Somatic Transcriptome Priming Gates Lineage-Specific Differentiation

ARTICLE

Received 27 Feb 2014 | Accepted 20 Oct 2014 | Published 3 Dec 2014 DOI: 10.1038/ncomms6605 Somatic transcriptome priming gates lineage-speciﬁc differentiation potential of human-induced pluripotent stem cell states

Jong-Hee Lee1, Jung Bok Lee1, Zoya Shapovalova1, Aline Fiebig-Comyn1, Ryan R. Mitchell1,2, Sarah Laronde1, Eva Szabo1, Yannick D. Benoit1 & Mickie Bhatia1,2

Human-induced pluripotent stem cells (hiPSCs) provide an invaluable source for regenerative medicine, but are limited by proficient lineage-specific differentiation. Here we reveal that hiPSCs derived from human fibroblasts (Fibs) versus human cord blood (CB) exhibit indistinguishable pluripotency, but harbour biased propensities for differentiation. Genes associated with germ layer specification were identical in Fib- or CB-derived iPSCs, whereas lineage-specific marks emerge upon differentiation induction of hiPSCs that were correlated to the cell of origin. Differentiation propensities come at the expense of other lineages and cannot be overcome with stimuli for alternative cell fates. Although incomplete DNA methylation and distinct histone modifications of lineage-specific loci correlate to lineage- specific transcriptome priming, transitioning hiPSCs into naive state of pluripotency removes iPSC-memorized transcriptome. Upon re-entry to the primed state, transcriptome memory is restored, indicating a human-specific phenomenon whereby lineage gated developmental potential is not permanently erased, but can be modulated by the pluripotent state.

1 McMaster Stem Cell and Cancer Research Institute, Faculty of Health Sciences, McMaster University, 1200 Main Street West, MDCL 5029, Hamilton, Ontario, Canada L8N 3Z5. 2 Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada. Correspondence and requests for materials should be addressed to M.B. (email: [email protected]).

NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605

xpression of a discrete and defined set of gene products and functional criteria routinely applied to characterize associated with pluripotency (OCT4, SOX2, c-MYC, KLF4, fully reprogrammed iPSCs, hCB-iPSCs and hFib-iPSCs ENANOG and LIN28) induces reprogramming of somatic are indistinguishable using established criteria for the human cells into self-renewing pluripotent cell types similar to human pluripotent state2,10. embryonic stem cells (ESCs)1,2. While morphology, pluripotent gene expression profiles and pluripotent markers in human- Equivalency in transcriptional profiles of three germ layers. induced pluripotent stem cells (hiPSCs) are suggestive of Recent reports indicate that incomplete DNA methylation equipotent developmental potential, recent studies reveal underlies transcriptional differences in iPSCs derived from dif- substantial molecular and functional differences among iPSCs ferent origins/tissues, suggesting that cellular memory persists in derived from distinct cell types. iPSCs produced different the iPSCs following reprogramming4, and that this phenomenon properties in teratoma formation that correlate with the tissue is unique to human iPSCs as mouse iPSCs lose this memory over of origin3 and moreover, persistence of the DNA methylation passaging5. To better understand whether previously observed status of the cell of origin has been linked with molecular differential propensities for one lineage versus another followed a aspects of hiPSC programming4. Aside from these molecular developmental paradigm (for example, could lineage descriptions, transcriptome analysis and functional measure- augmentation be due to an initial increased overall germ layer ments, such as lineage-specific maturation potential have yet to be propensity), we analysed gene expression patterns corresponding robustly examined. to the three germ layers (ectoderm, endoderm and mesoderm). Recently, iPSCs have been shown to retain a DNA methylation Hierarchical clustering on genes related to ectoderm, endoderm status akin to the cell of origin4–7. However, to date, no causal and mesoderm emergence revealed similar patterns across all evidence has been presented to suggest that incomplete PSCs from all cell sources, methods and clones of human iPSCs methylation is responsible for imposing memory, as opposed to (Fig. 2a,d,g). Furthermore, we directly compared germ layer- a consequence of unknown mechanisms, which drive the observed related expression between hCB- and hFib-iPSCs through GSEA, changes in epigenetic status of specific loci or whether this impacts which revealed no significant differences between hiPSC differentiation behaviour. One of the most promising applications lines (Fig. 2b,e,h), suggesting that somatic memory cannot of iPSCs is to model human disease, and is only limited by efficient be explained by inherent activation of transcription related and controlled differentiation of human PSCs into specific cell to the specification stage of germ layer commitment within the types. Here we generated hiPSCs from different sources, adult pluripotent state. As such, we next examined whether the dermal fibroblasts (Fibs) and CD34 þ umbilical cord blood (CB) apparent discrepancy in the differentiation capacity of hiPSCs cells, and functionally and quantitatively compared their from different somatic origins was due to a disparity in the differentiation potential along with transcriptional profiles in the initiation of early developmental programmes related to lineage- undifferentiated state. Cell fate decisions were shown to closely specific commitment. To assess this, we initiated differentiation in relate to the transcriptional profiles connected to lineage of origin, hiPSCs towards specific lineages, neural (ectoderm), hepatocytes and did not extend to the corresponding germ layer. We propose (endoderm), cardiomyocytes and hematopoietic (mesoderm), and that the differentiation potential of human iPSCs is channelled examined early lineage-associated markers. Early neural marker, towards the parental lineage, but this comes at the expense of PAX6 (Fig. 2c), and hematopoietic markers, brachyury (T) and differentiation to other cell types. MixL1 (Fig. 2i), showed marked differences in the initial stage of differentiation, wherein hCB-iPSCs displayed increased hemato- Results poietic and decreased neural marker expression compared with hFib-iPSCs (Fig. 2c,i). However, no clear expression differences Equivalency of pluripotent states of hiPSCs. Molecular and were observed between hCB- and hFib-iPSCs for endodermal functional properties of human hiPSCs derived from different markers, FoxA2, Gata4 and Sox17 (Fig. 2f; Supplementary Fig. 2), somatic sources were compared between hiPSCs generated from or the mesodermal marker smooth muscle actin (SMA) (Fig. 2i) CD34 þ CD45 þ umbilical CB cells and compared with iPSCs during early developmental progression into hepatocytic and from human adult dermal Fibs, a common source for iPSC cardiomyocytic lineages, respectively. This indicates that hiPSCs generation. Inclusion of CD45 was critical to assure cells used possess a biased lineage specification towards cell types closely were of a hematopoietic lineage, and not endothelial cells that also related to the tissue of origin. Overall, these results reveal that express CD34 (refs 8,9). To provide generalizable results, all pre-existing germ layer transcriptional activity is equivalent in hiPSC lines were derived by transduction with Oct4, Sox2, Nanog hiPSCs, but lineage-specific transcription becomes gradually and Lin28 or Oct4, Sox2, c-Myc and KIf4 expressing lentiviral unbalanced in a stepwise manner during early-induced vectors. Both hFib and hCB-derived iPSC lines were commitment stages of hPSC development. morphologically indistinguishable from hESCs and acquired typical pluripotent marker expression, similar among hiPSCs and hESCs (Fig. 1a). Both hFib- and hCB-iPSCs exhibited com- Distinct differentiation potential in hFib- versus hCB-iPSCs. parable transcript levels of the genes associated with pluripotency To functionally assess the consequence of differential activation (Dppa4, Tbx3, c-Myc, Oct4, Sox2 and Nanog), similar to hESCs of lineage-specific transcription between hiPSCs from different (Supplementary Fig. 1a,b) and produced teratomas containing the cellular origins, we further evaluated their in vitro differentiation three embryonic germ layers (Fig. 1b). Moreover, global gene potential starting with neural lineage specification. Neural lineage expression profiling followed by hierarchical cluster analysis on differentiation was evaluated in two stages; the initial analysis genes associated with pluripotency further indicated successful encompassed formation of neural precursor/progenitors followed molecular reprogramming to the pluripotent state (Fig. 1c). by differentiation into the three major cell types found in the Clustering of hiPSCs lines with hESCs by principal component central nervous system: neurons, oligodendrocytes and astro- analysis, and no significant differences between hFib- versus cytes11,12. Neurospheres, representing an early neural progenitor/ hCB-iPSCs in gene set-enrichment analysis (GSEA) provided precursor stage, were generated from dissociated hiPSCs, additional evidence that Fib- and CB-iPSCs are not expanded and then differentiated using previously optimized distinguishable by evaluating pluripotent status (Supplementary protocols11,12. In contrast to the comparable gene expression of Fig. 1c,d). Collectively, these results indicate that using molecular ectodermal lineage markers in undifferentiated cells, hCB-iPSCs

2 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. ae nPasnscreaincefiins ahrwrpeet gene. a represents row Each coefficients. correlation Pearson’s on based otmi hw ersnaieflwctmtyaayi o lrptn akr SA1 SA3 SA4 C4adTA16.Saebr,100 bars, Scale TRA-1-60. and the OCT4 At 200 33342. SSEA-4, bars, Hoechst SSEA-3, Scale or SSEA-1, mesoderm. DAPI and markers with endoderm pluripotent stained are for Nuclei analysis blood-iPSCs. cytometry cord ( flow and representative fibroblast- hESCs, shown in is TRA-1-60 bottom and OCT4 SSEA-4, SSEA-3, SSEA-1, markers iue1|Euvlnyo lrptn ttso iSsdrvdfo odbodadfibroblasts. and blood cord from derived hiPSCs of states pluripotent of Equivalency | 1 Figure 10.1038/ncomms6605 DOI: | COMMUNICATIONS NATURE AUECOMMUNICATIONS NATURE b eaoa rdcinb ECadhPClns amtxlnesnsandscin hwdfeetainit l he emlyr:ectoderm, layers: germ three all into differentiation show sections Haematoxylin–eosin-stained lines. hiPSC and hESC by production Teratomas )

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Cell of origin ARTICLE m m. 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 produced signiﬁcantly less neural precursor/progenitor cells after neurosphere-forming assays and displayed fewer nestin- and neural differentiation induction (Fig. 3a). hCB-iPSC-derived A2B5-positive cells compared with Fib-iPSCs (Fig. 3b,c). In precurser/progenitors were signiﬁcantly less proliferative during accordance with these differential outcomes of neural )1 )2 + + hESCs-2 hESCs-3 hESCs-1 Fibroblast 2 Fibroblast 1 hFibroblast-iPSC1-2 hFibroblast-iPSC1-3 hFibroblast-iPSC2-1 hFibroblast-iPSC2-2 hFibroblast-iPSC2-3 hFibroblast-iPSC2-4 hFibroblast-iPSC1-1 Cord blood (CD34 Cord blood (CD34 hCord blood-iPSC2-1 hCord blood-iPSC2-2 hCord blood-iPSC1-2 hCord blood-iPSC3-1 hCord blood-iPSC3-2 hCord blood-iPSC3-3 hCord blood-iPSC5-1 hCord blood-iPSC5-2 hCord blood-iPSC5-3 hCord blood-iPSC1-1

0.05 0.00 Neural –0.05

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Enrichment score (ES) –0.25 1

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Rank in ordered dataset Relative expression Rankend list menric (signal2Noise) Enrichment profile Hits Ranking metric scores 0.2 ** NES = –0.975 0 FDR = 0.5 PAX6 NOM P-value = 0.5 )1 )2 + + hESCs-1 hESCs-2 hESCs-3 Fibroblast 1 Fibroblast 2 hFibroblast-iPSC2-1 hFibroblast-iPSC1-1 hFibroblast-iPSC1-2 hFibroblast-iPSC2-2 hFibroblast-iPSC1-3 hFibroblast-iPSC2-3 hFibroblast-iPSC2-4 Cord blood (CD34 Cord blood (CD34 hCord blood-iPSC1-1 hCord blood-iPSC2-1 hCord blood-iPSC2-2 hCord blood-iPSC1-2 hCord blood-iPSC3-1 hCord blood-iPSC3-2 hCord blood-iPSC3-3 hCord blood-iPSC5-1 hCord blood-iPSC5-2 hCord blood-iPSC5-3

0.1 0.0 Hepatocytic –0.1

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Rankend list menric (signal2Noise) Hits NES = –1.32 0 FDR = 0.088 FOXA2 GATA4 SOX17 NOM P-value = 0.088 )1 )2 + + hESCs-1 hESCs-2 hESCs-3 Fibroblast 1 Fibroblast 2 hFibroblast-iPSC1-1 hFibroblast-iPSC1-2 hFibroblast-iPSC1-3 hFibroblast-iPSC2-1 hFibroblast-iPSC2-2 hFibroblast-iPSC2-3 hFibroblast-iPSC2-4 Cord blood (CD34 Cord blood (CD34 hCord blood-iPSC1-1 hCord blood-iPSC2-1 hCord blood-iPSC2-2 hCord blood-iPSC1-2 hCord blood-iPSC3-1 hCord blood-iPSC3-2 hCord blood-iPSC3-3 hCord blood-iPSC5-1 hCord blood-iPSC5-2 hCord blood-iPSC5-3

0.00 –0.05 –0.10 i –0.15 Cardiomyocytic Hematopoietic –0.20 –0.25 80 5 hFibroblast-iPSCs Mesoderm –0.30 ** –0.35 hCord blood-iPSCs –0.40 Enrichment score (ES) –0.45 4 60

3 5.0 CBiPS (positive correlated) ** 2.5 40 0.0 Zero cross at 10,419 2 SMA (%) –2.5 FibiPS (negative correlated) 20

0 2.500 5.000 7.500 10.000 12.500 15.000 17.500 20.000 Relative expression 1 Rank in ordered dataset

Rankend list menric (signal2noise) Enrichment profile Hits Ranking metric scores NES = –1.37 0 0 FDR = 0.085 No BMP4/ T MIXL1 NOM P-value = 0.085 treat activin A

4 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 ARTICLE differentiation, analyses of neural-specific gene expression in from CB, we next analysed undifferentiated hCB-iPSCs for undifferentiated hCB- and hFib-iPSCs revealed significantly transcriptional activity related to hematopoietic programmes. In downregulated expression of NF68 and NEUROD1 in cells line with the correlation between downregulated neural gene derived from CB compared with Fibs, further strengthening the expression and poor neural differentiation, hCB-iPSCs displayed correlation between phenotypic outcome and lineage-specific higher expression levels of critical hematopoiesis genes such as, transcriptional control (Fig. 3d). C/EBPa, Scl/Tal and GATA2 (ref. 14), compared with hFib-iPSCs Neural precursors from both CB- and Fib-iPSCs were further (Fig. 3n), demonstrating that hCB-iPSCs are predisposed towards differentiated into neurons, oligodendrocytes and astrocytes11,12. the hematopoietic fate. Moreover, GSEA of genes associated While both hiPSC lines were capable of generating all three with the hematopoietic lineage15,16 revealed a highly enriched neural cell types, quantitative analysis revealed that the reduced hematopoietic signature in hCB-iPSCs, further supporting that ability of hCB-iPSCs to produce neural progenitor cells translated differentiation potential is transcriptionally encoded in the into a reduced number of GFAP-, O4- and bIII-Tubulin-positive undifferentiated state (Fig. 3o). To validate the observed cells representative of astrocytes, oligodendrocytes and neurons, differentiation disparities present in hCB- and hFib-iPSCs, we respectively (Fig. 3e–g). These results suggest that differentiation analysed multiple clones from three independent CB and two towards the neural lineage is limited in hCB-iPSCs, but this independent Fib donors that were reprogrammed with two limitation does not affect the resulting progenitor differentiation distinct methodologies for both neural and hematopoietic potential. To evaluate whether this limitation in differentiation differentiation. Each individual clone clearly revealed biased potential was transcriptionally encoded in the undifferentiated differentiation propensity of hFib- versus hCB-iPSCs towards state, we performed GSEA using a neural lineage gene set (Fig. 3h, neural versus hematopoietic fates, respectively (Fig. 3p), right panel) between hCB- and hFib-iPSCs (Fig. 3h, left panel). suggesting that cellular origin affects differentiation propensity While gene expression profiles of ectodermal development were of hiPSCs regardless of reprogramming factors or the donor comparable between hiPSCs (Fig. 2b), neural gene expression in sample used (Supplementary Table 3). Moreover, unlike murine hCB-iPSCs was significantly inhibited (Fig. 3h), indicating that iPSC memory that is erased after 10 passages, this disparity in biological differentiation outcomes are likely controlled by differentiation potential was not altered by prolonged culture lineage-specific programming (not germ layer specification) examined (20 passages) (Supplementary Fig. 3b,c), implying within pluripotent cells. sustained functional discrepancy among hiPSCs, in contrast to To better understand predetermined cell fate decisions of mouse iPSCs4,6. undifferentiated hiPSCs, we broadened our comparison to evaluate in vitro hematopoietic differentiation, which represents the parental lineage of hCB-iPSCs. Human embryoid bodies Predetermined decision of hCB-iPSCs into hematopoietic (hEBs) were generated from cell lines of both Fib and blood lineage. To further evaluate the basis of maintained somatic origin, and the frequency of cells expressing hematopoietic lineage preferences in hFib- versus hCB-iPSCs, we expanded our markers was assessed13. Following hEB formation, an increase in analysis to other mesodermal cell types in addition to hemato- the frequency of primitive blood (CD34 þ CD45 þ ) cells was poietic differentiation, such as cardiomyocyte development. In observed in hCB-iPSCs when compared with hFib-iPSCs (Fig. 3i). contrast to hematopoietic differentiation capacity, cardiomyocytic Marked generation of primitive (CD34 þ CD45 þ ) and fate was limited in hCB-iPSCs, even in the presence of activating mature (CD34-CD45 þ ) blood cells also demonstrated an growth and inducing factors (Supplementary Fig. 4a). While both augmented response of hCB-hiPSCs to hematopoietic-specific hiPSC lines were capable of generating comparable frequencies of signals (Fig. 3j). As a consequence, the overall number of the smooth muscle marker SMA (Fig. 2i), the expression of the hematopoietic cells generated was highly enriched from hCB- mature cardiomyocyte markers, including isoform of Troponin T iPSCs (Fig. 3k). To further compare the multi-lineage potential of (cTnT), was markedly reduced in hCB-iPSCs (Supplementary hiPSC-derived hematopoietic progenitors from different cell Fig. 4b,c). Compared with GSEA of hematopoietic genes, origins, we performed hematopoietic colony-forming unit hCB-iPSCs displayed reduced expression of genes associated with (CFU) assays (Supplementary Fig. 3a). In line with increased cardiomyocytic fates (Supplementary Fig. 4d), highlighting that generation of CD34 þ CD45 þ cells, hCB-iPSCs produced more the differentiation propensity of pluripotent cells is governed by CFUs than hFib-iPSCs (Fig. 3l), indicating that enhanced the underlying somatic transcriptome associated with the cell of acquisition of the hematopoietic phenotype does not occur at origin. the expense of progenitor function. Analysis of specific Since differentiation potential is highly correlative to the hematopoietic developmental potential revealed biased transcriptional profiles related to the cellular origin of established differentiation of progenitors derived from hCB-iPSCs into hiPSCs, we generated hEBs in the absence of exogenous stimuli CFU-erythroid (CFU-E), while the frequency of CFU- conductive to a specific lineage to explore whether spontaneous macrophage (CFU-M) and CFU-granulocyte (CFU-G) was differentiation can be affected by the inherent transcriptome lower compared with hFib-iPSCs (Fig. 3m). Having confirmed distinctions among hiPSCs. Although the overall number of the expected hematopoietic differentiation bias of hiPSCs derived hematopoietic cells is reduced when compared with cells treated

Figure 2 | Equivalency in transcriptional profiles of three germ layers. (a) Hierarchical cluster analysis using genes associated with ectoderm lineage. (b) GSEA plot of enrichment of gene signature related to ectoderm lineage between fibroblast- versus cord blood-iPSCs. (c) Quantitative RT-PCR analysis of early neural marker, PAX6. Samples are purified at day 4 during neural differentiation. Individual reactions were normalized according to GAPDH. (n ¼ 6). (d) Hierarchical cluster analysis with genes of endoderm lineage. (e) GSEA plot of enrichment of gene signature associated with endoderm lineage between fibroblast- versus cord blood-iPSCs. (f) Frequency of FOXA2-, GATA4- and SOX17-positive cells for endoderm formation during hepatocyte generation (n ¼ 14). (g) Hierarchical cluster analysis with genes of mesoderm lineage. (h) GSEA plot of enrichment of gene signature related to mesoderm lineage. (i) (left) Percentage of smooth muscle actin (SMA)-positive cells at day 7 during cardiomyocyte differentiation, performed in hEBs medium supplemented with activating factors (BMP4 and Activin A) or not (n ¼ 5). (Right) quantitative RT-PCR analysis for mesodermal markers, MixL1 and Brachyury (T), during hematopoietic differentiation (day 4). Individual reactions were normalized according to GAPDH. (n ¼ 6). All data are represented as mean±s.e.m. **Po0.01, Student’s t-test.

NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications 5 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 with cytokines (Fig. 3k), the relative expansion of primitive blood iPSCs (Fig. 4b). Overall, these data support that hCB-iPSCs cells derived from hCB-iPSCs compared with hFib-iPSCs was possess enhanced hematopoietic differentiation potential that is increased ﬁvefold (Fig. 4a). Furthermore, irrespective of the predetermined, where the addition of exogenous hematopoietic complete absence of hematopoietic factors in vitro, functional factors can further augment blood development in hCB-iPSCs. In capacity of hCB-iPSCs to generate hematopoietic progenitors addition, our results reveal that differentiation potential of hCB- (CFUs) was higher as compared with hFib-iPSC-derived iPSCs is highly tuned towards the cell of origin, while the cells (Fig. 4a), suggesting that the hematopoietic lineage cardiomyocytic fate of shared germ layer origin is severely propensity is independent of hematopoietic stimuli and is cell compromised. autonomous. In addition, hCB-iPSCs preferentially differentiate On the basis of lineage propensities within a germ layer, we into the erythroid sub-lineage (CFU-E), compared with hFib- next explored whether hematopoietic differentiation

hFibroblast -iPSCs 35 35 35 100 100 40 hFibroblast-iPSCs 30 30 30 hCord Blood-iPSCs 80 35 ** 25 80 25 25 ** 60 60 30 20 20 ** 20 25 15 ** 40 15 40 15 ** 20 10 20 10 20 10 15 5 0 5 0 5 # Of A2B5 (×100K) # Of sphere per 1 K 10 100 101 102 103 104 # Of nestin (×100 K) 100 101 102 103 104 ** 0 0 0 # Of cells (×100 K) 5 Nestin A2B5 0 0 1st 2nd 3rd Isotype Isotype hCord blood-iPSCs (86±10%) hCord Blood-iPSCs (78±1.7%)

hCord blood -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs hFibroblast-iPSCs (99±0.5%) hFibroblast-iPSCs (95±4%) hFibroblast hFibroblast hFibroblast hCord blood hCord blood hCord blood

Astrocytes Oligodendrocytes Neurons DAPI+GFAP DAPI+O4 DAPI+B-III tubulin NF68 NeuroD 1.2 1.2 1 1 0.8 ** 0.8 ** 0.6 0.6 0.4 0.4 0.2 0.2 25 40 35 Relative expression 0 Relative expression 0 20 30 30 25 15 20 20 ** 10 ** 15 -iPSCs -iPSCs -iPSCs -iPSCs Neural differentiation 10 10 5 ** 5 hFibroblast hFibroblast hCord blood hCord blood

0 0 # Of cells (×100 K) 0 # Of cells (×100 K) # Of cells (×100 K) -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs hFibroblast hFibroblast hFibroblast hCord blood hCord blood hCord blood

0.10 0.05 0.00 –0.05 –0.10 –0.15 Genes related to neural differentiation –0.20 –0.25 –0.30

Enrichment score (ES) –0.35 –0.40 HAPLN1 RPS6KA5 COL4A2 CACNA1G CACNA1C COL14A1 ST8STA4 CACBA1H DPYSL2 CACNA1F ARHGDIA COL9A1 C5ORF23 ST8SIA2 COL29A1 ARHGEF12 NFATC1 EEF1A2 CACNA1D CYP26A1 COL1A1 COL9A3 GFRA4 CACNB4 CACNB3 CACNB1 CACNB2 EFNA2 ARL6IP5 LRRC4C CRABP1 CRABP2 KBTBD10 MAP2K2 NCAM1 CACNA1I NFATC4 NFATC2 KCNN2 CACNA1S DNAJC1 FAM89B AKR1C1 SPTAN1 MAPK1 COL6A1 COL3A1 COL1A2 COL6A2 COL6A3 COL5A1 COL5A2 COL9A2 COL2A1 CXCL12 CDC42 EPHB6 ATP1A2 EPHB1 EPHA7 GPR126 EFNB2 ARLIM3 CDH1 GRN IGFBP5 ABLIM2 HMGA1 GNRH1 EPHA6 CEBPD COL4A4 CXCR4 HOXB2 MAPK3 COBLL1 COL4A3 COL4A5 LIMK1 EGFR1 L1CAM EPHR3 EPHR4 EPHR2 ERBB3 GFRA2 CREB1 GSK3B EFNR1 CDH19 CHN2 EFNA3 EPHA4 EFNA5 EFNB3 EPHA5 EPHA8 EPHA1 BCHE SPTA1 NFAT5 CRYZ PSPN CNR1 CHP2 AP1S2 NFATC3 GNAI3 COL6A6 HOXA2 PTK2 ELK3 KCNK5 AGRN CHGA CFL2 ITGB1 CDK5 GDNF NCAN DPYD NGEF HRAS BMP5 GRB2 ARTN ENC1 NCK1 SPTB PHA2 DLC1 FNA1 RAF1 ABL1 GNAI2 CFL1 GFRA1 EPHA3 GYPC NRTN GAP43 IFI16 SOS1 RGN DCN DCC NCK2 GSN CHP GNAI1 COL4A1 EYN EAP FST EDNRA INA FES C9ORF95 hCord blood 5.0 -iPSCs CBiPS (positive correlated) 2.5 Zero cross at 10419 hFibroblast 0.0

(signal2noise) –2.5 NES = –1.74 -iPSCs Ranked list metric FibiPS (negative correlated) FDR ≤ 0.0001 Colour range 0 NOM P-value ≤ 0.0001 2,500 5,000 7,500 10,000 12,500 15,000 17,500 20,000 Rank in ordered dataset –2.5 0 2.5 Enrichment profile Hits Ranking metric scores

Figure 3 | Distinct differentiation potential in hiPSCs derived from fibroblasts and cord blood. (a) Quantitative results of neurosphere expansion (n ¼ 14). Right panel shows the images of neurosphere generated at the end of the 2nd expansion cycle of hiPSCs. Scale bars, 200 mm. (b) Quantification of the neurosphere generation capacity from 1 K numbers of cells (n ¼ 18). (c) Representative flow cytometry plots for neural stem cell marker nestin and the neural precursor marker A2B5 in neurosphere. Right panel shows total number of nestin- or A2B5-positive cells. (n ¼ 10). (d) Quantitative RT-PCR analysis of neural-specific genes from undifferentiated cells. Individual reactions were normalized to GAPDH. (n ¼ 6). Neural stem cells from hiPSCs were further differentiated into astrocytes as indicted by GFAP staining (e, top panel), oligodendrocytes as indicated by O4 staining (f, top panel) and neurons as indicated by b-III-tubulin staining (g, top panel). Scale bars, 50 mm(e,f) and 100 mm(g). Bottom panels for e–g are graphs for quantification of total number of differentiated cells of respective neural lineages (n ¼ 10). (h) GSEA plot of gene signatures related to neural lineage (right) and heat map of genes (left). (i) Representative fluorescence-activated cell sorting plot for the expression of primitive blood cells markers, CD34 and CD45, of hiPSCs hEBs after 15 days hEB culture. (j) Total number of differentiated primitive blood cells and mature blood cells in hEBs from hiPSCs over days 7, 10 and 15 (n ¼ 14). (k) Total number of differentiated CD34 þ CD45 þ cells from hiPSCs (n ¼ 10). (l) Total number of hematopoietic CFUs and number of colony subtypes generated from day 15 hEBs. Inset shows hematopoietic progenitor capacity of hEBs. (n ¼ 10). (m) Distribution of colony subtypes derived from hiPSC lines. (n) Quantitative RT-PCR analysis for blood-specific genes. Samples are purified from undifferentiated cells and individual reactions were normalized to GAPDH. (n ¼ 6). (o) GSEA plot of enrichment of gene signature related to hematopoietic lineage (right) and heat map of genes (left). (p) Quantification of expression of neural stem cell marker nestin during neural differentiation (left). Quantitative results of CD45 þ -positive cells during hematopoietic differentiation (right). All bars indicate average of all clones. All data are represented as mean±s.e.m. *Po0.05, **Po0.01, Student’s t-test.

6 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 ARTICLE

hFibroblast-iPSCs hCord blood-iPSCs Isotype Fold 4 4 4 hFibroblast-iPSCs 300 140 ± 10 10 10 140 ** ** 4.3 2.6 ** 2.3±1.9 7.7±1.1 hCord blood-iPSCs 120 3 120 250 3 10 10 103 (×1 K) 100 2 100 + 10 200 80 2 2 1 80 10 10 10 150 60 60 CD45 CD34 0 +

10 0 1 2 3 4 Total # of 40 101 1 10 10 10 10 10 100 10 40 20 5.7±1.5 8.2±1.3 20 50 100 100 * CD34 0 100 101 102 103 104 100 101 102 103 104 0 0 Mature blood cells (×1 K) CD45 Primitive blood cells (×1 K) D7 D10 D15 D7 D10 D15 -iPSCs -iPSCs hFibroblast hCord blood

CFU-E 1±0.5% hFibroblast-iPSCs 9±1% CFU-G C/EBPα SCL/TAL1 GATA2 hCord blood-iPSCs 1,000 900 ** CFU-M 3.5 14 800 CFU-GM 3.5 3 ** ± ** 700 ± 46 5.4% ** 12 600 34 6.6% GEMM 3 2.5 * 3 500 10 400 10±1.5% 2.5 2 2.5 300 2 2 8 # CFUs/100 K 200 100 hCord blood 1.5 6 ** 0 1.5 1.5 4±2% 2±2.9% -iPSCs 1 4 ** 1 1 21±4.5% 0.5 2 ** 0.5 0.5 ** 45±3.3% 0 0 0 Total # of CFUs (×1 K) 0 28±5.3% E G M GM Total

GEMM hFibroblast -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs -iPSCs

Hematopoietic differentiation -iPSCs hFibroblast hFibroblast hFibroblast hCord blood hCord blood hCord blood

Genes related to hematopoiesis 0.5 0.4 0.3 0.2 METAP2 EIF6 PFKP ERP44 CD28 CSDA RUVBL2 SSR2 MAPRE2 GALK1 WDR77 DBT SCHIP1 TMEM97 MCM7 RET1 G3BP1 NCL ICAM2 RUVBL1 GOT1 APEX1 SNRPA1 MCM3AP PDE7A RHOJ TYMK CTSZ MYB GUSB KLF9 BCL7C ASNS RSU1 IMMT NOLC1 AHCY MYRL2 SURF6 RRS1 MRPL15 RPN1 RCAT1 GTF3A DUT WEE1 NOP58 BID IDE CTPS MYLK MSH2 GABPB1 MPO ELP2 CEBPA AFP AP3S2 MUC13 GSTM1 NUCB2 IFRD2 C1QRP PARL HMGCR NEDD4 HDAC2 SCIN RRM1 UHRF1 MTM1 MRPS25 HOXA9 PLAC8 DNAJA3 0.1

Enrichment score (ES) 0.0 hFibroblast -iPSCs 5.0 CBiPS (positive correlated) hCord blood 2.5 Zero cross at 10,419 -iPSCs 0.0 –2.5 (signal2noise) FibiPS (negative correlated) NES = 2.08

Colour range Ranked list metric 0 FDR ≤ 0.0001 0 NOM P-value ≤ 0.0001 –2.5 0 2.5 2,500 5,000 7,500 10,000 12,500 15,000 17,500 20,000 Rank in ordered dataset Enrichment profile Hits Ranking metric scores ) ) + + 100 40 Clone 1 Clone 1 90 Clone 2 Clone 2 30 80 Clone 3 Clone 3 Clone 4 Clone 4 70 Clone 5 20 Clone 5 Clone 6 Clone 6 Neuronal 60 10 50 Hematopoietic

differentiation (% CD45 0

differentiation (% nestin 0 hCord blood hFibroblast hCord blood hFibroblast -iPSCs -iPSCs -iPSCs -iPSCs Figure 3 | Continued.

predominates in hCB-iPSCs even under stimuli for alternative cell not only a larger number of haemangioblast colonies, but the fates. Marked generation of primitive and mature blood cells clonal capacity measured by cellularity was greater compared during cardiomyocyte conducive differentiation suggest that with hFib-iPSC lines (Fig. 4d). In addition, hCB-iPSC-derived differentiation of hiPSCs is gated to the specific somatic cell fate haemangioblasts generated more CFUs that preferentially associated with their cellular origin, and in doing so restricts differentiate into CFU-E (Supplementary Fig. 5b,c). Although alternative haemangioblasts derived from hCB-iPSCs maintained endo- cell fates (Fig. 4c). Given that CB-hiPSCs retained the donor cell- hematopoietic differentiation potential, these cells appear specific molecular profiles even under stimuli for alternative biased towards hematopoietic lineages. Giving the over- cell fates, we examined whether those features can be applied whelming evidence that progenitors of hCB-iPSCs preferentially for generation of hematopoietic cells with definitive/adult differentiate into CFU-E (Fig. 3m; Supplementary Fig. 5c), we hematopoietic programmes that have been elusive from hPSCs further examined erythrocyte generation21. Increased numbers of at the functional protein level17 without stimulation of novel cells were obtained from hCB-iPSCs compared with hFib-iPSCs pathways18. Recently, an efficient method has been developed despite the same number of input haemangioblast cells (Fig. 4e). to generate large numbers of bipotential progenitors, known The expression of high levels of glycophorin A (CD235a) as haemangioblasts, which retain both hematopoietic and and the lack of CD45 on the surface confirmed the presence endothelial potential19,20. As predicted from previous reports, of erythrocytic features (Supplementary Fig. 5d). Further hiPSC lines could produce haemangioblasts that further maturation of hCB-iPSC-derived erythrocytes induced a differentiated into both endothelial and hematopoietic lineages definitive hematopoietic programme as indicated by human (Supplementary Fig. 5a). Consistently, hCB-iPSCs produced adult b-globin protein expression and enucleated erythroid cells

NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications 7 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605

No hematopoietic No hematopoietic cytokines 50 cytokines Fold ** 5±0.7 ) 50 60 40 hFibroblast-iPSCs + ** ** 40 50 hCord blood-iPSCs 30 CD45

+ 40 30 30 20 20 (×1 K) 20 10 10 10 Total # of CFUs (×100) ** * **

0 Total # of CFUs (×100) 0 0 Total # of (CD34 E G M GM GEMM

hFibroblast -iPSCs hFibroblast-iPSCs hCord-iPSCs blood hCord-iPSCs blood

Hemangioblasts hFibroblast hCord blood hCord blood hFibroblast -iPSCs -iPSCs -iPSCs -iPSCs 6 hFibroblast-iPSCs hCord blood-iPSCs ** ** 4 ** 2,000 ** 1,800 ** 60 300 1,600 2 ) ** 2 50 250 ** 1,400 Frequency (%) 40 200 1,200 1,000 0 30 150 Cardiac 800 –+ – + induction 20 100 600 Primitive blood Mature blood in erythrocytes 10 50 400 Colony size (mm # Colonies per 50 K

Total # of cells (×100 K) 200 0 0 from same initial # of cells 0

hFibroblast hFibroblast-iPSCs -iPSCshCord-iPSCs blood hCord-iPSCs blood hFibroblast -iPSCshCord-iPSCs blood

BF Beta globin 100 70 70 ** ** 60 80 60 50 60 50 40 40 40 30 30 20 20

20 Hoechst Merge in erythrocytes % Of beta globin 10

0 Beta globin (%) 10 100 101 102 103 104 0 0 Beta globin D16 D31 Isotype hFibroblast hCord blood-iPSCs (D16) -iPSCshCord-iPSCs blood hCord blood-iPSCs (D31) Figure 4 | Predetermined fate decision of hCB-iPSCs into hematopoietic lineage. (a) Total number of differentiated CD34 þ CD45 þ cells from hiPSCs in the absence of hematopoietic cytokines. Number indicates fold change between hiPSCs (left). Total number of hematopoietic CFUs generated from day 15 hEBs without activating factors (right). (n ¼ 8). (b) Total number of colony subtypes generated from day 15 hEBs without hematopoietic cytokines (n ¼ 6). (c) Maintenance of hematopoietic potentials in hCB-iPSCs under stimuli for alternative cell fates. Frequency of differentiated primitive blood cells and mature blood cells during cardiomyocyte differentiation (n ¼ 6). (d) Different size of haemangioblasts colony and colony numbers produced from different hiPSCs. (Top) is phase contrast images showing haemangioblasts derived from hiPSCs. Scale bars, 100 mm. (Bottom) quantification of haemangioblasts sizes and colony numbers (n ¼ 12). (e) Quantification of total number of differentiated erythroid cells from hiPSCs-haemangioblast cells (n ¼ 9). (Top) representative images of differentiated erythroid cells from hiPSCs. Scale bars, 200 mm. (f) Representative fluorescence-activated cell sorting plot of b-globin expression in erythorid cells at day 16 and day 31 of differentiation and maturation cultures. (Middle) is quantification of flow cytometry (n ¼ 9). (Right) is immunostaining of mature erythroid cells, derived from hUCB-iPSCs, with b-globin chain-specific antibody. Nuclei are stained with DAPI. (c) indicates b-globin-positive and DAPI-negative cells. Scale bars, 50 mm. (g) Quantification of adult b-globin-positive red blood cells from hiPSCs (n ¼ 6). All data are represented as mean±s.e.m. *Po0.05, **Po0.01, Student’s t-test.

(Fig. 4f,g), hence hCB-iPSCs show enhanced erythropoiesis within cells comprising hESCs22. By marking cells expressing differentiation capacity. Taken together, these results c-KIT (hematopoietic primed) and A2B5 (neural primed), demonstrate superior potential of hCB-iPSCs to generate adult we successfully subfractionated hESCs and demonstrated clear b-globin-positive red blood cells, providing insight for targeting propensities for hematopoietic and neural lineage differentiation, explicit somatic cell sources for hiPSC generation intended for respectively22. A signiﬁcant increase in the frequency of lineage-speciﬁc applications. c-KIT-positive cells and reduced A2B5-expressing cells were observed in hCB-iPSCs (Fig. 5a), which correlates with increased Differentially encoded differentiation potential in hiPSCs. hematopoietic and reduced neural lineage capacity shown Recently, we revealed that cell fate potential is encoded (Fig. 3), thereby supporting the notion that cell fate decisions

8 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 ARTICLE

c-KIT 70 A2B5 hFibroblast-iPSCs hCord blood-iPSCs 60 ** 104 104 50 ** 103 103 40

102 102 30 * A2B5 A2B5 Frequency (%) 101 101 20 ** 10 100 100 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 0 #4 #5 #3 #4 c-KIT hFibroblast-iPSCs hCord blood-iPSCs

MIXL1 T hFibroblast-iPSCs 5 ** 12 hCord blood-iPSCs

4 8 3

2 4

Relative enrichment 1 ** * ** 0 0 H3K4me3 H3K27me3 meDNA H3K4me3 H3K27me3 meDNA

PAX6 NF68 14 24 ** hFibroblast-iPSCs hCord blood-iPSCs ** 18 10.5

12 7 ** **

4 ** 3.5 Relative enrichment

0 0 H3K4me3 H3K27me3 meDNA H3K4me3 H3K27me3 meDNA

Blood specific Fibroblast specific

Primed Naive Primed Naive 4 4 4 4 3 3 3 743 0 2 2 2 2 1 1 1 1 0 0 0 0 –1 –1 –1 –1 –2 –2 –2 –2 –3 37–3 0 –3 –3 hFibroblast-iPSCs hFibroblast-iPSCs –4 –4 –4 –4 –4 –3 –2 –1 0 1 2 3 4 –4 –3 –2 –1 0 1 2 3 4 –4 –3 –2 –1 0 1 2 3 4 –4 –3 –2 –1 0 1 2 3 4 hCord blood-iPSCs hCord blood-iPSCs

3 2

1 4 Blood specific Fibroblast specific

0 ) 3 254 ) ) Naive 4 4 –1 (62/74) 2 3 3 –2 2 2 –3 1 1 1 –3 –2–10123 Re-primed

0 Re-primed Re-primed 3 0 0 Naive 2 –1 –1 Naive –1 112 –2 –2 1 –2 –3 –3 0 –3 (14/37)

hFib-iPSCs ( –4 –4 –1 hFib-iPSCs ( hFib-iPSCs ( –4 –4–3–2–101234 –4 –3 –2 –1 0 1 2 3 4 Re-primed –2 –4 –3 –2 –1 0 1 2 3 4 hCB-iPSCs (Re-primed) hCB-iPSCs (Re-primed) –3 –3 –2 –1 0 1 2 3 hCB-iPSCs (Re-primed) Primed Figure 5 | Differentially encoded cell differentiation potential in hiPSCs derived from human adult dermal fibroblast and CD34 þ umbilical cord blood cells. (a) Flow cytometry analysis of the expression of c-KIT and A2B5, associated with mesoderm progenitors and neural precursors, respectively, in undifferentiated hiPSCs. (Right) is percentages of c-KIT- and A2B5-positive cells in hiPSCs (n ¼ 5). (b,c) Single ChIP analysis of histone modification and DNA methylation on mesodermal lineage genes (b; T and MIXL1) and neural lineages genes (c;PAX6andNF68)loci.(n ¼ 3). (d) Loss of transcriptional somatic memory in naive conditions. Blood-specific (red) and fibroblast-specific (blue) genes are indicated in global gene expression comparison. (e,f) Conversion into original primed conditions reacquires initial transcriptional differences. All data are represented as mean±s.e.m. *Po0.05, **Po0.01, Student’s t-test.

NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications 9 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 are differentially predisposed within cells comprising hiPSCs based on cellular origins used to derive hiPSCs. This suggests from different somatic sources. To aid in determining the that determination and characterization of somatic cells for molecular basis of this lineage priming effect, we investigated reprogramming may provide enhanced differentiation for specific whether distinct epigenetic modifications of key lineage-specific research and therapeutic applications, and could be tailored regulatory genes correlated with lineage propensity demonstrated. accordingly. Similar to previous reports showing epigenetic memory relates to Mechanistically, it has previously been reported that iPSCs incomplete DNA methylation, we found distinct methylation retain DNA methylation profiles of the tissue from which they status in lineage-related genes, whereas hCB-iPSCs showed were derived5–7 as an explanation for iPSC memory. Our current decreased methylation in the hematopoietic marker, T (Fig. 5b), study supports these findings and further implicates epigenetic and increased methylation in the neural marker, PAX6 (Fig. 5c), modification of histone status as markers of cellular memory compared with hFib-iPSCs. Moving beyond the methylation within reprogrammed pluripotent cells. However, like DNA status, we investigated for evidence of distinct histone methylation, whether these histone modifications are causal modifications in lineage-specific gene loci for both neural and determinants or a consequence of additional processes not hematopoietic regulatory genes. hCB-iPSCs displayed enrichment defined in this study still remain to be elucidated. Beyond these of H3K4me3 activation marks on MIXL1 (Fig. 5b) and uncertainties, our results reveal that hiPSCs preferentially activate H3K27me3 repressive marks on NF68 (Fig. 5c), further transcriptional programmes related to the specific cell of origin strengthening the current hypothesis of epigenetic regulation from whence they were derived at the expense of other unrelated that governs distinct differentiation potentials. lineages. As current differentiation procedures are principally Despite displaying similar levels of pluripotent hallmark genes based on desired germ layer targeting, followed by lineages within and germ layer specification-related transcriptional programmes, that germ layer, this suggests that new procedures for hiPSCs that hiPSCs from different somatic sources retain a level of lineage- arise from different cells of origin may be unique. Moreover, we specific gene activation that results in efficient differentiation now show that like hESCs, cell fate potential is encoded within towards their cell of origin. Given that murine iPSCs are able to cells comprising hiPSCs. Phenotypic assessment of newly created erase this transcriptional memory after serial passaging, yet iPSC lines for fate-coding markers such as c-KIT and A2B5 could human cells do not, it is likely that distinct molecular help to provide insight into retained somatic memory and mechanisms are at play in the human scenario. Alternatively, eventual differentiation outcome. murine and human PSCs rely on different signalling pathways to Consistent differentiation propensity in the extended passage sustain self-renewal in the pluripotent state, and are cultured in suggests persistent preservation of molecular and cellular proper- completely different conditions from previous studies comparing ties in hiPSCs unlike their mouse counterparts, where serial iPSC memory; it is possible the ‘state’ of iPSCs (primed versus passaging attenuates their differences4,6. So far, epigenetic naive) is critical to memory status. Recent studies have memory (incomplete DNA methylation) is the most plausible demonstrated that human iPSCs normally considered to be in molecular explanation for somatic memory both of mouse and ‘primed pluripotent state’ can be converted into cells having human iPSCs, but fails to reconcile the causal differences between mouse ESC-like traits, called the ‘naive pluripotent state’23–25.As regulation of mouse and human memory. Our study proposes such, we transitioned our CB- and Fib-derived hiPSCs into the that the difference in human versus mouse memory is related naive PSC state and performed comparative transcriptome to primed versus naive states of pluripotency. We have analysis. Human iPSCs in the primed state maintain gene demonstrated that human naive culture conditions rewire and expression of their cell of origin, but surprisingly, under naive erase transcriptional memory of somatic cells retained in hCB- conditions, hiPSCs rewire and erase transcriptional memory of iPSCs, implicating, for the first time, that the pluripotent state somatic cells (Fig. 5d). In contrast to transcriptional retention of establishes memory but can be re-established by switching back the somatic cell of origin of hiPSCs in the primed state, regardless to the primed state of hiPSCs. These findings suggest that iPSC of cellular origin, similar transcriptional profiles were displayed memory is likely governed by mechanisms that go beyond once hiPSCs were placed in naive state. However, when incomplete methylation or modified histone status that allow for reversed from the naive state, back to primed conditions, unique marking and requiring transcriptional plasticity after hiPSCs reacquire transcriptional profiles similar to parental apparent erasure. This may form the basis for the distinguishing hiPSCs and surprisingly, re-established transcriptome memory feature of mouse versus human iPSCs in the context of somatic (Fig. 5e,f), implicating the pluripotent ‘state’ as a direct effector of memory, where serial passage of hiPSCs is unable to remove somatic memory for the first time. memory.

Discussion Methods Lentivirus preparation and induction of hiPSCs. Plasmids pSIN-EF2-OCT4-Pur, Efficient and controlled differentiation to specific lineages pSIN-EF2-SOX2-Pur, pSIN-EF2-Nanog-Pur, pSIN-EF2-LIN28-Pur, pSIN4-EF2- represents the major barrier for future clinical applications O2S, pSIN4-EF2-N2L and pSIN-CMV-K2M were gifts from James A. Thomson of hiPSCs, including cell replacement therapies and (University of Madison-Wisconsin). These vectors were transfected with second- drug discovery26. Therefore, a deeper understanding of the generation packaging plasmids (psPAX2 and pMD2.G) in a 293FT cell line. Viral supernatant were collected 48 h after transfection, concentrated with ultra- starting populations of hiPSCs is required to instruct proficient centrifugation and titred with HeLa cells2. Human dermal Fibs (ScienCell) or differentiation. Our study demonstrates that although the hiPSC human CB cells from healthy donors (two independent Fibs donors and three CB lines express pluripotent genes and support differentiation into samples) were transduced with concentrated lentiviral vectors in the presence of À 1 cell types of all three germ layers, measured by established assays 8 mgml polybrene (Millipore). Informed consent was obtained from all sample 27 donors in accordance with Research Ethics Board-approved protocols at McMaster for pluripotency , hiPSC lines possess unique functional University and the London Health Science Centre. CD34 þ CB cells, used for hCB- properties when induced to differentiate, where these distinct iPSCs generation, were isolated by immunomagnetic separation. Transduced cells properties are predetermined at the transcriptome level of were maintained on irradiated feeder mouse embryonic fibroblasts (MEFs) in undifferentiated hiPSC states. We propose that although human Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 20% Knockout Serum Replacement (Gibco), 100 mM b-mercaptoethanol, 100 mM iPSCs are molecularly and functionally defined as pluripotent, À 1 nonessential amino acid (Gibco), 1 mM L-glutamine (Gibco) and 10 ng ml basic hiPSCs are heterogeneous in their developmental potential and fibroblast growth factor (bFGF). hiPSCs were examined for the expression of are better defined by ‘lineage gated’ capacity for pluripotency pluripotent markers including OCT4, cell surface stage-specific antigen 3 (SSEA3),

10 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 ARTICLE

SSEA4 and TRA1-60. Specific primer sets were used to detect pluripotent genes evaluated by culturing the cells in the presence of 10 mgmlÀ 1 Alexa Fluor 594 Ac- and are listed in Supplementary Table 1. LDL (Invitrogen) for 8 h. The expression of endothelial markers and intracellular LDL staining was examined using fluorescence microscopy. Human ESCs and iPSCs cultures. The hESC line and Fibs-hiPSCs were cultured on Matrigel (BD Biosciences) in MEF-conditioned medium (MEF-CM) Neurosphere generation and neural differentiation. For neural differentiation, supplemented with 8 ng ml À 1 bFGF (Invitrogen) as previously described28. Fib the hiPSCs, cultured on iMEFs, were depleted of feeders, seeded onto Matrigel- hiPSCs and umbilical CB hiPSCs were cultured on irradiated feeder MEFs in coated plates and then cultured for 7 days with MEF-CM supplemented with DMEM/F12 (Gibco) with 20% Knockout Serum Replacement (Gibco), 100 mM 8ngmlÀ 1 bFGF and then EBs were generated with EB medium without cytokines b-mercaptoethanol, 100 mM nonessential amino acid (Gibco), 1 mM L-glutamine as previously described22. After 4 days in suspension culture, EBs were plated onto (Gibco) and 10 ng ml À 1 bFGF. Cells were fed with fresh medium daily and poly-L-lysine/laminin-coated plates (BD Biosciences) in neural differentiation confluent cultures were mechanically passed using 1 mg ml À 1 collagenase IV medium composed of DMEM/F12 with B27 (Gibco) and N2 (Gibco) supplements, (Invitrogen). Naive hiPSCs were derived based on previous reports23–25 and 25 ng ml À 1 EGF (R&D Systems), 2.5 ng ml À 1 IGF (R&D Systems), 25 ng ml À 1 maintained with 1 mM MEK inhibitor PD0325901, 3 mM GSK3 inhibitor platelet-derived growth factor (PDGF)-AA (R&D Systems) and 8 ng ml À 1 bFGF. CHIR99021 and 10 ng ml À 1 LIF. After 5 days under this condition, EBs were collected for fluorescence-activated cell sorting analysis. Neurosphere expansion and subsequent differentiation were Teratoma assay. The McMaster University Animal Care Council approved all carried out as previously described11,12. Briefly, the hiPSCs, cultured on iMEFs, procedures and protocols. Undifferentiated hiPSCs (1.0 Â 106 cells per mouse) were depleted of feeders, seeded onto Matrigel-coated plates and then cultured for were mechanically collected, resuspended in PBS and then injected intratesticularly 7 days with MEF-CM supplemented with 8 ng ml À 1 bFGF. hEBs were generated into male non-obese diabetic/severe combined immunodeficient (NOD/SCID)28. by suspension culture with neural-proliferation media (DMEM/F12 supplemented À À Mice were killed 8 weeks after initial injection. Teratomas were collected, with 1 Â N2, 1 Â B27, 20 ng ml 1 EGF and 20 ng ml 1 FGF-2). hEBs were embedded in paraffin, sectioned followed by deparaffinization in xylene and cultured for 7 days with a media change every other day. For neurosphere processing through a graded series of alcohol concentrations. Samples were stained formation and expansion, hEBs were collected and dissociated into single cells with with haematoxylin and eosin followed by dehydration and xylene treatment. Accutase (Sigma). Dissociated single cells were plated into low-attachment 6-well Images were acquired using ScanScope CS digital slide scanner (Aperio, CA, USA). plates (2 Â 105 cells per well) in neural-proliferation media to facilitate neurosphere formation over 7 days. Media was changed every 3 days. After 7 days, neurospheres were collected, dissociated into single cells and counted. To quantify neurosphere hEB formation and hematopoietic differentiation. hiPSCs cultured on irradiated generation capacity of hiPSCs, minimal numbers of cells (1,000) at the end of MEFs (iMEFs) were depleted of iMEFs, split and seeded onto Matrigel-coated expansion cycles were cultured with neural proliferating media and neurosphere À 1 plates, and then cultured for 7 days with MEF-CM supplemented with 8 ng ml were counted. Neural precursors were analysed for the neural stem cell marker bFGF (Invitrogen). To generate hematopoietic cells from hiPSCs, we generated nestin (R&D Systems) and the neural precursor marker A2B5 (R&D Systems) by 29 À 1 hEBs . Confluent undifferentiated hiPSCs were treated with 200 U ml flow cytometry. To differentiate neural precursors along the neuronal lineage, cells collagenase IV for 5–10 min and then transferred to 6- or 12-well ultralow were plated onto poly-L-lysine/laminin-coated plates (BD Biosciences) grown in attachment plates (Corning). Clumps of hiPSCs were incubated overnight to allow DMEM F12 supplemented with 1 Â N2, 1 Â B27, all-trans Retinoic acid (Sigma) hEB formation in the hEB-differentiation medium supplemented with at 2 mM and forskolin (Sigma) at 5 mM. To induce astrocytic differentiation, neural À 1 hematopoietic growth factors as follows: 50 ng ml granulocyte colony- precursors were cultured in DMEM/F12 supplemented with 1 Â N2, 1 Â B27 and À 1 stimulating factor (Amgen Inc., Thousand Oaks, CA, USA), 300 ng ml stem cell 5% fetal bovine serum. Oligodendrocyte differentiation was induced by culturing À 1 factor (SCF; Amgen), 10 ng ml interleukin-3 (R&D systems, Minneapolis, MN, cells in DMEM/F12, 1% N2, 1% B27 and IGF-1 (200 ng ml À 1). In all conditions, À 1 À 1 USA), 10 ng ml interleukin-6 (R&D systems), 25 ng ml BMP4 (R&D systems) cells were allowed to differentiate for 1 week. and 300 ng ml À 1 Flt-3 ligand (Flt-3L: R&D systems). hEBs were cultured for 7–15 days and the medium was changed every 3 days. In vitro hepatocytic differentiation. For hepatocyte differentiation, the hiPSCs Colony-forming unit assay. CFU assay was performed by plating single-cell seeded onto Matrigel-coated plates, and then cultured for 5 days with MEF-CM supplemented with 8 ng ml À 1 bFGF and then replaced with RPMI supplemented suspensions from hEBs into methylcellulose H4434 (Stem Cell Technologies, À 1 À 1 Vancouver, BC, Canada)13. hEBs were dissociated with collagenase B and with 1 Â B27, 100 ng ml activin A, 10 ng ml hepatocyte growth factor and m 1 mM CHIR99021 (GSK3 inhibitor) for 3 days of endoderm formation as pre- cell-dissociation buffer and then filtered through a 40- m cell strainer. Dissociated 30 hEBs were counted and plated into methylcellulose H4434 in 24- or 12-well viously described . Then cell medium was replaced with knockout-DMEM with ultralow attachment plates. For characterization of hematopoietic colony-forming 20% knockout-SR, 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM b- capability from haemangioblasts, 1 Â 103 cells were purified from blast cultures mercaptoethanol and 1% dimethyl sulfoxide for 4 days. For maturation, the cells m were cultured in Iscove’s modified Dulbecco’s medium (IMDM) containing at day 6 and resuspended in 20 l of Stemline II medium and mixed well À 1 À 1 with 0.5 ml of methylcellulose H4436 (Stem Cell Technologies) as previously 20 ng ml oncostatin M, 0.5 mM dexamethasone and 50 mg ml ITS premix. assayed19. After 14 days, differential colony counts were performed based on morphology. Flow cytometry analysis. Single-cell suspensions were stained using the following antibodies: CD31-PE (1:100) (BD Pharmingen), CD34-PE or -FITC (1:100) Generation of haemangioblasts. Haemangioblasts were generated as previously (Miltenyi Biotech, Bergisch Gladbach, Germany), CD45-APC (1:100) (Miltenyi described19. Briefly, hEBs were cultured in hEB-differentiation medium (Stemline Biotech), SSEA-1 (1:100), SSEA-3 (1:100), SSEA-4 (1:100) (Developmental Studies À II (Sigma) supplemented with 50 ng ml 1 BMP4 and vascular endothelial growth Hybridoma Bank), TRA1-60-Alexa Fluor 647 (1:500), TRA1-85-APC (1:500), factor (R&D Systems)) for 48 h, and then replaced with Stemline II medium OCT4 (1:200) (BD Biosciences), A2B5 (1:100), Nestin (1:500), Oligodendrocyte containing 50 ng ml À 1 BMP4, 50 ng ml À 1 vascular endothelial growth factor and Marker O4 (1:200), Neuron-specific beta-III Tubulin (1:200) (R&D Systems), À 20 ng ml 1 bFGF. After 24 h, hEBs were collected, dissociated into single-cell GFAP (1:200) (Sigma) and Haemoglobin b-PerCP (1:500) (Santa Cruz). Uncon- suspension and resuspended in Stemline II medium and then plated in blast jugated antibodies were visualized with appropriated Fluorochrome-conjugated growth medium (BGM) for up to 6 days. secondary antibody. Fluorescence-activated cell sorting analysis was performed on a FACSCalibur cytometer (Becton Dickinson Immunocytometry Systems) and analysed using FlowJo software (Tree Star Inc). Differentiation into erythroid cells. Erythroid differentiation and expansion was carried out as previously described21. Briefly, haemangioblasts at day 6 were collected and cultured in BGM medium supplied with 3 U ml À 1 erythropoietin Immunocytochemistry. Immunostaining was performed as previously descri- and cultured for 5–7 days until confluence. To further expand, cells were collected 13 and replated in BGM medium with 3 U ml À 1 erythropoietin. For further bed . Briefly, cells were fixed in 4% paraformaldehyde and stained with differentiation and maturation, expanded cells were cultured with BGM medium appropriate antibodies. If permeation was needed, cells were treated with 0.1% mixed with StemPro-34 SCF medium at 1:1 ratio. SCF (100 ng ml À 1)and saponin (BD Biosciences) prior to staining. Appropriate primary and 3 units ml À 1 erythropoietin were added to media. b-globin protein expression was fluorochrome-conjugated secondary antibodies were used. Cells were then counterstained with Hoechst 33342 (Invitrogen) or mounted with Vectashield detected after 6 days in culture. Plating in BGM medium after hEB dissociation was 0 denoted as day 0 of erythroid culture. Mounting Medium containing 4 ,6-diamidino-2-phenylindole (DAPI) (Vector Labs). The following antibodies were used: SSEA-1 (1:100), SSEA-3 (1:100), SSEA- 4 (1:100), OCT4 (1:200), TRA-1-60-Alexa Fluor 488 (1:100) (BD Biosciences), Differentiation into endothelial cells. Purified haemangioblasts were plated on GFAP (1:200), Oligodendrocyte Marker O4 (1:200), Neuron-specific beta-III fibronectin-coated plate with EGM-2 medium (Lonza) as previously described19. Tubulin (1:200) and Haemoglobin b (1:100) (Santa Cruz). Alexa Fluor 488- Endothelial cells were cultured for 30 days. Expression of the endothelial-specific conjugated secondary antibody was used for SSEA-1, SSEA-3, SSEA-4 and marker PECAM (BD Biosciences) on differentiated endothelial cells was confirmed Oligodendrocyte Marker O4, and Alexa Fluor 555-conjugated secondary antibody by immunofluorescence staining. Low-density lipoprotein (LDL) uptake was was used for OCT4, GFAP, Neuron-specific beta-III Tubulin and Haemoglobin b.

NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications 11 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605

Quantitative RT-PCR. Total RNA was extracted from cells using the RNeasy kit 8. Haase, A. et al. Generation of induced pluripotent stem cells from human cord (Qiagen). Complementary DNA (cDNA) was made with 1 mg of total RNA using blood. Cell Stem Cell 5, 434–441 (2009). the first-strand cDNA synthesis kit (Invitrogen) and subsequent quantitative real- 9. Wang, Z. Z. et al. Endothelial cells derived from human embryonic stem cells time PCR (Q-PCR) was carried out in triplicate, using SYBR Green reverse tran- form durable blood vessels in vivo. Nat. Biotechnol. 25, 317–318 (2007). scriptase (RT)-PCR reagents (Applied Biosystems) on an Mx3000P Q-PCR System 10. Takahashi, K. et al. Induction of pluripotent stem cells from adult human according to the manufacturer’s instructions (Stratagene). Amplifications were fibroblasts by defined factors. Cell 131, 861–872 (2007). performed using the following conditions: 95°C for 10 min, followed by 40 cycles of 11. Reynolds, B. A. & Weiss, S. Generation of neurons and astrocytes from isolated 95 °C for 15 s and 60 °C for 1 min. All data were normalized to glyceraldehyde cells of the adult mammalian central nervous system. Science 255, 1707–1710 3-phosphate dehydrogenase (GAPDH). Primer sequences used were shown in (1992). Supplementary Table 1. 12. Pollard, S. M. et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4, 568–580 (2009). Affymetrix analysis. Total RNA for microarrays was isolated using the RNA isolation kit (Invitrogen). RNA was hybridized to Affymetrix Human Gene 1.0 ST 13. Vijayaragavan, K. et al. Noncanonical Wnt signaling orchestrates early arrays (London Regional Genomics Centre, ON, Canada). Array data were nor- developmental events toward hematopoietic cell fate from human embryonic malized using the robust multichip averaging method and baseline transformed to stem cells. Cell Stem Cell 4, 248–262 (2009). the median of all samples (GeneSpring 12.0, Agilent Technologies). Hierarchical 14. Lengerke, C. et al. Hematopoietic development from human induced clustering using Pearson distance metric and centroid linkage rule was done on pluripotent stem cells. Ann. N.Y. Acad. Sci. 1176, 219–227 (2009). normalized expression values of all entities. Unsupervised GSEA was done on 15. Ivanova, N. B. et al. A stem cell molecular signature. Science 298, 601–604 normalized expression values of all entities between hCB- and hFib-iPSCs samples (2002). using GSEA software v2.07 (Broad Institute). Curated (C2v3.0) and gene ontology 16. Hess, J. L. et al. c-Myb is an essential downstream target for homeobox- (C5v3.0) gene sets from Molecular Signatures Database (MSigDB) were used. Gene mediated transformation of hematopoietic cells. Blood 108, 297–304 (2006). sets were filtered based on permutation on gene sets with n ¼ 1,000 permutations. 17. Doulatov, S. et al. Induction of multipotential hematopoietic progenitors from Enrichment plots were generate on statistically significant relevant functional gene human pluripotent stem cells via respecification of lineage-restricted sets: mesoderem, ectoderm, endoderm, heart development (Gene Ontology), precursors. Cell Stem Cell 13, 459–470 (2013). neuronal lineage (KEGG, Reactome, and previously reported data31), hepatocytic 18. McIntyre, B. A. et al. Gli3-mediated hedgehog inhibition in human pluripotent lineage32 and hematopoietic lineage15,16 from published data. stem cells initiates and augments developmental programming of adult hematopoiesis. Blood 121, 1543–1552 (2013). 19. Lu, S. J. et al. Generation of functional hemangioblasts from human embryonic Chromatin immunoprecipitation assays. Chromatin immunoprecipitation stem cells. Nat. Methods 4, 501–509 (2007). (ChIP) experiments were performed as previously described22. For each condition, 20. Kennedy, M., D’Souza, S. L., Lynch-Kattman, M., Schwantz, S. & Keller, G. 6 1 Â 10 cells (Fib-derived or CB-derived iPS) were collected and cross-linked using Development of the hemangioblast defines the onset of hematopoiesis in 1% formaldehyde. Chromatin was fragmented by sonication in buffer containing human ES cell differentiation cultures. Blood 109, 2679–2687 (2007). B 1% NP-40 to obtain fragments of 250 bp length. Sonicated DNA was subjected to 21. Lu, S. J. et al. Biologic properties and enucleation of red blood cells from human immunoprecipitation using anti-trimethyl H3K4 (07-473, Millipore), anti- embryonic stem cells. Blood 112, 4475–4484 (2008). trimethyl H3K27 (17-622 ChIPAb þ , Millipore) and anti-rabbit IgG (17-622 22. Hong, S. H. et al. Cell fate potential of human pluripotent stem cells is encoded ChIPAb þ , Millipore) antibodies. Immunoprecipitated DNA was further reverse by histone modifications. Cell Stem Cell 9, 24–36 (2011). cross-linked, purified and subjected to Q-PCR analysis to detect MixL1, Brachyury 23. Hanna, J. et al. Human embryonic stem cells with biological and epigenetic (T), Pax6 and NF68 promoter fragments. Sequence enrichment was calculated characteristics similar to those of mouse ESCs. Proc. Natl Acad. Sci. USA 107, relative to input signal. Specific primers used to amplify each promoter sequence 9222–9227 (2010). were described in Supplementary Table 2. 24. Li, W. et al. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4, 16–19 (2009). Methylated DNA-pulldown analysis. Fractionation of methylated double-stran- ded DNA was performed using the MethylMiner methylated DNA enrichment kit 25. Gafni, O. et al. Derivation of novel human ground state naive pluripotent stem according to manufacturer’s recommendations (ME10025, Invitrogen). Briefly, cells. Nature 504, 282–286 (2013). 1 Â 106 Fib-derived or CB-derived iPS cells were collected and genomic DNA was 26. Ellis, J. & Bhatia, M. iPSC technology: platform for drug discovery. Point. Clin. extracted using the DNeasy Blood and Tissue kit (#69504, Qiagen). Fragmentation Pharmacol. Ther. 89, 639–641 (2011). of genomic DNA was performed by sonication (B250 bp length) and methylated 27. Ellis, J. et al. Alternative induced pluripotent stem cell characterization criteria DNA portions were pulled down based on their interaction with the methyl-CpG- for in vitro applications. Cell Stem Cell 4, 198–199 (2009). binding domain (MBD) of human MBD2 protein immobilized on beads. Purified 28. Werbowetski-Ogilvie, T. E. et al. Characterization of human embryonic fragments were analysed by Q-PCR to assess for the enrichment of MixL1, Bra- stem cells with features of neoplastic progression. Nat. Biotechnol. 27, 91–97 chyury (T), Pax6 and NF68 promoter sections, relative to input material. Specific (2009). primers used to amplify each promoter sequence were described in Supplementary 29. Hong, S. H., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Lee, J. B. & Bhatia, M. Table 2. Multiparameter comparisons of embryoid body differentiation toward human stem cell applications. Stem Cell Res. 5, 120–130 (2010). 30. Chen, Y. F. et al. Rapid generation of mature hepatocyte-like cells from human Statistical analysis. All results were expressed as means±s.e.m. and generated induced pluripotent stem cells by an efficient three-step protocol. Hepatology from at least three independent experiments. Statistical significance was deter- 55, 1193–1203 (2012). mined using the Student’s t-test and results were considered significant or highly 31. Lee, G. et al. Isolation and directed differentiation of neural crest stem cells significant when Po0.05 or Po0.01, respectively. derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468–1475 (2007). 32. Chiao, E. et al. Isolation and transcriptional profiling of purified hepatic References cells derived from human embryonic stem cells. Stem Cells 26, 2032–2041 1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse (2008). embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). 2. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic Acknowledgements cells. Science 318, 1917–1920 (2007). This work was supported by grants from the Canadian Institute of Health Research 3. Miura, K. et al. Variation in the safety of induced pluripotent stem cell lines. (CIHR), and Canadian Cancer Society Research institute (CCS-RI) to M.B. M.B. is Nat. Biotechnol. 27, 743–745 (2009). supported by the Canadian Chair Programme and holds the Canada Research 4. Ohi, Y. et al. Incomplete DNA methylation underlies a transcriptional memory Chair in human stem cell biology. Y.D.B. is supported by fellowships from the of somatic cells in human iPS cells. Nat. Cell Biol. 13, 541–549 (2011). Canadian Institute of Health Research and the Fonds de Recherche en Sante´ du 5. Polo, J. M. et al. Cell type of origin influences the molecular and functional Que´bec. We would like to thank Seok-Ho Hong, Ruth M. Risuenõ and Kyle Salci properties of mouse induced pluripotent stem cells. Nat. Biotechnol. 28, for their invaluable insights and technical assistance during the preparation of this 848–855 (2010). manuscript. 6. Kim, K. et al. Epigenetic memory in induced pluripotent stem cells. Nature 467, 285–290 (2010). 7. Kim, K. et al. Donor cell type can influence the epigenome and differentiation Author contributions potential of human induced pluripotent stem cells. Nat. Biotechnol. 29, J.-H.L. designed and performed experiments and wrote the manuscript. J.B.L. performed 1117–1119 (2011). differentiation of hiPSCs. Z.S. carried out affymetrix analysis; A.F.-C. contributed to

12 NATURE COMMUNICATIONS | 5:5605 | DOI: 10.1038/ncomms6605 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6605 ARTICLE

teratoma formation and analysis. R.R.M. contributed to experimental design and writing Supplementary Information accompanies this paper at http://www.nature.com/ the manuscript. S.L. contributed to cell line maintenance and immunocytochemistry. E.S. naturecommunications contributed experimental design; Y.D.B. performed ChIP and methylated DNA-pulldown analysis; M.B. conceived the original idea, designed and interpreted results and Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. wrote the manuscript. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/

Additional information How to cite this article: Lee, J.-H. et al. Somatic transcriptome priming gates Accession codes: All array data have been uploaded to Gene Expression Omnibus lineage-speciﬁc differentiation potential of human-induced pluripotent stem cell states. (http://www.ncbi.nlm.nih.gov/geo/) under accession codes GSE62066. Nat. Commun. 5:5605 doi: 10.1038/ncomms6605 (2014).