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A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs

The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

controlling for cell-line clonality and sex. We find that transcriptional variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detected between isogenic hESCs and hiPSCs neither predicted functional outcome nor distinguished an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional comparisons of pluripotent cell lines, explaining some of the previously observed expression differences between unmatched hESCs and hiPSCs.
The question of whether hiPSCs, derived from somatic cells by overexpression of the transcription factors Oct4, Klf4, Sox2 and c-Myc (OKSM) 1 , are equivalent to hESCs, the gold standard of pluripotent cell lines, is becoming increasingly urgent as patient-specific hiPSCs are advanced toward clinical application [1][2][3][4] . Initial studies showed that hESC and hiPSC lines are fundamentally different at the transcriptional level, whereas subsequent work concluded that they are virtually indistinguishable when comparing larger sample sets [5][6][7] . More recent reports using refined gene expression analyses found small sets of differentially expressed genes (DEGs) [8][9][10] . However, the origins of these DEGs, their consistency across independent studies and their impact on the differentiation potential of hiPSC lines remain unclear. Transcriptional patterns are influenced by numerous biological and technical parameters that may confound results. The reprogramming method, including the choice of integrating versus non-integrating factor delivery systems, can alter gene expression in iPSCs [11][12][13] . Likewise, genetic background may influence transcriptional signatures in pluripotent cell lines since iPSCs derived from different individuals are reportedly more divergent than iPSCs derived from the same individual. The difference between the clonal origin of hiPSC lines, derived from single somatic cells, and the polyclonal origin of most hESC lines may also introduce transcriptional variation 14 . An additional consideration is the sex of cell lines and defects in X chromosome reactivation in female hiPSCs 17,18 . Some of these variables have been addressed in previous reports 11,12,15,16 , but, to our knowledge, no comparative study of hESCs and hiPSCs has accounted for all of them.
We previously showed that comparing genetically matched mouse ESC and integration-free iPSC lines eliminates most of the transcriptional variation observed between unmatched cell lines 16 . Although we could not identify consistent transcriptional differences between mouse ESC and iPSC lines, we discovered a small group of transcripts that was aberrantly silenced in a subset of iPSC lines, which adversely affected their developmental potential. Here we extend our analyses to the human system and ask whether molecular differences can be identified in hiPSC lines relative to hESC lines that cannot be attributed to the SeV reprogramming method, genetic background, clonal origin or sex, and whether any such differences impact functional outcomes.

Approach to generate isogenic hESCs and hiPSCs
To compare hESCs with genetically matched hiPSC lines devoid of viral integrations, we generated hiPSCs from in vitro-differentiated hESCs using a non-integrating Sendai virus (SeV)-based reprogramming system 19 ; SeV is an RNA virus that is diluted from infected cells in a replication-dependent manner, leaving no genetic footprint behind (Fig. 1A,B). We chose two well-characterized hESC lines, HUES2 and HUES3 20 , for these experiments. We selected male hESC lines because female iPSCs can exhibit defects in X chromosome reactivation 17,18 , which might confound subsequent interpretations 9,21 .
First, we subcloned each line in order to ensure genetic and epigenetic homogeneity of cells and to properly control for the clonal origin of hiPSCs (Fig. 1A). We differentiated one hESC subclone from each background by switching cells to serum-containing medium without basic fibroblast growth factor (bFGF), which is critical for the maintenance of hESCs, and sorting fibroblast-like cells based on CD90 + /TRA-1-81 − expression (Fig. 1A,C). These fibroblast-like cells, which resemble primary human fibroblasts by morphological criteria (Fig. 1C), did not form Alkaline Phosphatase (AP)-positive colonies in hESC media, indicating successful differentiation and the absence of residual pluripotent cells in the culture ( Supplementary Fig. 1A). Analysis of global gene expression by RNA-sequencing revealed that the fibroblast-like cells were highly similar to dermal fibroblasts but distinct from pluripotent stem cell lines ( Supplementary Fig. 1B). Pluripotency-associated promoters, such as POU5F1, LEFTY1, TDGF1, and SCNN1A, were re-methylated and decreased in expression levels whereas fibroblast-specific promoters such as TMEM173, EMILIN1, LMNA, and RIN2 were demethylated and regained expression in fibroblast-like cells (Fig. 1D). In a final step, the fibroblast-like cultures were reprogrammed into hiPSCs by infecting the cells with SeV vectors expressing OKSM, as previously reported 19 (Fig.   1A). Emerging colonies were isolated after ~3 weeks, expanded and confirmed to be positive for AP activity and endogenous OCT4 expression, indicating successful reprogramming (Fig. 1C). Moreover, we ensured loss of SeV expression in all lines, demonstrating reprogramming factor independent self-renewal ( Supplementary Fig. 1C,D).

Genetic background drives transcriptional variation
First we studied whether the SeV reprogramming method affects global transcription. The parental hESC subclones were infected with GFP-expressing SeV (SeV-GFP) and passaged until GFP fluorescence was no longer detectable before analyzing cell lines by RNAsequencing ( Fig. 1A and 2A). We found a common set of 63 genes that was differentially expressed between three uninfected hESC subclones (hESC SCs) and three SeV-GFP infected hESC subclones (hESC GFPs) from each genetic background, which demonstrates that viral infection itself leads to subtle but statistically significant transcriptional changes that persist after viral loss (Fig. 2B). This 63-DEG set consistently separated hESC SC lines from hESC GFP lines (Fig. 2C). Gene Ontology terms significantly enriched among these 63 DEGs are related to transcription, DNA binding, and development ( Supplementary Fig.  1E). Based on these observations, we decided to use expression data from hESC GFP lines as controls for all subsequent comparisons with SeV-generated hiPSC lines.
A comparison of the transcriptional profiles of hESC subclones (hESC SCs and hESC GFPs), in vitro-differentiated fibroblasts and derivative hiPSCs by unsupervised clustering showed the largest differences between pluripotent cell lines and differentiated cell types, consistent with previous observations 5,15,22,23 (Supplementary Fig. 2A). Likewise, global methylation analysis of representative samples by reduced representation bisulfite sequencing (RRBS) separated pluripotent cells from in vitro-differentiated fibroblasts, indicating distinct epigenetic states ( Supplementary Fig. 1F). Notably, we observed a clear segregation of all pluripotent samples into two transcriptionally related groups, irrespective of whether cell lines were infected with SeV or not (Fig. 2D, expanded from Supplementary Fig. 2A). This segregation could not be explained by the cellular origin of cell lines from embryos (hESCs) or somatic cells (hiPSCs) but instead correlated with the genetic background of each line. That is, HUES2-derived hESC subclones clustered with HUES2derived hiPSC lines whereas HUES3-derived hESC subclones clustered with HUES3derived hiPSC lines. Consistent with this finding, overall transcriptional variation between groups of genetically matched hESC and hiPSC lines was significantly lower than that between unmatched cell lines ( Supplementary Fig. 2B). Moreover, transcriptional variation within groups of genetically matched hiPSC or hESC lines was similar, indicating that hiPSCs and hESCs are equally variable ( Supplementary Fig. 2C). Of note, the number of promoters differentially methylated between unmatched pluripotent cell lines was approximately twice as high (2,610) as that between matched pluripotent cell lines (1,205), suggesting that genetic background also influences epigenetic patterns in hESCs and hiPSCs ( Supplementary Fig. 2D). We conclude that genetic background is a major driver of transcriptional and epigenetic differences between pluripotent cell lines, whereas the SeV reprogramming method introduces more subtle yet stable transcriptional changes in hiPSCs.

Expression differences between matched hESCs and hiPSCs
Although genetic background accounted for most transcriptional differences among the analyzed pluripotent cell lines, we noticed that hESCs clustered with each other and separately from hiPSCs within a given background, suggesting subtle but consistent transcriptional differences that reflect distinct cellular origins (Fig. 2D). To identify any DEGs that distinguish hESC from hiPSC lines independent of SeV infection and genetic background, we compared transcriptional profiles of hiPSC lines with those of genetically matched hESC GFP lines. This analysis revealed that 52 and 91 genes were up-and downregulated, respectively, in hiPSC lines derived from the HUES2 background, whereas 77 and 426 genes were up-and down-regulated in hiPSC lines derived from the HUES3 background, respectively. Forty-nine genes were commonly dysregulated in both genetic backgrounds (Fig. 3A). Considering the good depth of our RNA-seq data (~40 million mapped reads per sample on average) ( Supplementary Fig. 2E), it is highly unlikely that this small number of DEGs was due to low sensitivity. As expected, the 49-DEG signature reliably separated our hiPSC lines from our hESC lines (Fig. 3B).
We did not detect any Gene Ontology term that was significantly enriched among the 49 DEGs. A comparison of our DEG set with 8 different protein interaction databases, including BIND, DIP, MINT and REACTOME INTERACTION using DAVID, also showed no significant enrichment (data not shown). Notably, 48 of 49 DEGs were downregulated in hiPSCs relative to hESCs (Fig. 3C). This raised the possibility that the DEGs were silenced in fibroblast-like cells and were not properly reactivated in derivative hiPSCs. However, the expression levels of the DEGs in fibroblast-like cells did not show a consistent pattern, excluding incomplete reprogramming or the retention of 'epigenetic memory' (Fig. 3C).
We next asked whether the DEGs have functional consequences. We focused on two DEGs, LDHA and SLC2A1 (also known as GLUT1), because of their strong basal expression in hESCs and reduced expression in all hiPSCs (Fig.3C,D). Both gene products are involved in energy metabolism; LDHA plays an important role in glycolysis by catalyzing the conversion of pyruvate to lactate 24,25 , whereas SLC2A1 facilitates glucose uptake in cells 26,27 . Accordingly, LDHA and SLC2A1 are abundantly expressed in pluripotent cells, which produce energy through glycolysis 28 (Fig. 3C). Based on the down-regulation of these two genes in all examined hiPSC lines compared to hESC lines by RNA-seq and qPCR analyses (Fig. 3E), we hypothesized that hiPSC lines might be less glycolytic than hESC GFP lines. However, neither lactate production nor glucose uptake levels differed between isogenic hiPSC and hESC GFP lines (Fig. 3F). Further, there was no difference in LDHA protein levels despite the observed transcriptional differences (Fig. 3G). Thus, at least two of the 49 DEGs seem not to translate into functional differences, possibly owing to posttranscriptional compensatory mechanisms.
The low level of transcriptional differences between undifferentiated hESCs and hiPSCs does not exclude the existence of iPSC-specific aberrations that become detectable only after differentiation. We performed RNA-sequencing of fibroblast-like cells derived from 8 hESC subclones (2 hESC SC and 6 hESC GFP lines) and 6 hiPSC subclones using the same in vitro differentiation protocol as described above (Fig. 1A). Only two genes were consistently upregulated in hiPSC-derived fibroblast-like cells compared to hESC-derived fibroblast-like cells from both genetic backgrounds, and they did not overlap with the 49 DEGs between undifferentiated hESC and hiPSC lines ( Supplementary Fig. 3A,B). However, HUES2derived fibroblast-like cells tended to cluster together and apart from HUES3-derived fibroblast-like cells using PCA analysis ( Supplementary Fig. 1B), which is consistent with the segregation of undifferentiated cells by genetic background. We infer that genetic background also drives transcriptional variation in differentiated cell populations, and that any transcriptional differences observed between undifferentiated hESC and hiPSC lines do not persist in differentiated fibroblast-like cells.

Dysregulation of genes in a subset of hiPSC lines
As most of the DEGs between undifferentiated hESC GFP and hiPSC lines produced lowabundance transcripts that were not obviously connected through a common biological process (Fig. 3C), we examined genes that were dysregulated in only a subset of hiPSC lines, which we refer to as inconsistently differentially expressed genes (iDEGs) (Supplementary Fig. 3C). We have previously shown that iDEGs between isogenic mouse ESCs and iPSCs could predict full developmental potential of subsets of iPSC lines 16 . Applying the same principle to our human data set, we found that 34 genes were upregulated, whereas 27 genes were downregulated in some of the HUES2-derived hiPSC lines when compared to genetically matched hESC GFP lines. Similarly, 9 genes were upregulated and 32 genes were downregulated in some of the HUES3-derived hiPSC lines relative to matched hESC GFP controls ( Supplementary Fig. 3C). Only eight iDEGs were dysregulated in both genetic backgrounds, and these were thus selected for further analysis ( Fig. 4A and Supplementary Fig. 3C).
The iDEGs IRX2 and DPP10 have been linked to neural development or psychiatric disease [29][30][31][32] and IRX2 suppression reportedly impairs hESC differentiation into neural progenitors. Silencing of IRX2 and DPP10 in some of the hiPSC lines and none of the hESC lines ( Fig. 4B) was confirmed by qPCR (Fig. 4C). However, the iDEGs did not affect the cells' potential to differentiate into neuroectodermal cells using a published protocol 33 (Fig.  4D), as determined by RNA expression analysis for NESTIN, SOX1, PAX6, and FOXG1, well-established markers of neuroectoderm differentiation from human pluripotent stem cells 34 (Fig. 4E). Consistent with this, PAX6 and SOX1 were equally expressed at the protein level during neural differentiation from hiPSC and hESC GFP lines ( Fig. 4F and Supplementary Fig. 3D).
To determine whether hiPSCs exhibit biases in differentiation into other lineages, we evaluated their ability to generate ectodermal, endodermal and mesodermal derivatives by the Score card assay 6 . Briefly, hiPSC and hESC GFP lines from both genetic backgrounds were differentiated into embryoid bodies (EBs) before scoring for the expression of 77 developmental marker genes by qPCR. Hierarchical clustering of these data showed that all markers were expressed at similar levels in genetically matched cell lines (Fig. 4G). Thus, isogenic hESCs and hiPSCs appear to have equivalent potential to differentiate into cell types of the three germ layers.

Genetic background explains previous expression differences
We asked whether the 49 genes differentially expressed between our isogenic hESCs and hiPSCs are also dysregulated in hiPSC lines derived from primary somatic cells as well as in other published datasets. First, we reanalyzed a published set of unmatched hESC (n=18) and hiPSC (n=12) lines generated from primary fibroblasts using retroviral vectors, whose gene expression patterns were previously analyzed by microarrays 6 . Since many of the 49 DEGs were not covered in the available microarray data, we performed RNA-sequencing of these hESC and hiPSC lines, which offers increased sensitivity, especially for lowabundance transcripts 35,36 . However, unsupervised clustering was unable to separate these hESCs from hiPSCs (Fig. 5A). Although 3 DEGs (RP11-1, MEG3, AL1327) were identified between unmatched hESCs and hiPSCs, these were likely false positives based on permutation analysis. Indeed, supervised clustering of all samples with these 3 DEGs (data not shown) or an extended set of 16 DEGs using loosened criteria could not distinguish hESCs from hiPSCs ( Fig. 5A and Supplementary Fig. 4A,D). Notably, our stringently defined 49-DEG signature was also unable to segregate the transcriptomes of this extended set of hESC and hiPSC lines (Fig. 5B).
Next, we determined the potential overlap between DEGs identified within our isogenic and unmatched hESC/hiPSC lines, and two previously reported sets of DEGs 8,10 . There was little to no overlap between DEGs discovered by independent laboratories (Fig. 5C and Supplementary Fig. 4B) and these DEGs could not distinguish hiPSC and hESC lines from the respective other data sets using supervised clustering ( Supplementary Fig. 4C-I). Only two of our 49 DEGs (MT1E, S100A14) and two of our 8 iDEGs (IRX2 and DPP10) overlapped with DEGs reported in ref. 10. Collectively, these data support the view that other parameters, such as reprogramming method, genetic background or sex, account for the majority of previously reported transcriptional differences between hESCs and hiPSCs.
In agreement with this conclusion, DEGs reported in ref. 10 distinguished our hESC and hiPSC cell lines by genetic background rather than cellular origin (Fig. 5D, left panel). In that study, multiple hiPSC lines generated from one man were compared to male and female hESC lines of different genetic backgrounds. Conversely, genes that distinguish our HUES2derived and HUES3-derived pluripotent cell lines were able to separate the hESCs and hiPSCs in ref. 10 (Fig. 5D, right panel; Fig. 5E; Supplementary Fig. 4J,K).
In further support of the notion that genetic background profoundly influences transcriptional patterns in pluripotent cell lines, we found that DEGs that distinguish our HUES2-derived and HUES3-derived cell lines account for more transcriptional variation among our 30 unmatched hESCs and hiPSCs than do all genes or DEGs that distinguish our isogenic hESCs and hiPSCs or SeV-infected and uninfected hESCs (Fig. 5F). Taken together, these meta-analyses suggest that the main transcriptional differences between genetically unrelated hESC and hiPSC lines are primarily driven by genetic background rather than cellular origin or reprogramming method.

DISCUSSION
Here we show that isogenic male hESC and hiPSC lines are transcriptionally highly similar to one another, suggesting that genetic background variability and possibly sex differences account for most of the previously reported gene expression differences between hESCs and hiPSCs. This conclusion is particularly relevant to studies where only a limited number of hESC lines or a single iPSC donor individual was used, as imbalances in genetic background and sex may further inflate transcriptional differences 9,10,37,38 . Our finding that a previously reported set of DEGs between 4 hiPSC lines derived from a single individual and 4 hESC lines separated our hESC and hiPSC lines by genetic background rather than cellular origin supports this conclusion (Fig. 5D).
Our study reveals that a commonly used non-integrating reprogramming method can subtly but stably alter transcriptional patterns in iPSCs (Fig. 2B,C). However, the transcriptional signature introduced by SeV infection (63 DEGs) did not separate hESCs from hiPSCs previously generated with retroviral or episomal vectors, suggesting that each reprogramming system may introduce unique transcriptional alterations into iPSCs ( Supplementary Fig. 4F,I). Whereas the molecular mechanisms of this observation remain to be elucidated, our findings highlight the importance of controlling for the method of iPSC induction when studying transcriptional patterns in iPSCs. Indeed, a recent comparison of hiPSCs generated with different OKSM delivery systems showed that hiPSCs derived with integrating vectors (e.g., retroviral transgenes) more often exhibit expression, methylation and differentiation defects compared to hiPSCs produced with non-integrating approaches 39 .
We identified only 49 DEGs that could distinguish hESCs and hiPSCs and 8 iDEGs that were dysregulated in a subset of hiPSC lines (Fig. 3C and 4A). This small number of genes contrasts with previous studies, which identified much larger numbers of DEGs when comparing unmatched hESCs and hiPSCs using a similar cutoff [5][6][7]10,13,22,23 . Of note, we found no evidence that two of the tested DEGs (LDHA and SLC2A1) and two of the tested iDEGs (IRX2 and DPP10) predict functional outcome, i.e., energy production or differentiation potential into neural cells or EBs. These results therefore suggest that hESC and hiPSC lines are equivalent after accounting for genetic background differences. We surmise that the remaining DEGs we detected between isogenic hESCs and hiPSCs might represent transcriptional noise from weakly expressed genes. In support of this notion, the vast majority of the 49 DEGs was expressed at relatively low levels in our hESCs and hiPSCs and showed no overlap with previously reported gene expression signatures. However, we cannot exclude that the lack of an obvious phenotype with the abovementioned assays could be due to insufficient expression of the analyzed genes in undifferentiated hiPSCs or compensation by posttranscriptional mechanisms, as appears to be the case with LDHA (Fig. 3G). Alternatively, our metabolic and in vitro-differentiation assays may not have been sensitive enough to detect functional differences. Another possibility is that hiPSCs are distinguished from hESCs by epigenetic or genetic differences that do not manifest in the pluripotent state. However, our finding that fibroblast-like cells derived from all examined hESC and hiPSC lines show no discernable transcriptional differences argues against this explanation ( Supplementary Fig. 3A,B). The fact that isogenic hESC and hiPSC lines exhibit equivalent differentiation potentials using either a directed or spontaneous differentiation paradigm further supports this interpretation (Fig. 4D-G). Critically, hiPSCs were derived from in vitrodifferentiated fibroblasts in this study and, we can therefore not rule out that hiPSCs produced from primary cells accrue additional aberrations that cannot be recapitulated with our in vitro differentiation approach.
Our results may have implications for the use of iPSC technology in disease modeling approaches, where hiPSC lines from healthy individuals are usually compared to hiPSC lines from affected individuals. Because of the apparent influence of genetic background on gene expression patterns in both undifferentiated and differentiated cells, it will be critical to study a sufficient number of hiPSC lines to detect robust phenotypes; this is particularly relevant in complex diseases where the causal mutation(s) are not known. When studying monogenic diseases, it may be necessary to introduce mutations into wild-type hESCs or rescue mutations in patient-derived hiPSCs, as different backgrounds may mask subtle transcriptional differences 40 .

Cell culture
hESC lines and hiPSC lines were cultured with mouse embryonic fibroblasts (MEFs, Globalstem) pre-plated at 12-15,000 cells/cm 2 . Medium containing DMEM/F12, 20% knockout serum replacement, 1mM L-glutamine, 100 uM MEM non-essential amino acids, and 0.1 mM beta-mercaptoethanol was used. 10 ng/ml of FGF-2 was added after sterile filtration and cells were fed daily and passaged weekly using 6 U/mL dispase or mechanically.

RNA-sequencing
Undifferentiated hESC/hiPSC cells were sorted for hTRA-1-81 + to control for the homogeneity of cells before RNA extraction. The quality and quantity of total input mRNA was determined on an Agilent BioAnalyzer 2100 using Agilent RNA 6000 Nano kit. One microgram of total RNA from each sample was then used as input for library preparation using Illumina TruSeq RNA Sample Prep Kit, following manufacturer's instructions. Each paired-end library was prepared with an adaptor with unique index sequence. The size profile and quantity of resulting libraries were than determined on the BioAnalyzer 2100 with Agilent High Sensitivity DNA kit. These libraries were then pooled together at equal molar concentration and sequenced on an Illumina HiSeq 2000. All hESC and hiPSC samples for RNA-Seq analysis were prepared on the same day by the same person, and then sequenced simultaneously on the same run (except for hiPSC lines 1, 2 and 3; this did not affect the clustering). All fibroblasts samples were prepared and sequenced in the same manner as the pluripotent samples but on different days.
RNA-seq reads were mapped using Bowtie 0.12.7 allowing up to 2 mismatches, to the library of human transcriptome sequences obtained from ENSEMBL (GRCh37.67) reference chromosomes, then entries with identical gene symbols were merged. The transcriptome includes both protein-coding genes and non-coding genes such as lincRNAs. EMSAR was used to quantify the expression levels in TPM (transcripts per million) and to infer read counts for individual genes. Differentially expressed genes were identified using edgeR 3.4.2 and confirmed using DESeq 1.8.3.

Methylation analysis
Methylation of individual CpGs was derived by observing bisulfite conversion of unmethylated cytosines in RRBS reads when compared to the reference genome. Methylation maps of individual CpGs show the average methylation value obtained by dividing the number of reads on which the CpG was methylated by the total times the CpG was covered by a read. Promoters were defined as 1 Kb up-and downstream of Refseq gene transcription start sites. Methylation values of individual CpGs in promoters were pooled in a weighted manner (i.e. proportional to the number of reads covering that CpG).
To count differentially-methylated promoters that supported variance due to cellular origin or genetic background, within-sample methylation difference was compared to the between-sample methylation difference for each promoter in sets based on cellular origin (hESC/ hiPSC) and cell background (HUES2/HUES3). The promoter was assigned to the set with the lesser methylation difference, such that promoters in the hESC/hiPSC set showed greater methylation difference between hESCs and hiPSCs and lesser metylation difference between HUES2 and HUES3.
Global methylation clustering was performed by first pooling individual CpG methylation levels into 1 Kb non-overlapping tiles using weighted averages as with promoters, and then using Pearson's correlation to compute distance between samples. Ward's method was used for hierarchical clustering analysis. Analyses were performed using R and Perl.

Immunostaining
Immunostaining was performed using the following antibodies: α-hTRA

Lactate production assay
Lactate production assay was done according to Zhong et at 41

Glucose uptake assay
The glucose uptake assay was done according to Sebastián et al. 42 . Cells were grown under normal conditions for 24 hr and 100 mM 2-NBDG (Invitrogen) was added to the media for 2 hr. Fluorescence was measured in a FACSCalibur Analyzer (BD).

Neural differentiation
Neural induction was performed as previously reported 33 . Briefly, cells were dissociated to single cells using Accutase and plated on gelatin for 10 minutes to remove MEFs. Nonadherent cells were collected and plated on Geltrex-treated dishes at a density of 150-200k cells per well of a 24-well plate in the presence of MEF-conditioned hESC media containing 10 ng/ml of FGF-2 (Life Tech) and 10 uM of Y-27632 (Tocris). Neural differentiation was initiated when cells were confluent using KSR media containing 820 ml of Knockout DMEM (Life Tech), 150 ml Knockout Serum Replacement (Life Tech), 1 mM L-glutamine (Life Tech), 100 uM MEM non-essential amino acids (Life Tech), and 0.1 mM betamercaptoethanol (Life Tech) to inhibit SMAD signaling, 100 nM of LDN-193189 (Cat. no. ab142186, Abcam) and 5 uM of SB431542 (Cat. No. 13031, Cayman Chemical) were added on Days 0 through 9. Cells were fed daily, and N2 media (Life Tech) was added in increasing 25% increments every other day starting on Day 4 (100% N2 on Day 10).

Western blot analysis
For Western blot analysis of PAX6, 10 ug of whole cell lysates was loaded to 4-20% gradient SDS-PAGE gels and then transferred to nitrocellulose membranes (BIO-RAD) by using Trans-Blot ® Turbo™ Transfer System (BIO-RAD). Blocked membranes were incubated with antibodies against PAX6 (Cat. no. 5790, Abcam) or GAPDH (Cat. no. 2118, Cell Signaling), respectively. For Western blot analysis of LDHA, undifferentiated hESC/ hiPSC cells were sorted for hTRA-1-81 + in order to control for the homogeneity of the cells, and then the rest of the procedure ensued as above. LDHA (Cat. no. 2012S, Cell Signaling), β-ACTIN (Cat. no. MA5-15739-HRP, Thermo Scientific).

RNA extraction and qPCR
Total RNA was extracted from differentiating hESC/hiPSC lines using the TRIzol Reagent (Life Tech), and 0.51 ug of RNA was reverse transcribed by High Capacity cDNA Reverse Transcription Kit RT2 first strand kit (ABIQiagen). Primer sequences are provided below. qRT-PCR mixtures were prepared with SYBR Green PCR Master Mix Universal (Applied BiosystemsKapabiosystem) and reactions were done with the Eppendorf Realplex2.

EB scorecard assay
EB differentiation was performed as described previously 6 . On day 7, EBs were lysed and total RNA was extracted before analyzing differentiation markers using qPCR.