Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons

Miriam E Korsgen; Congxin Sun; Elena Seranova; Malgorzata Zatyka; Dewi Astuti; Tetsushi Kataura; Timothy Barrett; Viktor I Korolchuk; Sovan Sarkar

doi:10.1016/j.xpro.2023.102529

Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons

Miriam E Korsgen
, Congxin Sun^*
, Elena Seranova
, Malgorzata Zatyka
, Dewi Astuti
, Tetsushi Kataura
, Timothy Barrett
, Viktor I Korolchuk^*
, Sovan Sarkar^*

^*Corresponding author for this work

Cancer and Genomic Sciences

Research output: Contribution to journal › Article › peer-review

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Abstract

Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.

Original language	English
Article number	102529
Journal	STAR Protocols
Volume	4
Issue number	3
Early online date	25 Aug 2023
DOIs	https://doi.org/10.1016/j.xpro.2023.102529
Publication status	E-pub ahead of print - 25 Aug 2023

Bibliographical note

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Cell Biology
Cell Culture
Cell-Based Assays
Stem Cells
Cell Differentiation

Access to Document

10.1016/j.xpro.2023.102529Licence: Creative Commons: Attribution (CC BY)

KorsgenME2023AnalysisFinal published version, 4.71 MBLicence: Creative Commons: Attribution (CC BY)

Development of a novel repurposed drug treatment for neurodegeneration and diabetes in Wolfram syndrome
Nagy, Z. (Co-Investigator), Peet, A. (Co-Investigator), Brock, K. (Co-Investigator), Barrett, T. (Principal Investigator) & Martin, U. (Co-Investigator)
Medical Research Council
1/03/17 → 31/07/25
Project: Research Councils
Human cellular platforms for studying the regulation and therapeutic application of autophagy
Sarkar, S. (Principal Investigator)
The Wellcome Trust
1/07/16 → 31/12/18
Project: Research

Cite this

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title = "Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons",

abstract = "Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.",

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doi = "10.1016/j.xpro.2023.102529",

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T1 - Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons

AU - Korsgen, Miriam E

AU - Sun, Congxin

AU - Seranova, Elena

AU - Zatyka, Malgorzata

AU - Astuti, Dewi

AU - Kataura, Tetsushi

AU - Barrett, Timothy

AU - Korolchuk, Viktor I

AU - Sarkar, Sovan

PY - 2023/8/25

Y1 - 2023/8/25

N2 - Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.

AB - Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.

KW - Cell Biology

KW - Cell Culture

KW - Cell-Based Assays

KW - Stem Cells

KW - Cell Differentiation

U2 - 10.1016/j.xpro.2023.102529

DO - 10.1016/j.xpro.2023.102529

M3 - Article

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SN - 2666-1667

VL - 4

JO - STAR Protocols

JF - STAR Protocols

IS - 3

M1 - 102529

ER -

Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons

Abstract

Bibliographical note

UN SDGs

Keywords

Access to Document

Fingerprint

Projects

Development of a novel repurposed drug treatment for neurodegeneration and diabetes in Wolfram syndrome

Human cellular platforms for studying the regulation and therapeutic application of autophagy

Cite this