Autophagy modulates cell fate decisions during lineage commitment

ABSTRACT

Early events during development leading to exit from a pluripotent state and commitment toward a specific germ layer still need in-depth understanding. Autophagy has been shown to play a crucial role in both development and differentiation. This study employs human embryonic and induced pluripotent stem cells to understand the early events of lineage commitment with respect to the role of autophagy in this process. Our data indicate that a dip in autophagy facilitates exit from pluripotency. Upon exit, we demonstrate that the modulation of autophagy affects SOX2 levels and lineage commitment, with induction of autophagy promoting SOX2 degradation and mesendoderm formation, whereas inhibition of autophagy causes SOX2 accumulation and neuroectoderm formation. Thus, our results indicate that autophagy-mediated SOX2 turnover is a determining factor for lineage commitment. These findings will deepen our understanding of development and lead to improved methods to derive different lineages and cell types.

Abbreviations: ACTB: Actin, beta; ATG: Autophagy-related; BafA1: Bafilomycin A₁; CAS9: CRISPR-associated protein 9; CQ: Chloroquine; DE: Definitive endoderm; hESCs: Human Embryonic Stem Cells; hiPSCs: Human Induced Pluripotent Stem Cells; LAMP1: Lysosomal Associated Membrane Protein 1; MAP1LC3: Microtubule-Associated Protein 1 Light Chain 3; MTOR: Mechanistic Target Of Rapamycin Kinase; NANOG: Nanog Homeobox; PAX6: Paired Box 6; PE: Phosphatidylethanolamine; POU5F1: POU class 5 Homeobox 1; PRKAA2: Protein Kinase AMP-Activated Catalytic Subunit Alpha 2; SOX2: SRY-box Transcription Factor 2; SQSTM1: Sequestosome 1; ULK1: unc-51 like Autophagy Activating Kinase 1; WDFY3: WD Repeat and FYVE Domain Containing 3.

KEYWORDS:

• Autophagosome
• differentiation
• ectoderm
• endoderm
• mesoderm
• pluripotent stem cells
• SOX2

Acknowledgments

We thank Michael Munson for help with image analysis and Carina Knudsen for the illustrations. We thank Narasimha Swamy Telugu from MDC, Berlin for his help related to CRISPR experiments. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme (project number 262652 to AS and project number 262613 to GS), Helse EU program (project number 269881) and FRIPRO grants (project number 221831 to AS and project number 247624 to GS), and the Norwegian Cancer Society (project number 171318 to AS). We also acknowledge Prof Tormod Fladby and Prof Hilde Nilsen for help in doing experiments at Epigen, Akershus University Sykehus.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Kreftforeningen (NO) [171318]; Norges Forskningsråd [262652]; Norges Forskningsråd [247624]; Norges Forskningsråd [269881]; Norges Forskningsråd (NO) [221831]; Norges Forskningsråd [262613].