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Glucose metabolism has a crucial role for providing substrates required to generate ATP and regulate the epigenetic landscape. We reported that F9 embryonal carcinoma stem-like cells require cytosolic reactive oxygen species to differentiate into extraembryonic endoderm; however, mitochondrial sources were not examined. To extend these studies, we examined the metabolic proﬁle of early and late-passage F9 cells, and show that their ability to differentiate is similar, even though each population has dramatically different metabolic proﬁles. Differentiated early- passage cells relied on glycolysis, while differentiated late-passage cells transitioned towards oxidative phosphorylation (OXPHOS). Unexpectedly, electron transport chain protein stoichiometry was disrupted in differentiated late-passage cells, whereas genes encoding mitofusion 1 and 2, which promote mitochondrial fusion and favor OXPHOS, were upregulated in differentiated early-passage cells. Despite this, early-passage cells cultured under conditions to promote glycolysis showed enhanced differentiation, whereas promoting OXPHOS in late- passage cells showed a similar trend. Further analysis revealed that the distinct metabolic proﬁles seen between the two populations is largely associated with changes in genomic integrity, linking metabolism to passage number. Together, these results indicate that passaging has no effect on the potential for F9 cells to differentiate into extraembryonic endoderm; however, it does impact their metabolic proﬁle. Thus, it is imperative to determine the molecular and metabolic status of a stem cell population before considering its utility as a therapeutic tool for regenerative medicine. Introduction ATP, sufﬁcient glucose ﬂux in glycolysis compensates for 12–14 Metabolism provides substrates for energy expendi- the rate of ATP production . This categorization of 1–3 ture and can modulate the epigenome, thereby inﬂu- metabolic proﬁles is distinct in early mammalian 4–6 15 encing cell fate . Typically, somatic cells rely on embryos . Naive embryonic stem cells (ESCs) use gly- oxidative phosphorylation (OXPHOS) to generate ATP, colysis and OXPHOS, whereas primed ESCs, having whereas proliferative cancer and stem cells use glyco- structurally mature mitochondria capable of OXPHOS, 7–11 16,17 lysis . ATP requirements in proliferative cells are high transition from bivalent metabolism to glycolysis . and, although OXPHOS is more efﬁcient in generating Studies show that extraembryonic trophoblast stem cells preferentially use OXPHOS to produce ATP . However, the metabolic proﬁle of extraembryonic endoderm (XEN) Correspondence: Gregory M. Kelly (firstname.lastname@example.org) stem cells, which differentiate into primitive (PrE) or Department of Biology, Collaborative Graduate Specialization in parietal endoderm (PE) in a process recapitulated using F9 Developmental Biology, The University of Western Ontario, London, ON, Canada embryonal carcinoma stem-like cells (F9 cells), remains 19–21 Department of Paediatrics, The University of Western Ontario, London, ON, unknown . We reported that F9 cells require Canada increased levels of cytosolic reactive oxygen species (ROS) Full list of author information is available at the end of the article. Edited by A. Ruﬁni © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 2 of 16 22–24 to differentiate into PrE , but the role of the mito- Gata6 and Dab2 (Fig. S1C, D, respectively), and levels of chondria, a major source of ROS, has not been DAB2 (Fig. S1F, H) and KERATIN-8 (Fig. S1F, I) were investigated. evidence that F9 cells had differentiated into PrE. Finally, Mitochondria and metabolism have a key role in the signiﬁcant increases in Thrombomodulin (Thbd) expres- reprogramming of somatic cells to induced pluripotent sion (Fig. S1E) and in THBD levels (Fig. S1F, J) indicated stem cells (iPSCs). These events require a metabolic that cells had differentiated to PE. Collectively, these results support the ﬁndings that F9 cells differentiated to transition from OXPHOS to glycolysis in order for cells to sustain proliferation and to reset the epigenetic land- PrE and PE-like lineages when treated with RA and RDB, 25–27 20,21 scape . The acquisition of pluripotency is not respectively . immediate as iPSCs that have undergone few passages share a molecular and epigenetic signature reminiscent of Mitochondrial activity during late-passage differentiation their somatic counterparts, whereas prolonged passaging Although ROS generated from cytoplasmic sources 28–30 22–24 resets their proﬁle closer to ESCs . However, and accompanies F9 cell differentiation , mitochondrial 31,32 although not universal , ESCs passaged extensively sources were never examined. To address this, late- develop abnormal karyotypes, yet maintain pluripotency passage F9 cells were treated with DMSO-, RA-, or RDB, and differentiation potential . Although studies have and then stained with MitoSOX to detect mitochondrial focused on the metabolic status of stem cells or the effects superoxide (Fig. 1a–c) or with TMRM for mitochondrial of passaging on their ability to differentiate, an under- activity (Fig. 1e–g). PrE cells showed a signiﬁcant increase standing of how the two are linked is limited. in mitochondrial superoxide levels relative to controls To address this, two populations of F9 cells were (Fig. 1d), but not mitochondrial activity (Fig. 1h). Sig- investigated and results show that early and late-passage niﬁcant differences in superoxide levels and mitochon- cells had similar differentiation potential, but each have drial activity were also noted in PE cells (Fig. 1d, h, dramatically different metabolic proﬁles. These differ- respectively). These changes suggested that differentiation ences observed were due to changes in the expression and was accompanied by an increase in OXPHOS, which was protein levels of pyruvate dehydrogenase (PDH) kinases supported by the increase in ATP levels in these cells (PDKs), which regulate the activity of PDH complex, (Fig. 1i). Cells were also assayed for changes in metabo- thereby inﬂuencing the metabolic proﬁle of cells. In lites in the media, and results showed that overall, a sig- addition, genes encoding mitochondrial fusion proteins niﬁcant decrease in glucose uptake (Fig. 1j), but not were upregulated in early-passage F9 cells, while relative lactate production (Fig. 1k) occurred in PrE cells. To levels of mitochondrial electron transport chain (ETC) examine if these changes were due to altered expression of proteins were disrupted in late-passage cells. Surprisingly, glucose transporters , cells were differentiated, and then culturing either cell population under their preferred assayed for Glut1–4, 8, and 9 (Fig. S2). Glut1, 3, and 9 metabolic conditions enhanced the exit from pluripotency expression increased signiﬁcantly in PrE, whereas Glut3 and promoted PrE formation. More importantly, late- and 9 were signiﬁcantly upregulated in PE. Conversely, passage cells possessed an abnormal karyotype, resulting Glut4 transcript abundance decreased in PrE, whereas in increased proliferation rates, which were correlated to Glut2 and 8 expression remained unchanged. These signiﬁcant increases in the expression of cell cycle reg- results suggested that GLUT4 is likely the key glucose ulators. Together, these results demonstrate that early- vs. transporter during RA-induced differentiation of late- late-passage F9 cells retain their ability to differentiate passage F9 cells. into XEN; however, this ability to occur in cells that have different metabolic proﬁles and chromosomal composi- Metabolic changes accompanying late-passage tion, underpins the importance of monitoring the phy- differentiation siology of stem cell populations to ensure their quality as a To examine the metabolic proﬁle, late-passage F9 cells tool for regenerative medicine. were differentiated and then analyzed to detect transcripts encoding Lactate dehydrogenase A (LDHA), which pro- Results motes the conversion of pyruvate to lactate, and LDHB, Late-passage F9 cells differentiate to XEN-like cells which catalyzes the reverse reaction. Ldha expression was Undifferentiated late-passage F9 cells grew in compact signiﬁcantly downregulated in PrE and PE cells (Fig. 2a), colonies, while those induced to form PrE or PE adopted a but only in PE cells at the protein level (Fig. 2c, d). stellate-like phenotype (Fig. S1A). Oct4 expression in RA- However, Ldhb transcripts were only signiﬁcantly down- induced PrE was similar to controls (Fig. S1B), but protein regulated in PrE (Fig. 2a) and a similar trend was seen at levels were reduced signiﬁcantly (Fig. S1F, G). This was the protein level (Fig. 2c, e). The expressions of Pdk1–4 more dramatic in cells induced to PE by RA and db- (Fig. 2b), which encode PDK isoforms that phosphorylate cAMP (RDB; Fig. S1B, F, G). Increased expression of and inactivate the PDH complex, were examined (Fig. 2b). Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 3 of 16 MitoSOX DMSO RA RDB AB C D 1.5 *** *** 1.0 0.5 0.0 TMRM DMSO RA RDB EF G 2.0 *** 1.5 1.0 0.5 0.0 1.5 2.0 1.5 1.5 1.0 *** 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Treatment Treatment Treatment Fig. 1 Differentiated late-passage F9 cells display increased mitochondrial ROS production, membrane potential, and ATP levels. A–C Fluorescence images and quantiﬁcation of F9 cells (D) stained with MitoSOX to detect mitochondrial superoxide in undifferentiated F9 cells, primitive endoderm (RA treatment) and parietal endoderm (RDB treatment). E–G Fluorescence images and quantiﬁcation (H) of F9 cells stained with TMRM to detect mitochondrial membrane potential during F9 cell differentiation. I ATP levels, J relative glucose uptake, and K lactate production were also measured during differentiation. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was tested using a one-way * *** ANOVA followed by a Tukey’s test. P < 0.05, P< 0.001. (Scale bar, 20 μm) Results show that Pdk1 was signiﬁcantly downregulated To further examine the role of PDK1 in PrE differ- during differentiation, whereas Pdk2 was signiﬁcantly entiation, Pdk1-expressing stable lines were generated upregulated relative to controls (Fig. 2b). Similar results (Fig. 3). As expected, clones overexpressing PDK1 showed were seen at the protein level (Fig. 2f, g, h). Despite these signiﬁcantly higher levels of Pdk1 expression (Fig. 3a) and pSer232 pSer293 increases, PDH-E1α (Fig. 2f, i) and PDH-E1α protein levels (Fig. 3e, f) over controls. To test the func- pSer232 (Fig. 2f, j) levels were reduced signiﬁcantly, suggesting that tionality of the exogenous PDK1, PDH-E1α was PDK1 likely regulates the activity of the PDH complex assayed and results show its levels were signiﬁcantly during differentiation. higher than those in controls (Fig. 3e, g). Next, Oct4 Ofﬁcial journal of the Cell Death Differentiation Association DMSO RA RDB DMSO RA RDB DMSO RA RDB DMSO RA RDB DMSO RA RDB Relative ATP Levels Relative Glucose Uptake Relative Lactate Production RFU RFU Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 4 of 16 A B C D E F G H I J Fig. 2 Differentiated late-passage F9 cells downregulate transcripts and enzymes involved in inhibiting OXPHOS metabolism. Relative transcript abundance of A Ldha and Ldhb, and B Pdks during F9 cell differentiation. L14 was used a constitutive gene for qRT-PCR. C Representative immunoblot and densitometric analyses (D, E) of LDHA and LDHB, respectively, during F9 cell differentiation. F Representative immunoblot showing pSer232 pSer293 pSer232 PDK1, PDK2, PDH-E1α , PDH-E1α , and PDH-E1α levels, and densitometric analysis of G PDK1, H PDK2, I PDH-E1α , and J. PDH- pSer293 E1α levels. β-Actin served as a loading control. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was * ** tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05, P< 0.01 expression and protein levels were examined and found to PrE cells, there was no signiﬁcant difference when com- be reduced signiﬁcantly in control cells treated with RA pared to Pdk1-overexpressing cells (Fig. 3d). In converse, (Figs. 3b, h, respectively), but neither were affected by RA DAB2 protein levels were signiﬁcantly lower when Pdk1 in PDK1-overexpressing cells (Fig. 3b, h). Despite seeing was overexpressed (Fig. 3i). Lastly, overexpressing Pdk1 changes in OCT4 levels between the two clones, Gata6 attenuated RA-induced PrE differentiation as evident by expression did not differ (Fig. 3c). However, although the levels of KERATIN-8, which were not signiﬁcantly Dab2 expression was signiﬁcantly upregulated in control different from controls (Fig. 3j). Together, these results Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 5 of 16 A B 1.5 1.0 1 0.5 0.0 RA (nM) -+ - + RA (nM) -+ -+ pcDNA C1 DDK-MYC- pcDNA C1 DDK-MYC- mPdk1 C5 mPdk1 C5 C D NS 400 * 80 * 1.6 1.2 0.8 0.4 0.0 RA (nM) -+ - + RA (nM) -+ - + pcDNA C1 DDK-MYC- pcDNA C1 DDK-MYC- mPdk1 C5 mPdk1 C5 E F 2.0 DDK-MYC- * pcDNA C1 mPdk1 C5 1.5 RA (nM) -+ -+ 1.0 DDK-MYC-mPDK1 -48kDa WT PDK1 0.5 pSer232 -42kDa PDH-E1α 0.0 -+ - + RA (nM) -42kDa PDH-E1α pcDNA C1 DDK-MYC- mPdk1 C5 -42kDa OCT4 -96kDa DAB2 -54kDa KERATIN-8 β-ACTIN -43kDa -+ - + RA (nM) pcDNA C1 DDK-MYC- mPdk1 C5 H I J 40 * 25 * 2 5 0 0 -+ - + -+ - + RA (nM) -+ - + RA (nM) RA (nM) pcDNA C1 DDK-MYC- pcDNA C1 DDK-MYC- pcDNA C1 DDK-MYC- mPdk1 C5 mPdk1 C5 mPdk1 C5 Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Relative OCT4 Abundance normalized to -ACTIN Relative Gata6 Abundance Relative Pdk1 Abundance Relative DAB2 Abundance normalized to -ACTIN Relative Dab2 Abundance Relative Oct4 Abundance Ser232 Relative pPDH /PDH Relative PDK1 Abundance Abundance normalized to normalized to -ACTIN -ACTIN Relative KERATIN-8 Abundance normalized to -ACTIN Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 6 of 16 (see ﬁgure on previous page) Fig. 3 Overexpression of PDK1 attenuates RA-induced differentiation of late-passage F9 cells. qRT-PCR of relative abundance of A Pdk1, B Oct4, C Gata6, and D Dab2 in pcDNA 3.1 clone 1 (pcDNA C1) or DDK-MYC-mPDK1 clone 5 (DDK-MYC-mPDK1 C5)-overexpressing F9 cells treated with DMSO or RA for 96 h. L14 was used a constitutive gene for qRT-PCR. Representative immunoblot (E) and densitometric analysis of F PDK1, G PDH- pSer232 E1α , H OCT4, I DAB2, and J KERATIN-8 in pcDNA C1 or DDK-MYC-mPDK1 C5-overexpressing F9 cells treated with DMSO or RA for 96 h. β-Actin served as a loading control. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05 indicate that the overexpression of Pdk1, and inactivation treatment, but only in early-passage cells, suggesting that pSer293 of the PDH complex in F9 cells, maintains pluripotency the increase in PDH-E1α levels is due to PDK4. To and reduces the differentiation potential. test whether the PDH complex was inactive, lactate levels in the media and glucose uptake were measured in Passaging alters metabolism but not differentiation early-passage undifferentiating and differentiated cells potential (Fig. 4h, i, respectively). Media from PE cells showed As passage number inﬂuences stem cell quality and signiﬁcantly more lactate (Fig. 4h) when compared to 36–40 fate , we asked whether F9 cells at early passages (<20) controls, and this was accompanied by a signiﬁcant exhibited a differentiation and metabolic proﬁle similar to increase in glucose uptake (Fig. 4i). The relative abun- that in late-passage cells. Examining markers of differ- dance of Glut1–4, 8 and 9 in early-passage F9 cells was entiation (Gata6, Dab2, and Thbd) showed similar also examined, and while Glut1 and 3 expression was expression proﬁles between the two populations (Fig. S3a- signiﬁcantly upregulated in PrE, Glut2, and 4 expression c). Similarly, OCT4 levels were higher in the undiffer- were only upregulated signiﬁcantly in PE (Fig. S5). Glut8 entiated state, and KERATIN-8 levels higher in the dif- expression was downregulated signiﬁcantly in PrE ferentiated state (Fig. S3d-f). Thus, early- and late-passage (Fig. S5), unlike that seen in late-passage cells (Fig. S2). F9 cells exhibited similar differentiation proﬁles, and it Collectively, the results indicate that unlike late-passage was expected that the metabolic proﬁles would be similar. F9 cells, those that have not been passaged extensively To test this, the expression of Ldh transcripts and the transition from OXPHOS metabolism toward glycolysis levels of protein were examined (Fig. S4A and Fig. S4B, C, during differentiation. respectively). No signiﬁcant differences in the abundance of Ldha and Ldhb were detected in early-passage cells, Mitochondrial dynamics in early- vs. late-passage cells and at the protein level, both levels were signiﬁcantly As uncoupling proteins, which have been implicated in 41,42 reduced by RDB (Fig. S4B, C). Differences were seen, regulating stem cell differentiation , showed no dif- however, in the expression of Pdks, and while the ference between the two populations (Vorobieva and decreasing Pdk1 trend with differentiation was main- Kelly, unpublished), the focus turned to ETC proteins. tained between the two populations (Fig. 4a and Fig. 2b, Analysis revealed that undifferentiated and differentiated respectively), Pdk3 and Pdk4 expression was upregulated early-passage cells express comparable levels of all ETC signiﬁcantly only in early-passage cells (Fig. 4a). No sig- subunits, with the exception of succinate dehydrogenase niﬁcant difference in the abundance of Pdk2 was evident complex iron sulfur subunit B (SDHB) in complex II in early-passage cells (Fig. 4a), which contrasts that seen (Fig. 5a, b). Differentiation of late-passage cells to PrE or in late-passage cells (Fig. 2b). At the protein level, PDK1 PE caused a signiﬁcant decrease in the levels of MTCO1 levels dropped signiﬁcantly with differentiation (Fig. 4b, (complex IV), SDHB (complex II), and NDUFB8 (complex c), while PDK4 levels increased signiﬁcantly in PE (Fig. 4b, I; Fig. 5c, d). In order to explain the disruption of ETC e). Despite the increase in Pdk3 expression (Fig. 4a), PDK3 proteins, the expression proﬁles of genes involved in levels remained unchanged in differentiated early-passage mitochondrial ﬁssion and fusion were examined. Early- cells (Fig. 4b, d). These differences in PDK proﬁles passage cells showed no signiﬁcant change in Drp1, Opa1, prompted further investigation into the phosphorylation and Fis1 expression, which encode proteins that promote pSer293 status of PDH-E1α , which in late-passage F9 cells mitochondrial ﬁssion; however, Mfn1 and 2 expression decreased with differentiation (Fig. 2f, k). In early-passage was signiﬁcantly upregulated in PE (Fig. 5e). No obvious pSer293 cells, PDH-E1α levels increased signiﬁcantly, but changes were detected in late-passage cells (Fig. 5f), and only in PE (Fig. 4b, f). To explain the increase, PDH despite seeing elevated mitochondrial ROS levels and phosphatases (PDPs), which dephosphorylate serine resi- increased activity in differentiated late-passage cells dues and subsequently activate the PDH complex, were (Fig. 1a–h), the data suggests that the mitochondria in examined (Fig. 4g). Results show Pdp1 and Pdp2 expres- both early and late-passage cells are mature, fused and sion was signiﬁcantly downregulated in response to RA capable of OXPHOS metabolism. Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 7 of 16 AB CD E F HI Fig. 4 Differentiated early-passage F9 cells upregulate transcripts and enzymes involved in glycolysis. A qRT-PCR of Pdk1–4 expression, pSer293 representative immunoblot (B) of PDK1, PDK3, PDK4, PDH-E1α , and PDH-E1α, and densitometric analysis of C PDK1, D PDK3, E PDK4, and F pSer293 PDH-E1α during F9 cell differentiation. β-Actin served as a loading control. G qRT-PCR analysis of Pdp1 and Pdp2 transcripts, H lactate production, and I glucose uptake during F9 cell differentiation. L14 was used a constitutive gene for qRT-PCR. Values are presented as mean ± SEM of * ** *** at least three biological replicates. Signiﬁcance was tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05, P< 0.01, P< 0.001 Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 8 of 16 A Early Passage Early Passage DMSO RA RDB 1.5 -55kDa CV-ATP5A -48kDa CIII-UQCRC2 1.0 -40kDa CIV-MTCO1 0.5 -30kDa CII-SDHB 0.0 -20kDa CI-NDUFB8 Treatment Late Passage C D Late Passage DMSO RA RDB DMSO RA RDB 1.5 -55kDa CV-ATP5A -48kDa CIII-UQCRC2 1.0 -40kDa CIV-MTCO1 0.5 * -30kDa CII-SDHB -20kDa CI-NDUFB8 0.0 MTCO1 SDHB NDUFB8 E Early Passage F Late Passage DMSO RA RDB DMSO RA RDB 2.0 1.5 *** 1.0 0.5 0 0.0 Drp1 Opa1 Fis1 Mfn1 Mfn2 Drp1 Opa1 Fis1 Mfn1 Mfn2 Fig. 5 ETC components and mitochondrial dynamics are deregulated in differentiated late-passage F9 cells. Representative immunoblots and densitometric analyses of subunits in the ETC seen during the differentiation of A, B early- and C, D late-passage F9 cells. Relative transcript abundance of mitochondrial fusion proteins (Mfn1 and Mfn2) and mitochondrial ﬁssion proteins (Drp1, Opa1 and Fis1) during the differentiation of E early- and F late-passage F9 cells. L14 was used a constitutive gene for qRT-PCR. Values are presented as mean ± SEM of at least three biological * *** replicates. Signiﬁcance was tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05, P< 0.001 Ofﬁcial journal of the Cell Death Differentiation Association DMSO RA RDB Relative Transcript Abundance Relative Transcript Abundance Relative SDHB Abundance Relative Protein Abundance normalized to -ACTIN normalized to -ACTIN Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 9 of 16 Glycolysis promotes differentiation of early-passage F9 uptake transporter. Concentrations between 0.05 and 5 cells µM had no apparent effect on cell viability compared to If early-passage F9 cells transition towards glycolysis the control (data not shown). A 5 µM concentration was during differentiation, then promoting glycolysis in these selected, and results show that treatment in combination cells should induce differentiation. To test this, early- with RA caused a signiﬁcant reduction in the abundance passage cells were treated with UK5099, a non- of Oct4 relative to RA alone (Fig. 6a). Although these competitive inhibitor of the mitochondrial pyruvate results suggested cells were exiting the pluripotent state, ns 1.5 8 * * 1.0 0.5 0.0 -+ -+ RA (nM) -+ -+ RA (nM) UK5099 (µM) -- UK5099 (µM) -- + + + + -+ - + RA (nM) UK5099 (µM) -- + + OCT4 -42kDa -96kDa DAB2 KERATIN-8 -58kDa β-ACTIN -43kDa * ns D E F 1.5 * 3 5 1.0 0.5 1 0.0 0 -+ -+ -+ -+ RA (nM) -+ -+ RA (nM) RA (nM) UK5099 (µM) -- + UK5099 (µM) -- UK5099 (µM) -- + + + + + Fig. 6 Glycolysis promotes differentiation of early-passage F9 cells. qRT-PCR analysis of A Oct4 and B Dab2 in F9 cells differentiated without or with UK5099 to inhibit mitochondrial pyruvate transport. L14 was used a constitutive gene for qRT-PCR. C Representative immunoblot of DAB2, KERATIN-8 and OCT4, and densitometric analyses of D OCT4, E DAB2, and F KERATIN-8 levels in F9 cells treated with RA or RA and 50 μM UK5099. β- Actin served as a loading control. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05 ns, not signiﬁcant Ofﬁcial journal of the Cell Death Differentiation Association Relative OCT4 Abundance normalized to -ACTIN Relative Oct4 Abundance Relative DAB2 Abundance normalized to -ACTIN Relative Dab2 Abundance Relative KERATIN-8 Abundance normalized to -ACTIN Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 10 of 16 treatment alone or in combination with RA showed no Furthermore, the chromosome frequency distribution of additive effects on Dab2 expression (Fig. 6b). OCT4, early-passage cells was signiﬁcantly different from the DAB2, and KERATIN-8 levels were also analyzed, and as late-passage cells (χ = 42.868, df = 2, P < 0.001, Fig. 8b). expected OCT4 levels were signiﬁcantly lower in RA- Cell proliferation was examined as this is known to be treated cells, and even lower in the treatment with affected by chromosomal abnormalities . Results showed UK5099 (Fig. 6c, d). DAB2 levels, similar to the corre- early-passage cells proliferated at a signiﬁcantly lower rate sponding expression data, were not signiﬁcantly different than late-passage cells (Fig. 8c). An examination of var- between RA or RA and UK5099 treatments (Fig. 6c, e); ious cell cycle regulators including Cdkn1a, Cdkn1b, however, under the same conditions, KERATIN-8 levels Cdkn2a, Cdkn2c, Cdkn2d, Ccnd1, Cdk4, Cdk6, P53, and were signiﬁcantly higher (Fig. 6c, f). Thus, culturing early- Rb1 revealed that with the exception of Cdkn2a, the other passage cells under conditions to promote glycolysis cell cycle markers were upregulated in late-passage cells enhanced the exit from pluripotency and differentiation to (Fig. 8d). Together, this loss of genomic integrity could PrE. account for the differences in the proliferation rates and metabolic proﬁles seen between early- vs. late-passage OXPHOS enhances the exit of pluripotency in late-passage cells. cells As promoting glycolysis in early-passage F9 cells Discussion enhanced their ability to differentiate, then promoting Stem cells hold great potential for regenerative medi- OXPHOS in late-passage cells should augment differ- cine applications; however, understanding their physiol- entiation. To test this hypothesis, late-passage cells were ogy in vitro is crucial to ensure the delivery of quality cultured in increasing concentrations of DCA, a compe- cells to patients. We used F9 cells to determine the titive PDK inhibitor, and then assayed for changes in metabolic proﬁle during XEN-like differentiation, and metabolism and differentiation (Fig. 7). Cells cultured in how passaging may affect their metabolic proﬁle and the highest concentration of DCA (6 mM) showed a sig- differentiation potential. Two cell populations were niﬁcant reduction in cell viability (88 ± 1%; Fig. 7a); examined and although each differentiated and upregu- however, 6 mM DCA was selected, as it caused the most lated markers of PrE and PE (Figs. S1, 3), their metabolic pSer232 signiﬁcant reduction in PDH-E1α and PDH- proﬁles differed dramatically. During differentiation, late- pSer293 E1α levels (Fig. 7b–d), resulting in signiﬁcantly passage F9 cells transitioned from glycolysis to OXPHOS resulting in an increase in mitochondrial ROS levels higher ATP levels (Fig. 7e). As for differentiation, DAB2 and KERATIN-8 levels were signiﬁcantly higher than the and mitochondrial activity, both correlated with increased controls, but only in cells co-treated with RA and DCA ATP production (Fig. 1). The increase in OXPHOS dur- (Fig. 7f, g and Fig. 7f, h, respectively). As the results would ing differentiation is explained by the reduction in indicate that cell differentiation was augmented by DCA, PDK1 levels (Fig. 2f, g), resulting in reduced pSer232 pSer293 OCT4 levels in the cells should be dramatically decreased. PDH-E1α (Fig. 2f, i) and PDH-E1α (Fig. 2f, k) This was tested and results show OCT4 levels were sig- levels. Conversely, overexpressing Pdk1 attenuated RA- niﬁcantly reduced in RA-treated cells relative to controls induced differentiation in these cells (Fig. 3). A similar (Fig. 7f, i). As expected, these levels were even more trend occurs with iPSCs, human ESCs (hESCs), and reduced in cells treated with RA and DCA (Fig. 7f, i), mesenchymal stem cells (MSCs), which are glycolytic in suggesting that promoting OXPHOS in late-passage cells nature due to an inactive PDH complex shuttling pyruvate 25,26 not only enhanced their exit from pluripotency, but it also to lactate , but employ OXPHOS when differ- 27,47,48 promoted their differentiation. entiated . The opposite scenario occurs in the mouse early embryo as naïve ESCs exhibit bivalent Genomic integrity and cell cycle regulation in early- vs. metabolism, while differentiated primed ESCs use glyco- late-passage cells lysis , similar to differentiated early-passage cells as they Although metabolic changes can inﬂuence genomic increased lactate production (Fig. 4g), with elevated levels integrity , reports have shown that extensive passaging of of PDK4 (Fig. 4b, e), and concomitant increased levels of pSer293 adherent cells and ESCs can induce genetic abnormalities PDH-E1α (Fig. 4b, f). During the differentiation of 44,45 thereby affecting cellular metabolism . To address this, adult stem cells, elevated lactate levels and subsequent karyotyping was used to determine whether chromosomal increased Acetyl-CoA levels enhance histone H3K9 composition could explain the variations observed acetylation, a prelude required for differentiation . between early and late-passage cells. Analysis revealed However, this is not universal, and the fact that other approximately 60% of the early-passage F9 cells have the stem cells transition towards OXPHOS when they dif- proper karyotype, while less than 5% of the late-passage ferentiate , highlight cell-speciﬁc differences regulated by F9 cells had the optimal chromosome number (Fig. 8a). the intricacies of the metabolome. Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 11 of 16 B C D F G Fig. 7 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 12 of 16 (see ﬁgure on previous page) pSer232 Fig. 7 OXPHOS promotes differentiation of late-passage F9 cells. A MTT viability assay, representative immunoblot (B) of PDH-E1α , PDH- pSer293 pSer232 pSer293 E1α , and PDH-E1α, and densitometric analyses of C PDH-E1α and D PDH-E1α of F9 cells treated with 0, 2, 4, and 6 mM DCA. E Relative ATP levels in untreated and 6 mM DCA-treated F9 cells. F Representative immunoblot of DAB2, KERATIN-8, and OCT4, and densitometric analyses of G DAB2, H KERATIN-8, and I OCT4 in F9 cells treated with RA or RA and 6 mM DCA. β-Actin served as a loading control. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was tested using a one-way ANOVA followed by a Tukey’s test. P < 0.05, ** *** P< 0.01, P< 0.001 A B C D Fig. 8 Early-passage F9 cells display a genetically stable karyotype and proliferate slower than late-passage F9 cells. a Number of chromosome, b chromosomal frequency, and c growth curve of early and late-passage F9 cells. d Differential expression of genes encoding proteins involved in cell cycle regulation and progression between early- vs. late-passage F9 cells. e Schematic overview of the mechanisms regulating metabolism in both populations. Values are presented as mean ± SEM of at least three biological replicates. Signiﬁcance was tested using a one-way 2 *** ANOVA followed by a Tukey’s test. Chromosomal distribution frequency was tested by χ -test for the Goodness of Fit. P< 0.001 Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 13 of 16 The metabolome is directly affected by the mitochon- these characteristics are seen in late-passage MSCs and 51 56 dria , whose activity is linked to substrate availability and hESCs , and although they are attributed to abnormal structure . ETC protein stoichiometry dictates the ﬂow karyotypes, other cell lines retain proper chromosomal of electrons, which inﬂuences mitochondrial activity .As composition and show no apparent change in cell pro- described earlier, iPSCs upregulate glycolytic enzymes liferation rate or pluripotency potential . Thus, like early- 26,27 during the early stages of reprogramming and this and late-passage F9 cells, stem cells have properties that coincides with the downregulation of proteins in complex may or may not change with passaging, suggesting that I and IV, and the upregulation of proteins in complex II, culturing methods and passage number are not the only 54 57 III and V . Although the metabolic proﬁle of iPSCs is factors promoting chromosomal degradation . similar to differentiated early-passage cells, there were no Overall, our results clearly demonstrate that early versus detectable changes to complex I and IV in these cells late-passage F9 cells have the ability to differentiate into (Fig. 5a, b). In contrast, the reduction in protein levels in XEN, but they do so using different metabolic proﬁles these complexes were seen in differentiated late-passage (Fig. 8e). Moreover, culturing either population under cells, which favor OXPHOS during differentiation conditions that favor their proﬁle enhances their exit from (Fig. 5c, d). Nevertheless, the disruption of ETC stoi- pluripotency and promoted differentiation. Although chiometry in late-passage cells explains the elevation in several reports have documented the differentiation of F9 58–63 mitochondrial ROS (Fig. 1a–d), which together with cells into PrE , none have addressed the mechanisms cytosolic sources, activate the canonical WNT/β-catenin in reference to metabolic proﬁle or altered karyotypes that 22–24 pathway required for F9 cell differentiation . Sur- accompany extensive passaging. Thus, the desired differ- prisingly, these elevated mitochondrial ROS levels were entiation phenotype of cells may come at a cost if genomic not accompanied by changes in the levels of mitochon- integrity is compromised. Furthermore, this underpins drial dynamic proteins (Fig. 5f) that are typically asso- the importance of continually scrutinizing a stem cell ciated with mitochondria actively generating ATP . population to ensure best practices for regenerative Instead, an increase in the expression of Mfn1 and Mfn2, therapies. which encode mitochondrial fusion-promoting proteins, was seen in differentiated early-passage cells (Fig. 5e). Materials and methods Similarities exist with primed ESCs and hESCs, which Cell culturing conditions possess oval-shaped mitochondria with dense matrix, F9 embryonal carcinoma stem-like cells (Sigma) were prominent cristae, and high mtDNA copy number, but cultured at 37 °C and 5% CO on 0.1% gelatin-coated have reduced mitochondrial respiration rates due to plates in Dulbecco’s modiﬁed Eagle’s medium (Lonza) deﬁciencies in in complex I and IV . These incon- supplemented with 10% heat-inactivated fetal bovine sistencies in the literature confounded the interpretation serum (Thermo Fisher Scientiﬁc) and 1% of our data and details to explain the differences in penicillin–streptomycin (Thermo Fisher Scientiﬁc). All metabolic proﬁle seen between early- vs. late-passaged F9 cultures were tested for mycoplasma and cells under 20 cells led us to examine other possibilities. passages were classiﬁed as being early. To induce PrE Genomic integrity was one possible explanation, as differentiation, cells were treated daily for 72 h with 100 long-term passaging of stem cells can promote chromo- nM All-trans retinoic acid (RA; Sigma), or treated daily somal abnormalities resulting in loss of pluripotency and with 100 nM RA and 10 mM db-cAMP (RDB; Sigma) to low contribution to chimeras . In our report, early- and induce PE. Cells treated with dimethyl sulfoxide (DMSO) late-passage F9 cells shared similar pluripotency and dif- served as a negative control. ferentiation proﬁles (Fig. S1, S3); however, based on a comparison with other stem cell lines, we were unable to Generation of PDK1-stable cell line assign candidates that would implicate their involvement F9 cells were plated as described above and reverse in the metabolic differences seen in the two F9 cell transfected using Lipfectamine 2000 (Thermo Fisher populations. It is known that mESC lines cultured for Scientiﬁc). Brieﬂy, 200,000 cells were seeded in 35 mm prolonged periods develop abnormal karyotypes, yet gelatin-coated plates already containing 4 μg of DNA maintain pluripotency and differentiate when grown as plasmid. Culture media was replaced 6 h post transfection embryoid bodies . Our results revealed dramatic differ- and cells were allowed to grow for 24 h. Following incu- ences in the karyotypes of early- and late-passage F9 cells bation, media was changed and cells were selected with 2 (Fig. 8a, b), and these would have profound effects on the mg/ml G418 (Gemini Bio-Products) for 2 weeks. Cells physiology of each population. Differences included were trypsinized and seeded at low density allowing single altered levels of ROS (Fig. 1a–d) and elevated CDKNA1 colony formation. Single clones were selected and pro- and p53 levels (Fig. 8d), the latter affecting the prolifera- pagated for downstream analysis. pcDNA 3.1 plasmid was tion rate in late-passage F9 cells (Fig. 8c). Interestingly, generously provided by Dr. Robert C. Cumming and Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 14 of 16 DDK-MYC-mPDK1 overexpression construct was pur- diamidino-2-phenylindole mounting media containing chased from Origene. ProLong™ Gold Antifade Mountant and examined using an Axio Imager A1 microscope (Carl Zeiss). Thirty RNA isolation and quantitative reverse transcription-PCR representative images were taken of each cell and chro- analysis mosomes were counted manually for statistical analysis. Total RNA, isolated at 72 h from cells using a RNeasy Mini kit (Qiagen), was reverse transcribed into ﬁrst strand Detection of mitochondrial ROS and membrane potential cDNA using the High-Capacity cDNA Reverse Tran- Mitochondrial ROS and membrane potential were scription kit (Applied Biosystems). Quantitative reverse detected using MitoSOX™ Red Mitochondrial Superoxide transcription-PCR (qRT-PCR) reactions, containing 500 indicator (MitoSOX; Thermo Fisher Scientiﬁc) and Tet- nM of each primer (Supplementary Table 1), SensiFAST ramethylrhodamine, methyl ester perchlorate (TMRM; SYBR Mix (FroggaBio), and cDNA, were carried out using Thermo Fisher Scientiﬁc), respectively. Brieﬂy, cells were a CFX Connect Real-Time PCR Detection System (Bio- seeded onto 0.1% gelatin-coated plates and allowed to Rad). Results were analyzed using the comparative cycle differentiate as described above. At 96 h, cells were −ΔΔCt threshold (2 ) method with L14 serving as the washed with phosphate-buffered saline and incubated internal control. with 100 nM MitoSOX or TMRM for 30 min at 37 °C and 5% CO . Images were captured on an Axio Observer A1 Immunoblot analysis Inverted microscope (Carl Zeiss) equipped with a Retiga Protein lysates were harvested using RIPA buffer (10 1300 camera (QImaging). Relative ﬂuorescence intensity mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% was quantiﬁed using ImageJ 1.48 V software (NIH). Triton X−100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl) supplemented with HALT™ protease Measuring ATP levels, glucose uptake, and lactate cocktail inhibitor (Thermo Fisher Scientiﬁc). Protein production concentrations were determined using a DC™ Protein ATP levels were measured using a CellTiter-Glo Assay (Bio-Rad) and 5–40 μg of lysates were prepared in Luminescent Cell Viability Assay (Promega). Brieﬂy, cells 5X SDS loading buffer (300 mM Tris-Hcl pH 8.0, 10% were cultured and differentiated as described above. At SDS, 20 mM EDTA, 0.1% bromophenol blue, and 50% 72 h, cells were trypsinized, suspended in 100 µl of media, glycerol) and 10% β-mercaptoethanol. Proteins were and added to a 96-well plate. After adding 100 µl of separated on 5–15% polyacrylamide gels for 2 h at 100 V CellTiter-Glo reagent, cells were lysed for 2 min and and then transferred onto polyvinylidene diﬂuoride incubated for 10 min in the dark at room temperature. membranes (Bio-Rad) for 2 h at 250 mA and 4 °C. Mem- Luminance was recorded using a Modulus™ II microplate branes were placed in Tris-buffered saline with 0.1% multimode system (Promega) and an integration time of Tween-20 (TBS-T) containing 5% w/v skim milk powder 1.0 s. For glucose uptake and lactate production, cells and shaken at room temperature for 30 min. Membranes were cultured and differentiated as above, and at 72 h were then incubated overnight at 4 °C with a primary media was removed and centrifuged at 4 °C for 10 min at antibody (Supplementary Table 2). After extensive 10,000 r.p.m. Media was analyzed for glucose and lactate washing with TBS-T, membranes were incubated with using a BioProﬁle 400 Chemical Analyzer (Nova Biome- secondary antibodies (Supplementary Table 2) for 2 h at dical) at GCRC Metabolomics Core Facility (McGill room temperature and signals were detected using a University). Protein concentration was used to normalize Immobilon Classico Western HRP substrate (Milllipore values. Sigma). Images were collected using a ChemiDoc™ Touch Imaging System (Bio-Rad); for densitometric analysis, Cytotoxicity assay images were analyzed using Image Lab™ (Bio-Rad). F9 cells were cultured in 0, 2, 4, and 6 mM dichlor- oacetate (DCA) and assayed for viability using a MTT (3- Karyotype analysis (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- Early- and late-passage F9 cells were cultured on 0.1% mide) assay (Sigma). Brieﬂy, media was removed 24 h post gelatin-coated coverslips and allowed to reach 80% con- treatment and MTT reagent was added to cells cultured ﬂuency. Cells were treated with 0.2 μg/ml colcemid for 6 h at 37 °C and 5% CO . To dissolve formazan crys- (Cayman Chemical) for 2 h at 37 °C, followed by trypsi- tals, DMSO was added to cells, which were shaken in the nization and suspension in pre-warmed 0.075 M KCl dark overnight at room temperature. Absorbance values solution for 20 mins at 37 °C. Cells were ﬁxed in acetic at 570 nM with a reference wavelength at 650 nM were acid-methanol solution and then transferred onto pre- collected using the Modulus™ II microplate system chilled glass slides. Chromosomes were stained with 4′,6- described above. Ofﬁcial journal of the Cell Death Differentiation Association Gatie and Kelly Cell Death Discovery (2019) 5:42 Page 15 of 16 Scanning electron microscopy References 1. Ito, K. & Ito, K. Metabolism and the control of cell fate decisions and stem cell F9 cells were cultured and differentiated as mentioned renewal. Annu. Rev. Cell. Dev. Biol. 32,399–409 (2016). above. Brieﬂy, cells were washed in 0.1 M phosphate 2. Ito, K. & Suda, T. Metabolic requirements for the maintenance of self-renewing buffer and ﬁxed with 2% glutaraldehyde for 30 mins, fol- stem cells. Nat. Rev. Mol. Cell Biol. 15,243–256 (2014). 3. Mathieu, J. & Ruohola-Baker, H. Metabolic remodeling during the loss and lowed by osmium tetroxide for 2 h at 4 °C. 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Mol. Syst. Biol. 7, 538 (2011). Ofﬁcial journal of the Cell Death Differentiation Association
Cell Death Discovery – Springer Journals
Published: Sep 26, 2018
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