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

STAR Protoc. 2023 Sep 15;4(3):102529. doi: 10.1016/j.xpro.2023.102529. Epub 2023 Aug 25.

Authors

Miriam E Korsgen¹, Congxin Sun², Elena Seranova¹, Malgorzata Zatyka¹, Dewi Astuti¹, Tetsushi Kataura³, Timothy Barrett⁴, Viktor I Korolchuk⁵, Sovan Sarkar⁶

Affiliations

¹ Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
² Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address: scongxin2019@gmail.com.
³ Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.
⁴ Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; Department of Endocrinology, Birmingham Women's and Children's Hospital, Steelhouse Lane, Birmingham B4 6NH, UK.
⁵ Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE4 5PL, UK. Electronic address: viktor.korolchuk@newcastle.ac.uk.
⁶ Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Electronic address: s.sarkar@bham.ac.uk.

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.

Keywords: Cell Biology; Cell Culture; Cell Differentiation; Cell-Based Assays; Stem Cells.

MeSH terms

Autophagy / genetics
Cell Differentiation / genetics
Human Embryonic Stem Cells*
Humans
Neurons / metabolism

Grants and funding

WT_/Wellcome Trust/United Kingdom