CHIR-99021 Regulates Mitochondrial Remodelling Via Β-Catenin Signalling

RESEARCH ARTICLE CHIR-99021 regulates mitochondrial remodelling via β-catenin signalling and miRNA expression during endodermal differentiation Yuejiao Ma, Minghui Ma, Jie Sun, Weihong Li, Yaqiong Li, Xinyue Guo and Haiyan Zhang*

ABSTRACT been closely linked to cell fate determination and development, and Mitochondrial remodelling is a central feature of stem cell evidence has demonstrated that mitochondrial biogenesis is differentiation. However, little is known about the regulatory essential for the successful differentiation of stem cells (Varum mechanisms during these processes. Previously, we found that a et al., 2011; Chen et al., 2012; Folmes et al., 2012; Michel et al., pharmacological inhibitor of glycogen synthase kinase-3α and -3β, 2012; Xu et al., 2013; Wanet et al., 2015). An increased CHIR-99021, initiates human adipose stem cell differentiation into mitochondrial mass and mitochondrial DNA (mtDNA) copy human definitive endodermal progenitor cells (hEPCs), which were number, together with the elongation of the mitochondrial directed to differentiate synchronously into hepatocyte-like cells after network and maturation of the cristae ultrastructure, has been in vitro further treatment with combinations of soluble factors. In this study, observed during the differentiation of stem cells (Chen et al., we show that CHIR-99021 promotes mitochondrial biogenesis, the 2008; Varum et al., 2011; Wanet et al., 2014, 2017). The expression of PGC-1α (also known as PPARGC1A), TFAM and morphology, localization, abundance and function of NRF1 (also known as NFE2L1), oxidative phosphorylation mitochondria could potentially be used as key markers for the capacities, and the production of reactive oxygen species in differentiation of stem cells into specialized cell types (Varum et al., hEPCs. Blocking mitochondrial dynamics using siRNA targeting 2011; Wanet et al., 2017). However, little is known about the DRP1 (also known as DNM1L) impaired definitive endodermal regulatory mechanisms connecting mitochondrial structural and differentiation. Downregulation of β-catenin (CTNNB1) expression functional remodelling to the differentiation of stem cells. Thus, a weakened the effect of CHIR-99021 on the induction of mitochondrial better understanding and control of mitochondrial remodelling remodelling and the expression of transcription factors for during the differentiation process in the laboratory should translate mitochondrial biogenesis. Moreover, CHIR-99021 decreased the into enhanced efficiency and increased fidelity in the resulting cells. expression of miR-19b-2-5p, miR-23a-3p, miR-23c, miR-130a-3p Previously, we reported that the pharmacological inhibition of α β α β and miR-130a-5p in hEPCs, which target transcription factors for glycogen synthase kinase-3 and -3 (GSK-3 , GSK-3 , also mitochondrial biogenesis. These data demonstrate that CHIR-99021 known as GSK3A and GSK3B) with specific inhibitors (CHIR- plays a role in mitochondrial structure and function remodelling via 99021 and CHIR-98014) initiates the efficient differentiation of activation of the β-catenin signalling pathway and inhibits the human adipose stem cells (hASCs) to human definitive endodermal expression of miRNAs during definitive endodermal differentiation. progenitor cells (hEPCs) by upregulating the transcription factors GATA4, FOXA2 and SOX17 via activation of the Wnt/β-catenin This article has an associated First Person interview with the first pathway (Huang et al., 2017). CHIR-99021 has also been reported author of the paper. as an important cocktail component of endodermal differentiation inducers for pluripotent stem cells (Blauwkamp et al., 2012; Lian KEY WORDS: Human adipose stem cells, Mitochondrial biogenesis, et al., 2014; Teo et al., 2014; Bao et al., 2015; Morrison et al., 2016). Endodermal differentiation, PGC-1α, Oxidative phosphorylation CHIR-99021-induced GSK-3 inhibition during differentiation further resulted in the inhibition of T cell factor 3 (TCF3, also INTRODUCTION known as transcription factor 7-like 1, Tcf7l1), which in turn Mitochondria are highly specialized and dynamic organelles inside relieved the repression of FoxA2 in mouse embryonic stem cells mammalian cells that play crucial roles in several cellular pathways, (Morrison et al., 2016). The inhibition of TCF4 (also known as such as the production of energy in the form of ATP via oxidative Tcf7l2), another member of the transcription factor 7-like family, phosphorylation, apoptosis and reactive oxygen species (ROS) was sufficient to increase the expression of peroxisome proliferator- signalling (Friedman and Nunnari, 2014; Chandel, 2015). To meet activated receptor gamma (PPARγ) coactivator-1α (PGC-1α, also the different energy demands of distinct cell types, cells modulate known as PPARGC1A), the master regulator of mitochondrial mitochondrial numbers and activity through biogenesis and through biogenesis (Ventura-Clapier et al., 2008) and oxidative dynamic events (Eisner et al., 2018). Recently, mitochondria have phosphorylation activity during the early step of human bone marrow mesenchymal stem cell commitment to hepatic differentiation (Wanet et al., 2017). We therefore anticipated that Department of Cell Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China. the effects of CHIR-99021 on endodermal induction may be attributable to the recapitulation of mitochondrial biogenesis and *Author for correspondence ([email protected]) glycolysis-oxidative phosphorylation shifts. Y.M., 0000-0001-7237-0527; H.Z., 0000-0001-9005-5774 Mitochondrial biogenesis and oxidative phosphorylation activity are accomplished by the coordinated expression of genes from the

Received 14 January 2019; Accepted 17 June 2019 nuclear and mitochondrial genomes. Several transcription and/or Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948 replication factors have been reported to directly regulate gene of PGC-1α, TFAM and NRF1 in hEPCs were significantly expression in mitochondrial biogenesis. Nuclear respiratory factor 1 increased compared to hASCs (Fig. S2). Next, two fluorescent (NRF1, also known as NFE2L1) and NRF2 (also known as probes were used to assess the mitochondrial mass (MitoTracker NFE2L2) control all ten nucleus-encoded cytochrome oxidase Green) and mitochondrial membrane potential (TMRM) in hASCs subunits, and the orphan nuclear hormone receptor, ERRα (also and hEPCs. The relative intensities of MitoTracker Green FM and known as ESRRA), controls the medium chain acyl-coenzyme A TMRM were calculated, and the relative intensity of TMRM dehydrogenase (MCAD) promoter (Huss and Kelly, 2004). The fluorescence was normalized to MitoTracker Green FM in each cell. PGC-1 family of regulated coactivators [PGC-1α, PGC-1β (also The results showed that the mitochondrial mass and mitochondrial known as PPARGC1B) and PRC (also known as PPRC1)] targets membrane potential in the hEPCs were significantly higher than multiple transcription factors, including NRF1, NRF2, and ERRα those in the hASCs (Fig. 1A,B). plays a central role in a regulatory network governing the To further demonstrate changes in mitochondrial numbers and transcriptional control of mitochondrial biogenesis (Wu et al., structures, we performed ultrastructural analyses using transmission 1999; Scarpulla, 2011; Wanet et al., 2017). PGC-1 binds to and electron microscopy (TEM). Upon definitive endodermal coactivates the transcriptional function of NRF1 on the promoter for differentiation, the hEPCs showed a higher number and density of mitochondrial transcription factor A (TFAM), a direct regulator of mitochondria than did the hASCs (Fig. 1C,D). Mitochondria in mitochondrial DNA replication and/or transcription (Virbasius and hASCs contained functionally immature mitochondria with a Scarpulla, 1994; Picca and Lezza, 2015). Hence, CHIR-99021 may globular shape, poorly developed cristae and perinuclear regulate mitochondrial biogenesis and activities, at least in part, localization, all indicative of a less-active mitochondrial state, through modulating the amounts and/or functions of these key sets while the hEPCs possessed a complex morphology, with developed of regulators. Here, we investigated whether CHIR-99021 can cristae, a denser matrix, and an elongated appearance (Fig. 1E,F). regulate certain mitochondrial regulatory factors to control To investigate aerobic metabolism activity levels during mitochondrion biogenesis and oxidative phosphorylation activities definitive endodermal differentiation, we quantified ATP during the definitive endodermal differentiation of hASCs. production rates by measuring the two major production pathways in mammalian cells; glycolysis and oxidative phosphorylation were RESULTS analysed using Agilent Seahorse XF technology. Differentiating hEPCs induced by CHIR-99021 possess multilineage cells undergo a metabolic switch from highly glycolytic metabolism differentiation potential to active mitochondrial aerobic metabolism and drive ATP Previously, we reported that the efficient differentiation of hASCs to production though oxidative phosphorylation (Khacho and Slack, hEPCs was initiated by activin A, Wnt3a, or the pharmacological 2017). As shown in Fig. 1G, the ATP production rate in hEPCs was inhibition of GSK-3 (CHIR-99021 and CHIR-98014) separately. higher than that in hASCs. Moreover, the proportional The hEPCs were directed to differentiate synchronously into mitochondrial oxidative phosphorylation pathway ATP (mitoATP) hepatocyte-like cells after further combinations of soluble factors production rate in hEPCs was ∼63.4%, while the proportional by a reproducible three-stage method (Li et al., 2014; Huang et al., mitoATP production rate in hASCs was only ∼29.2%. The 2017). Among these factors, CHIR-99021 exhibited high efficiency proportional glycolytic pathway ATP (glycoATP) production rate for upregulating endodermal-specific transcription factors, in hEPCs was ∼36.6%, while the proportional glycoATP including FOXA2, GATA4, SOX17, and C-X-C motif chemokine production rate in hASCs was ∼71.8%. The increased oxygen receptor 4 (CXCR4) (Huang et al., 2017). consumption rate (OCR) and extracellular acidification rate (ECAR) Definitive endoderm is the common progenitor to epithelial cells values in hEPCs represented mitochondrial oxidative in diverse internal organs, including the liver, pancreas and intestines phosphorylation instead of glycolysis (Fig. 1H,I). This finding (Tremblay and Zaret, 2005). To examine their multilineage indicates that ATP production in hEPCs mainly depends on differentiation potential, we cultured hEPCs in pancreatic (Aigha oxidative phosphorylation in mitochondria, while ATP production et al., 2018) or intestinal (Spence et al., 2011) induction medium for in hASCs mainly depends on glycolysis. These results suggest that four days and then evaluated the cells for expression of pancreatic mitochondrial biogenesis and oxidative phosphorylation capacities lineage-specific transcription factors pancreatic and duodenal are increased as cells undergo a cell fate transition during the homeobox1 (PDX1) (Offield et al., 1996) and caudal type definitive endodermal differentiation of hASCs. homeobox 2 (CDX2), a member of the caudal-related homeobox transcription factor gene family. CDX2 is a major regulator of Disruption of mitochondrial dynamics impairs definitive intestine-specific genes involved in cell growth and differentiation endodermal differentiation (Gao et al., 2009). Immunocytochemistry analyses confirmed that Growing evidence suggests that the mitochondrial dynamics expression levels of PDX1 in pancreatic lineage differentiated cells involved in fission and fusion enable mitochondria to divide and (Fig. S1A) and of CDX2 in intestinal lineage differentiated cells help ensure proper organization of the mitochondrial network (Fig. S1B) were significantly increased compared to hASCs. These during biogenesis (Ventura-Clapier et al., 2008). To investigate the data confirmed that hEPCs induced by treatment with CHIR-99021 role of mitochondrial dynamics in definitive endodermal possess multilineage differentiation potential. differentiation of hASCs, mitochondrial fission was impaired with genetic inhibition of dynamin 1-like (DRP1, also known as Mitochondrial biogenesis and oxidative phosphorylation DNM1L) (Agarwal et al., 2016) in hASCs, prior to the onset of activities are increased during definitive endodermal endodermal differentiation. As shown in Fig. S3, the mRNA and differentiation protein level of DRP1 in cells treated with siRNA targeting DRP1 To investigate mitochondrial biogenesis during definitive were successfully reduced by 83.1% and 74.9%, respectively, endodermal differentiation of hASCs, the expression of the compared to the control siRNA group. After reduction of the DRP1 transcription factors PGC-1α, TFAM and NRF1 in hEPCs and expression level, morphology of the mitochondria became long and hASCs was determined. The results showed that the mRNA levels thin (Fig. 2A) during definitive endodermal differentiation. The Journal of Cell Science

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Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. Mitochondrial biogenesis and ATP production rate increased (succinate dehydrogenase complex flavoprotein subunit A, SDHA) during definitive endodermal differentiation. (A) Mitochondrial mass and (Bezawork-Geleta et al., 2018), which is nDNA-encoded, were both membrane potential in hASCs and hEPCs were determined by MitoTracker significantly increased in CHIR-99021-treated cells compared to Green FM and TMRM staining analysis. Scale bars: 25 μm. (B) Relative fluorescence intensities of MitoTracker Green, and of TMRM normalized to levels in vehicle control-treated cells (Fig. 3G). MitoTracker Green, in each cell were analysed using ImageJ. n=200 cells. As shown in Fig. 3H, the real-time ATP production rate in CHIR- (C,E). Mitochondrial morphology and structure in hASCs and hEPCs were 99021-treated cells was higher than that in the vehicle control- determined using TEM. White boxes in C show areas magnified in E. Scale treated cells. However, there was no difference in the proportional bars: 5 μminC,1μm in E. (D) Relative mitochondrial mass in cells (as imaged mitoATP production rate between the two groups. The proportional in C) was analysed using ImageJ. (F) Relative numbers of mitochondrial mitoATP production rate in CHIR-99021-treated cells was ∼63.4%, cristae in cells (as imaged in E) were analysed using ImageJ. (G) ATP while the proportional mitoATP production rate in the vehicle production rate as determined using the Seahorse XF Real-Time ATP rate ∼ assay. The mitoATP and glycoATP production rates in hASCs and hEPCs control-treated cells was 59.37%. The OCR and ECAR values in were calculated. (H,I) The kinetic profile of the OCR (H) and ECAR (I) were CHIR-99021-treated cells were also higher than those in the vehicle measured in hASCs and hEPCs using the Seahorse XF Real-Time ATP control-treated cells (Fig. 3I,J). rate assay. Green lines show times of treatment with oligomycin (Oligo), The production of ROS in CHIR-99021-treated cells was rotenone (Rot) and antimycin A (AA). Statistical significance compared to significantly higher than in the vehicle control-treated cells after ’ hASC group, **P<0.01 by unpaired two-tailed Student s t-test. Data tert-butyl hydrogen peroxide (TBHP) treatment (Fig. S4A). These represented as mean±s.d. data show that CHIR-99021 upregulates mitochondrial biogenesis and oxidative phosphorylation activities during definitive mitochondrial mass and membrane potential in cells treated with endodermal differentiation of hASCs. DRP1 siRNA were significantly lower than those in the control siRNA cells (Fig. 2B). Meanwhile, inhibition of DRP1 significantly CHIR-99021 upregulates the expression of transcription decreased the mRNA levels of PGC-1α, TFAM and NRF1 factors involved in mitochondrial remodelling (Fig. 2C), and protein levels of TFAM in DRP1 siRNA-treated In an attempt to understand the mechanism by which CHIR-99021 cells were decreased compared to levels in control siRNA-treated promotes mitochondrial biogenesis and oxidative phosphorylation cells (Fig. 2D). This result indicates that inhibition of DRP1 activities, we next examined the expression of transcription factors significantly affects mitochondrial dynamics and mitochondrial PGC-1α, TFAM and NRF1 in CHIR-99021-treated hASCs and biogenesis during definitive endodermal differentiation. vehicle control-treated hASCs. These transcription factors are Furthermore, mRNA levels of FOXA2, GATA4 and SOX17 in involved in mitochondrial biogenesis and oxidative phosphorylation DRP1 siRNA-treated cells were significantly decreased compared to activities. Real-time RT-PCR analyses showed that mRNA levels of those in control siRNA-treated cells (Fig. 2E). Immunocytochemistry PGC-1α, TFAM and NRF1 in CHIR-99021-treated cells were analyses showed that the relative fluorescence intensities of GATA4 significantly higher than those in the vehicle control group in DRP1 siRNA-treated cells were also significantly decreased (Fig. 4A). Western blotting analyses showed that the protein level compared to control siRNA-treated cells (Fig. 2F). These results of PGC-1α in CHIR-99021-treated cells was higher than that in the suggest that mitochondrial dynamics play a key role in the definitive vehicle control-treated group (Fig. 4B). Immunocytochemistry endodermal differentiation of hASCs. analyses showed that the relative fluorescence intensities per cell of TFAM (Fig. 4C) and NRF1 (Fig. 4D) in CHIR-99021-treated CHIR-99021 promotes mitochondrial biogenesis and cells were also higher than those in the vehicle control-treated oxidative phosphorylation activities during definitive cells. These findings suggest that CHIR-99021 increases the endodermal differentiation expression of transcription factors involved in mitochondrial To investigate the role of CHIR-99021 in mitochondrial biogenesis biogenesis and oxidative phosphorylation activities. and oxidative phosphorylation during differentiation, hASCs were treated with 2 µM CHIR-99021 or vehicle control (DMSO) for 24 h. CHIR-99021 regulates mitochondrial remodelling through Insulin-transferrin-selenium (ITS) was then added to the medium β-catenin signalling for another 48 h. The properties of mitochondrial biogenesis were To assess whether the effect of CHIR-99021 on the induction of first compared between the two groups. The results showed that the mitochondrial remodelling depends on the β-catenin signalling mitochondrial mass and mitochondrial membrane potential were pathway, we disrupted the signalling pathway by delivering siRNA significantly increased in CHIR-99021-treated cells compared to to knock down expression of the β-catenin gene in hASCs. As vehicle control-treated cells (Fig. 3A,B). The mitochondrial shown in Fig. S5, the mRNA and protein levels of β-catenin in β- morphology of CHIR-99021-treated cells exhibited developed catenin siRNA-treated cells were successfully reduced by 83.1% cristae and a more dense matrix compared to the vehicle control- and 50.1%, respectively, compared to the control siRNA-treated treated cells (Fig. 3C,D). group. After reducing β-catenin expression using siRNA, To further demonstrate changes to the mitochondrial components mitochondrial biogenesis and oxidative phosphorylation activities of DNA and protein, mtDNA copy numbers and protein subunits of were assessed in β-catenin siRNA-treated and control siRNA- the respiratory chain were assessed in CHIR-99021-treated cells treated cells. The results showed that mitochondrial mass and and vehicle control-treated cells. The mtDNA content in CHIR- mitochondrial membrane potential were significantly decreased in 99021-treated cells increased ∼1.57-fold over the content in vehicle β-catenin siRNA-treated cells compared to control siRNA-treated control-treated cells (Fig. 3E). The mRNA levels of cytochrome c cells (Fig. 5A,B). The mitochondrial morphology in β-catenin oxidase (COX) subunit 6C (COX6C) and COX8A in CHIR-99021- siRNA-treated cells exhibited a loss of integrity of the outer treated cells were significantly higher than in the vehicle control- membrane and undeveloped cristae compared to the control siRNA- treated cells (Fig. 3F). The protein levels of COX subunit 1 (also treated cells (Fig. 5C,D). The mtDNA content in β-catenin siRNA- known as MTCO-1) (Dennerlein and Rehling, 2015), which is treated cells showed a decrease of ∼25% compared to the control mtDNA-encoded, and subunit A of mitochondrion complex II siRNA-treated cells (Fig. 5E). The protein levels of SDHA and Journal of Cell Science

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Fig. 2. Disruption of mitochondrial dynamics impairs definitive endodermal differentiation. (A) Mitochondrial mass and membrane potential in DRP1 siRNA-treated and control siRNA-treated hASCs were determined by MitoTracker Green FM and TMRM staining analysis. Scale bars: 25 μm. (B) Relative fluorescence intensities of MitoTracker Green, and of TMRM normalized to MitoTracker Green, in each cell were analysed using ImageJ. n=200 cells. (C) Relative mRNA levels of PGC-1α, TFAM and NRF1 in the DRP1 siRNA-treated and control siRNA-treated hASCs were determined by real-time RT-PCR. (D) Protein levels of TFAM in the DRP1 siRNA-treated and control siRNA-treated hASCs were determined by immunofluorescence staining. Scale bars: 25 μm. Relative fluorescence intensity of TFAM in each cell was analysed using ImageJ. n=200 cells. (E) Relative mRNA levels of FOXA2, GATA4, CXCR4 and SOX17 in DRP1 siRNA-treated and control siRNA-treated hASCs were determined by real-time RT-PCR. (F) Protein levels of GATA4 in the DRP1 siRNA-treated and control siRNA-treated hASCs were determined by immunofluorescence staining. Scale bars: 25 μm. Relative fluorescence intensity of GATA4 in each cell was analysed using ImageJ. n=200 cells. Statistical significance compared to the control siRNA group. *P<0.05, **P<0.01 by unpaired two-tailed Student’s t-test. Data represented as mean±s.d.

MTCO-1 significantly decreased in the β-catenin siRNA-treated treated cells. The proportional mitoATP production rate in β-catenin cells compared to those in the control siRNA-treated cells (Fig. 5F). siRNA-treated cells was ∼65.2%, while the proportional mitoATP As shown in Fig. 5G, the ATP production rate in the β-catenin production rate in vehicle control-treated cells was ∼72.9%. The siRNA-treated cells was slightly lower than in control siRNA- OCR values in the β-catenin siRNA-treated cells were also lower Journal of Cell Science

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Fig. 3. See next page for legend. Journal of Cell Science

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Fig. 3. CHIR-99021 promotes mitochondrial biogenesis and ATP (Fig. S4B). These findings suggest that the CHIR-99021 regulation production rate during definitive endodermal differentiation. of mitochondrial biogenesis and oxidative phosphorylation (A) Mitochondrial mass and membrane potential in CHIR-99021-treated and activities during definitive endodermal differentiation of hASCs vehicle control-treated hASCs were determined by MitoTracker Green β FM and TMRM staining analysis. Scale bars: 25 μm. (B) Relative fluorescence depends on the -catenin signalling pathway. intensities of MitoTracker Green, and of TMRM normalized to MitoTracker To further assess whether the induction of transcription factors by Green, in each cell were analysed using ImageJ. n=200 cells. CHIR-99021 depends on the β-catenin signalling pathway, mRNA (C) Mitochondrial morphology and structure in CHIR-99021-treated and and protein levels of PGC-1α, TFAM and NRF1 were determined in vehicle control-treated hASCs were determined using TEM. Scale bars: 1 μm. β-catenin siRNA-treated and control siRNA-treated hASCs. The (D) Relative mitochondrial mass and numbers of mitochondrial cristae in cells results showed that the mRNA and protein levels of NRF1 in β- (as imaged in C) was analysed using ImageJ. (E) Relative mitochondrial DNA content (measured as mitochondrial tRNA) in hASCs was determined by real- catenin siRNA-treated cells were decreased compared to those in the time PCR. (F) Relative mRNA levels of mitochondrial cytochrome c oxidase control siRNA-treated cells (Fig. 6A,B). The mRNA level of PGC- subunit in hASCs were determined by real-time RT-PCR. (G) Relative protein 1α was not changed, but the protein level of PGC-1α in β-catenin levels of mtDNA-encoded MTCO-1 or nDNA-encoded SDHA in hASCs were siRNA-treated cells was decreased compared to control siRNA- determined by western blotting. The relative band density of each protein was treated cells (Fig. 6A,C). While the mRNA level of TFAM was analysed using ImageJ. (H) ATP production rates as determined using the increased in β-catenin siRNA-treated cells, the protein level was Seahorse XF Real-Time ATP Rate Assay, and the mitoATP and glycoATP decreased compared to control siRNA-treated cells (Fig. 6A,D). production rate in CHIR-99021-treated and vehicle control-treated hASCs were calculated. (I,J) Kinetic profiles of OCR (I) and ECAR (J) were measured These findings indicate that the regulation of transcription factors by in CHIR-99021-treated and vehicle control-treated hASCs using the Seahorse CHIR-99021 through increasing the expression of mRNAs during XF Real-Time ATP Rate Assay. Green lines show times of treatment the definitive endodermal differentiation of hASCs partly depends with oligomycin (Oligo), rotenone (Rot) and antimycin A (AA). Statistical on the β-catenin signalling pathway. significance compared to vehicle control, *P<0.05, **P<0.01 by unpaired ’ two-tailed Student s t-test. Data represented as mean±s.d. CHIR-99021 regulates transcription factor expression by decreasing the expression of miRNAs than those in the vehicle control-treated cells (Fig. 5H,I). However, MicroRNAs (miRNAs) are key components of a broadly conserved the production of ROS in β-catenin siRNA-treated cells was not post-transcriptional regulatory mechanism that controls gene different to control siRNA-treated cells after treatment with TBHP expression by targeting mRNAs (Ong et al., 2015). Previous

Fig. 4. CHIR-99021 upregulates the expression of transcription factors involved in mitochondrial biogenesis and oxidative phosphorylation activities during definitive endodermal differentiation. (A) Relative mRNA levels of PGC-1α, TFAM and NRF1 in CHIR-99021-treated and vehicle control-treated hASCs were determined by real-time RT-PCR. (B) Protein levels of PGC-1α in CHIR-99021-treated and vehicle control-treated hASCs were determined by western blotting. Relative band density of PGC-1α was analysed using ImageJ. (C,D) The relative protein levels of TFAM (C) and NRF1 (D) in CHIR-99021-treated and vehicle control-treated hASCs were determined by immunofluorescence staining. Relative immunofluorescence staining intensity of TFAM and NRF1 in each cell was analysed using ImageJ. Scale bars: 25 μm. Statistical significance compared to vehicle control, **P<0.01 by unpaired two-tailed Student’s t-test. Data represented as mean±s.d. Journal of Cell Science

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Fig. 5. See next page for legend. Journal of Cell Science

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Fig. 5. Depletion of β-catenin in hASCs attenuates the ATP production As sites of cellular respiration and energy production, rate and the effect of CHIR-99021 on mitochondrial biogenesis and mitochondria display a characteristic ultrastructure. We observed oxidative phosphorylation activities. (A) Mitochondrial mass and potential elongation of the mitochondrial network and maturation of the in β-catenin siRNA-treated and control siRNA-treated hASCs were determined by MitoTracker Green FM and TMRM staining analysis. Scale bars: 25 μm. cristae ultrastructure in hEPCs differentiated from hASCs. Healthy (B) The fluorescence intensities of MitoTracker Green, and of TMRM mitochondrial membranes maintain a difference in electrical normalized to MitoTracker Green, in each cell were analysed using ImageJ. potential between the interior and exterior of the organelle, n=200 cells. (C) Mitochondrial morphology and structure in β-catenin siRNA- referred to as membrane potential. TMRM analysis showed that treated and control siRNA-treated hASCs were determined by TEM. Red the mitochondrial membrane potentials in hEPCs were higher than μ arrows indicate mitochondrial outer membrane. Scale bars: 1 m. (D) Relative those in hASCs. mitochondrial mass and numbers of mitochondrial cristae in cells (as imaged in C) were analysed using ImageJ. (E) Relative mitochondrial DNA content In addition to morphological and ultrastructural changes, we (measured as mitochondrial tRNA) in hASCs was determined by real-time found that hEPCs exhibited elevated active mitochondrial metabolic PCR. (F) The protein levels of SDHA and MTCO-1 in cells were determined by function. Mitochondria are notably recognized for their generation western blotting. Relative band densities of protein were analysed using of ATP through oxidative phosphorylation. In addition to ATP ImageJ. (G) ATP production rate as determined using the Seahorse XF Real- generation through mitochondrial respiration, the formation of ATP Time ATP rate assay, and the mitoATP and glycoATP production rates in the β- through glycolysis is an essential cellular energy metabolic process catenin siRNA-treated and control siRNA-treated hASCs were calculated. in cells. Energy production in stem cells mainly results from a high (H,I) Kinetic profiles of the OCR (H) and ECAR (I) were measured in the β-catenin siRNA-treated and control siRNA-treated hASCs using the Seahorse glycolysis rate, leading to increased lactate production, whereas XF Real-Time ATP rate assay. Green lines show times of treatment with oxidative phosphorylation activity is limited, leading to reduced oligomycin (Oligo), rotenone (Rot) and antimycin A (AA). Statistical oxygen consumption and ROS production (Wanet et al., 2015). significance compared to vehicle control, *P<0.05, **P<0.01 by unpaired two- Therefore, measuring the rate of ATP production from both tailed Student’s t-test. Data represented as mean±s.d. pathways simultaneously in live cells enables a view into cellular mitochondrial metabolic function that is not provided by simply research has found that CHIR-99021 decreased the level of mature measuring the amount of ATP level in the cell. We found that hEPCs miRNAs of most miRNA species in mouse embryonic stem cells exhibited elevated oxygen consumption, ATP production (ESCs) (Wu et al., 2015). To further decipher the possible role of predominantly through oxidative phosphorylation activity, and CHIR-99021 in miRNA expression, we investigated the effects elevated ROS production. In contrast, hASCs exhibited lower of miRNAs on the regulation of transcription factors. Six candidate oxygen consumption, ATP production predominantly through miRNAs that potentially target the 3′-UTR of PGC-1α were predicted glycolysis activity, and lower ROS production. These results by TargetScan and PicTar (Table S3), and were selected for further indicate mitochondrial structural and metabolism remodelling investigation using qRT-PCR assay. The results showed that the upon definitive endodermal differentiation (Wanet et al., 2015), expression levels of miR-19b-2-5p, miR-23a-3p, miR-23c, which is in agreement with data showing that mitochondrial miR-130a-3p and miR-130a-5p (Fig. S6A) were significantly biogenesis occurs during osteogenic differentiation (Chen et al., decreased in CHIR-99021-treated cells. Among these, miR-19b-2- 2008; Quinn et al., 2013), adipogenic differentiation (Hofmann 5p was also found to target key mitochondrial biogenesis transcription et al., 2012; Quinn et al., 2013; Zhang et al., 2013) and hepatogenic factors, including TFAM and NRF1 (Table S4, Fig. S6B) (Saini et al., differentiation of mesenchymal stem cells (Wanet et al., 2014). 2018). These results suggest that miR-19b-2-5p may be a key miRNA Growing evidence suggests that the mitochondrial dynamics that regulates mitochondrial biogenesis. These results also suggest involved in fission and fusion enable mitochondria to divide and that CHIR-99021 regulates mitochondrial biogenesis and oxidative help ensure proper organization of the mitochondrial network phosphorylation activities by increasing the expression of during biogenesis (Ventura-Clapier et al., 2008). Fission is transcription factors, a process that is mediated by miRNAs and in mediated by DRP1 and fission protein 1 (FIS1) (Detmer and a β-catenin-dependent manner. Understanding the mitochondrial Chan, 2007). DRP1, a master fission mediator, is a cytosolic biogenesis and metabolic regulation of stem cell differentiation will dynamin GTPase that is translocalized to the mitochondrial outer have great value in manipulating the increased efficiency of cellular membrane, forming constricting spirals around mitochondria to differentiation potential. facilitate mitochondrial division. During differentiation, treatment of hASCs with DRP1 siRNA caused extensive elongation of DISCUSSION mitochondria, which coincided with decreased expression of The crucial role of mitochondria and the bioenergetic function in mitochondrial biogenesis transcription factors, including PGC-1α, stem cells has recently come to light, and the importance of TFAM and NRF1, and endodermal-specific transcription factors mitochondria in mediating stem cell fate is increasingly including FOXA2, GATA4 and SOX17. These results suggest an recognized (Wanet et al., 2015; Zhang et al., 2018). The precise essential role of mitochondrial dynamics in successful definitive mechanism by which mitochondrial remodelling is regulated endodermal differentiation progression, similar to previous work during stem cell differentiation has not been elucidated. In the from Kim et al. (2013) reporting that inhibition of DRP1-dependent present study, we found that CHIR-99021 promotes mitochondrial mitochondrial division impairs myogenic differentiation. biogenesis and oxidative phosphorylation activities during Based on our results, we propose that CHIR-99021 may definitive endodermal differentiation of hASCs via activation of participate in the regulation of mitochondrial biogenesis. Next, the β-catenin signalling pathway. We also demonstrated that the properties of mitochondrial biogenesis were compared between CHIR-99021 decreases the expression of miR-19b-2-5p, miR- CHIR-99021-treated cells and vehicle control-treated cells. The results 23a-3p, miR-23c, miR-130a-3p, and miR-130a-5p in hEPCs confirmed that mitochondrial components, including mtDNA copy differentiated from hASCs. These results suggest that CHIR- number, amount of protein subunits and mitochondrial oxidative 99021 may be one of the key factors that mediates mitochondrial phosphorylation activity were significantly increased following structural and functional remodelling during the differentiation of treatment with CHIR-99021. Mitochondrial biogenesis in eukaryotic stem cells in vitro. cells requires a set of transcription factors (Scarpulla, 2011). Among Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948

Fig. 6. Depletion of β-catenin downregulates the expression of transcription factors involved in mitochondrial biogenesis during definitive endodermal differentiation. (A) Relative mRNA levels of PGC-1α, TFAM and NRF1 in β-catenin siRNA-treated and control siRNA-treated hASCs were determined by real-time RT-PCR. (B,D) The protein levels of NRF1 (B) and TFAM (D) in β-catenin siRNA-treated and control siRNA-treated hASCs were determined by immunofluorescence staining. Scale bars: 25 μm. Relative immunofluorescence staining intensity of NRF1 and TFAM in each cell was analysed using ImageJ. (C) The protein levels of PGC-1α in β-catenin siRNA-treated and control siRNA-treated hASCs were determined by western blotting. The relative band density of PGC-1α was analysed using ImageJ. Statistical significance compared to vehicle control, *P<0.05, **P<0.01 by unpaired two-tailed Student’s t-test. Data represented as mean±s.d. these, PGC-1α is considered to be a crucial regulator of mitochondrial endodermal differentiation from hASCs, apart from the β-catenin biogenesis, as it can physically dock with and coactivate transcription signalling pathway. factors such as TFAM and NRF1, which modulate the expression of During embryonic development, cells within embryos are exposed genes encoding mitochondrial proteins (Virbasius and Scarpulla, to numerous ligands and morphogens of several signalling pathways. 1994; Wu et al., 1999; Ventura-Clapier et al., 2008). Consistent with miRNAs may sharpen morphogen gradients in the developing the mitochondrial component and function increase, the expression embryo or serve as a positive regulator by amplifying signal levels of PGC-1α, TFAM, and NRF1 were increased in CHIR-99021- strength in duration to allow cell responsiveness to subthreshold treated cells. This finding indicates that the increase in mitochondrial stimuli (Inui et al., 2010). Recent studies have shown that many biogenesis in hEPCs correlates with upregulation of the expression of miRNAs that are involved in the mitochondrial biogenesis of key master transcription factors. different cell types are thought to regulate the expression of Earlier studies showed that lithium increases PGC-1α expression PGC-1α (Ong et al., 2015; Wu et al., 2015; Ai et al., 2016; Cha and mitochondrial biogenesis in primary bovine aortic endothelial et al., 2017; Jiang et al., 2017; Portius et al., 2017; Saini et al., cells (Struewing et al., 2007) and megakaryocytes (Undi et al., 2018). A study found that CHIR-99021 decreased the mature 2017) by regulating β-catenin signalling. To understand the miRNA levels of most miRNA species in mouse ESCs (Wu et al., mechanism of CHIR-99021 on the induction of mitochondrial 2015). In the present study, we found that the expression levels biogenesis, we disrupted the β-catenin signalling pathway by of miR-19b-2-5p, miR-23a-3p, miR-23c, miR-130a-3p, and delivering siRNA to knock down the expression of β-catenin in miR-130a-5p were decreased upon treatment with CHIR-99021. hASCs. The results showed that downregulating the expression of Among these, miR-19b-2-5p targets PGC-1α, TFAM and NRF1, β-catenin impairs the effect of CHIR-99021 on the induction of which is consistent with a detailed bioinformatics analysis from mitochondrial metabolism and remodelling upon definitive Saini et al. (2018). Based on our data and the bioinformatics endodermal differentiation of hASCs. However, mRNA levels of analysis, we suggest that miR-19b-2-5p could be a key to the PGC-1α and TFAM did not decrease in β-catenin siRNA-treated regulation of mitochondrial gene expression and biogenesis. cells. These findings indicate that there are other factors involved in In conclusion, we observed fragmentation of the mitochondrial regulating PGC-1α and TFAM mRNA levels during definitive network morphology, elevated oxidative phosphorylation Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948 capacities, and increased production of reactive oxygen species 20-min incubation at room temperature, the complexes were added to the cells following the pharmacological inhibition of GSK-3α and GSK-3β at a final siRNA concentration of β-catenin (25 nM), DRP1 (50 nM). The with CHIR-99021, which initiated the definitive endodermal medium was replenished with antibiotic-free basal medium 24 h post- differentiation of hASCs. We demonstrated that CHIR-99021 transfection. The culture medium was changed every 2 days for the duration promotes mitochondrial biogenesis via activation of mitochondrial of the experiment. biogenesis transcription factors, including PGC-1α,TFAMand Mitochondrial labelling and imaging using live confocal NRF1, through the β-catenin pathway, and inhibits the expression of fluorescence microscopy miRNAs during definitive endodermal differentiation of hASCs. Cells were seeded into confocal chambers (NEST) at a density of 40,000 Impairment of the mitochondrial dynamic balance or downregulation cells per well. For mitochondria and nuclei staining, the cells were incubated of β-catenin expression weakens the effect of CHIR-99021 on the with Hoechst 33342 (0.2 µg/ml, Thermo Fisher Scientific) for 20 min at induction of mitochondrial remodelling and the expression of key 37°C, then 25 nm TMRM (I34361, Thermo Fisher Scientific) or 50 nM transcription factors for mitochondrial biogenesis. Further MitoTracker Green FM (M7514, Thermo Fisher Scientific) (Karbowski and research is still required to unravel the mechanisms involved in Youle, 2003; Medeiros, 2008) was added. The cells were incubated for the interplay between these two pathways. Understanding how 30 min at 37°C and then washed three times with PBS. After washing, the these pathways are regulated and determining whether they images of fluorescently labelled mitochondria and nuclei were captured regulate each other is of great interest to researchers working to using a confocal laser scanning microscope (Leica). The emission improve the directed differentiation of stem cells into specific cell wavelengths for the individual dyes were 490 nm for MitoTracker Green FM and 574 nm for TMRM. The density of mitochondria in each cell was types. Understanding the role played by mitochondria in stem cell assessed using ImageJ as described previously (Arena et al., 2017). The data differentiation may therefore be useful in regenerative medicine are presented as mean±s.d. and pharmacological testing. Calculation of mitochondrial relative masses and membrane MATERIALS AND METHODS potential Cell culture and differentiation The relative intensities of MitoTracker Green FM and TMRM were All human tissues and cells were obtained with donor consent, and calculated in each cell using ImageJ software. ImageJ was used to set experimental protocols were approved and carried out in accordance with mitochondria as the first threshold and remove the influence of cytosolic or the relevant guidelines and regulations of the Ethics Committee of Capital background signals. Pictures were converted into bitmap as a binary colour Medical University (2011SY08), China. All clinical investigations were (colour=1, no colour=0). The value of the calibration or scale was adjusted. conducted according to the principles expressed in the Declaration of Helsinki. The threshold was adjusted to separate the red in the picture from the hASCs from four different donors were developed, as described in our background. Different ranges were selected depending on the outline of previous report (Li et al., 2014). Cells from passages six to nine were used in every cell. At least 200 cells per group of each parameter were compared this study. Definitive endodermal differentiation was performed as among the treatment groups by unpaired two-tailed Student’s t-tests using described in our previous report (Huang et al., 2017). Briefly, hASCs the statistical software SPSS 11.5 (IBM Corporation). The calculation were plated on dishes (Nunc) coated with collagen I (Invitrogen) and method was performed as previously described (Shprung et al., 2009). cultured in DMEM/F-12 (Invitrogen) supplemented with mesenchyme stem cell-screened 10% foetal bovine serum (FBS-MSCS, HyClone), 100 U/ml Immunofluorescence staining penicillin and 100 µg/ml streptomycin at 37°C with 5% CO2. Once the cells Immunofluorescence analysis was performed as previously described (Li reached 90% confluence, they were washed twice with PBS and incubated et al., 2014). Briefly, the cells were fixed with 4% paraformaldehyde for with serum-free DMEM/F-12 medium for 48 h. The cells were then 20 min at room temperature, followed by permeabilization with 0.3% Triton incubated with DMEM/F-12 containing 0.5 mg/ml albumin fraction V X-100 in PBS for 5 min. The cells were rinsed and blocked with 10% goat (Sigma-Aldrich) with or without CHIR-99021 (2 µM, Selleckchem) for serum (Zsgb-Bio) for 60 min at room temperature. The cells were then 24 h, One percent insulin-transferrin-selenium (ITS) (Sigma-Aldrich) was incubated with the primary antibodies, which are listed in Table S2, at 4°C added to the medium beginning on the second day, and cells cultured overnight. Following three 5-min washes in PBS with gentle agitation, an for 48 h. Alexa Fluor-conjugated secondary antibody (Invitrogen) at 1:500 was added, For differentiation of hEPCs into pancreatic lineage cells, the cells were and the samples were incubated for 1 h at 37°C. The nuclei were counter- incubated with MCDB 131 basal media (Invitrogen) containing 50 ng/ml stained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Noggin (Sigma-Aldrich), 50 ng/ml FGF10 (Peprotech) and 2 µM CHIR-99021 for 2 days. Then the cells were cultured with DMEM Quantitative real-time RT-PCR medium (Invitrogen) containing 50 ng/ml FGF10, 50 ng/ml Noggin and Quantitative real-time RT-PCR was performed as previously described 1% B27 supplement (Invitrogen) for 2 days. (Guo et al., 2017). Total cellular RNA was extracted from 2.0×105 cells with For differentiation of hEPCs into intestinal lineage cells, the cells were the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s incubated with RPMI 1640 media (Invitrogen) containing 0.5 mg/ml BSA, instructions. For PCR analysis, 0.5 μg of RNA was reverse-transcribed to 1% ITS, 100 ng/ml Activin A (Peprotech), 500 ng/ml FGF4 (Peprotech) and cDNA using Superscript III reverse transcriptase and random hexamer 3 µM CHIR-99021 for 4 days. primers (Invitrogen). Real-time PCR analysis was performed on a Thermo Fisher Scientific applied Biosystems QuantStudio 5 system (Applied siRNA transfection Biosystems) using the SYBR Green PCR Master Mix (Applied hASCs were plated at 20,000 cells/cm2 in antibiotic-free basal medium 24 h Biosystems). The reaction consisted of 10 μl of SYBR Green PCR Master prior to transfection. siRNA transfection was performed following the Mix, 1 μlofa5μM mix of forward and reverse primers, 8 μl of water, and manufacturer’s protocol, as previously described (Huang et al., 2017). Briefly, 1 μl of template cDNA in a total volume of 20 μl. Cycling was performed ON-TARGET SMARTpool siRNAs directed against β-catenin (L-003482- using the QuantStudio Design Analysis Software. The relative expression of 00-0005, Dharmacon) or non-targeting siRNAs (D-001810-10-05, each gene was normalized against 18S rRNA. The data are presented as the Dharmacon) were mixed with Transfection DharmaFECT 1 (Dharmacon). mean±s.d. The primers used are listed in Table S1. DRP1 siRNA (5′-GGCCAAUAGAAAUGGAACATT/UGUUCCAUUUC- For miRNA qRT-PCR analysis, the total RNA was extracted from 2.0×105 UAUUGGCCTT-3′) or control siRNA (5′-UUCUCCGAACGUGUCACG- cells with the RNeasy Mini Kit (QIAGEN), and 1 μgwasreversetranscribed UTT/ACGUGACACGUUCGGAGAATT-3′) (SyngenTech) were mixed using a miRcute Enhanced miRNA cDNA First Strand Synthesis Kit with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). After a (KR211, TIANGEN). Real-time PCR was performed in triplicate for each Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948 sample using the miRcute Enhanced miRNA SYBR Green PCR Kit (FP411, by DCFDA Cellular ROS Detection Assay Kit, Abcam), and 200 μM TBHP TIANGEN). The primers used were designed by TIANGEN. RNA RNU6 (Abcam) was added for 2 h at 37°C in the dark. The intracellular levels of was used as the endogenous reference RNA to normalize the amount of ROS were immediately measured by a fluorescence plate reader template added. The data are presented as the mean±s.d. (PerkinElmer), with excitation and emission spectra of 495 nm and 529 nm, respectively. Determination of mtDNA copy number Total DNA from cells was obtained using the QIAamp DNA Mini Kit Statistical analysis (QIAGEN). Quantitative real-time PCR was performed to quantify the At least three independent determinations of each parameter were compared mtDNA content. Primers used for this experiment were specific to mt-tRNA among the treatment groups by unpaired two-tailed Student’s t-test using the (Leu) (UUR), and the 18S rRNA unique gene sequences (Table S1) were statistical software SPSS 11.5 (IBM Corporation). The differences were used for the nuclear genomes. considered significant if P<0.05.

Transmission electron microscopy analysis Acknowledgements The ultra-structural analysis was performed as previously described (Li The authors thank Chenguang Zhang, Jun Deng, Man Ji and Hua Wei for advice in et al., 2014). The samples were examined using a HT7700 transmission performing these experiments. electron microscope (Hitachi). Competing interests The authors declare no competing or financial interests. Western blots Cells were lysed on ice using a Nuclear and Cytoplasmic Extraction Reagent Author contributions Kit (NE-PER) (Thermo Scientific). The samples were normalized for Conceptualization: H.Z.; Methodology: Y.M., M.M., J.S., Y.L., X.G., H.Z.; Software: protein concentration using the BCA protein assay. Each sample (15–20 µg) M.M., J.S.; Validation: Y.M., H.Z.; Formal analysis: Y.M., M.M.; Investigation: was analysed using 10% SDS-PAGE (Invitrogen) and transferred to a PVDF Y.M., H.Z.; Resources: M.M., W.L.; Data curation: M.M., J.S., Y.L., X.G.; Writing - membrane (Merck Millipore). The membranes were blocked in 5% BSA in original draft: Y.M., H.Z.; Writing - review & editing: Y.M., H.Z.; Visualization: TBST, and were incubated overnight at 4°C with the specific primary Y.M., J.S., Y.L.; Supervision: W.L., H.Z.; Project administration: W.L.; Funding antibodies (Table S2). Glyceraldehyde-phosphate dehydrogenase acquisition: H.Z. (GAPDH) and β-tubulin were used as internal reference. The membranes were washed with TBST and incubated with IRDye- Funding This work was supported by grants from the National Natural Science Foundation conjugated secondary antibodies (Table S2) for 1 h at room temperature. of China (grant 81770616), and the Beijing Municipal Natural Science Foundation The membranes were scanned with the Odyssey detection system (Li- (grant 5172009). COR). Relative densities of proteins were quantitatively assessed using ImageJ. The data are presented as the mean±s.d. Supplementary information Supplementary information available online at Real-time ATP rate assay http://jcs.biologists.org/lookup/doi/10.1242/jcs.229948.supplemental The rate of ATP production from the two key energy pathways was detected simultaneously in live cells using the Agilent Seahorse XF Real-Time ATP References rate assay (Seahorse Bioscience). Briefly, hASCs were plated in seeding Agarwal, S., Yadav, A., Tiwari, S. K., Seth, B., Chauhan, L. K., Khare, P., Ray, medium in collagen I-coated 24-well XFe24 cell culture microplates, with R. S. and Chaturvedi, R. K. (2016). Dynamin-related protein 1 inhibition mitigates bisphenol a-mediated alterations in mitochondrial dynamics and neural stem cell 20,000 or 25,000 (for differentiation) cells per well, at 37°C with 5% CO 2 proliferation and differentiation. J. Biol. Chem. 291, 15923-15939. doi:10.1074/ for definitive endodermal differentiation. At the end of the definitive jbc.M115.709493 endodermal induction step (day 5), cells were washed twice with warmed Ai, Z., Shao, J., Wu, Y., Yu, M., Du, J., Shi, X., Shi, X., Zhang, Y. and Guo, Z. DMEM assay medium (XF assay-modified DMEM supplemented with (2016). CHIR99021 enhances Klf4 expression through beta-catenin signalling 10 mM XF glucose, 1 mM XF pyruvate, 2 mM XF glutamine, pH 7.4) and and miR-7a regulation in J1 mouse embryonic stem cells. PLoS ONE 11, e0150936. doi:10.1371/journal.pone.0150936 incubated at 37°C in a non-CO2 incubator for 45–60 min prior to the assay to allow cells to pre-equilibrate with the assay medium. Before starting the XF Aigha, I. I., Memon, B., Elsayed, A. K. and Abdelalim, E. M. (2018). Differentiation assay, the medium was removed and replaced with fresh, warmed assay of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors. Stem Cell Res. Ther. 9, 83. doi:10.1186/s13287-018-0834-0 medium. The OCR and ECAR measurements were then assessed using a Arena, E. T., Rueden, C. T., Hiner, M. C., Wang, S., Yuan, M., Eliceiri, K. W. Seahorse XF Real-Time ATP Rate Assay Kit (Seahorse Bioscience), (2017). Quantitating the cell: turning images into numbers with ImageJ. Wiley according to the manufacturer’s instructions, and using 1.5 µM oligomycin Interdiscip Rev. Dev. Biol. 6, e260. doi:10.1002/wdev.260 and 0.5 µM rotenone/antimycin A. 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37°C in the dark. Next, the cells were washed twice with 1×buffer (provided 1634/stemcells.2007-0509 Journal of Cell Science

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