Investigating the causes and consequences of mitotic errors in human pluripotent stem cells - implications for cell therapy
G Gelezauskaite(1) S E McClelland(3) M Fellows(2) I Barbaric(1)
1:The University of Sheffield; 2:AstraZeneca; 3:Barts Cancer Institute QMUL
For human pluripotent stem cell (hPSC)-derived therapy to enter routine clinical use, it must be both efficacious and safe. However, during in vitro expansion, hPSCs can acquire recurrent, chromosomal abnormalities, which result in altered cell behaviour, such as increased proliferation and/or reduced propensity for differentiation, akin to cancerous cells. Chromosomally abnormal variants raise concerns surrounding tumorigenicity, and the ability to derive functional cell types for clinical use. As such, it is vital to understand the mechanisms driving variant hPSCs, to eliminate them as a therapy risk.
One likely mechanism of variant hPSC emergence is through errors in mitosis. Recent work by Barbaric and Godek labs have identified an increased mitotic error rate in hPSCs compared to somatic and differentiated cells. This suggests that the pluripotent state is associated with an impaired spindle assembly checkpoint and/or altered propensity for aneuploidy, which may be critical for variant formation. Nevertheless, the mechanisms and consequences of mitotic errors in hPSCs have not been fully explored. In this project, we have adopted chemical and genetic (CRISPR/dCas9) manipulation to experimentally induce chromosome mis-segregation in pluripotent and isogenic differentiated cells, to study how cells respond to mitotic errors and understand how aneuploid variants arise in culture. We utilise high-content microscopy to track mis-segregating cell fate, and have successfully implemented the aforementioned techniques to derive novel aneuploid hPSC lines, to study the safety implications of chromosome-specific aneuploidies. Finally, we assess the potential bias of chromosome mis-segregation in PSCs, to further elucidate the importance of chromosome identity upon cell division.