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Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution

Sungmin Kim, Edward Ren, Paola Marco Casanova, View ORCID ProfileEugenia Piddini, View ORCID ProfileRafael Carazo Salas
doi: https://doi.org/10.1101/2021.01.30.428961
Sungmin Kim
1School of Cellular & Molecular Medicine, University of Bristol, BS8 1TD Bristol, UK
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Edward Ren
1School of Cellular & Molecular Medicine, University of Bristol, BS8 1TD Bristol, UK
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Paola Marco Casanova
2The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
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Eugenia Piddini
1School of Cellular & Molecular Medicine, University of Bristol, BS8 1TD Bristol, UK
2The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
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  • ORCID record for Eugenia Piddini
Rafael Carazo Salas
1School of Cellular & Molecular Medicine, University of Bristol, BS8 1TD Bristol, UK
3Pharmacology Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
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  • ORCID record for Rafael Carazo Salas
  • For correspondence: rafael.carazosalas@bristol.ac.uk
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ABSTRACT

Live imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 31, 2021.
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Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution
Sungmin Kim, Edward Ren, Paola Marco Casanova, Eugenia Piddini, Rafael Carazo Salas
bioRxiv 2021.01.30.428961; doi: https://doi.org/10.1101/2021.01.30.428961
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Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution
Sungmin Kim, Edward Ren, Paola Marco Casanova, Eugenia Piddini, Rafael Carazo Salas
bioRxiv 2021.01.30.428961; doi: https://doi.org/10.1101/2021.01.30.428961

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