Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells.


Titles and Legends for Supplementary Figures
Supplementary Figure S1 Microscope setting for single-cell lineage tracking analysis An 8-well chambered coverglass was placed on a microscope stage. DIC images were acquired using a 40x oil objective every 10 min. In each well, a two dimensional image acquisition array (filed of views: FOVs) was made to cover the area of interest. This figure illustrates an example of the movement of microscope stage. For example, the stage moves from FOV1 of well 1 (W1) to FOV15 of well 8 (W8).  Table 1 for categorization). c. Correlation between the doubling time of individual cells and the number of surviving progeny at 130 h was determined. Prism 6 was used to calculate r and p values. Means and SDs are shown.

Supplementary Figure S7 In depth analysis of cell-lineage database to identify mortal and immortal cells using 58 h after the first cell division of Pr-Progenitors and GD-progenitors, and to analyze cells that underwent MD, CF and/or cell death
In silico synchronization analysis of cell cycle was performed and data was arranged as described in the legend of Fig. 5. a. The number of surviving progeny cells found after 58 h of culture from the first cell division of Pr-Progenitor cells was determined and results were arranged based on the number of surviving progeny. b. The data were reassembled according to the groups (Group A-G) as in a. The number of surviving progeny cells of GD-progenitor cells found after 58 h of culture from the first cell division was determined and results were arranged based on the number of surviving progeny cells. a and b. The numbers shown in each column are the average number of progeny cells. In Group G column, Pr-Progenitors or GD-Progenitors, which produced ≥ 7 surviving progeny cells, are highlighted by blue column. The percentages of those cells within the entire cell population were calculated. Means and SD are shown (at the right side of blue box). c. The number of Pr-Progenitor and/or their progeny cells, which underwent MD and/or CF in the period of 66 h was determined. d. The number of Pr-Progenitor and/or their progeny cells, which underwent cell death in the period of 66 h was determined.
Supplementary Figure S8 The analysis of cells, which retain reproductive ability, and of cellular events that occurred prior to cell death a. The number of Group a-e and Group f cells at each time point was determined. Cells, which retained reproductive ability, were selected (cells that were capable to undergo DD). The percentages of Group a-e and Group f are shown. b. The number of cellular events occurred prior to cell death was determined. DD: Dipolar cell division. MD: Multipolar cell division. CF: Cell fusion. M: Mitosis.

Supplementary Figure S9 Single-cell indirect immunofluorescence
After performing live cell imaging, cells were fixed and p14 ARF was stained using indirect immunofluorescence. Then, DIC (a) and fluorescent (b) images of cells were acquired. The nucleus of each cell was identified (a) and the expression levels of p14 ARF in each nucleus were determined (b).

Supplementary Figure S10 Change in expression of p14 ARF in HeLa S3 cells
The expression levels of p14 ARF in progeny are indicated at the end of lineage lines (color coded bars). The values at the right side of the bars represent an average of relative expression level of p14 ARF . The white arrow indicates a DD (dipolar cell division) where the change in expression of p14 ARF would occur. HLCONT-21 (a), HLCONT-76 (b) and HLCONT-37 (c) were analyzed.

Supplementary Movie S1 Growth of HeLa cells on microscope stage
The movie is composed of 963 frames (160.5 h of live cell imaging).

Supplementary Movie S2 Tripolar cell division (TD)
Representative movie of TD is shown. Cell No. C2 underwent TD.