The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

We show a longitudinal profile of NRF2 activity during iPSC reprogramming peaking at day 8 prior to initiation of a HIFα-mediated glycolytic shift and thereafter decreasing to basal levels. In contrast to the existing dogma, we show that in the early stages of reprogramming, highly proliferative cells actually increase mitochondrial respiration as well as channeling glucose to the pentose phosphate pathway (PPP) to manage increased nucleotide synthesis demands. The peaks in cell proliferation, oxidative phosphorylation (OXPHOS), and PPP all correlate with maximal NRF2 activity. Glycolysis increases in response to a transient HIFα peak, which is in itself dependent on NRF2 activity. Our data indicate that NRF2 activity is primarily affected through increased ROS production in this context and can be reversed by KEAP1 overexpression, which inhibits metabolic reprogramming and results in drastically reduced iPSC colony formation. We conclude that NRF2 acts at a critical nexus between coordinating the distribution of glucose between catabolism and anabolism while managing the stress response and initiating the metabolic switch during the initiation stages of iPSC reprogramming.

From an initial screen of eight candidate transcription factors or cell signaling pathways known to play a role in iPSC reprogramming, we found a reproducible temporal wave of nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity prior to a distinct HIFα peak, which correlated with the metabolic shift toward glycolysis. NRF2, which is upregulated within 2 days of iPSC reprogramming, is a master regulator of the stress response, particularly to reactive oxygen species (ROS), and its activation is complex and multifactorial. Under conditions of homeostasis, NRF2 forms proteasomal degradation complexes with two E3 ubiquitin ligase adaptors: Kelch-like ECH-associated protein 1 (KEAP1) and β-TrCP. Whereas p62/SQSTM1 competes with NRF2 for binding to KEAP1, thus activating NRF2 signaling (), glycogen synthase kinase-3β (GSK-3β) increases the binding of β-TrCP to NRF2, thus resulting in ubiquitination and proteasomal degradation of NRF2 (). ROS exposure causes cysteine modifications in KEAP1, allowing newly translated NRF2 to evade ubiquitination and thus mediate activation of genes containing antioxidant response elements in their promoters ().

Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity.

A major limitation in the study of transcription factor activity driving metabolic reprogramming during iPSC generation, stem cell differentiation, or tumor initiation is the ability to quantitate activity in living cells. To date, only end-point or semiquantitative fluorescent protein analyses have been employed in mechanistic investigations of iPSC reprogramming (). Here we utilize a dual-reporter system where secreted NanoLuc luciferase (NLuc) and eGFP are expressed under the conditional control of a transcription factor activated reporter (TFAR) and normalized for cell proliferation against a second constitutively active secreted Vargula luciferase (VLuc). Using this method, we are able to monitor transcription factor activity in live cell cultures throughout iPSC reprogramming.

Many stem cells, including hESCs, maintain quiescence and potency in a physiologically hypoxic niche in vivo (). Furthermore, iPSC reprogramming () and the maintenance of hESC lines () are enhanced under hypoxic conditions. Hypoxia inducible factor-α (HIFα) transcription factor activity stimulates glycolytic gene expression in adult stem cells () and cancer stem cells () and occurs during iPSC reprogramming (), with two recent studies indicating that HIFα activation is integral to the upregulation of glycolysis in the initiation stages of iPSC reprogramming independent of oxygen tension (). Specifically,show that ectopic expression of the isoform HIF1α throughout iPSC reprogramming promotes colony formation, whereas HIF2α overexpression enhances the early stages but is inhibitory in the later phases.

The ability to genetically reprogram a somatic cell to an induced pluripotent stem cell (iPSC) represented a paradigm shift in stem cell research upon its first description () and provides great promise for regenerative medicine, but the process remains inefficient. It has been proposed that iPSC reprogramming is a stochastic process (), but there is emerging evidence that it is deterministic with initiation, stabilization, and maturation stages () involving the coordinated temporal activation and repression of cell signaling pathways (). Reprogramming cells undergo profound changes in morphology, function, and metabolic activity with somatic cells that predominantly rely on mitochondrial respiration to produce ATP, switching to glycolysis (). The opposite transition has also been shown to occur during differentiation of human embryonic stem cells (hESCs;) and involves mitochondrial biogenesis. However, upon reprogramming, human dermal fibroblast (hDF) mitochondria acquire immature morphological features typical of those observed in hESCs (), although their relative density as a ratio to cytoplasmic volume remains broadly the same ().

Our data indicated a significant role for ROS-induced NRF2 in modulating the metabolic shift that occurs during iPSC reprogramming, so we generated a KEAP1-overexpressing lentiviral vector (KEAP1 O/E) to selectively inhibit NRF2 activity in transduced cells. The ability of KEAP1 O/E to decrease both NRF2 activity ( Figure 4 Ai) and target gene expression ( Figure 4 Aii) was confirmed in hDFs. We then subjected KEAP1 O/E and control empty vector transduced (LNT CTL) cells to iPSC reprogramming. KEAP1 O/E significantly inhibited HIFα TFAR activity at day 11 of reprogramming ( Figure 4 Bi) and reduced transcript levels of HIFα targets ( Figure 4 Bii). Furthermore, HIFα TFAR activity was significantly enhanced by activation of NRF2 either with deta NONOate, which induces mitochondrial ROS production ( Figures S4 i–iii) or an NRF2-overexpressing adenovirus (NRF2 O/E; Figures S4 iv–vi). KEAP1 O/E also resulted in significantly lower levels of glycolysis, as assessed by luciferase ATP assay after inhibition with IAA ( Figure 4 Ci) and lactate production by day 14 of reprogramming ( Figure 4 Cii), whereas NRF2 activation either by deta NONOate ( Figure S4 vii) or by NRF2 O/E ( Figure S4 viii) resulted in early increases in the level of lactate production. Critically, KEAP1 O/E also resulted in a 5-fold decrease in iPSC colony formation ( Figure 4 D). Taken together, these data indicate that NRF2 promotes the metabolic shift from OXPHOS to glycolytic energy production during iPSC reprogramming via HIFα activation.

(C) (i) ATP and (ii) lactate assays to show decreased levels of glycolysis in KEAP1 O/E cells compared to LNT CTL cells at day 14 of iPSC reprogramming.

(B) (i) Luciferase assay data to show decreased levels of HIFα TFAR activity in KEAP1 O/E cells compared with LNT CTL cells at day 11 of iPSC reprogramming. (ii) qPCR to show decreased levels of HIFα target gene expression in KEAP1 O/E cells compared with LNT CTL cells at day 11 of iPSC reprogramming.

(A) (i) Luciferase assay data to show decreased levels of NRF2 activity in hDFs transduced with a KEAP1 O/E lentivirus compared with a control lentivirus (LNT CTL). (ii) qPCR to show decreased expression of NRF2 targets in hDFs transduced with a KEAP1 O/E lentivirus.

Interestingly, this increase in OXPHOS at day 8 of iPSC reprogramming is supported by our RNA-seq data within which there is a substantial enrichment of transcripts encoding OXPHOS-related proteins at this time point ( Figure 3 C). Intriguingly, we also observed decreases in glycolysis by both analysis of ATP production when glycolysis is blocked by idoacetate (IAA; Figure 3 Di) and assessment of the rate ofO production from 3-H-glucose ( Figure 3 Dii) after day 8 of iPSC reprogramming. This would be consistent with glucose being shuttled away from the glycolytic pathway and toward the PPP. PPP activity was quantified by assessment of the difference betweenCOproduction from [1-C]-glucose (which decarboxylates through the 6-phosphogluconate dehydrogenase-catalyzed reaction) and that of [6-C]-glucose (which decarboxylates through the tricarboxylic acid cycle), as previously described (). PPP flux increased concomitantly with the decrease in glycolytic flux after day 8 in pre-iPSCs compared with control cells ( Figure 3 E). Consistent with a programmed metabolic shift, increases in glycolysis in iPSCs became significant at day 14, after the HIFα TFAR peak, and decreases in OXPHOS only become significant by day 17 ( Figures 3 Fi and ii).

If the observed elevated ROS levels were due to increased mitochondrial respiration during the early stages of iPSC reprogramming, we would expect OXPHOS-mediated ATP production to be increased. We used a luciferase assay to determine levels of ATP produced when ATP synthase (Complex V), and therefore ATP production by OXPHOS, was inhibited using oligomycin A. We observed significantly higher levels of OXPHOS in reprogramming cells compared with control cells at day 8 of reprogramming ( Figure 3 A). This was also demonstrated by the increased rates of routine and maximal oxygen consumption, after injection of the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), observed in pre-iPSCs compared with controls at day 8 of iPSC reprogramming ( Figure S3 viii). This increase in mitochondrial OXPHOS activity and capacity early in iPSC reprogramming correlated with a significant increase in cell proliferation ( Figure 3 B) and is consistent with associated increased metabolic demands.

n = 3 for all. Error bars represent SEM for three biological replicates.p < 0.05p < 0.005,p < 0.001. PRODH, proline dehydrogenase (oxidase) 1; PDHX, pyruvate dehydrogenase complex component X; GRPEL1, GrpE-like 1; COX15, cytochrome c oxidase assembly homolog 15; COX5A, cytochrome c oxidase subunit Va; CYCS, cytochrome c; AK4, adenylate kinase 4; MARS2, methionyl-tRNA synthetase 2; CLPB, ClpB caseinolytic peptidase B; PDSS1, prenyl (decaprenyl) diphosphatase synthase, subunit 1; ADCK3, aarF domain-containing kinase 3; FOXRED1, FAD-dependent oxidoreductase domain containing 1; ACSS1, acyl CoA synthetase short-chain family member 1; CAT, catalase; BCL2L13, BCL2-like 13; SLC22A4, solute carrier family 22, member 4; COX7A1, cytochrome c oxidase subunit VIIA, polypeptide 1 (muscle). See also Figure S3

We hypothesized that the early increase in NRF2 activity was in response to elevated ROS generated from high levels of mitochondrial activity in reprograming cells, so we analyzed ROS levels using flow cytometry for 2′,7′-dichlorofluorescin diacetate (DCF-DA) at day 8 of iPSC reprogramming. Levels of ROS were indeed higher in reprogramming cells compared with control cells ( Figure 2 C). In addition to ROS, NRF2 can be activated by the autophagy-associated p62 protein. There was no quantifiable difference in p62 protein in lysates from iPSC reprogramming cells either at day 2 or day 8 and no quantifiable change in the autophagy-associated ATG5 protein at day 2 ( Figures 2 D–2E). Additionally, we found no difference in the levels of transcript expression of the NRF2 repressor protein KEAP1 at this time point ( Figure S3 vii), thus suggesting that KEAP1 regulation is post-translational. This is consistent with our hypothesis that modification of cysteine residues of KEAP1 by ROS causes NRF2 activation at day 8 of iPSC reprogramming.

In this study, we focused on the peak in NRF2 activity at day 8 of iPSC reprogramming since NRF2 is the master regulator of the antioxidant response. At this time point, NRF2 is localized in both the cytoplasm and nucleus in reprogramming cells but is largely excluded from the nucleus in control cells ( Figure 2 Ai). An RNA-seq comparison of reprogramming and control cells at day 8 also showed NRF2 target gene transcripts to be significantly upregulated in reprogramming cells ( Figure 2 Aii). This was also confirmed at the molecular level since the NRF2 target genes thioredoxin 1 (TRX1), NAD(P)H dehydrogenase quinone 1 (NQO1), sulfiredoxin 1 (SRXN1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase catalytic (GCLC) subunit were significantly upregulated in reprogramming cells compared with control cells at day 8 ( Figure 2 Aiii). Consistent with our TFAR data during iPSC reprogramming, HIF1α and its glycolytic target GLUT1 were significantly upregulated at day 11 compared with controls. Interestingly, HIF2α transcript expression was not significantly altered in reprogramming cells compared with control cells at day 11 of reprogramming ( Figure 2 B). These data are consistent with the observations of

n = 3 for all. Scale bars represent 100 μm.p < 0.05,p < 0.01. Error bars represent SEM for three biological replicates. ADAM22, A disintegrin and metalloprotease domain 22; BMP4, bone morphogenetic protein 4; c10orf105, chromosome 10 open reading frame 105; RNF114, ring finger protein 114; SEMA6A, semaphorin-6A; TRIM9, tripartite motif containing 9; TTYH,: Tweety family member 1; CRIM1, cysteine-rich transmembrane BMP regulator 1; RPS6KA2, ribosomal protein S6 kinase; TRX1, thioredoxin 1; NQO1, NAD(P)H dehydrogenase quinone 1; SRXN1, sulfiredoxin 1; HO-1, heme oxygenase 1; GCLC, glutamate-cysteine ligase catalytic subunit. See also Figures S2 and S3

(A) (i) Immunofluorescent cell staining to show NRF2 is localized in the nucleus of pre-iPSCs but largely excluded from the nucleus of control cells at day 8 of reprogramming. (ii) Heatmap to show significantly altered NRF2 target gene expression in iPSCs and control cells at day 8 of reprogramming by RNA-seq. (iii) qPCR to show upregulation of NRF2 target genes at day 8 of iPSC reprogramming compared to control cells.

TFAR activity was assayed throughout iPSC reprogramming using the protocol shown in Figure 1 C. NLuc/VLuc activity was quantified in conditioned medium and expressed as a fold change over NLuc/VLuc activity in control cultures transfected with equivalent molar quantities of empty episomal plasmid. Quality control was determined by the required emergence of more than ten colonies per 1 × 10cells after 25-days post-transfection. FOXO and ICAM1 reporters showed no significant changes in activity, but NFAT and NOTCH both showed persistent repression during iPSC reprogramming compared with controls from day 11 ( Figures S3 i–iv). Most intriguingly, we observed early and significant increases in NF-κB, AP-1, NRF2, and HIFα TFAR activity ( Figure 1 Di–v), transcription factors previously associated with the stress/antioxidant response (). Increased activity of these TFARs was validated by the concomitant increased expression of established target genes at day 2 ( Figure S3 v) and the observation of nuclear localization of c-Fos protein at day 4 in reprogramming cells ( Figure S3 vi).

We designed and produced seven TFAR lentiviral vectors containing synthetic promoters activated by cell signaling pathways previously implicated in iPSC reprogramming ( Figure S1 Di; sequences and validation can be found in). For clarity, the “AP-1” synthetic promoter consists of eight repeats of the sequence TGAGTCAG; thus, the TFAR can be activated by either c-Fos/c-Jun heterodimers or c-Jun/c-Jun homodimers (). We also included a reporter vector with a truncated version of the ICAM1 promoter becausehave previously reported a critical temporal role for ICAM1 expression in the early stages of mouse iPSC reprogramming. The lentiviral expression cassettes express secreted NLuc and are based on our previously described vectors (). In order to control for cell proliferation when using genome-integrating vectors, we developed a second constitutively active lentiviral vector expressing the secreted VLuc ( Figure S1 Dii). NLuc and VLuc have unique non-overlapping substrates whose activity is independent of ATP. Specificity of our TFAR was confirmed in transduced hDFs (<10 multiplicity of infection [MOI]) exposed to relevant pathway agonists and antagonists ( Table S1 ). Expression of eGFP fluorescence and NLuc/VLuc luciferase activity was assayed in conditioned medium over 72 hr ( Figures 1 Bi–iv and S2 i–viii). All TFARs demonstrated modulation of eGFP expression and significant changes in NLuc/VLuc ratio within this timeframe.

In this study, we chose to use the latest iteration of the Yamanaka iPSC reprogramming methodology employing episomally maintained plasmids (). iPSCs generated using this protocol were shown to exhibit pluripotent morphology ( Figure S1 Ai), pluripotency-associated gene expression ( Figure S1 Aii), and protein expression ( Figure 1 A). iPSCs from this protocol also formed embryoid bodies in vitro ( Figures S1 Bi–iii) and teratomas in NOD/SCID mice ( Figures S1 Ci–iii) containing tissues representative of all three germ layers.

Discussion

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Jin Y. Foxd3 suppresses NFAT-mediated differentiation to maintain self-renewal of embryonic stem cells. iPSC reprogramming is a fascinating biological phenomenon that we still know very little about. It remains debatable whether iPSC reprogramming is a stochastic series of events that concludes in colony formation or occurs in a deterministic stage-wise fashion. In this longitudinal study of transcription factor activity in hDF cell cultures during iPSC reprogramming, we assessed the activity of seven transcription factors and ICAM1 gene regulation. ICAM1 was included due to the observations ofthat mouse embryonic fibroblasts obtaining a CD44/ICAM1phenotype during iPSC reprograming more efficiently transition to NanogiPSC colonies. In contrast to this group, we did not observe modulation of the ICAM1 promoter during human iPSC reprogramming. This may be becausequantified cell surface protein rather than transcriptional activation or may be due to species differences between mouse and human cells. Interestingly, we also did not detect any modulation of FOXO activity despite it being implicated in establishing the pluripotent state in hESCs (). However, we did observe significant changes in six TFARs; four increased and two decreased their activity during iPSC reprogramming compared with controls. Levels of NFAT and NOTCH activity were lower during iPSC reprogramming compared with control sham reprogramming, which is consistent with previous reports demonstrating that inhibition of these pathways promotes pluripotency or self-renewal (). Most strikingly, we observed a reproducible temporal wave of NF-κB, AP-1, NRF2, and HIFα activity. All four TFARs were significantly upregulated by day 2 with NF-κB and HIFα dropping to control levels by day 4, while AP-1 remained significantly elevated compared with controls throughout. NRF2 and HIFα activity peaked at days 8 and 11, respectively, prior to falling back to control levels.

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et al. Mitochondrial metabolism transition cooperates with nuclear reprogramming during induced pluripotent stem cell generation. Our data imply that metabolic reprogramming occurs between day 8 and day 14 of iPSC reprogramming in this system. This is the period in which both NRF2 (day 8) and HIFα (day 11) activity peaks. NRF2 peak activity preceded that of HIFα, so we sought to investigate a functional link between the two transcription factors. Our data are consistent with ROS/KEAP1-mediated NRF2 activation, so we modulated NRF2 activity by genetic overexpression of KEAP1. Indeed, KEAP1 O/E resulted in a significant reduction in expression of NRF2 target genes and, importantly, a 56% inhibition of the HIFα TFAR peak during iPSC reprogramming compared with controls at day 11. In support of our hypothesis, either NRF2 overexpression or ROS activation with deta NONOate resulted in increased HIFα activation during reprogramming. These data therefore place NRF2 upstream of HIFα activation during iPSC reprogramming. Furthermore, KEAP1 O/E-mediated NRF2 inhibition resulted in reduced glycolytic activation, whereas NRF2 activation resulted in increased glycolysis. This demonstrates that NRF2-HIFα co-operation promotes the metabolic switch to glycolytic energy production. Moreover, KEAP1 O/E-mediated NRF2 inhibition reduced iPSC colony formation consistent with the observations of, who showed that NRF2 shRNA transcript knockdown decreased reprogramming efficiency. NRF2 has been shown to activate HIF1α via TRX1 in lung adenocarcinoma A549 cells (). We speculate that the same mechanism may be occurring during iPSC reprogramming, as we show here that TRX1 is significantly increased during this process. Our demonstration of the central role of NRF2 in iPSC reprogramming is consistent with the observation that cells with higher levels of OXPHOS reprogram more readily (), possibly due to increased NRF2 activity in these cells.

In summary, we present evidence that the metabolic changes occurring throughout iPSC reprogramming are more complex than a simple switch from one predominant form of energy production to another. Instead, they constitute a series of intermediate steps involving an initial increase in OXPHOS and diversion of glucose from glycolysis to the PPP, before the well-documented “metabolic switch” in which cells increase glycolysis and decrease OXPHOS. We also demonstrate unequivocally that molecules controlling the cellular redox state and metabolic states work together to facilitate the ultimate transition from somatic to pluripotent cellular metabolism. Elucidation of the molecular interactions between NF-κB, AP-1, NRF2, and HIFα transcription factors and their potential roles in the metabolic switch therefore warrants further investigation since manipulation of the redox state using small molecules has potential to improve iPSC reprogramming efficiency. Finally, these data collectively add weight to the emerging concept that, at least in the pre-colony forming initiation stages, iPSC reprogramming is a stage-wise deterministic process with quality-control checkpoints. This process shows intriguing similarities with tumor initiation that warrants further investigation in order to guarantee the development of safe and efficacious regenerative medicine approaches.