A large-scale survey and characterisation of spontaneous cell division defects in human Pluripotent Stem Cells and their impact on cell proliferation and early cell differentiation

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Human pluripotent stem cells (hPSCs) represent a groundbreaking technology with significant
implications for Regenerative Medicine. Despite hPSCs’ vast therapeutic potential, concerns
about their poorly understood tumourigenic potential has limited their application. Recent
evidence suggests that genomic instability may contribute to hPSCs’ tumourigenic potential.
In this thesis, we developed a multi-day, multi-colour, time-lapse high-throughput microscopy
pipeline to investigate spontaneous chromosome segregation errors (CSEs) in hPSCs, and
used that approach to carry out a large-scale human-curated survey of spontaneous mitotic
defects in hPSCs, the largest such survey to date. Specifically, we analysed 49,505 mitoses in
hPSCs and 51,200 mitoses across different early differentiation cell types to investigate the
possible causes, features and outcomes of CSEs during pluripotency and early differentiation.
I demonstrate that hPSCs display the same types of CSEs observed in cancer cells including
lagging chromosomes, chromatin bridges and multipolar mitoses, and that spontaneous
CSEs in hPSCs generate micronuclei - thought to play key roles in chromothripsis and
tumourigenic potential in cancer cells, suggesting they could play similar roles in hPSCs. I
further demonstrate that CSE-bearing hPSCs produce mostly viable progeny with seemingly
normal cell cycle dynamics, suggesting that progeny with genomic instability may evade cell
cycle checkpoint detection and propagate throughout cell populations. Moreover, I show that
hPSCs sometimes display central spindle defects, a potential cause for cytokinetic failure, and
describe attempts to visualise ‘live’ cytokinetic failure and binucleation as potential routes
to multipolar cell division. Strikingly, I demonstrate that CSEs persist and increase during
early differentiation, suggesting that the possible CSEs-associated tumourigenic potential
may endure beyond pluripotency into early differentiation, with implications for tissue design.
Finally, I show how a semi-automated image analysis pipeline can be used to detect and
characterise CSEs in a screening setting, enabling future large-scale screens to identify factors
controlling tumourigenic potential in hPSCs.
Date of Award7 May 2024
Original languageEnglish
Awarding Institution
  • University of Bristol
SponsorsAstra Zeneca Pharmaceuticals
SupervisorRafael E Carazo Salas (Supervisor)

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