Heme oxygenase-1 deficiency promotes embryonic stem cell differentiation

[[abstract]]Pluripotent embryonic stem cells (ESCs) are promising donor sources in cell therapies for various diseases. Many signals including cytokines, growth factors, and oxidative stress influence ESC survival and differentiation. The stress response protein heme oxygenase-1 (HO-1) has been shown to protect tissues from injury via its antioxidative and anti-inflammatory functions. Given that ESCs are highly proficient in antioxidant defense, we hypothesized that HO-1 might contribute to ESCs' antioxidant defense and differentiation. To test this hypothesis, we isolated HO-1-deficient (HO-1–/–) ESCs from mouse blastocysts. As with D3 wild type ESCs, HO-1–/– ESCs exhibit high levels of pluripotent markers Oct3/4 and SSEA-1, strong alkaline phosphatase activity, and low levels of reactive oxygen species (ROS). Since elevated levels of ROS can initiate differentiation, we used an embryoid body (EB) model and harvested EBs at day 3, 5, and 7 to first examine ROS levels. ROS levels increased during ESC differentiation, reaching maximum levels at day 5. However, HO-1 deficiency enhanced ROS levels at day 3. We next examined differentiation marker gene expression by quantitative RT-PCR. The endoderm marker GATA6 increased during the course of differentiation; interestingly, HO-1 deficiency enhanced GATA6 levels by 9-fold at day 3. Furthermore, the mesoderm marker Brachyury was 50-fold higher than D3 at day 3. However, no difference was observed in ectoderm marker FGF5 between D3 and HO-1–/– ESCs at day 3. Collectively, these results suggest that HO-1 might modulate ESC differentiation, particularly endoderm and mesoderm, via regulating cellular ROS levels