Abstract
BackgroundCell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity.ResultsWe utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827.ConclusionsWe demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents.
Highlights
Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time
Doublestranded barcodes were cloned into the non-coding region of a self-inactivating lentiviral vector upstream of the enhanced green fluorescent protein transgene expressed from a ubiquitin C (UBC) promoter
As with the original K562 experiments, we saw only a small number of major clones that recurred in different biological replicates, and a number of major clones that were unique to a single population. These results indicate that the HeLa and HEK-293 T-cell lines, as with K562 cells, show significant clonal dynamics even under ideal culture conditions
Summary
Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Even under ideal growth conditions, cultured cells exhibit genetic heterogeneity. It is valuable, technically challenging, to track the behavior and interplay of clones within a cellular population. We aimed to develop a sensitive and quantitative method for tracking the clonal dynamics within populations of cells with minimal disruption to both individual cells and the population as a whole.
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