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

Abstract

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.

Figure 1. Outline of single-cell tracking analysis.

(a,b) Representative images of Time point 1 (a) and Time point 850 (b) are shown. Images acquired from each FOV were stitched together and every cell (progenitor) recorded in the image of Time point 1 was numbered (a). Cell numbers were assigned all progeny cells produced from progenitors (b). A progenitor and its progeny cells were identified with a unique colors (a,b). (c) Still images shown in (c) illustrate how cell tracking was performed. The times and the types of all cellular events occur to progenitors and the progeny cells were recorded from Time point 1 till the end of the movie (typically Time point 850–950). For example, a cell (C14) identified at Time point 223 underwent TD at Time point 224, producing 3 progeny cells, C15 (F1a), C16 (F1b) and C6 (F1c). Then, progeny cells produced by the TD were tracked, time points, at which cellular events occurred, and types of events were recorded until the end of the movie. In this example, at Time point 424, C6 (F1c) underwent DD, producing 2 progeny cells, C10 (F1c/F2a) and C4 (F1c/F2b). Those progeny cells continue to proliferate and some of those underwent two more cell divisions. (d) An example of cell-lineage map is shown. Expression levels of p14^ARF in progeny cells were included in the map. UD: Undetectable. DD: Dipolar cell division. TD: Tripolar cell division. CF: Cell fusion. CD: Cell death.

Figure 2. Population expansion curves.

The number of cells at each time point was determined by using data stored in the cell-lineage database to draw cell population expansion curves. Four independent experiments (experiment number 1, 2, 3 and 4) were performed. The initial number of cells was normalized to 100 cells. The analyses were performed with data of all cells recorded in the cell-lineage database (Whole) or with data of selected group of cells (Selected), which belong to Group d–f (see Table 1). The average cell numbers were calculated (Average, bold line). The means and SDs of the number of cells at Time point 200, 400, 600 and 780 were shown and population doubling times for Whole and Selected were indicated. The average coefficient of variation was 9.45 (Whole) and 8.43% (Selected), suggesting that reproducible and quantitative cell biological analysis with long-term live cell imaging can be performed on a microscope stage.

Figure 3. Cell doubling time.

Cell doubling time of each cell was determined using data stored in the cell-lineage database. (a) All cells recorded in the cell lineage database. (b) Cells, which underwent cell division between 0–66 h. (c) Cells, which underwent cell division between 66–130 h. Cell doubling times of Group a–f cells (see Table 1 for categorization) are shown. (d) Cells of Group a. (e) Cells of Group b. (f) Cells of Group c. (g) Cells of Group d. (h) Cells of Group e. (i) Cells of Group f. Average cell doubling times and SDs, which were calculated using nonlinear regression-Gaussian equation (GraphPad Prism 6), are shown.

Figure 4. Change in the composition of HeLa cell culture in the observation period of 130 h.

(a) Change in the composition of cells that belong to Group a–f (see Table 1) during the observation period of 130 h is shown. The total cell number at Time 1 is set as 100. The numbers at the left side of bars at 33, 66, 100 and 130 are the percentage of Group a+b, Group c and Group d–f, and the numbers in the parentheses is the number of cells. Passage of HeLa culture is generally performed 3–4 days (72–96 h) after cell plating (Passage). The line (light purple) indicates 96 h after cell plating. Because live cell imaging was started after 12 h of cell plating, 84 h of imaging is equivalent to 96 h after cell plating. (b) The composition of Group a–f cells at 96 h (84 h of imaging) is shown. (c) Frame (c) illustrates that HeLa cells show similar growth profile every after cell passage. The data shown in this frame is identical with that in frame (a).

Figure 5. An outline of in depth analysis of cell-lineage database.

(a,b) An example of in depth analysis of cell-lineage database is shown. (a) A progenitor cell (Pr-Progenitor, black arrow) of a cell lineage underwent the first cell division at the time point indicated by blue arrowhead. (b) When in silico synchronization of cell cycle was performed, the time when the Pr-Progenitor cell undergoes the first division is now defined as Time point 1 (blue arrowhead). Pr-progenitor cells were categorized by the number of surviving progeny cells found after 130 h of culture. Pr-progenitor cells produced 0, 1–4, 5–8, 9–12, 13–16, 17–20 and ≥21 surviving progeny were categorized as Group A, B, C, D, E, F and G, respectively. As the Pr-Progenitor cell produced 23 surviving progeny cells after the 130 h of culture (black line) from the first cell division, the Pr-Progenitor cell shown in (b) belongs to Group G and gave rise to 7 surviving progeny cells after 66 h of culture (blue line) from the first cell division. Its 4 granddaughter cells (GD-Progenitors) are indicated by green arrows and those cells undergo the first cell division at the time point indicated by red arrowheads. The GD-Progenitor cells gave rise to 3–7 surviving progeny cells after 66 h of culture (red line) from the first division.

Figure 6. In depth analysis of cell-lineage database to identify mortal and immortal cells.

(a) In depth analysis was performed on all cell lineages and the data were reassembled according to the groups (Group A–G) designated as Pr-Progenitor. The number of surviving progeny cells found after 66 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. The Pr-Progenitor cell shown in Fig. 5b is an example cells that belongs to Group G and, as the cell produced 7 surviving progeny cells after 66 h of culture, the example cell was categorized as a class, which produces ≥7 progeny cells (black column). (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 66 h of culture from the first cell division was determined and results were arranged based on the number of surviving progeny cells. The GD-Progenitor cell shown in Fig. 5b is an example belongs Group G and, as the cells produced 4–8 surviving progeny cells after 66 h of culture, the example cells were categorized as classes, which produce 4 and ≥7 progeny cells (white and black column). (a,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 and means and SD are shown (at the right side of blue box).

Figure 7. A model for the growth profile of HeLa cells.

(a) A classical model: Illustration for the assumption that cell lines comprise putatively homogeneous clonal cells. Although there are some variations in growth rate among cells and in phenotypic characteristics (illustrated by different gray colors), these cells are clonally expand every after cell passage. (b) A model proposed based on the analysis with the cell-lineage database: Small number of immortal cells play a role in maintaining HeLa cell line and produce mortal HeLa cell population. Mortal cells have reduced reproductive ability (groups with arrow) and eventually terminate their growth (block ends). Phenotypic changes also occur during HeLa cell growth (illustrated by different gray colors).