**4. Accelerated senescence of human fibroblasts induces senescing tetraploid cells with transient self-renewal potential**

In one of our recent experimental systems, embryonal lung human fibroblasts (IMR90 cells) were grown in normoxia (20%) and 5% CO2 and reached full senescence (proliferative arrest with zero mitotic index) after 32–34 passages. Senescence was characterized by flat morphology of enlarged cells, nuclear positivity of p21CIP1 and cytoplasmic accumulation of p16INK4A; at the terminally stage nuclei were swelling and p16 entered the cell nuclei [43]. DNA cytometry revealed an accumulation of a portion of the prematurely senescing cells in the G2 compartment (**Figure 1A, B**) which were also overcoming the tetraploidy barrier with formation of a few (4–6%) tetraploid cells, which sometimes entered aberrant mitoses. These cells with large polyploid nuclei began to express both senescence markers (p21CIP1 and p16INK4A) with the self-renewal marker NANOG; these cells were also positive for DNA double strand breaks (DSB) (**Figure 1C–F**). The acquisition of bi-potentiality by a small proportion of normal

**Figure 1.** Characteristics of IMR90 human embryonal fibroblasts undergoing pre-senescence. Cells were cytospun, fixed and stained for DNA image cytometry. DNA content was determined for at least 200 cells in each condition and is represented as the percentage in a state of (**A**) logarithmic growth; (**B**) presenescence with some accumulation of cells in the G2M (4C) checkpoint and some increase of >4C DNA cell numbers. (**C**) shows the results from immunofluorescence studies staining for γ-H2AX, NANOG, P21CIP1 or p16INKA4A proteins with positive staining discriminated with respect to nuclei size; (**D**–**F**) – typical immunofluorescence patterns showing combinations of self-renewal (NANOG) and senescence (p21CIP1 and p16INK4A) markers in the same cells with large (>4C nuclei as measured by DAPI) nuclei, and positivity for DSBs γ-H2AX. Bars= 10 μm. Republished from Ref. [43].

cells overcoming the G2M DNA damage checkpoint is reminiscent of stem cell activity; with the same lack of arrest in the G1/S checkpoint and weak G2M checkpoint. Nevertheless, these cells did not persist in the culture and NANOG expression was lost in later passages.

These observations heightened our interest in the role of senescence in the CSC model.

with zero mitotic index) after 32–34 passages. Senescence was characterized by flat morphology of enlarged cells, nuclear positivity of p21CIP1 and cytoplasmic accumulation of p16INK4A; at the terminally stage nuclei were swelling and p16 entered the cell nuclei [43]. DNA cytometry revealed an accumulation of a portion of the prematurely senescing cells in the G2 compartment (**Figure 1A, B**) which were also overcoming the tetraploidy barrier with formation of a few (4–6%) tetraploid cells, which sometimes entered aberrant mitoses. These cells with large polyploid nuclei began to express both senescence markers (p21CIP1 and p16INK4A) with the self-renewal marker NANOG; these cells were also positive for DNA double strand breaks (DSB) (**Figure 1C–F**). The acquisition of bi-potentiality by a small proportion of normal

48 Senescence - Physiology or Pathology

**Figure 1.** Characteristics of IMR90 human embryonal fibroblasts undergoing pre-senescence. Cells were cytospun, fixed and stained for DNA image cytometry. DNA content was determined for at least 200 cells in each condition and is represented as the percentage in a state of (**A**) logarithmic growth; (**B**) presenescence with some accumulation of cells in the G2M (4C) checkpoint and some increase of >4C DNA cell numbers. (**C**) shows the results from immunofluorescence studies staining for γ-H2AX, NANOG, P21CIP1 or p16INKA4A proteins with positive staining discriminated with respect to nuclei size; (**D**–**F**) – typical immunofluorescence patterns showing combinations of self-renewal (NANOG) and senescence (p21CIP1 and p16INK4A) markers in the same cells with large (>4C nuclei as measured by DAPI) nuclei,

and positivity for DSBs γ-H2AX. Bars= 10 μm. Republished from Ref. [43].
