Dataset Information

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.

SUBMITTER: Korsgen ME

PROVIDER: S-EPMC10474488 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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

Korsgen Miriam E ME Sun Congxin C Seranova Elena E Zatyka Malgorzata M Astuti Dewi D Kataura Tetsushi T Barrett Timothy T Korolchuk Viktor I VI Sarkar Sovan S

STAR protocols 20230825 3

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<sup>-/-</sup>) 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 experimenta ...[more]

PMID: 37624702

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Project description:Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Dataset Information

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

Publications

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

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