Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Arens Taga; Christa W. Habela; Alexandra Johns; Shiyu Liu; Mollie O'Brien; Nicholas J. Maragakis

doi:10.3791/62726

Estabelecimento de uma plataforma eletrofisiológica para modelagem da ELA com astrócitos e neurôn...

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JoVE Journal Neuroscience

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Estabelecimento de uma plataforma eletrofisiológica para modelagem da ELA com astrócitos e neurônios derivados de células-tronco pluripotentes humanas regionalmente específicas

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11:52 min

August 26, 2021

DOI: 10.3791/62726-v

Arens Taga*¹, Christa W. Habela*¹, Alexandra Johns¹, Shiyu Liu¹, Mollie O'Brien¹, Nicholas J. Maragakis¹

¹Department of Neurology,Johns Hopkins University

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Overview

This study describes a method for differentiating spinal cord human induced pluripotent stem cell (iPSC)-derived astrocytes and neurons, facilitating their co-culture for electrophysiological recordings. The developed technique enables researchers to analyze neuronal activity influenced by astrocytes, contributing to the investigation of spinal cord diseases.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Electrophysiology

Background

Spinal cord neurons play a critical role in neurodegenerative diseases.
Astrocytes influence neuronal behavior and health.
Human iPSC technology provides a relevant model for studying these interactions.
The method aids in understanding diseases such as amyotrophic lateral sclerosis (ALS).

Purpose of Study

To create spinal cord neuron and astrocyte models for electrophysiological analysis.
To assess the effects of neuronal factors and astrocyte interactions.
To aid in the study of spinal cord specific diseases.

Methods Used

Cell culture methods to generate human iPSC-derived astrocytes and neurons.
Neural differentiation protocols with specific media and incubation conditions.
Co-culture techniques for simultaneous recording of neuronal activity influenced by astrocytes.
Electrophysiological recording after establishing co-cultures over set timelines.

Main Results

The co-culture method enables robust electrophysiological recording of neuron activity.
Observations allow for studying cellular interactions relevant to disease mechanisms.
Critical influences of astrocytic support on neuronal health and function were noted.
Lay the groundwork for exploring therapeutic interventions targeting neurons and astrocytes.

Conclusions

This method enhances the understanding of spinal cord cell interactions and their implications in disease.
It enables a detailed investigation of cellular responses under different conditions.
The findings support further exploration into neurodegenerative diseases and potential treatments.

Frequently Asked Questions

What are the advantages of using human iPSC-derived cells?

Human iPSC-derived cells provide a relevant model system that closely mimics human physiology and disease conditions, allowing for more accurate experimental insights.

How is the differentiation into neural progenitor cells achieved?

Differentiation involves the use of specific induction media and carefully controlled incubation conditions to promote the growth of neural progenitor cells from human iPSCs.

What types of data are obtained from this co-culture system?

Electrophysiological data is collected to analyze neuronal activity, influenced by the presence and support from co-cultured astrocytes, providing insights into cellular interactions.

How can the method be adapted for other research questions?

The co-culture method can be modified by introducing different growth factors or inhibitors to examine various pathways and interactions in neuronal health and disease.

What are the key limitations of this approach?

Limitations may include variability in differentiation efficiency and potential challenges in maintaining cell viability over extended culture periods, which can affect experimental outcomes.

Descrevemos um método para diferenciar astrócitos e neurônios derivados pluripotentes induzidos por humanos da medula espinhal e sua co-cultura para registro eletrofisiológico.

A técnica que apresentamos hoje permite a análise da atividade dos neurônios da medula espinhal como resultado do fator neuronal intrínseco e da influência dos astrócitos circundantes. Esta técnica gera de forma confiável neurônios humanos e astrócitos humanos que são específicos da medula espinhal e usa uma medida de saída funcional que pode ser dimensionada para reprodutibilidade. A técnica que apresentamos permite investigar doenças específicas dos neurônios da medula espinhal, como a esclerose lateral amiotrófica, usando neurônios de pacientes com doença esporádica ou genética.

Para começar, examine as placas iPSC humanas em busca de colônias suficientemente densas, de modo que o centro das colônias exiba áreas brancas dispersas. Em seguida, para começar a embalar as células, desenhe linhas de grade na superfície externa inferior das placas com um marcador para facilitar a cobertura precisa de toda a placa. Use um microscópio de contraste de fase de ampliação de 10x em um campo de cultura para separar colônias diferenciadas raspando manualmente com uma ponta de pipeta de 200 microlitros.

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