Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line

Abstract

Cancer stem cells resemble normal tissue-specific stem cells in many aspects, such as self-renewal and plasticity. Like their non-malignant counterparts, cancer stem cells are suggested to exhibit a relative quiescence. The established cancer cell lines reportedly harbor slow-proliferating cells that are positive for some cancer stem cells markers. However, the fate of these cells and their progeny remains unknown. We used time-lapse microscopy and the contrast-based segmentation algorithm to identify and monitor actively dividing and non-dividing cells in human osteosarcoma MG-63 cell line. Within the monitored field of view the non-dividing cells were represented by three cells that never divided, and one cell that attempted to divide, but failed cytokinesis, and later, after significantly prolonged division, produced the progeny with enlarged segmented nuclei, thus pointing to a possible mitotic catastrophe. Together, these cells initially constituted about 6.2% of the total number of seeded cells, yet only 0.02% of all cells at the end of the observation period when cells became confluent. Non-dividing cells were characterized by rounded shape, dark nuclei, random cytoplasmic streaming and subtle oscillatory movement, however, they did not migrate and rarely formed cell-cell contacts as compared to actively dividing cells. Our data indicate that the observed non-dividing MG-63 cells do not have a growth advantage over other cells and, therefore, they do not contribute to the cancer stem cells pool.

Keywords: Non-dividing; aneuploidy; cancer; cell-cell interaction; quiescent; stem cells; time-lapse microscopy.

Figure 1.

Actively dividing (AD) and non-dividing (ND) MG-63 cells. A, C – the regular view, and B, D – the tracking view mode. A, B – the image taken at 2 min (the starting culture), C, D – the image taken at 124 hours and 52 min (full confluence). Scale bars – 200 µm. See also Supplementary Data.

Figure 2.

Failure of cytokinesis. A: Non-dividing cell #1 (ND1) is starting to divide (14 h 58 min); B: ND1 cell has divided without cytoplasmic abscission (15 h 30 min); C: Division is aborted, two daughter cells merged into the single one (15 h 56 min); D: ND1 cell resumes cytokinesis (23 h 20 min); E: ND1 in the process of division (cytokinesis) without cytoplasmic abscission. Arrows point to the nuclear segments, which may represent either several nuclei, or the single multi-lobed nucleus (around 56 hours and 40 min); F: Completion of ND1 cell division by 75 h 46 min. Scale bars – 25 µm. See also Supplementary Data.

Figure 3.

Motility and morphology of non-dividing cells. A, B – 8 hours, C, D – 40 hours, E, F – 80 hours, and G, H – 92 hours and 6 min of time-lapse microscopic observation. Red circles indicate non-dividing cells, which did not migrate and maintained their morphology as rounded with dark nuclei. Scale bars – 200 µm. See also Supplementary Data.

Figure 4.

Cell-cell contacts. A, B: Red circles show non-dividing (ND3 and ND4) cells, white arrow indicates the actively dividing (AD7) cell. C: The number of observed cell-cell contacts in the early phase (before 70 h 50 min); D: 3 h 32 min, no cell-cell contacts; E: 3 h 34 min, cell-cell contact between AD7 and the other cultured cell (OC); F: 3 h 44 min, interaction of ND4 and AD7 cells with the OC; G: 5 h 08 min, interaction of ND3 cell with the OC; H: 70 h 52 min, prolonged interaction of the ND4 cell with the progeny of AD7 cell, which was lasting for 38 min. White arrows point to the sites of cell-cell interaction. Scale bars: A – 200 µm, B-H – 50 µm. See also Supplementary data.