Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells

Abstract

Stem cell cytogenetic abnormalities constitute a roadblock to regenerative therapies. We investigated the possibility that reactive oxygen species (ROSs) influence genomic stability in cardiac and embryonic stem cells. Karyotypic abnormalities in primary human cardiac stem cells were suppressed by culture in physiological (5%) oxygen, but addition of antioxidants to the medium unexpectedly increased aneuploidy. Intracellular ROS levels were moderately decreased in physiological oxygen, but dramatically decreased by the addition of high-dose antioxidants. Quantification of DNA damage in cardiac stem cells and in human embryonic stem cells revealed a biphasic dose-dependence: antioxidants suppressed DNA damage at low concentrations, but potentiated such damage at higher concentrations. High-dose antioxidants decreased cellular levels of ATM (ataxia-telangiectasia mutated) and other DNA repair enzymes, providing a potential mechanistic basis for the observed effects. These results indicate that physiological levels of intracellular ROS are required to activate the DNA repair pathway for maintaining genomic stability in stem cells. The concept of an "oxidative optimum" for genomic stability has broad implications for stem cell biology and carcinogenesis.

Figure 1. Karyotyping data of human cardiosphere-derived cells by G-banding analysis after culture under different conditions

Each bar represents a histogram of one sample of stem cells; blue denotes cells with a normal karyotype. Compared with culture in traditional 20% O₂ incubator (95% room air/5% CO₂), the number of cells with DNA breaks or translocations (colored green) and losses or gains of chromosomes (red) was decreased by culture under 5% O₂ (p=0.007), but increased by culture in traditional 20% O₂ incubator with the addition of a commercial antioxidant supplement in 1000-fold dilution (Antioxi. A) or a homemade antioxidant cocktail at 100 μM (Antioxi. B) (p<0.001).

Figure 2. Intracellular ROS levels in human cardiosphere-derived cells after 24 hours culture under 20% O₂ with different concentrations of antioxidants, catalase, and H₂O₂

The same cells were initially used and maintained in traditional 5% CO₂/20% O₂ culture condition. Intracellular ROS levels in cells cultured in 96-well plates were measured by the fluorescence intensity after staining with DCF-DA for 60 minutes using a multiple counter. The intracellular ROS levels were decreased with the addition of 100–1,000,000-fold diluted antioxidant supplement (A), 0.1–1000 μM homemade antioxidant cocktail (B), and 0.1–1000 unit/ml catalase (C), but increased with the addition of 0.1–1000 μM H₂O₂ (D), in a dose-dependent manner. The “0” in each group test the baseline level of ROS. Compared to baseline, the DCF fluorescence was decreased by antioxidants (A–C), but increased by oxidative stress stimulator of H₂O₂. Results are means and s.d. for six separated experiments by using different twice-passaged CDCs. a.u.: arbitrary units. * p<0.01, † p<0.05 vs the baseline levels.

Figure 3. Intracellular ROS concentration in human cardiosphere-derived cells with long-term culture under 20% O₂, 20% O₂, or under 20% O₂ with added antioxidants

A. representative histograms show that the intracellular ROS was lower in cells cultured under 5% O₂ than under 20% O₂. Intracellular ROS was decreased to very low levels by the addition of 1000-fold diluted antioxidant supplement (Antioxi. A) or 100 μM homemade antioxidant cocktail (Antioxi. B). B. DCF fluorescence intensity measured by a multiple counter also showed a significant decrease of intracellular ROS levels in cells cultured under 5% O₂, which was decreased even further by the addition of antioxidants. C. Representative images of γ-H2AX foci in CDCs (arrows) cultured for long-term under different conditions. D. Compared to traditional 20% O₂ culture, quantitative data showed that γ-H2AX foci in CDCs was significantly decreased in 5% O₂ culture, but increased by the supplement with antioxidants. * p<0.01 vs other groups, † p<0.01 vs 5% O₂ and 20% O₂.

Figure 4. DNA damage in human cardiosphere-derived cells and human embryonic stem cells after 24 hours culture under 20% O₂ with antioxidants, catalase, and H₂O₂

DNA damage was evaluated by the formation of γ-H2AX foci, a marker of DNA double-strand breaks, in cells using immunostaining analysis. A. The percentages of CDCs with γ-H2AX foci (bar graph) were decreased at low doses (10,000–100,000-fold dilution), but increased by high doses (100–1000-fold dilution) of antioxidant supplement (Antioxidant A). Similar results were also observed in ES cells (red crosses and line). B. The percentages of CDCs with γ-H2AX foci (bar graph) were decreased at low doses (1–20 μM), but conversely increased by high doses (100–1000 μM) of homemade antioxidant cocktail (Antioxidant B). Similar results were also observed in human ES cells (red crosses and line). C. The percentages of CDCS (bar graph) and ES cells (red crosses and line) with γ-H2AX foci were decreased at low doses (1–10 units/ml), but increased at high doses (100–1000 units/ml) of catalase. D. The percentages of CDCs (bar graph) and ES cells (red crosses and line) with γ-H2AX foci were increased by H₂O₂, in a dose-dependent manner. Results are means and s.d. for six separate experiments using different twice-passaged CDCs.

Figure 5. ATM protein levels in human cardiosphere-derived cells after 24 hours culture under 20% O₂ with antioxidants, catalase, and H₂O₂

A. The protein levels of ATM in CDCs were decreased at high doses (≤1000-fold dilution), but not at low doses (≥10,000-fold dilution) of antioxidant supplement (Antioxidant A). B. ATM in CDCs decreases at ≥100 μM, but not at ≤10 μM, of homemade antioxidant cocktail (Antioxidant B). C. Catalase also decreases ATM protein level in CDCs at ≥100 units/ml, but not at ≤10 units/ml. D. The expression of ATM in CDCs was slightly increased at a high dose (≥100 μM) of H₂O₂. Quantitative data are means and s.d. for four separate experiments using different twice-passaged CDCs.

Figure 6. Expressions of DNA repair-related factors in human cardiosphere-derived cells with 1–2 months long-term culture under different conditions

A. Compared with cells cultured under traditional 20% O₂, ATM expression did not obviously change in cells cultured under 5% O₂, but was significantly decreased by the addition of 1000-fold diluted antioxidant supplement (Antioxi. A) or 100 μM homemade antioxidant cocktail (Antioxi. B). B. Compared with cells cultured under traditional 20% O₂, the expression of ATR, Rad50, Rad51, Chk 1, and Chk2 did not obviously change in cells cultured under 5% O₂. However, many of these factors were decreased in cells cultured under traditional 20% O₂ with the addition of 1000-fold diluted antioxidant supplement (Antioxi. A) or 100 μM homemade antioxidant cocktail (Antioxi. B).

Figure 7. Schematic of our hypothesis regarding the links between intracellular ROS levels and genomic stability

Left panels: Excessive suppression of ROS undermines DNA repair pathways in a novel manifestation of reductive stress. Center panels: Optimal ROS levels maintain competent DNA repair and minimize DNA damage. Right panels: Excessive ROS levels lead to DNA damage by conventional oxidative injury.