondrial transport assessed in this protocol?

Mitochondrial transport is assessed via live cell imaging, using fluorescent dyes to visualize and track mitochondrial dynamics along the axons.

What outcomes can be measured with this protocol?

Key outcomes include mitochondrial length, area, transport velocity, and motility characteristics, which are crucial for understanding neuronal health.

Can this method be adapted for other types of neurons?

Yes, this methodology can be adapted for other neuronal types by using appropriate differentiation protocols and imaging techniques specific to those neurons.

What are some limitations of this study?

Limitations may include the inability to fully replicate in vivo conditions and potential variability in stem cell differentiation outcomes.

How does this study contribute to the understanding of neurodegenerative diseases?

By using human-derived neurons to study mitochondrial dysfunction, the findings enhance our understanding of the cellular mechanisms underlying neurodegenerative diseases.

ミトコンドリアの輸送および形態の障害は、様々な神経変性疾患に関与している。提示されたプロトコルは、遺伝性痙攣対麻痺におけるミトコンドリア輸送および形態を評価するために、誘導多能性幹細胞由来前脳ニューロンを使用する。このプロトコルは、軸索に沿ったミトコンドリア人身売買の特徴付けとその形態の分析を可能にし、神経変性疾患の研究を容易にする。

ミトコンドリアの機能不全は、多くの神経変性疾患の根底にあります。我々のプロトコルは、軸索のミトコンドリアダイナミクスを調べるための重要なツールを提供し、軸索変性を伴う神経疾患の研究を促進する。ミトコンドリア標識、生きた細胞イメージング、誘導多能性幹細胞技術を組み合わせることで、当社のプロトコルを使用して、ヒト軸索に沿ったミトコンドリア人身売買を特徴付け、その形態を解析することができます。

ミトコンドリアの輸送および形態の障害は、これらの疾患を治療するための潜在的な治療標的を提供する神経変性疾患の幹細胞培養および動物モデルにおいて観察することができる。培養の35日目の後、ヒト誘発多能性幹細胞と分化した神経球を、摂氏37度で2分間、1mg/mlの細胞剥離液を有する小さなクラスターに解離する。インキュベーションの終わりに遠心分離によって細胞クラスターを収集し、NDMの1ミリリットルでペレットを再懸濁します。

View the full transcript and gain access to thousands of scientific videos