Establishing a High-Content Microscopy Workflow to Study Human Pluripotent Stem Cell Biology Quantitatively in Vitro

Abstract

The discovery and use of human pluripotent stem cell (hPSCs) technologies has revolutionised our approach to regenerative medicine. However, for hPSC derived tissues to become a robust and safe therapeutic reality, key issues must be solved. In particular, we need to better understand why only certain subsets of cells efficiently follow instructive orders to become an intended target tissue, and why hPSC derived cells and tissues often harbour tumourigenic potential. Such aspects of cell fate choice can likely only be understood by visualising how single-cells and cell populations make cell proliferation and fate decisions in real time using 'live' imaging approaches. Such approaches have mostly been lacking. The aim of this project is to establish a live high-content microscopy workflow to visualise and investigate hPSC proliferation and cell biology quantitatively in vitro. First, we approached the methodological issues by establishing high-content microscopy and computational pipelines, capable of imaging and tracking colonies of live hESCs over multiple days. Second, we approached the biological issues by assessing hPSC proliferation with the FUCCI reporter system, to study the live hPSC cell cycle dynamics of individual cells and colonies. To probe the capabilities of this approach to reveal new biology, we used our automated pipelines and tracked colonies of human embryonic stem cells (hESCs) across multiple days. Strikingly, we found that ~50% of colonies displayed apparent cell cycle synchronisation among their cells. When plotted for individual colonies, the proportion of hPSCs displaying each FUCCI colour oscillated, a phenomenon dubbed as a 'cell cycle wave'. These waves had a period of ~15 hours, comparable to the cell cycle length of hPSCs. This contradicts the notion that hPSC divisions are not synchronised unless directly driven to do so, a notion born of bulk, single-time point data. Cell cycle synchronisation persists for many days and is independent of cell-culture substrate or medium. The cell cycle waves could be observed in hESCs and hiPSCs but not reliably in HeLa or HEK293 cells. Furthermore, the degree of synchronisation positively correlated with levels of OCT4 expression, and persisted into early neuroectodermal differentiation, suggesting possible roles for synchrony in proliferation and differentiation regulation, laying the groundwork for future studies in those directions. Finally, we established a quantitative pipeline to perform chemical screens to study the control of this cell cycle synchronisation in hESCs, and as a proof of principle, we tested several chemical compounds which targeted factors that could have be involved in the control of cell cycle synchronisation. In doing so, we gained preliminary evidence that elements of the circadian clock, WNT signalling and CX-45/-45 may help maintain the synchronisation. Such an approach could be used in the future to mechanistically dissect the origin, maintenance and impact of cell cycle synchronisation on hPSC differentiation control.

Date of Award	28 Sept 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Rafael E Carazo Salas (Supervisor)