Establishing the human naïve pluripotent state

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ScienceDirect Establishing the human naı¨ve pluripotent state Yair S Manor, Rada Massarwa and Jacob H Hanna Pluripotency is first assembled within the inner-cell-mass of developing pre-implantation blastocysts, and is gradually reconfigured and dismantled during early post-implantation development, before overt differentiation into somatic lineages ensues. This transition from pre-implantation to postimplantation pluripotent states, respectively referred to as naı¨ve and primed, is accompanied by dramatic changes in molecular and functional characteristics. Remarkably, pluripotent states can be artificially preserved in a selfrenewing state in vitro by continuous supplementation of a variety of exogenous cytokines and small molecule inhibitors. Different exogenous factors endow the cells with distinct configurations of pluripotency that have direct influence on stem cell characteristics both in mice and humans. Here we overview pluripotent states captured from rodents and humans under different growth conditions, and provide a conceptual framework for classifying pluripotent cell states on the basis of a combination of multiple characteristics that a pluripotent cell can simultaneously retain. We further highlight the complexity and dynamic nature of these artificially isolated in vitro pluripotent states in humans. Address The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel Corresponding authors: Massarwa, Rada ([email protected]) and Hanna, Jacob H ([email protected])

Current Opinion in Genetics & Development 2015, 34:35–45 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Amander T Clark and Thomas P Zwaka

http://dx.doi.org/10.1016/j.gde.2015.07.005 0959-437/# 2015 Elsevier Ltd. All rights reserved.

Introduction Pluripotency pertains to describing a transient embryonic developmental state which has the potential to give rise to all embryonic germ-layers and primordial germ cells, but not the extra-embryonic trophoblast lineage [1]. Recent lines of research have begun to provide experimental explanation and delineate relationships between two distinct states of pluripotency in rodents, with unique www.sciencedirect.com

genetic and epigenetic configuration in vivo and in vitro [2,3,4,5]. Murine naı¨ve pluripotency

In the pre-implantation mouse inner cell mass (ICM), the epiblast initiates expression of Nanog pluripotency factor, a process that coincides with global DNA hypomethylation and reactivation of the inactive X allele in females [6]. Murine embryonic stem cells (ESCs) with characteristics similar to the pre-implantation ICM were first isolated from limited strains of mice, typically from 129 pure or mixed genetic background, that were permissive for yielding ESCs in undefined conditions [2]. Lif/ Stat3 signaling emerged as a key pathway that allows capture of such mouse ESCs and endows them with indefinite renewal capacity in vitro, while maintaining many features of ICM pluripotent cells [7,8]. Alternative defined growth conditions that typically involve ERK1/2 inhibition and LIF/Stat3 stimulation enhanced ESC derivation efficiency and boosted their naı¨ve features [9,10]. Noteworthy, MEK and FGFR4 receptor small molecule inhibitors firstly together with GSK3b blocking (termed as 3i conditions), allowed maintaining mouse naı¨ve pluripotency independent of LIF/Stat3 signaling, albeit at reduced growth rate [11]. The use of LIF stimulation together with ERK1/2 and GSK3 inhibition (known as 2i/LIF) conditions emerged later as a robust condition for defined expansion of mouse naı¨ve pluripotent cells and as suboptimal for rat naı¨ve ESCs [12]. Supplementation of additional small molecule inhibitors of aPKC, TGFR and/or p38 signaling has been established as boosters for rat and mouse naı¨ve PSC (iPSC and ESC) stability [13,14,15]. Collectively, the above findings support the notion that different exogenous supplements can stabilize rodent PSCs, and that these exogenous factors may not authentically reflect requirements for pluripotency formation in vivo (e.g. LIF/Stat3 signaling) [11,16]. Moreover, these observations justify screening for additional factors and signaling pathways that may promote maintaining pluripotency in vitro and in vivo [17]. While naı¨ve pluripotent cells captured in vitro recapitulate to a large extent the epigenetic features of ICM cells, adaptation and alteration in gene expression occur upon explanting the cells in vitro [18]. This was emphasized in several single cell transcription analysis of explanted ICM cells that documented profound differences between the two, and enhanced expression of genes that endow ESCs with tumorigenic self renewal properties that are required Current Opinion in Genetics & Development 2015, 34:35–45

36 Cell reprogramming, regeneration and repair

in vitro [19]. Such properties may not be important for in vivo self renewal, as naı¨ve pluripotency is normally transient during early in vivo development (e.g. Eras [20]). Noteworthy, while stringent naı¨ve conditions endow homogeneity in expression of a selected number of well characterized genes like Nanog and Rex1, it remains to be defined whether such conditions render the cells homogenous at the global transcriptional and epigenetic levels [18,19]. Murine primed pluripotency

The naı¨ve ICM-like pluripotent state is quickly resolved in the post-implantation epiblast stage, where Oct4+ pluripotent cells reduce expression of Nanog, accumulate H3K27me3 over developmental genes, upregulate global DNA methylation and inactivate one of the X chromosomes [21,22]. This configuration is receptive to gradients of inductive signals that prime lineage commitment and differentiation of the embryonic cells according to their location in the egg-cylinder shaped post-implantation epiblast [23]. EpiSCs or primed pluripotent cells are classically stabilized in vitro by exogenous supplementation of bFGF and Activin A/Nodal pathways, that maintain Oct4 expression and confer in vitro self-renewal [3,24]. While much of our knowledge is focused on murine naı¨ve pluripotent cells, key regulators of primed state are starting to emerge (e.g. Otx2, Mettl3) [23–25]. Safeguarding adequate transition from naı¨ve to primed pluripotency in vivo has been recently shown to be critical for embryo viability [23]. Interestingly, functional differences between naı¨ve cells induced for short time (2–4 days) into primed conditions (termed EpiLCs) versus primed cells that are maintained long-term in primed conditions, have also been described [26]. Further, alternative growth conditions for maintaining murine EpiSCs that can further render them more similar to specific regions of the post-implantation epiblast, are emerging. This is typically induced by combinatorial regulation of WNT, FGF, and Nodal signaling components [27,28]. A summarizing list of key differences between murine naı¨ve and primed pluripotent PSCs is shown in Figure 1.

Defining the identity and stability of murine pluripotent states While the above summary pertains to highlighting the influence of exogenous signaling factors on the identity and stability of pluripotent states, the genetic background appears to provide endogenous determinants that dictate pluripotent state identity and stability in vitro under different growth conditions [29]. Before the application of MEK and GSK3 inhibitors in ES cultures, several studies had derived stem cells from non-129 mouse strains and rats that corresponded to EpiSCs, rather than ESCs [30]. Since such EpiSC-like cells were explanted from the pre-implantation ICM, it was largely presumed that these in vitro differences in stem cell line Current Opinion in Genetics & Development 2015, 34:35–45

Figure 1

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OXPHOS & Glycolytic Metabolism

Glycolytic Metabolism

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Upregulation of priming markers (e.g. Otx2,Zic2)

High competence as initial donor cells for PGCLC induction

No/Lowcompetence as initial donor cells for PGCLC induction

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High competence for TSC induction

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Predominant Nuclear localization of TFE3

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Efficient colonization of host ICM and contribution to chimeric animals

Inefficient colonization of host ICM and contribution to chimeric animals

Downregulation of priming markers (e.g.Otx2, Zic2)

Current Opinion in Genetics & Development

Naı¨ve and primed pluripotency characteristics in rodent PSCs. A summary of properties of pluripotency and their designation as either corresponding to the naı¨ve or primed pluripotent configuration. This list of features might be helpful for systematically describing new pluripotent states isolated in unique conditions and from different species, including humans.

characteristics reflect in vivo differences in the parental pluripotent cells giving rise to such ‘ESC’ lines, either at the molecular configuration level or origin of cellular lineage. www.sciencedirect.com

Human naı¨ve pluripotency Manor, Massarwa and Hanna 37

However, by using the non-obese diabetogenic (NOD) mouse as a model for ‘non-permissive’ genetic background for naı¨ve ESC derivation, we demonstrated that preserving naı¨ve pluripotency from such relatively ‘nonpermissive’ mice in vitro, requires additional boosting with exogenous factors. Naı¨ve NOD ESCs and iPSCs could be propagated in 2i/LIF or by continued constitutive expression of Klf4 and c-Myc in classical LIF only conditions [2]. In the absence of these additional exogenous factors, the naı¨ve state identity of NOD PSCs, even if initially isolated from the ICM, is masked by in vitro acquisition of pluripotent state that is nearly indistinguishable from EpiSCs in a process that probably imitates in vivo differentiation of naı¨ve epiblast into the postimplantation primed epiblast [2,31]. Genetic marking

experiments have demonstrated clonal relationship between the two states and ruled out the possibility that mixed populations constantly co-exist in culture in vitro or originate from different and independent in vivo embryonic progenitors [2]. Rather, this conclusively proved that pluripotent cells exist in a bi/metastable equilibrium between naı¨ve and primed ground states, where one can be inter-converted into the other when provided with signals that support one pluripotent state and destabilizes the other [2]. Largely, the primed state has antagonistic signaling dependence properties in comparison to naı¨ve ESCs in mice (Figure 2). Primed EpiSCs require functional signaling of ERK1/2 pathway and are destabilized by its inhibition.

Figure 2

Rodent Pluripotency Bmp4 Ins

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Current Opinion in Genetics & Development

Influence of defined signaling pathways on the stability of primed and naı¨ve pluripotency states. Upper panel shows summary of characterized signaling pathways that influence murine naı¨ve and primed pluripotent cells, either positively or negatively. It is important to highlight that possibly other important pathways may yet to be revealed and that alternative novel conditions may be devised and optimized in the future. While it is early days in our understanding of the signaling pathways that regulate human naı¨ve pluripotency, the lower panel highlights pathways so far involved in recent publications. It is important to note some potential differences than those observed in mouse naı¨ve pluripotency regulation. Early studies suggest potentially different roles of low dose FGF2, TGFB/ACTIVIN/NODAL and/or BMP4 (gray shaded boxes) signaling in mouse versus human naı¨ve pluripotency. www.sciencedirect.com

Current Opinion in Genetics & Development 2015, 34:35–45

38 Cell reprogramming, regeneration and repair

The pluripotency of naı¨ve mouse ESCs is enhanced by inhibition of ERK1/2 signaling, and in fact this manipulation enhances derivation efficiency of ESCs from embryos [5]. BMP4 signaling stabilizes naı¨ve pluripotency, but induces differentiation of EpiSCs. TGFB1/ Activin A and bFGF induce priming of naı¨ve ESCs, and support in vitro renewal of EpiSCs (Figure 2). Delineating these signaling dependence differences and their downstream effectors has become instrumental to defining the relationships and dictating interconversion efficiency and kinetics between naı¨ve and primed PSCs [32].

As the concepts and conclusions relating to the stability of rodent pluripotent states were evolving, a key question that emerged was whether a murine ICM-like (i.e. naı¨ve pluripotency) is also a core feature of early primate embryonic development and whether human cells that share defining features with mouse ESCs could be obtained in vitro [39]. The clonal bi/metastable pluripotency paradigm, as observed in NOD pluripotent cells, substantiated a scenario where appropriate conditions may have not been devised to allow isolation of naı¨ve stem cells from a range of species that have yielded thus far primed or EpiSC-like cells, including humans [39].

The isolation and interconvertability of both ESC and EpiSC-like pluripotent cells from multiple mouse and rat strains, underscored the inherent bi-stability of pluripotent states obtained from different genetic backgrounds that can assume two distinct states of pluripotency in vitro, primed and naı¨ve (or ESC-like and EpiSC-like), depending on the conditions applied rather than pluripotent cell origin (ICM, post-implantation epiblast or via direct somatic cell reprogramming into iPSCs) [2,33]. The stability of these states is regulated by endogenous genetic determinants and can be modified by defined exogenous factors. The naı¨ve and primed pluripotent states isolated in vitro correspond to some extent to in vivo counterparts, and that the same pluripotent states can be stabilized by different growth conditions that can yield a highly similar pluripotent state. The difference between the requirement for exogenous factors that stabilize naı¨ve pluripotency between different strains and species (at least when relating to mice, rats, and humans) is not predominantly due to difference in the response to signaling pathways or to parental origin of the donor cells (ICM, post-implantation epiblast, EPISCs or via direct reprogramming into iPSC), but rather due to the requirement for modulation of a greater number of naı¨ve pluripotency promoting pathways simultaneously [2,29].

It is important to indicate that conventional human ESCs/ iPSCs are not identical to mouse EpiSCs and share several features with murine naı¨ve pluripotent cells. This includes expression of E-CADHERIN and absence of FGF5. Human PSCs express high levels of NANOG and PRDM14 and their pluripotency is compromised upon ablation of these factors, while mouse EpiSCs nearly lack these factors and are unaffected upon their complete genetic ablation [40,41]. The above findings may suggest that human ESCs are relatively less primed than murine EpiSCs, however they still retain many characteristic features of murine primed PSCs. This includes, lack of a stable XaXa state, high levels of global DNA methylation, accumulation of H3K27me3 over developmental regulators, low nuclear preferential localization of TFE3, low competence to generate primordial germ cell-like cells (PGCLCs), etc. [42,43]. These observations suggest that human ESCs retain many features of primed pluripotency and perhaps can be toggled toward a more naı¨ve pluripotent state.

Conventional human ESCs The fact that human ESCs were initially established from the human ICM cells [34], and not the human postimplantation epiblast, have caused confusion when attempting to developmentally annotate conventional human ESCs and led to attributing their stark difference from their mouse ICM-derived ESCs and iPSC counterparts, solely to intrinsic genetic species differences [35]. However, as shown for NOD ESCs, naı¨ve ICMs and iPSCs, such a naı¨ve identity can progress to become primed upon derivation if stringent and adequate naı¨ve pluripotent conditions are not applied [2]. Indeed, recent studies mapping X chromosome and epigenetic changes of human ICM shortly following derivation in conventional FGF/TGFB conditions, support this scenario and document in vitro inactivation of X chromosome in female cell lines and increase in DNA methylation within as early as 1–2 passages [36–38]. Current Opinion in Genetics & Development 2015, 34:35–45

The quest and evidence for a naı¨ve human pluripotent state Unlike rodent cells, 2i/LIF conditions are not sufficient to maintain human pluripotent cells in culture, even when exogenous KLF4, KLF2, NANOG or c-MYC are individually expressed as constitutively expressed factors [31,39,44]. Thus, a more aggressive screening approach that involved introducing multiple reprogramming factors and/or small molecules was needed to stabilize the human naı¨ve pluripotent state. This led to in vitro stabilization of a novel pluripotent cell state that retained its pluripotency independent of MEK/ERK signaling. This was achieved from iPSCs and already established ESCs via cooperative action of over-expressing KLF2/KLF4 or OCT4/KLF4 together with applying 2i/LIF [39]. These results demonstrated direct evidence for a previously unidentified transgene dependent and ERK independent naı¨ve state of pluripotency in humans that is more similar to mouse naı¨ve PSCs (ESCs and iPSCs). Recently, these results were expanded by inducing exogenous expression of both KLF2 and NANOG in 2i/LIF conditions that also yielded unique transgene dependent human naı¨ve pluripotent cells [39,44,45]. www.sciencedirect.com

Human naı¨ve pluripotency Manor, Massarwa and Hanna 39

While such transgene dependent naı¨ve pluripotent states might still be of considerable interest and applicative potential [44,45], a crucial next challenge was to define conditions for isolating human naı¨ve pluripotent stem cells, where genetically unmodified cells can be grown indefinitely in defined growth conditions and the absence of feeder cells. Embarking on this challenge was carried by multiple groups despite the possibility that since pluripotency is a transient non self-renewing developmental state in vivo, it may not be possible to artificially render to self-renew and become indefinitely stable in vitro from non-rodent species at all or at least without permanent transgenic modifications [46].

Genetically unmodified human naı¨ve pluripotent cells Our group was the first to identify conditions that allow derivation of distinct and genetically unmodified human pluripotent cells capable of growing in 2i/LIF containing conditions (termed NHSM) [42], from human ICM, previously isolated primed hESCs and iPSCs. These conditions render such pluripotent cells distinct from conventional human ESCs in multiple aspects, and more similar to murine naı¨ve ESCs (although not identical) [42]. In addition to 2i/LIF, NHSM conditions utilize applying P38i, JNKi, aPKCi, ROCKi together with low doses of FGF2 and TGFB1 (or Activin A). These cells could be expanded in the absence of feeder cells, and retained some defining features of murine ground state naı¨ve pluripotency — maintenance of pluripotency independent of MEK signaling, exclusive nuclear retention of TFE3 and cleansing of H3K27me3 over developmental genes [42]. Transcriptionally, these cells down regulated expression of lineage commitment markers like OTX2, ZIC2 and CD24 [47] and moderately upregulated pluripotency genes (Klf5, Klf4, Klf2, Klf17, Tfcp2l1) [42]. The cells exhibited X chromosome reactivation [48] and only a mild global DNA hypomethylation [42]. Importantly, Cynomolgus monkey naı¨ve ESCs have been derived in NSHM conditions, and yielded the first chimera competent ESCs, that following ICM-microinjection, yielded chimeric monkey fetuses with contribution in all three germ layers (ectoderm, mesoderm, and endoderm) and to the germ cell lineage [49]. The latter provides an ultimate proof for endowing naı¨ve pluripotency features by NHSM conditions in non-human primates. Since the initial discovery by Gafni et al. [42], several important publications have devised alternative conditions that yield human ERK independent naı¨ve pluripotent cells, each producing different cellular molecular and epigenetic properties whose nature and importance remain to be fully elucidated [44,50–54]. TESR/3i/L conditions described by NG and colleagues, contains the following signaling pathway modulators: 2i/LIF, ROCKi, BMPRi, high doses of FGF2 and TGFB1 [50]. Human www.sciencedirect.com

PSCs in these conditions can be maintained only in the presence of MEFs, demonstrated an impressive upregulation in GATA6, DNMT3L and STELLA, and down regulated H3K27me3 over several developmental genes [50]. X chromosome remained inactive in female cells under these conditions, and the DNA methylation profile was not examined [50]. Obligatory feeder dependent 5i/LA conditions described by Theunissen et al. [44] comprises of 2i/LIF, BRAFi, ROCKi, SRCi, and Activin A (with or without FGF2 and JNKi). In comparison to the previous two studies, cells in 5i/LA conditions demonstrated a more pronounced upregulation of pluripotency markers like TFCP2L1, KLF2, KLF4, and DNMT3L [44]. However, these conditions nearly exclusively yielded chromosomally abnormal cells, upregulated XIST in female (and not male) cell lines indicative of X chromosome inactivation, and had a profound expression of early pre-implantation genes like (OOEP and CD70) that are not expressed in the human ICM [44]. Furthermore, the cells failed contribute to mouse chimeric embryos following microinjection to mouse pre-implantation blastocysts [44], although sensitive methods for detection of low-contribution chimerism were not applied (i.e. histological sectioning or in toto confocal imaging as conducted by Gafni et al. [42]). Under 5i/LA (6i/LA or 5i/LAF) conditions human PSCs cannot be maintained in feeder free conditions and, as such, whether they are FGF signaling independent remains to be proven [44], as the cells were not stringently analyzed for their ability to maintain growth and selfrenewal with FGF receptor signaling inhibitors (FGFRi). DNA methylation landscape for 5i/LA cells has not been analyzed, however downregulation in DNMT3A and DNMT3B were not observed in these conditions. Smith and colleagues described ectopic expression of KLF2 and NANOG together with 2i/LIF/aPKCi conditions as a mean to generate naı¨ve PSCs that can be expanded only in the presence of MEFs [45]. These cells exhibited more extensive DNA demethylation than observed by Gafni et al. [42], and more pronounced upregulation of naı¨ve markers like TFCP2L1, KLF2, and KLF4. They did not upregulate non-ICM early preimplantation genes (OOEP and CD70) as seen by Theunissen et al. [44]. However, as KLF2 is not expressed in the human ICM [55] and as transgene independent cells remain to be validated in 2i/LIF/aPKCi condition, it is unclear whether this state is rather artificial or indefinitely stable without retaining leaky transgenes and/or MEFs [45]. Further, careful reexamination of DNA methylation of such cells has highlighted aberrant global loss of imprinting and excessive hypomethylation of endogenous retroviral genes [56]. Finally, while these conditions do not contain exogenous FGF or TGFB/Activin A, the use of MEF feeders while applying short-term inhibition of FGFR/TGFR signaling is not a sufficient evidence to Current Opinion in Genetics & Development 2015, 34:35–45

40 Cell reprogramming, regeneration and repair

conclude FGF and TGF/Activin A signaling independence [45], as typically seen with rodent naı¨ve PSCs (Figure 2). Ware and colleagues reported that 2i/LIF conditions are sufficient on feeder cells to maintain human naı¨ve pluripotent cells [51], however it is important to note that, as stated in the article, they systematically avoided complete blocking of MEK/ERK signaling and could only use low levels of ERK inhibitor (only up to 0.4 mM of PD0325901). This indicates that cells described by Ware et al. are destabilized by complete ERK inhibition (1.0 mM of PD0325901) [51]. Excitingly, new studies and methodologies implicating new modulators, like Forskolin and HDAC inhibitors, in deriving novel MEK independent human naı¨ve pluripotent cells continue to emerge and provide new leads and anecdotes [51–54]. Collectively, the different components described in the published studies reviewed above are likely to help optimize robust conditions for human naı¨ve cells that are increasingly more similar to murine naı¨ve pluripotent cells and human ICM cells, under transgene free and feeder free conditions.

Relativity of naı¨vety within the naı¨ve to primed pluripotency spectrum The recent studies on human naı¨ve pluripotency have provoked the discussion on how to classify human pluripotent cells, which is inherently more complicated than in mice, since embryo chimerism analysis with human embryos cannot and must not be conducted. While it is highly complex and limiting to assign pluripotent states as naı¨ve or primed on the basis of a single property, in our opinion, one major molecular criterion that can be considered for making this separation is the PSCs ability to maintain their pluripotent state in the complete absence of MEK-ERK1/2 activity (Figure 3 — dashed black line). Within the naı¨ve or primed pluripotent state, it maybe still difficult to describe the pluripotent state of the cells in absolute terms, as naı¨ve cells can have, to some extent, primed pluripotency features, and vice versa. For instance, murine 129 ESCs expanded in FBS/LIF conditions retain hallmark naı¨ve pluripotency features like pre-x inactivation state, tolerating loss of Dnmt1 and high-chimerism contribution ability into host embryos [57]. Yet, naı¨ve mouse ESCs in FBS/LIF

Figure 3

ESC (2i/LIF F/ aPKCi) ESC ?? (2i/L IF F)

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Som a Diff tic Line eren a tiati ge on Current Opinion in Genetics & Development

Relativity of naı¨vety within the naı¨ve to primed pluripotency spectrum. One major molecular criterion that can be considered for separating naı¨ve from primed pluripotent cells is their ability to maintain and stabilize their pluripotent state in the absence of MEK-ERK1/2 activity (dashed black line). Within the naı¨ve or primed pluripotent state, it maybe still difficult to describe the pluripotent state of the cells in absolute terms, as naı¨ve cells can have, to some extent, primed pluripotency features. Similarly, within the primed pluripotency spectrum, primed PSCs expanded in different conditions have different features and varying degrees of naı¨vety. Finally, it is possible that supplementation of small molecule like aPKCi might further consolidate naı¨ve pluripotency, particularly from other rodents like rats whose stability in 2i/LIF feeder free conditions should be further improved.

Current Opinion in Genetics & Development 2015, 34:35–45

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Human naı¨ve pluripotency Manor, Massarwa and Hanna 41

simultaneously retain several primed pluripotency features (e.g. H3K27me3 marked developmental genes and lack of global DNA hypo-methylation) [57]. Transfer of naı¨ve ESCs expanded in FBS/LIF into 2i/LIF conditions, consolidates the naı¨vety of murine pluripotent cells by endowing them with additional naı¨ve pluripotency features that they lacked in FBS/LIF conditions (e.g. profound global DNA hypomethylation and cleansing of H3K27me3 over developmental genes) [57]. To facilitate detailed dissection of pluripotent cells expanded in different conditions, we propose to refer to mouse naı¨ve ESCs expanded in 2i/LIF and primed EpiSCs expanded in FGF2/Activin A conditions, as two extremes of naı¨ve and primed pluripotency spectrum, respectively (Figures 1 and 2). By using the latter states as a reference, we deduce a list of naı¨ve and primed properties that can be individually assigned to a variety of pluripotent cells isolated in different conditions and from different species (Figure 1). By applying this in depth analysis, it becomes immediately evident that many naı¨ve mouse pluripotent cells in FBS/LIF and BMP4/LIF conditions have a mixture of these features [58]. As highlighted earlier, human conventional/primed ESCs retain a variety of naı¨ve features and are distinct from murine primed EpiSCs, further emphasizing the relativity concept when referring to studying configurations of pluripotent states. The examples and classification properties proposed above may be highly relevant for understanding and dissecting recently described human ERK independent naı¨ve pluripotent cells expanded in different conditions, each endowing them with a distinct set of features. Importantly, their degree of naı¨vety varies and can possibly be enhanced by devising alternative conditions and supplementing other factor like inhibitors for SRC or aPKC signaling (Figures 2 and 3) [15,16]. Finally, this list sets a framework for comparing different human naı¨ve pluripotency conditions devised thus far, and highlights future goals for closing the gap between robust murine naı¨ve pluripotent cells and those isolated from other species, including humans (Figure 1).

Human naı¨ve pluripotency: who cares?? Importantly, this poorly understood novel naı¨ve human pluripotent configuration, not only comes in different molecular flavors, but also appears to have different functional properties. Our group was the first to show that human MAPK independent naı¨ve pluripotent cells have a greatly enhanced potential to recapitulate a certain differentiation protocol, that until recently was successful only when applied on mouse naı¨ve PSCs, but poorly with conventional human primed PSCs [43]. Specifically, primordial germ cell-like cells (PGCLCs) can be induced in vitro from mouse ESCs/iPSCs maintained in stringent naı¨ve ‘2i/LIF’ medium, after they acquire ‘priming’ for germ cell fate following two days culture in basic www.sciencedirect.com

fibroblast growth factor (FGF2) and Activin A, followed by BMP4 stimulation [26]. While reconstituting the PGC lineage with conventional human PSCs in vitro has been extremely inefficient and aberrant, when using human naı¨ve pluripotent cells expanded in 2i containing media (NHSM conditions with or without aPKCi), we were able to reconstitute human early PGCLCs in vitro that are similar to human in vivo embryo derived early migratory PGCs [43]. Understating the molecular basis for generating such lineage competence, both in mice and in humans, is of extreme importance, as it might be relevant for improving cell quality and reproducibility in other differentiation assays. Human conventional ESCs/iPSCs display great level of heterogeneity in gene expression, including differential expression of lineage commitment genes [59]. Careful comparative analysis for directed differentiation into specific lineages uncovered dramatic heterogeneity that was evident by marked differences in differentiation propensity among human PSC line [60]. It is plausible that these properties are direct result of the cells used corresponding to EpiSC stage, where the cells become highly sensitive to exogenous differentiating cues and initiate partial lineage commitment randomly. It will be valuable to test whether and which naı¨ve pluripotent cell conditions can display reduced heterogeneity upon differentiation of human iPSCs among clones obtained whether from the same donor or from multiple donors. As naı¨ve pluripotency correlates with reduced deposition of epigenetic repressive marks [57,61] that have been implicated in retention of epigenetic memory in human iPSCs, it is worthwhile to check whether naı¨ve conditions will facilitate adequate resolution of such residual memory in human iPSCs [62–65]. Previous attempts to microinject conventional/primed human and monkey ESCs into host mouse and monkey blastocysts, respectively, have not generated chimeric embryos with significant tissue contribution [66]. However, given that even mouse primed pluripotent cells are not efficient in generating chimerism after injection in pre-implantation host mouse embryos [3], this has raised the possibility that failure to do so with conventional human iPSCs is rather related to them being in a primed state, rather than the cross-species differences. Indeed, human and Rhesus Monkey naı¨ve iPSCs, showed integration in mouse in vitro developed ICMs at E3.5 [42,45,67], consistent with their enhanced E-CADHERIN expression and single cell survival. Further, mouse blastocysts obtained after microinjection with human/Rhesus monkey naı¨ve iPSCs were implanted in pseudo-pregnant female mice and allowed to develop for 7–14 additional days in vivo, after which they were dissected and systematically imaged. Remarkably, multiple mouse embryos showed chimerism with naı¨ve human/Rhesus monkey iPSC-derived cells integrated Current Opinion in Genetics & Development 2015, 34:35–45

42 Cell reprogramming, regeneration and repair

at different locations [42,67]. However, in depth functional and developmental analysis of the in vivo integrating human naı¨ve iPSC derived cells, and how these settings can be coaxed for meaningful modeling of human cell differentiation, is of great scientific interest and needs to be thoroughly addressed. The latter will involve developing cutting edge embryo handling and ex vivo manipulation systems, and advanced live imaging techniques (e.g. ex vivo in toto live embryo imaging, ex vivo whole mouse embryo roller culture systems and Light-Sheet Fluorescent Microscopy) [68].

transcriptional level [73,74], where key pluripotency factors establish a dense cross-regulatory network positively regulating other pluripotency factors either by direct binding or indirectly, and thus minimize exogenous factor supplementation requirement in comparison to other species. Additionally, while binding site for pluripotency factors might be conserved across different genetic backgrounds, the levels of binding may vary considerably and contribute to these differences. Such a mechanism has been proposed to explain divergence between closely related drosophila species [75].

Pluripotent states: of mice and men

Collectively, a molecular definition of core-transcriptional circuitry and epigenetic characteristics, both of bulk human naı¨ve cell cultures and at the single cell level, might provide insights into these cardinal questions. In addition, such endeavors will probably facilitate making human naı¨ve PSCs as the gold standard starting material for future therapeutic applications and might enhance their applied flexibility and utility in basic research and personalized therapy.

None of the recently devised conditions for expanding human naı¨ve PSCs generate cells that are identical to murine naı¨ve ESCs or human ICM [42], and it should not be assumed that it would definitely be attainable. It may be possible that rodents are unusually permissive for giving rise to robust naı¨ve pluripotent cells with relatively minimal exogenous intervention, while in other species like human and other primates, they may require an extensive milieu of exogenous manipulations and possibly genetic modifications. It is also plausible that differences in requirements for maintenance of naı¨ve and primed pluripotent cells in different species, and their altered characteristics in a variety of conditions, relate to in vivo developmental differences. The human post-implantation embryo lacks the classical extra-embryonic ecto-placental cone, which is a major source for patterning cytokines in mice [69]. Moreover, rodent embryos are exceptional in forming egg-cylinder shaped epiblasts, while most other mammals, including humans, obtain a bilaminar-disk shaped post-implantation epiblast [69]. The difference in genetic elements may also dictate signaling requirements for isolation of distinct pluripotent states from different species, and will be relevant to understand mechanisms for naı¨ve pluripotency maintenance. A number of genetic differences could underlie alteration in exogenous naı¨ve PSC requirements among different species. Genetic factors positively affecting susceptibility to testicular germ cell tumors in certain mouse strains have been shown to correlate with their permissiveness to yield naı¨ve ESCs in LIF only conditions [70]. Notably, it is also well established that human somatic fibroblast are more resistant to tumor transformation with defined factors than in mouse [71]. Furthermore, it is possible that a variety of tumorigenic factors, like ERAS which regulates rapid growth of mouse ESCs and supports Nanog expression via activating PI3K/AKT pathway and is not expressed in humans [20,72], could influence the ability to stabilize naı¨ve pluripotency in humans. We also speculate that the transcriptional circuitry of the rodent naı¨ve pluripotency is highly interconnected at the Current Opinion in Genetics & Development 2015, 34:35–45

Acknowledgements J.H.H is supported by a generous gift from Ilana and Pascal Mantoux; the New York Stem Cell Foundation (NYSCF), Flight Attendant Medical Research Institute (FAMRI), the Kimmel Innovator Research Award, the ERC-Stg program (StG-2011-281906), Leona M. and Harry B. Helmsley Charitable Trust, Moross Cancer Institute, the Israel Science Foundation Regular research program, the ICRF Foundation, Helen and Martin Kimmel Institute for Stem Cell research (HMKISCR), the Benoziyo Endowment fund, an HFSPO research grant. J.H.H. is a New York Stem Cell Foundation — Robertson Investigator. We thank members of the Hanna lab for discussions, particularly Leehee Weinberger, Muneef Ayyash and Noa Novershtern. We apologize to those whose work could not be directly cited due to space limitations.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Orkin SH, Hochedlinger K: Chromatin connections to pluripotency and cellular reprogramming. Cell 2011, 145:835850.

Hanna J, Markoulaki S, Mitalipova M, Cheng AW, Cassady JP, Staerk J, Carey BW, Lengner CJ, Foreman R, Love J et al.: Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 2009, 4:513-524. Manuscript demonstrated clonal bi/metastability between naı¨ve and primed cells, and defined factors that influence stability and identity of PSCs from 129 and NOD mice.

2. 

3.

Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RDG: New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007, 448:196-199.

4. 

Brons I, Clarkson A, Ahrlund-Richter L, Pedersen RA et al.: Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 2007, 448:191-195. Paper describes the generation of primed pluripotent cells from postimplantation mouse embryos with distinct characteristics from murine ESCs. 5.

Nichols J, Smith A: Naive and primed pluripotent states. Cell Stem Cell 2009, 4:487-492. www.sciencedirect.com

Human naı¨ve pluripotency Manor, Massarwa and Hanna 43

6.

Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S: The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003, 113:631-642.

7.

Yang J, van Oosten AL, Theunissen TW, Guo G, Silva JCR, Smith A: Stat3 activation is limiting for reprogramming to ground state pluripotency. Cell Stem Cell 2010, 7:319-328.

8.

Niwa H, Ogawa K, Shimosato D, Adachi K: A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 2009, 460:118-122.

9.

Lodge P, McWhir J, Gallagher E, Sang H: Increased gp130 signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts. Cloning Stem Cells 2005, 7:2-7.

10. Dunn SJ, Martello G, Yordanov B, Emmott S, Smith AG: Defining an essential transcription factor program for naive pluripotency. Science 2014, 344:1156-1160. 11. Ying Q-L, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J,  Cohen P, Smith A: The ground state of embryonic stem cell self-renewal. Nature 2008, 453:519-523. Describes 3i conditions (3 inhibitors for MEK, GSK3 and FGF receptor) that generate LIF/Stat3 independent ESCs. 12. Chen Y, Blair K, Smith A: Robust self-renewal of rat embryonic stem cells requires fine-tuning of glycogen synthase kinase-3  inhibition. Stem Cell Reports 2013, 1:209-217. See annotation below Ref. [13]. 13. Rajendran G, Dutta D, Hong J, Paul A, Saha B, Mahato B, Ray S,  Home P, Ganguly A, Weiss ML et al.: Inhibition of protein kinase C signaling maintains rat embryonic stem cell pluripotency. J Biol Chem 2013, 288:24351-24362. Manuscripts [12,13] implicate atypical PKC signaling inhibition in promoting naı¨ve mouse and rat ESC/iPSC generation. 14. Kawamata M, Ochiya T: Generation of genetically modified rats from embryonic stem cells. Proc Natl Acad Sci USA 2010, 107:14223-14228. 15. Dutta D, Ray S, Home P, Larson M, Wolfe MW, Paul S: Selfrenewal versus lineage commitment of embryonic stem cells: protein kinase C signaling shifts the balance. Stem Cells 2011, 29:618-628. 16. Shimizu T, Ueda J, Ho JC, Iwasaki K, Poellinger L, Harada I, Sawada Y: Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signalling. Stem Cells 2012, 30:1394-1404. 17. Li X, Zhu L, Yang A, Lin J, Tang F, Jin S, Wei Z, Li J, Jin Y: Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos. Cell Stem Cell 2011, 8:46-58. 18. Kumar RM, Cahan P, Shalek AK, Satija R, Jay Daley Keyser A, Li H, Zhang J, Pardee K, Gennert D, Trombetta JJ et al.: Deconstructing transcriptional heterogeneity in pluripotent stem cells. Nature 2014, 516:56-61. 19. Tang F, Barbacioru C, Bao S, Lee C, Nordman E, Wang X, Lao K, Surani MA: Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis. Cell Stem Cell 2010, 6:468-478. 20. Takahashi K, Mitsui K, Yamanaka S: Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 2003, 423:541-545. 21. Greber B, Wu G, Bernemann C, Joo JY, Han DW, Ko K, Tapia N, Sabour D, Sterneckert J, Tesar P et al.: Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell 2010, 6:215-226. 22. Smith ZD, Chan MM, Mikkelsen TS, Gu H, Gnirke A, Regev A, Meissner A: A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 2012, 484:339-344. 23. Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS www.sciencedirect.com

et al.: m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 2015, 47:10021006. 24. Buecker C, Srinivasan R, Wu Z, Calo E, Acampora D, Faial T, Simeone A, Tan M, Swigut T, Wysocka J: Reorganization of enhancer pattern transition from naı¨ve to primed pluripotency. Cell Stem Cell 2014, 14:838-853. 25. Acampora D, Di Giovannantonio LG, Simeone A: Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition. Development 2012, 140:43-55. 26. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M: Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011, 146:519-532. 27. Kim H, Wu J, Ye S, Tai C-I, Zhou X, Yan H, Li P, Pera M, Ying Q-L: Modulation of b-catenin function maintains mouse epiblast  stem cell and human embryonic stem cell self-renewal. Nat Commun 2013, 4:1-11. Study describes an alternative and unique condition to expand mouse and human primed cells in the absence of exogenous FGF2 and Activin A. 28. Wu J, Okamura D, Li M, Suzuki K, Luo C, Ma L, He Y, Li Z, Benner C, Tamura I et al.: An alternative pluripotent state confers interspecies chimaeric competency. Nature 2015, 521:316-321. 29. Hanna JH, Saha K, Jaenisch R: Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues. Cell 2010, 143:508-525. 30. Leitch HG, Blair K, Mansfield W, Ayetey H, Humphreys P, Nichols J, Surani MA, Smith A: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state. Development 2010, 137:2279-2287. 31. Guo G, Yang J, Nichols J, Hall JS, Eyres I, Mansfield W, Smith A: Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 2009, 136:1063-1069. 32. Yeo J-C, Jiang J, Tan Z-Y, Yim G-R, Ng J-H, Go¨ke J, Kraus P, Liang H, Gonzales KAU, Chong H-C et al.: Klf2 is an essential factor that sustains ground state pluripotency. Cell Stem Cell 2014, 14:864-872. 33. Najm FJ, Chenoweth JG, Anderson PD, Nadeau JH, Redline RW, McKay RDG, Tesar PJ: Isolation of epiblast stem cells from preimplantation mouse embryos. Cell Stem Cell 2011, 8:318325. 34. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA,  Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 1998, 282:1145-1147. First study to successfully derive primed pluripotent cells from human embryos. 35. De Los Angeles A, Loh Y-H, Tesar PJ, Daley GQ: Accessing naı¨ve human pluripotency. Curr Opin Genet Dev 2012, 22:272-282. 36. Lengner CJ, Gimelbrant AA, Erwin JA, Cheng AW, Guenther MG, Welstead GG, Alagappan R, Frampton GM, Xu P, Muffat J et al.: Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 2010, 141:872-883. 37. Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, Eggan K, Meissner A: DNA methylation dynamics of the human preimplantation embryo. Nature 2014, 511:611-615. 38. O’Leary T, Heindryckx B, Lierman S, van Bruggen D, Goeman JJ, Vandewoestyne M, Deforce D, de Sousa Lopes SMC, De Sutter P: Tracking the progression of the human inner cell mass during embryonic stem cell derivation. Nat Biotechnol 2012, 30:278282. 39. Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F,  Cassady JP, Muffat J, Carey BW, Jaenisch R: Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci USA 2010, 107:9222-9227. Study described first evidence for transgene dependent human ERK independent naı¨ve pluripotent cells. Current Opinion in Genetics & Development 2015, 34:35–45

44 Cell reprogramming, regeneration and repair

40. Yamaji M, Ueda J, Hayashi K, Ohta H, Yabuta Y: PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. Cell Stem Cell 2013, 12:368-382. 41. Chia N-Y, Chan Y-S, Feng B, Lu X, Orlov YL, Moreau D, Kumar P, Yang L, Jiang J, Lau M-S et al.: A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 2010, 468:316-320. 42. Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben Yosef D, Kalma Y, Viukov S, Maza I, Zviran A et al.: Derivation of novel human ground state naive pluripotent stem cells. Nature 2013, 504:282-286. Manuscript describes the first generation of genetically unmodified ERK independent human naı¨ve pluripotent cells form multiple cell sources, and the ability to generate developmentally advanced mouse–human crossspecies chimeric embryos that have undergone organogenesis. 43. Irie N, Weinberger L, Tang WWC, Kobayashi T, Viukov S,  Manor YS, Dietmann S, Hanna JH, Surani MA: SOX17 is a critical specifier of human primordial germ cell fate. Cell 2015, 160:253-268. This study showed that human PSCs expanded in 2i containing conditions have altered functional differences than conventional/primed ESCs, as demonstrated by heir enhanced ability to differentiate into PGCLCs (as seen with mouse naı¨ve cells). 44. Theunissen TW, Powell BE, Wang H, Mitalipova M, Faddah DA, Reddy J, Fan ZP, Maetzel D, Ganz K, Shi L et al.: Systematic identification of growth conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014, 15:471-487. 45. Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W et al.: Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 2014, 158:1254-1269. 46. Nichols J, Smith A: Pluripotency in the embryo and in culture. Cold Spring Harb Perspect Biol 2012, 4:a008128. 47. Shakiba N, White CA, Lipsitz YY, Yachie-Kinoshita A, Tonge PD, Hussein SMI, Puri MC, Elbaz J, Morrissey-Scoot J, Li M et al.: CD24 tracks divergent pluripotent states in mouse and human cells. Nat Commun 2015, 6:1-11.

preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 2013, 20:1131-1139. 56. Gkountela S, Zhang KX, Shafiq TA, Liao W-W, HarganCalvopin˜a J, Chen P-Y, Clark AT: DNA demethylation dynamics in the human prenatal germline. Cell 2015, 161:1425-1436. 57. Marks H, Kalkan T, Menafra R, Denissov S, Jones K,  Hofemeister H, Nichols J, Kranz A, Francis Stewart A, Smith A et al.: The transcriptional and epigenomic foundations of ground state pluripotency. Cell 2012, 149:590-604. This study uncovered defining differences between naı¨ve and primed pluripotency/PSCs relating to H3K27me3 deposition over developmental regulatory genes. 58. Ying Q-L, Nichols J, Chambers I, Smith A: BMP induction of id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003, 115:281-292. 59. Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, Smith ZD, Ziller M, Croft GF, Amoroso MW, Oakley DH et al.: Reference maps of human ES and iPS cell variation enable highthroughput characterization of pluripotent cell lines. Cell 2011, 144:439-452. 60. Osafune K, Caron L, Borowiak M, Martinez RJ, Fitz-Gerald CS,  Sato Y, Cowan CA, Chien KR, Melton DA: Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 2008, 26:313-315. This study carefully documented differentiation biases among human ES lines expanded under similar primed growth conditions. 61. Leitch HG, McEwen KR, Turp A, Encheva V, Carroll T, Grabole N, Mansfield W, Nashun B, Knezovich JG, Smith A et al.: Naive pluripotency is associated with global DNA hypomethylation. Nat Struct Mol Biol 2013, 20:311-316. 62. Krupalnik V, Hanna JH: Stem cells: the quest for the perfect reprogrammed cell. Nature 2014, 511:160-162. 63. Ma H, Morey R, O’Neil RC, He Y, Daughtry B, Schultz MD, Hariharan M, Nery JR, Castanon R, Sabatini K et al.: Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 2014, 511:177-183.

48. Barakat TS, Ghazvini M, de Hoon B, Li T, Eussen B, Douben H, van der Linden R, van der Stap N, Boter M, Laven JS et al.: Stable X chromosome reactivation in female human induced pluripotent stem cells. Stem Cell Reports 2015, 4:199-208.

64. Ohi Y, Qin H, Hong C, Blouin L, Polo JM, Guo T, Qi Z, Downey SL, Manos PD, Rossi DJ et al.: Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells. Nat Cell Biol 2011, 13:541-549.

49. Chen Y, Niu Y, Li Y, Ai Z, Kang Y, Shi H, Xiang Z, Yang Z, Tan T,  Si W et al.: Generation of cynomolgus monkey chimeric fetuses using embryonic stem cells. Cell Stem Cell 2015, 17:116-124. This study documents the first success to generate non human primate chimeric embryos with naı¨ve monkey ESCs expanded in naı¨ve (NHSM) conditions devised by Gafni et al. [42].

65. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LIR et al.: Epigenetic memory in induced pluripotent stem cells. Nature 2010, 467:285-290.

50. Chan Y-S, Go¨ke J, Ng J-H, Lu X, Gonzales KAU, Tan C-P, Tng WQ, Hong Z-Z, Lim Y-S, Ng H-H: Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 2013, 13:663-675. 51. Ware et al.: Derivation of naı¨ve human embryonic stem cells. PNAS 2014, 111:4484-4489. 52. Yang Y, Adachi K, Sheridan MA, Alexenko AP, Schust DJ, Schulz LC, Ezashi T, Roberts RM: Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure. PNAS 2015, 12:2337-2346. 53. Chen H, Aksoy IEN, Gonnot F, Osteil P, Aubry M, Hamela C, Rognard CE, Hochard A, Voisin S, Fontaine E et al.: Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency. Nat Commun 2015, 6:1-17. 54. Duggal G, Warrier S, Ghimire S, Broekaert D, Van der Jeught M, Lierman S, Deroo T, Peelman L, Van Soom A, Cornelissen R et al.: Alternative routes to induce naı¨ve pluripotency in human embryonic stem cells. Stem Cells 2015 http://dx.doi.org/ 10.1002/stem.2071. (Epub ahead of print). 55. Yan L, Yang M, Guo H, Yang L, Wu J, Li R, Liu P, Lian Y, Zheng X, Yan J et al.: Single-cell RNA-Seq profiling of human Current Opinion in Genetics & Development 2015, 34:35–45

66. James D, Noggle SA, Swigut T, Brivanlou AH: Contribution of human embryonic stem cells to mouse blastocysts. Dev Biol 2006, 295:90-102. 67. Fang R, Liu K, Zhao Y, Li H, Zhu D, Du Y, Xiang C, Li X, Liu H, Miao Z et al.: Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts. Cell Stem Cell 2014, 15:488-496. 68. Rivera-Pe´rez JA, Jones V, Tam PPL: Culture of Whole Mouse Embryos at Early Postimplantation to Organogenesis Stages: Developmental Staging and Methods. Elsevier Inc.; 2010:. (Chapter 11). 69. Hackett JA, Surani MA: Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 2014, 15:416-430. 70. Anderson PD, Nelson VR, Tesar PJ, Nadeau JH: Genetic factors on mouse chromosome 18 affecting susceptibility to testicular germ cell tumors and permissiveness to embryonic stem cell derivation. Cancer Res 2009, 69:91129117. 71. Rangarajan A, Hong SJ, Gifford A, Weinberg RA: Species- and cell type-specific requirements for cellular transformation. Cancer Cell 2004, 6:171-183. 72. Kameda T, Thomson JA: Human ERas gene has an upstream premature polyadenylation signal that results in a truncated, noncoding transcript. Stem Cells 2005, 23:1535-1540. www.sciencedirect.com

Human naı¨ve pluripotency Manor, Massarwa and Hanna 45

73. Boyer LA, Gifford DK, Melton DA, Jaenisch R, Young RA: Core  transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122:947-956. See annotation below Ref. [74]. 74. Loh Y-H, Wu Q, Chew J-L, Vega VB, Zhang W, Chen X, Bourque G,  George J, Leong B, Liu J et al.: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006, 38:431-440.

www.sciencedirect.com

Two studies [73,74] that described the foundation of pluripotent transcriptional regulatory circuitry and its interconnectivity in human primed and mouse naı¨ve ESCs. 75. Bradley RK, Li X-Y, Trapnell C, Davidson S, Pachter L, Chu HC, Tonkin LA, Biggin MD, Eisen MB: Binding site turnover produces pervasive quantitative changes in transcription factor binding between closely related Drosophila species. PLoS Biol 2010, 8:e1000343.

Current Opinion in Genetics & Development 2015, 34:35–45