Monitoring the induction of ferroptosis following dissociation in human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) are vulnerable to cell death upon dissociation. Thus, dissociation is an obstacle in culturing, maintaining, and differentiating of hESCs. To date, apoptosis has become the focus of research into the nature of cell death triggered by cellular detachment; it remains baffling whether another form of cell death can occur upon dissociation in hESCs. Here, we demonstrate that iron accumulation and subsequently lipid peroxidation are responsible for dissociation-mediated hESC death. Moreover, we found that a decrease of glutathione peroxidase 4 because of iron accumulation promotes ferroptosis. Inhibition of lipid peroxidation (ferrostatin-1) or chelating iron (deferoxamine) largely suppresses iron accumulation-induced ferroptosis in dissociated hESCs. The results show that P53 mediates the dissociation-induced ferroptosis in hESCs, which is suppressed by pifithrin α. Multiple genes involved in ferroptosis are regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2). In this study, solute carrier family 7 member 11 and glutathione peroxidase 4 are involved in GSH synthesis decreased upon dissociation as a target of Nrf2. In conclusion, our study demonstrates that iron accumulation as a consequence of cytoskeleton disruption appears as a pivotal factor in the initiation of ferroptosis in dissociated hESCs. Nrf2 inhibits ferroptosis via its downstream targets. Our study suggests that the antiferroptotic target might be a good candidate for the maintenance of hESCs.

Keywords: Nrf2; ferroptosis; glutathione peroxidase; human embryonic stem cells; iron.

Figure 1

The dissociated single hESCs undergo iron-dependent cell death.A, the cellular iron content of hESCs was measured in the colonies and dissociated hESCs, increasing intracellular iron level in a time-dependent manner. B, MDA assay was used to quantify lipid peroxidation in the colonies and dissociated hESCs, increasing MDA level in a time-dependent fashion. C, GPX4 enzyme activity was assayed in the colonies and dissociated hESCs. D, GPX4 mRNA expression between individualized and colonies from hESCs was quantified by qRT–PCR. E, GPX4 protein expression was measured using Western blot. F, densitometric analysis of GPX4 protein levels; immunoblotting was performed 24 h after dissociation of hESCs. All data are shown as mean ± SEM (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 in comparison to the colony group. GPX4, glutathione peroxidase 4; hESC, human embryonic stem cell; MDA, malondialdehyde; qRT–PCR, quantitative RT–PCR.

Figure 2

Ferroptosis inhibitors promote hESC survival and proliferation.A, diagram of MTS assay. B, phase images of hESCs treated with Y-27632, or 1, 10, and 100 μM DFO in combination with Y-27632 or 2, 20, and 100 μM fer-1 in combination with Y-27632. The scale bar represents 200 μm. C, viability of hESCs exposed to (1, 10, and 100 μM) DFO in the presence of Y-27632 or (2, 20, and 100 μM) fer-1 in combination with Y-7632 was analyzed using MTS assay. D, graphic quantification of crystal violet staining of hESC colonies showing dramatic differences in colony formation in the presence of (1 μM) DFO along with Y-27632 and (20 μM) fer-1 in combination with Y-27632 compared with Y-27632 treated alone. E, photograph of alkaline-positive staining of hESCs exposed to (1 μM) DFO in the presence of Y-27632 or (20 μM) fer-1 in combination with Y-7632. F, the ratio of ALP-positive colonies produced per seeded hESCs was used to assess cloning efficiency. The optimum dose of DFO (1 M) or fer-1 (20 M) was employed in combination with Y-27632 for the first 24 h following culturing of dissociated single hESCs until colonies formed. The cells were cultured for 8 days. All data are shown as mean ± SEM (n = 3). †p < 0.05, ††p < 0.01 compared with the Y-27632 group. ALP, alkaline phosphatase; DFO, deferoxamine; Fer-1, ferrostatin-1; hESC, human embryonic stem cell; MTS, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 3

Ferroptosis inhibitors decreased dissociation-induced iron overloading in hESCs.A, schematic figure showing the ferroptosis pathway and the functions of relevant inhibitors. B, intracellular iron level of hESCs treated with 1 μM of DFO or 20 μM of fer-1 along with Y-27632 for 4 h. C, MDA assay was used to detect lipid peroxidation in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632. D, GPX4 enzyme activity was assayed in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632 in dissociated hESCs. All data are shown as mean ± SEM (n = 3). ∗p < 0.05, ∗∗∗p < 0.001 compared with the untreated group and #p < 0.05, ##p < 0.01 compared with colonies. DFO, deferoxamine; Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; hESC, human embryonic stem cell; MDA, malondialdehyde.

Figure 4

p53 stimulates the dissociation-induced ferroptosis in hESCs.A, schematic figure showing p53-mediated activation of ferroptosis. B, p53 and SLC7A11 mRNA expression was determined by qRT–PCR in dissociated hESCs and colony culture. C, Western blot analysis of p53 expression in dissociated hESCs time-dependently. D, densitometric analysis of p53 protein levels; immunoblotting was performed 0–4 h after dissociation of hESCs. E, Western blot analysis of p53 expression in hESCs treated with Pft-α (10 μM). Dissociated cells were treated with Pft-α (10 μM) for 2 h and then dissociated into single cells and cultured for 1 h. F, densitometric analysis of p53 protein levels. G, Western blot analysis of xCT and GPX4 expression in hESCs treated with Pft-α (10 μM). H, densitometric analysis of xCT and GPX4 protein levels. I, mRNA expression of p53 was assessed by qRT–PCR in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632 in dissociated hESCs. GAPDH was used as a loading control. J, Western blot analysis of p53 expression in hESCs treated with DFO (1 μM) or fer-1 (20 μM) in combination with Y-27632. K, densitometric analysis of p53 protein levels; immunoblotting was performed 4 h after dissociation of hESCs. β-actin was used as an internal control. All the experiments were done 4 h after the dissociation of hESCs. All data are shown as mean ± SEM (n = 3). ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 compared with the untreated group and, ####p < 0.0001 in comparison to colonies. ††††p < 0.0001 compared with 0 h. $$p < 0.01 compared with 1 h. Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; hESC, human embryonic stem cell; Pft-α, pifithrin α; qRT–PCR, quantitative RT–PCR; xCT, the cystine–glutamate antiporter.

Figure 5

Nrf2 and its downstream targets (SLC7A11 and GPX4) promote the resistance of hESCs to ferroptosis in the dissociated single hESCs.A, schematic figure showing the role of Nrf2 in GSH synthesis through its target genes. B, GSH content was measured in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632 in dissociated hESCs. C, qRT–PCR was applied to measure Nrf2 mRNA expression in dissociated hESCs and colonies culture. D, Nrf2 mRNA expression was assessed by qRT–PCR in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632 in dissociated hESCs. E, expression of SLC7A11 was assessed by qRT–PCR in the presence of DFO (1 μM) or fer-1 (20 μM) along with Y-27632 in dissociated hESCs. F, qRT–PCR was applied to analyze GPX4 expression in the presence of DFO (1 μM) or fer-1 (20 μM) in combination with Y-27632 in dissociated hESCs. GAPDH was used as a loading control. G, Western blot analysis of Nrf2, xCT, and GPX4 expression in hESCs treated with DFO (1 μM) or fer-1 (20 μM) along with Y-27632. β-actin was used as an internal control. All the experiments were performed 4 h after the dissociation of hESCs. H, densitometric analysis of Nrf2, SLC7A11, and GPX4 protein levels; immunoblotting was performed 4 h after dissociation of hESCs. All data are shown as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 compared with the Y-27632 group and ###p < 0.001 compared with colonies. DFO, deferoxamine; fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; hESC, human embryonic stem cell; Nrf2, nuclear factor erythroid 2–related factor 2; qRT–PCR, quantitative RT–PCR; xCT, the cystine–glutamate antiporter.

Figure 6

Overview of cell death in dissociated hESCs. After hESC dissociation, the Rho/ROCK signaling pathway is activated, which leads to anoikis. Inhibition of Rho/ROCK signaling via Y-27632 suppresses hESC death; however, cloning efficiency is still low in many cases. Moreover, dissociation results in cytoskeleton perturbation leading to iron accumulation, which forms hydroxyl/peroxyl radicals via the Fenton reaction. Hydrogen atoms from polyunsaturated fatty acids (PUFAs) combine to form a lipid radical, which interacts quickly with oxygen to form lipid peroxide. This process can be inhibited by ferrostatin-1 (fer-1). Lipid peroxides can be degraded into reactive aldehydes, such as malondialdehyde (MDA). Decreased iron overload by iron chelators such as deferoxamine (DFO) inhibits dissociation-induced ferroptosis in hESCs. Activation of p53 is required for ferroptosis in dissociated hESCs. Direct transcriptional suppression of SLC7A11, a major component of system XC⁻, is required for ferroptosis. The xCT antiporter (consisting of two subunits SLC7A11 and SLC3A2) is responsible for maintaining redox homeostasis through importing cystine, subsequently GSH synthesis. Reduced GSH is converted to oxidized GSH by glutathione peroxidase 4 (GPX4), which also reduces lipid hydroperoxides to their corresponding alcohols or free hydrogen peroxide to water. Through the elevation of SLC7A11 and GPX4 expression, which enables cystine intracellular uptake, Nrf2 is thought to be a critical negative regulator of ferroptosis. hESC, human embryonic stem cell; Nrf2, nuclear factor erythroid 2–related factor 2; ROCK, Rho kinase.