Uncovering the Metabolic and Stress Responses of Human Embryonic Stem Cells to FTH1 Gene Silencing

Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs’ response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.


Introduction
An impaired intracellular balance between reactive oxygen species (ROS) production and clearance is responsible for establishing an oxidative stress status, eventually leading to lethal consequences [1] for the cells. ROS are able to elicit a wide spectrum of responses; slight variations in their concentration may produce profound and opposite effects in distinct cell types [2]. High levels of ROS, predominantly produced by mitochondrial respiration, are classically linked to cellular damage, DNA damage, and apoptosis. At the same time, a large body of data recognizes ROS as signaling messengers involved in many biological processes, including DNA synthesis, modulation of gene expression, cellular respiration, protein-protein interactions [3], as well as activation of critical pathways such as protein kinase B (AKT) and NF-κB signaling [4]. In hESCs, constant exposure to high levels of ROS was shown to induce cell cycle arrest and apoptosis [5]; however, by regulating the redox state, ROS also target important aspects of pluripotency, such as metabolism, clear evidence of an overactivation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway, which emerges as the major player and safeguard against iron-mediated oxidative stress in response to downregulation of ferritin expression. From a metabolic point of view, we also identified pentose phosphate pathway (PPP) activation as the primary metabolic response to maintain the redox status in hESCs.

Lentiviral Production and Generation of Stable Knock-Down ESCs
Lentiviral particles were produced in 293T cells following the standard procedure. Briefly, sub-confluent 293T cells were transfected with shFTH1 or shSCR control lentiviral vector plasmids (Sigma-Aldrich, St. Louis, MO, USA) along with pCMV-DR9-9 and pCMV-VSVG plasmids (Sigma-Aldrich). After 48 h, supernatants were harvested, filtered through a 0.45 µM filter, and used for ESCs infection, previously seeded in a 6-well plate at a cell density of 2 × 105 cells/well. Infected cells were incubated with shFTH1 and shSCR control lentiviral particles [Multiplicity of Infection (MOI) = 10] in the presence of 8 µg/mL of Polybrene (Sigma-Aldrich) using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Two days after transduction, transduced cells were selected with puromycin (1 µg/mL) for 10 days. Knock-down efficiency was assessed via both Western blot and qRT-PCR analysis ( Figure S1).

RNA Isolation and Quantitative Real-Time PCR
Cell pellets were collected after 48 h of culture and total RNA was extracted with TRIzol Reagent (Thermo Fisher Scientific, 15596018) and reverse transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA, 4368814) according to manufacturer's instructions. Real-time quantitative PCR (qRT-PCR) was performed according to StepOnePlus system's protocol (Applied Biosystems) with SensiFAST SYBR Hi-ROX Kit (Meridian Bioscience, Cincinnati, OH, USA, BIO-92020). Ct values for each target gene were normalized to GAPDH Ct values. See Supplement Information Table S1 for the primer sequences used in this study.

Measurement of Intracellular Iron, Total ROS and Mitochondrial Superoxide Species
Intracellular iron concentration was measured using the Iron Assay kit (Sigma-Aldrich, MAK025) according to the manufacturer's instructions. Total intracytoplasmic ROS as well as specific mitochondrial superoxide species were assessed by flow cytometry using the fluorescent probes 2 -7 -Dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific) and MitoSOX™ Red Mitochondrial Superoxide Indicator, respectively. For the quantification of mitochondrial superoxide levels, cells were cultured for 72 h and then incubated with 2 mM MitoSOX™ Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) for 30 min at 37 • C. Intracellular ROS levels were instead assayed by staining the cells with 1.5 mM CM-H2DCFDA (Thermo Fisher Scientific) for 30 min at 37 • C. Fluorescence intensity was measured by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorimeter (BD Biosciences, Bedford, MA, USA). The results were analyzed with FlowJo software (Tree Star, Inc., Williamson Way Ashland, OR 97520, USA). Three independent experiments were conducted.

Analysis of Mitochondrial Membrane Potential (∆Ψ)
Mitochondrial membrane potential (∆Ψ) was measured using 0.1 µM ∆Ψ fluorescent indicator TMRM (tetramethylrhodamine methyl ester) (Thermo Fisher Scientific). Cells were harvested after 72 h of culture and TMRM fluorescence intensity was measured after 30 min of incubation at 37 • C by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorimeter (BD Biosciences). The results were analyzed with FlowJo software (Tree Star, Inc.). Three independent experiments were conducted.

Alkaline Phosphatase Assay
After 48 h of culture, cells were fixed with 3.7% formaldehyde (FA) solution (Sigma Aldrich, 252549-100ML) for 15 min at room temperature. Alkaline phosphatase assay was performed using 1-Step NBT/BCIP (Thermo Fisher Scientific, 34042) according to the manufacturer's instructions.

Metabolic and Bioenergetic Function Profile
Total ATP production rates in H9-shFTH1 and shSCR control cells were measured with the Agilent Seahorse analyzer using the XFp Real-Time Rate Assay Kit. Simultaneous quantification of ATP fractions produced by mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis were detected. For bioenergetics quantification, cells were dissociated into single cells with Accutase and seeded onto Agilent Seahorse XFp Cell Culture Miniplates at a cell density of 2 × 10 3 /well in brew medium supplemented with 10 µM Y27632 ROCK inhibitor. After 24 h, the growth medium was exchanged with unbuffered media and incubated for 30 min at 37 • C, allowing temperature and pH to reach their equilibria before starting the baseline measurements. Once the basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were obtained, cells were metabolically perturbed by the addition of three different compounds: oligomycin (1.5 µM), rotenone, and antimycin A (0.5 µM). Finally, the template was loaded into the Seahorse Analyzer for ATP measurement. The same procedure for cell preparation was used for the glycolytic rate assay and measurement performed according to the manufacturer's instruction. Rotenone/antimycin A were used to block mitochondrial activity while 2-DG was used to inhibit glycolysis. Results were uploaded to Agilent Seahorse Analytics to calculate parameters relative to ATP production and XF glycolytic rate assays. GraphPad Prism software was used to create graphs.

Apoptosis Analysis
For the identification of apoptotic cells, a double staining with Annexin V and PI was performed using Alexa Fluor ® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. 72 h after seeding, cells were harvested and stained with Annexin V and PI, alone and in combination. After an incubation of 15 min at room temperature, each tube was diluted with 400 µL of annexin binding buffer and cells were analyzed by flow cytometry using BD LSRFortessa TM X-20 cytofluorometer (BD Biosciences).

Statistics
All statistical data were generated from at least three independent biological replicates and are represented as mean ± standard error of the mean (SEM). Data were analyzed with PRISM version 7.0 (GraphPad Software Inc., San Diego, CA, USA). The significance of differences (p-value) was analyzed using Student's t-test and presented with the following levels of significance: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Effects of FTH1-Silencing on ESCs Pluripotency
Iron homeostasis is involved in many biological processes, including maintenance of pluripotency in human PSCs. Results from previous work found that depletion of intracellular iron is associated with impairment of pluripotency and self-renewal due to a significant reduction in NANOG expression [22]. Cells maintain a balanced iron pool and ferritin, as the major iron storage protein, plays a crucial role in this regulation. Therefore, to speculate on the role of ferritin in the context of hESCs, we first generated phosphatase (AP) activity was reduced in these cells ( Figure 1A). Nevertheless, FTH1-KD hESCs colonies were positive for pluripotency markers Oct4, TRA-1-60, and Sox2, while the expression of Nanog was slightly increased ( Figure 1B). This last finding was further confirmed by immunoblot analysis, demonstrating that Nanog expression is indeed higher in FTH1-silenced hESCs. ( Figure 1C and Supplementary Figure S2A). At the transcriptional level, NANOG, OCT4, and SOX2 resulted in evenly increased FTH1-KD hESCs ( Figure 1D). We further asked whether FTH1 knockdown could, either directly or indirectly, promote spontaneous differentiation of hESCs. Analysis of the transcript levels of AFP, Nkx2.5, and PAX6, specific to endoderm, mesoderm, and ectoderm germ layers, respectively, revealed an enhanced expression of these genes in FTH1-KD hESCs ( Figure 1E, left panel). Next, we induced both SCR control and FTH1-KD hESCs to differentiate using the embryoid bodies (EBs) formation assay (Supplementary Figure S2B). On day 28 of differentiation, EBs were harvested and analyzed for endodermal-(GATA4, FOXA2), mesodermal-(HAND1, CD31), and ectodermal-(NESTIN, PAX6) associated markers. Interestingly, while endodermal and mesodermal transcript levels resulted in upregulated FTH1-KD-derived EBs compared to control EBs, the ectodermal genes NESTIN and PAX6 were significantly downregulated in EBs derived from silenced cells ( Figure 1E, right panel). Immunofluorescence analysis for Sox17 (endodermal marker), Brachyury (mesodermal marker), and Nestin further confirmed that the percentage of Nestin + cells is lower in FTH1-silenced hESCs compared to SCR control ( Figure 1F and Supplementary Figure S2C). We do not have a clear-cut explanation for the contradictory findings reported here; we hypothesize that the initial response of ESCs to FTH1 gene silencing might be overcome during EB formation, in particular towards mesodermal and endodermal differentiation. The molecular mechanisms underlying this controversial phenomenon are still under deep investigation by our group.
significant reduction in NANOG expression [22]. Cells maintain a balanced iron pool and ferritin, as the major iron storage protein, plays a crucial role in this regulation. Therefore, to speculate on the role of ferritin in the context of hESCs, we first generated FTH1silenced cells (FTH1-KD) via lentiviral transfection with short hairpin RNA (shRNA), confirming the successful knock-down both at the mRNA and protein level of the transfected hESC (Supplementary Figure S1B-E). Morphologically, colonies originated from FTH1-KD hESCs were more flattened than those of shSCR control (Supplementary Figure S1A), and alkaline phosphatase (AP) activity was reduced in these cells ( Figure  1A). Nevertheless, FTH1-KD hESCs colonies were positive for pluripotency markers Oct4, TRA-1-60, and Sox2, while the expression of Nanog was slightly increased ( Figure 1B). This last finding was further confirmed by immunoblot analysis, demonstrating that Nanog expression is indeed higher in FTH1-silenced hESCs. ( Figure 1C and Supplementary Figure S2A). At the transcriptional level, NANOG, OCT4, and SOX2 resulted in evenly increased FTH1-KD hESCs ( Figure 1D). We further asked whether FTH1 knockdown could, either directly or indirectly, promote spontaneous differentiation of hESCs. Analysis of the transcript levels of AFP, Nkx2.5, and PAX6, specific to endoderm, mesoderm, and ectoderm germ layers, respectively, revealed an enhanced expression of these genes in FTH1-KD hESCs ( Figure 1E, left panel). Next, we induced both SCR control and FTH1-KD hESCs to differentiate using the embryoid bodies (EBs) formation assay (Supplementary Figure S2B). On day 28 of differentiation, EBs were harvested and analyzed for endodermal-(GATA4, FOXA2), mesodermal-(HAND1, CD31), and ectodermal-(NESTIN, PAX6) associated markers. Interestingly, while endodermal and mesodermal transcript levels resulted in upregulated FTH1-KD-derived EBs compared to control EBs, the ectodermal genes NESTIN and PAX6 were significantly downregulated in EBs derived from silenced cells ( Figure 1E, right panel). Immunofluorescence analysis for Sox17 (endodermal marker), Brachyury (mesodermal marker), and Nestin further confirmed that the percentage of Nestin + cells is lower in FTH1-silenced hESCs compared to SCR control ( Figure 1F and Supplementary Figure  S2C). We do not have a clear-cut explanation for the contradictory findings reported here; we hypothesize that the initial response of ESCs to FTH1 gene silencing might be overcome during EB formation, in particular towards mesodermal and endodermal differentiation. The molecular mechanisms underlying this controversial phenomenon are still under deep investigation by our group. (B) Undifferentiated SCR and FTH1-KD hESCs were stained for Oct4 (green), Nanog (red), TRA-1-60 (green), and Sox2 (red) expression, while nuclei were counterstained with 4 .6-diamidino-2-phenylindole (DAPI). Scale bars, 50 µm. No significant differences were detected between the two groups with the exception of Nanog, which shows a slight positivity in FTH1-silenced cells; (C) The levels of Nanog protein were assessed by immunoblotting (IB). Band intensity analysis shows a higher level in FTH1-KD cells compared to SCR control. Actin levels were evaluated to confirm equal loading control; Cells 2021, 10, 2431 7 of 18 (D) mRNA levels of pluripotency-associated genes NANOG, OCT4, and SOX2 were measured in undifferentiated SCR and FTH1-KD hESCs via qRT-PCR; (E) left panel: mRNA levels of endodermal (AFP), mesodermal (Nkx2.5), and ectodermal (PAX6) genes were measured in undifferentiated SCR and FTH1-silenced hESCs via qRT-PCR analysis; AFP and Nkx2.5 expression levels were higher in shFTH1 cells, while the expression levels of PAX6 were significantly diminished in silenced cells; (E) right panel: mRNA levels of endodermal (GATA4, FOXA2), mesodermal (HAND1, CD31), and ectodermal (NESTIN, PAX6) genes were measured in SCR and FTH1-KD embryoid bodies (EBs) on day 28 of differentiation by qRT-PCR; (F) Sox17, Brachyury, and Nestin, endodermal, mesodermal, and ectodermal markers, respectively, were stained via immunofluorescence in SCR and shFTH EBs on day 28 of differentiation; scale bar, 50 µm. Results of all qRT-PCR shown were normalized to value in SCR cells and are represented as mean ± SEM (n = 3); significance is calculated by t-test: * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001.

ROS Levels Are Reduced upon FTH1-Gene Silencing in hESCs
The role of ferritin in preventing iron-mediated oxidative stress has been reported in several studies [34]. Downregulation in FTH1 expression leads to excess labile iron, in turn promoting the formation of oxygen-derived free radicals [35,36]. To speculate on the effects of FTH1 silencing in hESCs, we measured the levels of intracellular iron in these cells; moreover, we performed a measurement of total and mitochondrial ROS levels together with an analysis of the mitochondrial membrane potential (∆ΨM) on three biological replicates of SCR vs. FTH1-KD hESCs, using DCF, MitoSox, and TMRM dyes, respectively (Supplementary Figure S3B Figure 2C). Altogether, these results led us to conclude that hESCs activate a powerful and effective antioxidant mechanism and are thus capable of protect themselves, at least to some extent, from shFTH1-mediated iron toxicity. Alkaline phosphatase (AP) staining of colonies in SCR control and in hESCs transfected with lentiviral shFTH1 shows that FTH1-silenced ES colonies contain less AP+ cells; scale bar, 250 µm; (B) Undifferentiated SCR and FTH1-KD hESCs were stained for Oct4 (green), Nanog (red), TRA-1-60 (green), and Sox2 (red) expression, while nuclei were counterstained with 4′.6-diamidino-2-phenylindole (DAPI). Scale bars, 50 µm. No significant differences were detected between the two groups with the exception of Nanog, which shows a slight positivity in FTH1-silenced cells; (C) The levels of Nanog protein were assessed by immunoblotting (IB). Band intensity analysis shows a higher level in FTH1-KD cells compared to SCR control. Actin levels were evaluated to confirm equal loading control; (D) mRNA levels of pluripotency-associated genes NANOG, OCT4, and SOX2 were measured in undifferentiated SCR and FTH1-KD hESCs via qRT-PCR; (E) left panel: mRNA levels of endodermal (AFP), mesodermal (Nkx2.5), and ectodermal (PAX6) genes were measured in undifferentiated SCR and FTH1-silenced hESCs via qRT-PCR analysis; AFP and Nkx2.5 expression levels were higher in shFTH1 cells, while the expression levels of PAX6 were significantly diminished in silenced cells; (E) right panel: mRNA levels of endodermal (GATA4,i FOXA2), mesodermal (HAND1, CD31), and ectodermal (NESTIN, PAX6) genes were measured in SCR and FTH1-KD embryoid bodies (EBs) on day 28 of differentiation by qRT-PCR; (F) Sox17, Brachyury, and Nestin, endodermal, mesodermal, and ectodermal markers, respectively, were stained via immunofluorescence in SCR and shFTH EBs on day 28 of differentiation; scale bar, 50 µm. Results of all qRT-PCR shown were normalized to value in SCR cells and are represented as mean ± SEM (n = 3); significance is calculated by t-test: * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001.

ROS Levels Are Reduced upon FTH1-Gene Silencing in hESCs
The role of ferritin in preventing iron-mediated oxidative stress has been reported in several studies [34]. Downregulation in FTH1 expression leads to excess labile iron, in turn promoting the formation of oxygen-derived free radicals [35,36]. To speculate on the effects of FTH1 silencing in hESCs, we measured the levels of intracellular iron in these cells; moreover, we performed a measurement of total and mitochondrial ROS levels together with an analysis of the mitochondrial membrane potential (ΔΨM) on three biological replicates of SCR vs. FTH1-KD hESCs, using DCF, MitoSox, and TMRM dyes, respectively (Supplementary Figure S3B Figure 2C). Altogether, these results led us to conclude that hESCs activate a powerful and effective antioxidant mechanism and are thus capable of protect themselves, at least to some extent, from shFTH1-mediated iron toxicity.  staining showed that total cellular ROS levels are reduced in FTH1-silenced hESCs (light blue curve and bar plot) with respect to control cells; (B) Mitochondrial superoxide was significantly lower in FTH1-silenced hESCs (red curve and bar plot); (C) FTH1-KD cells presented a decrease in mitochondrial membrane depolarization (green curve and bar plot) compared to SCR cells. All data are represented as mean ± SEM (n = 3); ** p ≤ 0.01, t-test. Small dots refer to SCR replicates, while squares refer to shFTH1 triplicates.

Nrf2 Signaling Pathway Drives the Antioxidant Response in hESCs
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor ubiquitously expressed in most eukaryotic cells. Nrf2-Keap1 (Kelch-like ECH-associated protein 1) signaling pathway was shown to play a central role in protecting cells against oxidative stress [37,38]. Under basal conditions, Nrf2 is bound to its negative regulator Keap1, which directs it to proteasomal degradation. In the presence of high levels of intracellular ROS, Keap1 is oxidized and releases Nrf2, which is free to translocate into the nucleus, where it binds to antioxidant response elements (AREs) and induces the expression of antioxidant target genes, such as HMOX1, NQO1, GST, and GPX [39]. Besides its role as a fine regulator of redox and metabolic homeostasis, Nrf2 also acts as a pluripotency master gene: Nrf2 activation in hESCs was shown to enhance NANOG transcriptional activity by delaying Nanog protein degradation through POMP-mediated proteasome ubiquitination [40,41]. In this study, we found that FTH1-KD hESCs express higher levels of Nrf2 transcript and protein, assessed via qRT-PCR ( Figure 3A), immunoblot ( Figure 3B and Supplementary Figure S4A), and immunofluorescence ( Figure 3C and Supplementary Figure S4F), respectively. Similarly, we could also observe an increased expression of p62/Sqstm1 ( Figure 3D and Supplementary Figure S4B) which, in the non-canonical Nrf2-Keap1 pathway, is known to bind Keap1, allowing Nrf2 to migrate into the nucleus [42]. Moreover, the p62 protein yields a feedback loop that amplifies the Nrf2 system [43]. In addition to increased expression of Nrf2 in FTH1-silenced hESCs, we observed the overexpression of major antioxidant enzymes, such as glutathione peroxidases GPX2, GPX3 ( Figure 3E), Gpx4 ( Figure 3F and Supplementary Figure S4C), and superoxide dismutases (SOD) 1 and 2 ( Figure 3G). Additionally, several Nrf2-regulated genes (HMOX1, NQO-1, HIF1α, HIF2α) ( Figure 3H) and proteins (HIF1α, Ftl) ( Figure 3I,J respectively and Supplementary Figure S4D,E) were found to be highly expressed in FTH1-KD hESCs. Altogether, these findings suggest a crosstalk between FTH1 silencing and overactivation of the Nrf2 signaling pathway and its cognate effectors in hESCs.

Effects of FTH1 Knock-Down on Apoptosis
Labile iron accumulation is a well-known cell damage effector and pro-apoptotic factor [44][45][46]; intracellular iron overload by ferric ammonium citrate (FAC) treatment leads to ferroptosis, an iron-regulated cell death [47]. To check the effects of FTH1-silencing on apoptosis, expression analysis of intrinsic pro-apoptotic genes such as BAX and BIM, as well as CASP9 and CASP3 was performed, revealing a significant upregulation in FTH1-KD hESCs compared to SCR control ( Figure 4A,B). Similarly, immunoblot analysis of cleaved Caspase3 protein confirmed its overexpression in FTH1-silenced hESCs ( Figure 4C, Supplementary Figure S5A,E). Further, flow cytometry-based analysis of apoptosis showed a significantly higher percentage of late apoptotic cells in FTH1-KD cells compared to SCR (Supplementary Figure S7). It is by now generally accepted that Akt and Erk1/2 pathways are both linked to apoptosis, but with opposite effects: Erk promotes apoptosis, both intrinsically and extrinsically and its activity can be driven by the presence of ROS [48]. By contrast, Akt is linked to cell survival, therefore its activity mediates the suppression of apoptosis [49]. Based on these observations, we speculated whether Erk and Akt could be involved in our FTH1-KD hESCs system; therefore, we measured their expression levels by immunoblot analysis and found that the active, phosphorylated Akt protein (pAkt) expression was reduced ( Figure 4D, Supplementary Figure S5B,F), while the expression of pErk1/2 resulted in a significant increase ( Figure 4E, Supplementary Figure S5C,G) in FTH1-silenced hESCs. Collectively, these findings clearly suggest that an active cell death program occurs in hESCs when ferritin is stably downregulated. In addition, based on the evidence that high ROS levels and FTH1 modulation also associate with DNA damage [50][51][52][53], we measured the expression of some DNA damage-associated genes. In line with previous studies, here we show that BRCA1, DMC1, PCNA, and POLQ are indeed significantly upregulated in FTH1-KD hESCs ( Figure 4F). Similarly, we could also observe an increased expression of Parp1 protein, a substrate of activated caspase 3, in FTH1-silenced hESCs ( Figure 4G, Supplementary Figure S5D,H). Overall, these results provide strong evidence that FTH1 silencing induces apoptosis in hESCs and triggers the activation of DNA-damage response programs, which are efficiently counteracted by the survival mechanisms described above.
we show that BRCA1, DMC1, PCNA, and POLQ are indeed significantly upregulated in FTH1-KD hESCs ( Figure 4F). Similarly, we could also observe an increased expression of Parp1 protein, a substrate of activated caspase 3, in FTH1-silenced hESCs ( Figure 4G, Supplementary Figure S5D and H). Overall, these results provide strong evidence that FTH1 silencing induces apoptosis in hESCs and triggers the activation of DNA-damage response programs, which are efficiently counteracted by the survival mechanisms described above.   CASP3 and CASP9 (B) showing increased expression levels of apoptotic marker in FTH1-KD hESCs compared to SCR control; (C) Activation of apoptotic signaling in FTH1-KD hESCs is demonstrated also by immunoblot analysis for full length and cleaved form of caspase-3; (D) Western blot analysis of Akt1 and phosphorylated Akt1 (Ser473, pAkt) protein expression; (E) Immunoblot for total Erk1/2 and phosphorylated Erk1/2 (Thr202/Tyr204, pErk1/2) in SCR control and FTH1-KD hESCs; (F) qRT-PCR analysis for DNA damage related genes (BRCA1, DMC1, PCNA, POLQ) in control and silenced hESCs; (G) Western blot analysis for Parp1 protein expression in SCR and FTH1-silenced cells. For all Western blots shown, actin levels were evaluated as loading control. Results of qRT-PCR were normalized to measurements in SCR cells (n = 3); * p ≤ 0.05; ** p ≤ 0.01, t-test, *** p ≤ 0.001. Small dots refer to SCR replicates, while triangles refer to shFTH1 triplicates.

Effects of FTH1 Repression on Cellular Metabolism
Under oxidative stress conditions, a metabolic shift from OXPHOS to glycolysis was reported to be occurring [54], indicating a clear and intimate correlation between oxidative stress and metabolic changes. It is also well known that hPSCs mainly rely on glycolysis while OXPHOS takes place as soon as these cells enter differentiation [33,55]. Using the Seahorse analyzer, we first quantified the fraction of ATP generated by glycolysis and by OXPHOS, demonstrating an overall reduction in total ATP production from both metabolic sources in FTH1-silenced hESCs compared to SCR control. Therefore, we further sought to speculate on the total glycolytic activity in both cell lines. A lower basal and compensatory glycolytic rate was detected in FTH1-silenced hESCs, suggesting that mitochondrial respiration is somehow inhibited in these cells, which retain the capability to manage energy demand despite the downregulation of the ferritin gene ( Figure 5B and Supplementary Figure S6B). For a more comprehensive metabolic investigation, we analyzed the expression levels of glycolysis-specific genes such as ALDOA, PGK1, ENO1, and PKM, along with gluconeogenesis-related genes G6PC1 and FBP1. In line with the results obtained by the glycolytic rate analysis, we could not observe significant changes in the expression of glycolytic genes between SCR and FTH1-silenced hESCs, with the exception of the PKM (pyruvate kinase M1/2) gene, encoding an ATP-generating enzyme, which results in significantly downregulated silenced cells ( Figure 5C), strengthening the result obtained with the Seahorse analyzer quantification of total ATP production. On the other hand, the expression of G6PC1 (Glucose-6-Phosphatase Catalytic Subunit 1) resulted in significantly higher FTH1-silenced cells ( Figure 5D). G6PC1 encodes one of the key enzymes responsible for glucose production from glucose-6-phosphate, and therefore it is critically involved in glucose homeostasis. This finding perfectly matches with the fact that high glucose levels are required for the proper functionality of detox systems, such as the pentose phosphate (PPP), glucuronidation, and glutathione biosynthesis pathways, all of which are finely tuned by Nrf2 [56,57]. In support of this, we found that G6PD (glucose-6-phosphate dehydrogenase) and PGD (6-Phosphogluconate dehydrogenase), two key enzymes of the PPP pathway, were significantly increased in FTH1-silenced cells ( Figure 5E), strengthening the concept that the PPP may represent a crucial metabolic target through which the Nrf2 signaling potentiates the antioxidant response in hPSCs. The expression of PDK1 (pyruvate dehydrogenase kinase 1) was also significantly enhanced in FTH1-silenced hESCs ( Figure 5F); its expression is positively regulated by HIF1α, previously shown to participate in ROS-induced metabolic reprogramming, where PDK1 prevents the pyruvate from entering the TCA cycle (tricarboxylic acid cycle or Krebs cycle) [58]. We finally tested the expression level of UCP2 (Uncoupling protein-2), a protein of the inner mitochondrial membrane, involved in uncoupling OXPHOS from ATP synthesis. Analogously to PDK1, UCP2 expression was significantly higher in FTH1-silenced hESCs ( Figure 5G). As PDK1, UCP2 blocks pyruvate entry into the Krebs cycle and shunts it towards the PPP pathway [19,59,60]. Altogether, our findings are indicative of a metabolic rewiring occurring in response to iron-mediated oxidative stress in hESCs. Quantification of total cellular ATP production (from glycolysis and mitochondrial oxidative phosphorylation) rate using a Seahorse XF analyzer. ATP production was calculated by measuring the oxygen consumption and acidification rate. Each sample (SCR and shFTH1-hESCs) was measured in at least three biological replicates. Seahorse analysis shows a lower ATP production (from both sources) in FTH1-KD cells compared to SCR control; (B) Glycolytic rate measurements in SCR and FTH1-silenced cells calculated by subtracting extracellular acidification rate (ECAR). As shown, FTH1-KD hESCs do not undergo metabolic switch. Since mitochondrial acidification is blocked, the approach measures glycolytic-ECAR only correlating to extracellular lactate accumulation. Glycolytic rate was reduced in FTH1-silenced hESCs; (C) qRT-PCR analysis for glycolytic enzymes (ALDOA2, PGK1, ENO1, and PKM) mRNA expression; (D) Expression of G6PC and FBP1 gluconeogenesis genes in SCR and shFTH1-hESCs measured by qRT-PCR analysis; (E) Pentose phosphate pathway (PPP) genes G6PD and PGD were significantly up-regulated shFTH1 cells with respect to SCR control cells; (F,G) The PDK1 gene, responsible for inactivation of pyruvate dehydrogenase (F) and the gene UCP2 (G), Figure 5. Metabolic rearrangement in FTH1-KD hESCs. (A) Quantification of total cellular ATP production (from glycolysis and mitochondrial oxidative phosphorylation) rate using a Seahorse XF analyzer. ATP production was calculated by measuring the oxygen consumption and acidification rate. Each sample (SCR and shFTH1-hESCs) was measured in at least three biological replicates. Seahorse analysis shows a lower ATP production (from both sources) in FTH1-KD cells compared to SCR control; (B) Glycolytic rate measurements in SCR and FTH1-silenced cells calculated by subtracting extracellular acidification rate (ECAR). As shown, FTH1-KD hESCs do not undergo metabolic switch. Since mitochondrial acidification is blocked, the approach measures glycolytic-ECAR only correlating to extracellular lactate accumulation. Glycolytic rate was reduced in FTH1-silenced hESCs; (C) qRT-PCR analysis for glycolytic enzymes (ALDOA2, PGK1, ENO1, and PKM) mRNA expression; (D) Expression of G6PC and FBP1 gluconeogenesis genes in SCR and shFTH1-hESCs measured by qRT-PCR analysis; (E) Pentose phosphate pathway (PPP) genes G6PD and PGD were significantly up-regulated shFTH1 cells with respect to SCR control cells; (F,G) The PDK1 gene, responsible for inactivation of pyruvate dehydrogenase (F) and the gene UCP2 (G), which uncouples OXPHOS from ATP synthesis, both resulted in significantly up-regulated in FTH1-KD cells compared to SCR cells. For all qRT-PCR graphs, results were normalized to measurements in SCR cells (n = 3); * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, t-test compared to SCR. Small dots refer to SCR replicates, while triangles refer to shFTH1 triplicates.

Discussion
Pluripotent stem cells (PSCs), including ESCs and iPSCs, possess unique and extraordinary self-renewal capabilities, while maintaining their pluripotency [61]. In addition to these properties, PSCs also have the ability to give rise to almost any cell type, both in vivo and in vitro. On the basis of these key features, PSCs technology holds enormous potential in many fields of biomedical research, including drug screening, development and repurposing [62][63][64][65], disease modelling [66][67][68][69][70], cell therapy [71], aging [72], and regenerative medicine [73]. Although significant progress has been made, many hurdles still need to be overcome to push PSCs technology towards the road to clinical practice. Apart from immunogenicity and genomic instability, undoubtedly representing the two major challenges, the successful differentiation and functional maturation of PSC derivatives must faithfully recapitulate all aspects of somatic cells and tissues, including the metabolic function. For instance, little is known about the role of ferritin heavy chain (FTH1), the major ferroxidase protein, in human ESCs homeostasis. Ferritin is composed of 24 heavy and light chains, whose core is able to sequester up to 4500 iron atoms [74]. Ferritin heavy chain, in particular, is responsible for the oxidation of ferrous iron [Fe(II)] to less reactive ferric iron [Fe(III)] [75]. Even though there is a wide literature suggesting that FTH1 is a major player in protecting cells against oxidative stress [76], so far, its role in embryonic stem cells has not been fully elucidated. As mentioned earlier in this work, ROS play an important role as second messengers in embryo development [77,78], and their effects, beneficial or deleterious, depend on the embryo developmental stage. Compared to most terminally differentiated cells, ESCs are extremely resistant to hypoxic stress as they live and greatly proliferate at low oxygen concentrations [79,80], suggesting that they are equipped with a highly efficient antioxidant system [5,10]. Because of its strong ferroxidase activity, FTH1 is supposed to be one of the major players in this system, maintaining iron in a safe and bioavailable form. To gain insights on the effects of FTH1-mediated control of ROS-induced cellular stress, here we stably silenced the FTH1 gene in H9 human embryonic stem cell line using the shRNA strategy. Intracellular ROS production, measured in shFTH1-hESC by flow cytometry together with mitochondrial superoxide species, revealed that silenced cells have decreased levels of ROS accumulation. Similarly, mitochondrial membrane potential detected by TMRM assay, showed that FTH1 silencing produces a reduction in mitochondrial membrane depolarization in shFTH1-hESCs compared to SCR control. By monitoring self-renewal and pluripotency, interesting findings emerged: (i) morphologically, shFTH1 hESCs were more flattened and had less AP+ cells compared to SCR control cells; (ii) the core pluripotency genes OCT4, NANOG, and SOX2 were significantly overexpressed in FTH1-silenced cells; (iii) immunoblot analysis of Nanog protein revealed an increased expression in silenced cells. As pointed out elsewhere in the text, NANOG expression is under the control of the nuclear factor erythroid 2-related factor 2 (Nrf2), a stress-activated transcription factor involved in the cellular response to multiple injuries, among which oxidation is the most prominent. In a recent review, Dai and Coll. [81] summarize Nrf2-Keap1 signaling in the context of stem cell state and function, suggesting that it can serve as a master regulator of stem cell redox and metabolic homeostasis. Nrf2, in fact, can efficiently modulate ESCs and iPSCs pluripotency status by finely tuning NANOG and OCT4 expression through direct binding to upstream regions of these genes and delaying their proteasome-mediated ubiquitination [41]. Our data showed clear evidence of the increased Nrf2 expression in hESCs, both at mRNA and protein level, upon FTH1 silencing. Overexpression of Nrf2 is therefore responsible, in our model, for the upregulation of NANOG, as well as for the activation of the Gpx4 (glutathione peroxidase 4) ROS scavenger protein. A correlation between activation of Nrf2 and induced expression of FTH1 has been previously reported in mouse embryonic fibroblasts (MEFs) exposed to beta-naphthoflavone and chemo preventive dithiolethiones, providing a mechanistic link between regulation of the iron storage protein ferritin and the cancer chemo preventive response [82]. Ferritin is also known to function as a powerful anti-apoptotic protein [83]. Accordingly, our results show that FTH1-silencing triggers the activation of apoptosis-associated genes BAX, BIM, CASP3, and CASP9; we further deepened our analysis by studying the AKT and ERK1/2 pathways, which undergo significant perturbations during apoptosis. Previous studies have reported conflicting results, showing that MAPK/ERK and PI3/AKT signaling pathways can be either activated [84] or decreased [85] in mESCs and hESCs upon ROS exposure. Our data clearly indicate that ferritin silencing produces a reduction in the levels of the active, phosphorylated form of Akt, while ERK1/2 signaling is activated. With respect to cell metabolism, most of the studies have focused on glycolytic metabolism and on its role in maintaining pluripotency, as well as the importance of a metabolic shift required for somatic cells in order to enter pluripotency during reprogramming. In this study we went beyond previous reports, speculating whether modulation of FTH1 by repression may impact on hESCs homeostasis and metabolic properties. Interestingly, extensive analysis showed that FTH1-silenced hESCs undergo significant metabolic reorganization. More specifically, we observed that FTH1-KD hESCs cells respond to the loss of ferritin heavy chain by activating a strong antioxidant response that entails the expression of major ROS scavengers such as SODs, CAT, and GPXs. Moreover, FTH1-silenced hESCs had an increased expression of the G6PD gene, which is one of the most important sources of NADPH, essential for maintaining redox homeostasis [86]. Together with G6PD, PDK1 and UCP2 were upregulated as well in silenced cells, suggesting a rewiring of metabolites from glycolysis to PPP and corroborated by a reduced glycolytic flux in shFTH1-hESCs. Consistent with the idea that PPP flux leads to an elevated reduction of NADP+ to NADPH [87,88], we suggest that an increased PPP flux is favored in hESCs with repressed FTH1 expression to drive the ROS clearance.

Conclusions
To our knowledge, this is the first study shedding light on the role of ferritin modulation by repression in human ESCs. In agreement with our work, previous studies have reported that ESCs are very resistant to oxidative stress. Interestingly, here we provide evidence of a potential interplay between the Nrf2 axis and oxidative pentose phosphate pathway (PPP) as the molecular mechanism underlying the antioxidant response in hESCs under shFTH1-induced stress conditions ( Figure 6). Future investigations are necessary to validate the conclusions drawn from this study. modulation by repression in human ESCs. In agreement with our work, previous studies have reported that ESCs are very resistant to oxidative stress. Interestingly, here we provide evidence of a potential interplay between the Nrf2 axis and oxidative pentose phosphate pathway (PPP) as the molecular mechanism underlying the antioxidant response in hESCs under shFTH1-induced stress conditions ( Figure 6). Future investigations are necessary to validate the conclusions drawn from this study.  (1) is the master scavenger molecule for intracellular iron homeostasis and its inactivation leads to imbalanced redox homeostasis due to intracellular iron accumulation (2) and ROS production (3). Surprisingly, in hESCs, a reduced expression of FTH1 expression is not associated with oxidative stress and ROS production. This phenomenon can be explained by three crucial findings: first, FTH1-silenced hESCs showed an enhanced transcriptional activity of Nrf2 (4-5) on key antioxidant target genes such as HIF-1, HMOX-1, NQO-1, GPX2/3/4, SOD1/2, FTL (6); second, we observed the activation of an alternative metabolic program from glycolysis (7)(8) to pentose phosphate pathway (PPP) where the activity of the G6PD enzyme ensures the conversion of NADP+ to NADPH (9) involved in ROS neutralization; third, the mitochondrial metabolism was found to be inactive (10) in FTH1-silenced hESCs, as demonstrated by the upregulation of PDK1 and UCP2 genes (11), specifically involved in preventing pyruvate from entering the tricarboxylic acid cycle (TCA) (12).

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Figure S1: Assessment of FTH1 silencing efficiency, Figure S2: SCR and shFTH1 hESCs pluripotency characterization, Figure S3: Iron level quantification and flow cytometric assessment of intracellular ROS levels and mitochondrial membrane potential (ΔΨM), Figure S4: Study of Nrf2 signaling activation upon FTH1 silencing, Figure S5: Evaluation of FTH1 silencing effect on apoptosis and DNA damage, Figure S6: Analysis of metabolic changes occurring in FTH1 hESCs. Table S1: List of primers used for qRT-PCR experiments.   (1) is the master scavenger molecule for intracellular iron homeostasis and its inactivation leads to imbalanced redox homeostasis due to intracellular iron accumulation (2) and ROS production (3). Surprisingly, in hESCs, a reduced expression of FTH1 expression is not associated with oxidative stress and ROS production. This phenomenon can be explained by three crucial findings: first, FTH1-silenced hESCs showed an enhanced transcriptional activity of Nrf2 (4-5) on key antioxidant target genes such as HIF-1, HMOX-1, NQO-1, GPX2/3/4, SOD1/2, FTL (6); second, we observed the activation of an alternative metabolic program from glycolysis (7)(8) to pentose phosphate pathway (PPP) where the activity of the G6PD enzyme ensures the conversion of NADP+ to NADPH (9) involved in ROS neutralization; third, the mitochondrial metabolism was found to be inactive (10) in FTH1-silenced hESCs, as demonstrated by the upregulation of PDK1 and UCP2 genes (11), specifically involved in preventing pyruvate from entering the tricarboxylic acid cycle (TCA) (12).

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/cells10092431/s1, Figure S1: Assessment of FTH1 silencing efficiency, Figure S2: SCR and shFTH1 hESCs pluripotency characterization, Figure S3: Iron level quantification and flow cytometric assessment of intracellular ROS levels and mitochondrial membrane potential (∆ΨM), Figure S4: Study of Nrf2 signaling activation upon FTH1 silencing, Figure S5: Evaluation of FTH1 silencing effect on apoptosis and DNA damage, Figure S6: Analysis of metabolic changes occurring in FTH1 hESCs. Table S1: List of primers used for qRT-PCR experiments. Writing-review and editing, F.S.C. and G.C. All co-authors have reviewed the manuscript and approved its content prior to submission. All co-authors confirm that the manuscript has been submitted solely to this journal and is not published, in press, or submitted elsewhere. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All the data presented in this study are available from the corresponding author upon reasonable request.