Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Barbara Calabrese; Regina M. Powers; Alexandria J. Slepian; Shelley Halpain

doi:10.3791/59305

Resposta pós-diferenciação de neurônios derivados de células-tronco pluripotentes humanas para tr...

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Neuroscience

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JoVE Journal Neuroscience

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Resposta pós-diferenciação de neurônios derivados de células-tronco pluripotentes humanas para triagem de alto conteúdo de Neuritogênese e maturação de sinapse

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06:50 min

August 28, 2019

DOI: 10.3791/59305-v

Barbara Calabrese^1,2, Regina M. Powers^1,2, Alexandria J. Slepian^1,2, Shelley Halpain^1,2

¹Division of Biological Sciences,University of California, San Diego, ²Sanford Consortium for Regenerative Medicine

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Overview

This protocol outlines a procedure for resuspending and culturing human stem cell-derived neurons that were differentiated in vitro. The method allows for imaging-based assays of neurites, synapses, and neuronal markers, suitable for high-content screening applications.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Cell Culture Techniques

Background

Human pluripotent stem cell-derived neurons are critical for studying synapses.
These neurons require extensive culture periods for maturation.
Improving neuronal viability during culture is essential for accurate assays.
The procedure enhances cell recovery and viability post-replating.

Purpose of Study

To create a reliable method for culturing human stem cell-derived neurons.
To demonstrate effective replating for high-content screening.
To optimize protease incubation time for improved cell health.

Methods Used

Cell culture method involving human pluripotent stem cell-derived neurons.
Utilized a detailed protease treatment for gentle detachment of cells.
Incorporated visual checks to monitor neuron detachment during protocols.
Followed a strict timeline for incubation and replating processes.
The method is adaptable for other neuronal types and applications.

Main Results

Extended protease incubation improved neuronal survival and reduced cell death.
Demonstrated enhanced neuride outgrowth and viability over several days after replating.
Markers for synaptic proteins were observed within a week of replating.
Electrical activity was detectable, indicating functional neuronal networks.

Conclusions

The study provides a reproducible protocol for neuronal culture and assay applications.
Enhanced survival and neurite growth facilitates further research on neuronal functionality.
Findings contribute to understanding neuronal mechanisms and potential therapeutic avenues.

Frequently Asked Questions

What are the advantages of using human stem cell-derived neurons?

These neurons allow for the investigation of human-specific pathways and mechanisms in synaptic function and development.

How is the dissociation of neurons performed?

Neurons are gently detached using proteolytic enzymes, which require careful monitoring to optimize cell viability.

What types of data can be obtained from these cultured neurons?

Data on synaptic activity, neurite outgrowth, and cell viability can be obtained, suitable for both imaging and electrophysiological assays.

How can the method be adapted for other applications?

The protocol can be customized for various assays, such as imaging growth patterns or functional measurements using multielectrode arrays.

Are there any limitations to this protocol?

The success of the protocol highly depends on accurately timing protease treatment and monitoring neuronal health during culture.

Este protocolo descreve um procedimento detalhado para ressusciar e cultivar os neurônios derivados humanos da pilha de haste que foram diferenciados previamente dos progenitores neural in vitro por semanas múltiplas. O procedimento facilita os ensaios com base em imagens de neuritos, sinapses e marcadores neuronais de expressão tardia em um formato compatível com microscopia de luz e triagem de alto conteúdo.

Este protocolo facilita o uso de neurônios pluripotentes humanos derivados de células-tronco para aplicações de triagem de alto conteúdo. É particularmente adequado para avaliar sinapses, que em neurônios humanos requerem longos períodos de cultura. Demonstramos a replação de neurônios de pratos de grande formato em multi-poços compatíveis com HCS de forma a preservar sua viabilidade.

Contra-intuitivamente, mostramos que estender a incubação de protease antes de resuscupar e replacar produz melhor sobrevida neuronal. Os neurônios derivados de células-tronco pluripotentes humanos são cada vez mais relevantes nas áreas de pesquisa básica, desenvolvimento de medicamentos e medicina degenerativa. Este método de titulação suave pode ser usado para resuspensar qualquer grande mono-camada de células com uma rede espessa de processos.

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