Human Reproduction Update Advance Access published June 18, 2015 Human Reproduction Update, Vol.0, No.0 pp. 1– 11, 2015 doi:10.1093/humupd/dmv028

The post-inner cell mass intermediate: implications for stem cell biology and assisted reproductive technology Margot Van der Jeught 1, Thomas O’Leary 1,3, Galbha Duggal 1,4, Petra De Sutter 1, Susana Chuva de Sousa Lopes 1,2†, and Bjo¨rn Heindryckx1,*†

*Correspondence address. E-mail: [email protected]

Submitted on December 31, 2014; resubmitted on April 13, 2015; accepted on June 1, 2015

table of contents

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Introduction Methods The human post-ICM intermediate: the key intermediate from ICM to primed hESCs How to increase the rate of epiblast and PICMI formation? How to recognize the PICMI? What patient characteristics influence PICMI formation? Results The mouse PICMI: the key intermediate from ICM to naı¨ve mESCs From naı¨ve mESCs to primed EpiSCs and back again From primed hESCs to naı¨ve hESCs: redefining human pluripotency Human regenerative medicine: which pluripotency state should be used? Application of human pluripotent stem cells towards stem cell-derived gametes Conclusion

background: Until recently, the temporal events that precede the generation of pluripotent embryonic stem cells (ESCs) and their equivalence with specific developmental stages in vivo was poorly understood. Our group has discovered the existence of a transient epiblast-like structure, coined the post-inner cell mass (ICM) intermediate or PICMI, that emerges before human ESC (hESCs) are established, which supports their primed nature (i.e. already showing some predispositions towards certain cell types) of pluripotency. methods: The PICMI results from the progressive epithelialization of the ICM and it expresses a mixture of early and late epiblast markers, as well as some primordial germ cell markers. The PICMI is a closer progenitor of hESCs than the ICM and it can be seen as the first proof of why all existing hESCs, until recently, display a primed state of pluripotency. results: Even though the pluripotent characteristics of ESCs differ from mouse (naı¨ve) to human (primed), it has recently been shown in mice that a similar process of self-organization at the transition from ICM to (naı¨ve) mouse ESCs (mESCs) transforms the amorphous ICM into a rosette of polarized epiblast cells, a mouse PICMI. The transient PICMI stage is therefore at the origin of both mESCs and hESCs. In addition, several groups have now reported the conversion from primed to the naı¨ve (mESCs-like) hESCs, broadening the pluripotency spectrum and opening new opportunities for the use of pluripotent stem cells. †

These authors contributed equally to this work.

& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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1 Ghent Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, Ghent 9000, Belgium 2Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, The Netherlands 3 Present address: Coastal Fertility Specialists, 1375 Hospital Drive, Mt Pleasant, SC 29464, USA 4 Present address: Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK

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conclusions: In this review, we discuss the recent discoveries of mouse and human transient states from ICM to ESCs and their relation towards the state of pluripotency in the eventual stem cells, being naı¨ve or primed. We will now further investigate how these intermediate and/or different pluripotent stages may impact the use of human stem cells in regenerative medicine and assisted reproductive technology. Key words: post-inner cell mass intermediate / pluripotent states / embryonic stem cells / regenerative medicine

Introduction

Methods The human post-ICM intermediate: the key intermediate from ICM to primed hESCs Although human embryonic stem cells (hESCs) are derived from preimplantation embryos (Thomson et al. 1998), their characteristics are more similar

How to increase the rate of epiblast and PICMI formation? Small molecules, both agonists and antagonists of specific signalling pathways, have been shown to influence lineage fate decisions during early embryo development in mice and humans (Nichols and Smith, 2009; Yamanaka et al., 2010). In mice, the culture of 8-cell stage embryos for 3 days in 2i containing medium (PD0325901 and CHIR99021) directs the ICM towards a state in which all cells become NANOG positive and GATA4 (Gata-binding protein 4) negative, thus inhibiting hypoblast formation (Nichols and Smith, 2009; Yamanaka et al., 2010). In humans, the culture of 8-cell stage (Day 3) embryos up to Day 6, to the blastocyst stage, in 2i medium significantly increased the number of NANOG-positive cells in the ICMs of good quality blastocysts, however, it did not affect the number of GATA6- (Gatabinding protein 6) positive hypoblast cells (Van der Jeught et al., 2013) (Fig. 2). In bovine, embryo culture from Day 5 to Day 8 in the presence of 2i resulted in a proportional increase in the number of NANOG-positive cells and a decrease in the number of GATA6-positive hypoblast cells (Kuijk et al., 2012). The inhibition of transforming growth factor (TGF)b/ActivinA (ActA) receptors, and therefore the inhibition of TGFb signalling, by the small molecule SB431542 (SB) is known to have a positive effect on the self-renewal of mESC (Maherali and Hochedlinger, 2009; Galvin et al., 2010; Hassani et al., 2012). Treatment of human 8-cell stage (Day 3) embryos with SB for 3 more days, to the blastocyst stage, significantly increased the number of NANOG-positive epiblast cells in the human ICMs in both good and poor quality blastocysts, while the number of GATA6-positive hypoblast cells remained unchanged (Van der Jeught et al., 2013). Regulating the fibroblast growth factor (FGF), wingless-related MMTV integration site (WNT) or TGFb signalling pathways for 3 days between the 8-cell stage embryo (Day 3) and the blastocyst stage (Day 6) did not seem to affect hypoblast formation, but significantly influenced the number of epiblast cells. This suggests that other signalling pathways may regulate hypoblast formation during human preimplantation development. It was tested whether the increase in the number of epiblast cells in the blastocyst

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Pluripotency is a cellular state characterized by the capacity to differentiate in vivo or in vitro to derivatives of the three germ layers, ectoderm, mesoderm and endoderm. The blastomeres of the preimplantation embryo lose their totipotency after the first differentiation event when the cells are directed to either the trophectoderm (TE) or inner cell mass (ICM). The current idea is that cells that comprise the ICM have lost the ability to generate TE cells (and vice versa) and are considered pluripotent instead of totipotent. However, it has been recently shown that isolated TE cells from Day 5 blastocysts are able to develop into blastocysts with ICM cells expressing NANOG (Nanog homeobox), and most TE cells can integrate within the ICM when repositioned in the centre of the blastocyst (De Paepe et al., 2013). As development progresses through peri- and post-implantation stages, the cells of the ICM continue to differentiate into the embryonic part (ectoderm, mesoderm, endoderm and germline), and contribute as well to the extra-embryonic part of the embryo (placenta, amnion, chorion and umbilical cord). Alternatively, in vitro cultured ICM cells can generate self-renewing and pluripotent embryonic stem cells (ESCs) (Evans and Kaufman, 1981; Martin, 1981). Interestingly, despite the shared traits of self-renewal and pluripotency, stem cells can differ vastly in transcriptional profile, epigenetic profile, morphology and culture requirements. It is becoming clear that the differences observed between ESC types is the result of differing developmental stages of the ICM precursor cells that give rise to the stem cells. In mice, different types of self-renewing and pluripotent stem cell populations can be generated in culture from post-implantation embryonic day (E) 5.5–7.5 mouse embryos (Brons et al. 2007; Tesar et al. 2007; Bao et al. 2009), the best characterized being the epiblast stem cells, mEpiSCs. Despite the shared traits of self-renewal and pluripotency between mouse ESCs (mESCs) and mEpiSCs, including transcriptional profile, epigenetic profile, morphology and culture conditions, there are vast differences between these two different stem cell types. It hasthus become clear that pluripotency is not a fixed state, but exists in at least two distinct forms (Nichols and Smith 2009, 2011). Stem cells with ICM-like mESC characteristics are termed ‘naı¨ve’ and stem cells with mEpiSCs characteristics are designated ‘primed’, already showing some predisposition towards certain cell types. It has been shown that the derivation of naı¨ve mESCs from non-permissive mice strains is dependent on the small molecules PD0325901 and CHIR99021 (known as the 2 inhibitor or 2i condition), inhibiting the mitogen-activated protein kinase (Erk1/2) and glycogen synthase kinase 3b (GSK3b) pathway, respectively (Nichols and Smith 2009; Ying et al. 2008).

to post-implantation mEpiSCs (Brons et al., 2007; Tesar et al., 2007). Therefore, speculation arose that the human ICM progresses in vitro through an epiblast-like state before hESCs emerge in culture. Detailed analysis of the morphological changes of the human ICM during the hESC derivation process indeed revealed the formation of a transient structure that emerged 2 – 3 days after blastocyst plating (Fig. 1). Importantly, the specific development of this transient epiblast-like structure or post-ICM intermediate (PICMI) in culture proved to be predictive for the derivation of hESCs (O’Leary et al., 2012b, 2013). It should be noted that the PICMI is not a stable pluripotent state, but a transient structure that is formed just before the emergence of hESCs. Further investigations on the characteristics of the PICMI, an obligatory hESC-precursor, will help to get a more clear understanding of the human pluripotent states. To date, the PICMI formation rate is rather low, or mostly not reported on. About 40% of plated blastocysts showed signs of initial ICM organization, while only 21.3% formed a PICMI (O’Leary et al., 2013). Optimizing culture conditions, by inhibition or activation of key signalling pathways important for pluripotency, could help in increasing this efficiency.

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same efficiency as embryos that did not receive such treatment (Van der Jeught et al., 2013, 2014). Even though an enhanced number of epiblast cells did not lead to a higher derivation efficiency of novel hESC lines using a limited number of available embryos, an upgraded combination of small molecules should be capable of enhancing PICMI formation rates as well as subsequent hESC derivation. Fan et al. described a higher blastocyst formation and quality by adding both human recombinant leukemia inhibitory factor (LIF) and human basic FGF (bFGF). This combination increased the derivation efficiency of hESCs from poor quality embryos (Fan et al., 2010). Similarly, it has been shown that treatment with insulin increases the epiblast cell number in mice, leading to a higher mESC derivation efficiency (Campbell et al., 2013). Hence, it was suggested that insulin might improve the derivation of hESC from poor quality embryos (PQEs) as well.

How to recognize the PICMI?

Figure 2 The number of NANOG-positive epiblast cells can be increased using small molecules. (A) Human embryos were cultured from the 8-cell stage (Day 3) in the presence of regulators of the fibroblast growth factor (FGF) and wingless-related MMTV integration site (WNT) or transforming growth factor b (TGFb) signalling pathways, for a period of 3 days. (B) After 3 days of culture in medium containing 2i, the (good quality) blastocyst embryos showed a significantly increased number of NANOG-positive epiblast cells (green) in the inner call mass (ICM), while the number of GATA6-positive hypoblast cells (red) was unaffected. Nuclei of all cells were stained using 4′ ,6-diamidino-2-phenylindole (DAPI (blue)). Scale bars are 50 mm.

embryo increased the derivation efficiency of hESCs. Both culture with 2i and SB431542 of 8-cell stage (Day 3) human embryos until blastocyst stage (Day 6) allowed PICMI formation and subsequent hESC derivation, however, at

The PICMI is an epithelialized structure that can grow either flat or ball like and is surrounded by a layer of endoderm separated by a basement membrane. A healthy PICMI grows significantly during the following days (from Day 2 until Day 7) after plating (Fig. 3A). Mechanical subtraction of the entire PICMI structure and transfer onto a new layer of feeders results in flattening of the PICMI and the appearance of hESC-like outgrowths after a few days (O’Leary et al., 2013). The PICMI displays a unique transcriptional profile of a mix of early and late epiblast markers as well as early germ cell markers (O’Leary et al., 2012b). It is not known yet whether individual PICMI cells fluctuate between early and late epiblast stages, are co-expressing both types of marker genes, or if the PICMI cells contain a mixed population of cells at both early and late epiblast stages. O’Leary et al. (2012b) reported a trimethylated state of histone 3 on lysine 27 (H3K27me3) in all the female PICMIs analysed, indicating X chromosome inactivation (Xi), a pattern that is similar to the one observed in mouse epiblast cells (Nichols and Smith, 2009, 2011). However, the analysis of X chromosome inactivation status after subsequent hESC derivation revealed that some lines showed reactivation of the inactivated X chromosome (XaXa), which was attributed to the hypoxic conditions the hESCs

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Figure 1 Derivation of human embryonic stem cells (hESCs). Human blastocysts (Day 6 of development) are plated on mouse embryonic fibroblasts (MEFs) and cultured for 1 week in medium containing Knockout replacement serum (KOSR) and basic fibroblast growth factor (bFGF). During this period, a transient structure emerges in culture, the post-inner cell mass intermediate (PICMI). If 6 – 7 days after the plating of the blastocyst a defined PICMI is observed, this can be mechanically excised and placed onto fresh MEFs. After 2 – 4 days, an initial hESC outgrow from the PICMI is clearly visible and within 5 – 7 days it can be mechanically passaged. The newly derived hESC line can undergo unlimited propagation.

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were derived in, whereas other lines showed a mixed phenotype (with and without H3K27me3 Xi-indicative nuclear spots) (O’Leary et al., 2012b).

What patient characteristics influence PICMI formation? During assisted reproductive technology (ART), the patient undergoes controlled hormonal ovarian hyperstimulation, aimed towards retrieving a number of oocytes that is high enough to create a cohort of embryos, from which the ones with highest implantation potential are selected to be transferred to the patient directly or for cryopreservation to be transferred at a later stage (Alpha Scientists in Reproductive and Embryology, 2011; Willis et al., 2011). The embryos with characteristics associated with low implantation potential (such as high fragmentation (≥25%) on Day 2 or Day 3 of development, multinucleated blastomeres on Day 2, delayed development and/or abnormal fertilization (0, 1 or ≥3 pronucleii) on Day 1), termed PQEs, are typically discarded (O’Leary et al., 2011, 2013). However, these PQEs are still very useful for the derivation of hESCs, which can give rise to genomically stable hESC lines following extensive passaging (Raya et al., 2008; Venu et al., 2010; O’Leary et al., 2011). Moreover, it was reported that blastocysts originating from PQEs are as efficient in generating hESCs as the ones originating from good quality embryos (Strom et al., 2010). This could be explained by the presence of good quality ICMs in blastocysts with compromised implantation potential due to an inferior TE quality (della Ragione et al., 2007). In addition to the correlation between embryo morphology and blastocyst implantation competency and hESC derivation potential (O’Leary et al., 2011), the patient-specific characteristics (maternal age, responsiveness to ovarian stimulation etc.) have also been shown to be of

importance (Broekmans et al., 2007; van Loendersloot et al., 2010). O’Leary et al. reported a reduced developmental potential and ICM quality in blastocysts originating from PQEs from older women (.37 years) or from cycles that did not result in pregnancy (O’Leary et al., 2012a). Moreover, the capacity of a good quality ICM blastocyst to successfully generate novel hESCs was also dependent on the age of the mother (O’Leary et al., 2012a). In fact, good quality ICM blastocysts that originated from PQEs from older women (.37 years) were unable to give rise to hESCs (O’Leary et al., 2012a). As hESC generation is dependent on the successful formation of a PICMI (O’Leary et al., 2012b), the selective plating of embryos coming from patients with parameters shown to be beneficial for hESC derivation will further aid in increasing the efficiency of PICMI formation and subsequent hESC derivation.

Results The mouse PICMI: the key intermediate from ICM to naı¨ve mESCs In the mouse, the transcriptional profile of single ICM cells changes during naı¨ve ESC derivation in media containing serum and LIF (Tang et al., 2010). Moreover, naı¨ve mESCs exhibit a high degree of heterogeneity, clearly marked by the metastable expression of important pluripotency markers, including Stella/Dppa3 (Developmental pluripotency-associated protein 3) or Nanog (Chambers et al., 2007; Hayashi et al., 2008). It remains unclear whether the potency of each single ICM cell to generate mESCs, at least those ICM cells that express Nanog, is in fact similar.

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Figure 3 Different states of pluripotency in hESCs. (A) Plating human blastocysts onto a feeder layer of MEFs can give rise to post-ICM intermediates (PICMIs, black arrows) after 4 – 6 days of culture. These PICMIs allow for further hESC derivation in the primed state of pluripotency (B), which can be converted towards the naı¨ve state later on (C). Scale bars are 100 mm.

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Role of post-inner cell mass intermediate in pluripotency

Figure 4 PICMI: the mother of all stem cells in mouse and human. Cells of the ICM or epiblast (EPI), from both mouse and human, seem to undergo a transient stage, the PICMI, on the way to becoming ESCs, both in the naı¨ve and primed state.

of the culture conditions used (Najm et al., 2011; Boroviak et al., 2014) and, as in human, the mouse PICMI is necessary and sufficient to derive new pluripotent lines. This view would suggest that given the appropriate culture conditions it would be possible, as in mouse, to generate naı¨ve hESC from the human PICMI (Fig. 4).

From naı¨ve mESCs to primed EpiSCs and back again With the discovery and derivation of mouse EpiSCs (Brons et al., 2007; Tesar et al., 2007) an important step was taken in the stem cell field to link human and mouse pluripotency. The isolation of EpiSCs, that were derived and cultured under similar culture conditions as hESCs, sharing some of the morphological properties and molecular/cellular markers, resulted in a strong unification of the two animal models used in the stem cell field. Moreover, it also broadens the view of the ‘pluripotency state’ not as a single state but as a gradient of states (Kuijk et al., 2011). In parallel, this view of different ‘grades’ of pluripotent states also emerged from the observation that mESCs were not a homogeneous population of cells. Instead, mESCs are in a metastable state transiting between more naı¨ve pluripotency to more primed pluripotency states in culture (Chambers et al., 2007; Hayashi et al., 2008). There is still controversy about the biological significance of such heterogeneity in culture and some argue that these transient states may reflect intrinsic variety that allows the mESCs to respond differently to differentiation cues (‘lineage priming’) (Silva and Smith, 2008; Abranches et al., 2014). Interestingly, this metastable heterogeneity, at least in mice, seems to exist also at the level of DNA methylation. The DNA methylation landscape seems to be continuously remodelled by Tet and Dnmt enzymes, transiently ‘priming’ the cells for cell fate decisions (Lee et al., 2014a). A novel field of investigating the transcriptional profile and DNA methylation of cells to a single-cell resolution will hopefully shed more light on the significance of heterogeneity (Grun et al., 2014; Smallwood et al., 2014). It has become possible to ‘push’ mESCs from the naı¨ve state to a more primed state, as this is the ‘natural direction’ of events during development, towards a more differentiated state (reviewed in Festuccia et al., 2013). In fact, mESCs seem to need Wnt signalling activation to prevent their differentiation towards EpiSCs (ten Berge et al., 2011). The conversion from mEpiSCs to mESCs has been achieved by several

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In this respect, Boroviak et al. (2014) found that almost all E4.5 epiblast could become ESCs. To generate mESCs, the ICM cells of mouse blastocysts (or of blastocyst outgrowths) are dissociated and single cells (and/or small cell clumps) are plated so that after several days of culture self-renewing colonies emerge for subsequent single cell passaging. Any precursors of EpiSCs present in those cultures would probably differentiate and disappear naturally as EpiSCs do not survive well after single cell passaging. Avoiding complete single cell dissociation and using manual dissociation instead of trypsinization, it was shown indeed that mouse ICMs could be cultured to give rise to both mESCs and EpiSCs (Najm et al., 2011). During the preimplantation period of mouse development, two important lineage segregation steps lead to the formation of the epiblast (reviewed in Kuijk et al., 2011). The first lineage segregation to occur is the segregation of TE cells and ICM cells. At this stage, the TE cells form the surface of the embryo that contacts directly the zona pellucida and express the marker Cdx2; whereas ICM cells are localized inside the embryo and are Oct4 (Octamer-binding transcription factor 4) positive. The second segregation step involves the segregation of Gata6-positive primitive endoderm (PrE) cells and of Nanog-positive epiblast (EPI) cells that emerge from the (Oct4 positive) ICM cells of the expanding blastocyst. At least in mice, these two cell lineages (PrE and EPI) segregate in a scattered ‘salt and pepper’ fashion and rearrange themselves so that the PrE cells locate to the surface of the ICM interfacing with the blastocoelic cavity, whereas the EPI cells group together between the TE and PrE cells (reviewed in Kuijk et al., 2011). To use the term ‘epiblast’ at the expanded blastocyst stage can be misleading. In the mouse, epiblast is usually used for the polarized columnar or epithelialized cells of the ‘egg cylinder’ (or ‘epiblast’) of the implanting embryo at E5.0–6.5, instead of a subpopulation of the ICM in the preimplantation blastocyst. Interestingly, the polarization of the EPI cells in the implanting embryo is accompanied by a process of cavitation that leads to the formation of the proamniotic cavity. This cellular rearrangement in the embryo, during early implantation, does not seem to involve apoptosis, but instead involves the activation of signalling pathways by the PrE and its derivative visceral endoderm, including the bone morphogenetic protein (BMP) signalling pathway (Coucouvanis and Martin, 1999; Bedzhov and ZernickaGoetz, 2014). Recently, it has also been proposed that the transmembrane receptor b1 integrin expressed by the basal domain of the EPI cells plays a role driving epithelialization (Bedzhov and Zernicka-Goetz, 2014). In agreement, b1 integrin deficient mouse embryos are able to implant in the uterus, but fail to form the epiblast (Fassler and Meyer, 1995; Stephens et al., 1995). This model proposes that cells of the extraembryonic lineages secrete extracellular matrix proteins that form a basal membrane enveloping the EPI, and thereby creates a niche for its maturation during implantation (Bedzhov and Zernicka-Goetz, 2014). The transition that the EPI cells undergo during implantation is very much reminiscent of the PICMI formation in vitro during the derivation of hESCs from human blastocysts (O’Leary et al., 2012b), where selforganization of the ICM is followed by epithelialization in culture. In agreement, it has been shown that naı¨ve mESCs are not derived from the early ICM cells, but originate from late ICM cells after EPI specification (Boroviak et al., 2014). During the initial steps of mESC derivation, those specified EPI cells from the ICM may also go through a transient postimplantation ICM intermediate state (or PICMI) of self-organization as in humans (Fig. 3A). This transient stage with unique properties in vitro seems to be the origin of both the naı¨ve mESCs and the EpiSCs depending

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From primed hESCs to naı¨ve hESCs: redefining human pluripotency Until recently, hESCs were standardly derived in ‘primed’ culture conditions (Fig. 3B and C). As diapaused mouse blastocysts improve the derivation success of mESCs (Brook and Gardner, 1997; Nichols and Smith, 2012), it was thought for a long time that the absence of diapause in human embryo development was one of the possible causes of the failure to derive naı¨ve hESCs. However, for clinical applications, the derivation of hESCs in the naı¨ve state of pluripotency is of great interest, as the derivation of this type of cells will open new opportunities for patientspecific, disease-relevant research. There are three possible routes to achieve naı¨ve pluripotent state in humans: (1) directly from preimplantation embryos, (2) by reprogramming somatic cells and (3) by conversion of existing primed hESCs. To date, several different groups have reported various culture conditions needed to sustain the culture and maintenance of naı¨ve hESCs (Table I; Fig. 3B and C; Duggal et al., 2015b). The first successful efforts to convert primed hESCs to mESC-like naı¨ve hESCs were made in 2010 by ectopic expression of OCT4, KLF (Kru¨ppel-like factor)4 and KLF2 combined with the addition of LIF and inhibitors of GSK3b and ERK1/2 in hESCs (Hanna et al., 2010). Moreover in that study, Forskolin, a protein kinase A pathway stimulator that induces expression of KLF4 and KLF2, was shown to transiently substitute for the need for ectopic transgene expression. Thereafter, it was reported that modifications to the culture conditions were sufficient for a rapid conversion of primed hESCs to mESC-like hESCs (Gu et al., 2012). The combination of 2i, ascorbic acid and SB421542 (an inhibitor of the TGFb/ActA pathway), bFGF and LIF (Table I) seemed to eliminate the need for ectopic transgene expression. The converted lines were called mESC-like hESCs (mhESCs) and displayed several characteristics similar to those of mESCs. However, mhESCs could be maintained only for several passages. In line with this study, two other groups reported that the combined action of 2i and bFGF was sufficient to maintain naı¨ve-like human stem cells in the presence (Valamehr et al., 2014) or absence (Ware et al., 2014) of human LIF (hLIF). The need for bFGF may reflect a species-specific difference between the naı¨ve state in mouse and human ESCs. Valamehr et al. generated naı¨ve state pluripotent stem cells from somatic cells by episomal reprogramming (Oct4, Sox2 (SRY (sex

determining region Y)-box 2), SV40LT (SV40 large T) in ‘fate reprogramming medium’ for enhanced non-integrative cellular reprogramming of human induced pluripotent stem cells (hiPSCs), not hESCs, followed by ‘fate maintenance medium’ (FMM) culture for long-term maintenance. The FMM contained 2i, bFGF and hLIF, in combination with Rho-associated protein kinase (ROCK) inhibition (ROCKi) (Table I). ROCKi is known to promote cell attachment, survival and proliferation of ESCs (Watanabe et al. 2007). Ware et al. converted primed hESCs to naı¨ve hESCs by reverse toggling in medium containing the histone deacetylase inhibitors sodium butyrate and suberoylanilide, followed by culture in 2i and bFGF (Table I). In a more systematic approach to convert primed hESCs/hiPSCs to naı¨ve hESCs/hiPSCs, the addition of different combinations of 16 inhibitors and growth factors to the culture medium was tested (Gafni et al., 2013). This resulted in a chemically defined culture medium, termed NHSM (naı¨ve human stem cell medium), composed of PD0325901 and CHIR99021 (2i), hLIF, SB20 (p38 inhibitor), SP (JNK inhibitor), bFGF and TGFb1 (Table I), enabling both the conversion and for the first time also derivation of karyotypically normal naı¨ve hESC/hiPSC lines. In a different study (Chan et al., 2013), 11 different compounds were screened for enhanced expression of NANOG in mTeSR1, medium that contains high levels of bFGF and TGFb/ActA, plus the addition of hLIF, 2i and dorsomorphin (BMP inhibitor) (3i/L) (Table I). Using 3i/L resulted in a higher expression of genes expressed during human preimplantation , including KLF4, DPPA3 and TBX3 (T-box transcription factor TBX3) (Chan et al., 2013). Theunissen et al. used an iterative chemical screening, in hESCs overexpressing NANOG and KLF2, and identified a combination of five kinase inhibitors: inhibitors of b-RAF, LKC/SRC and ROCK pathways and the 2i that in combination with hLIF, bFGF and ActA (5i/L/A), converted primed to the naı¨ve state (Theunissen et al., 2014). More recently, it has been reported that short-term over expression of NANOG and KLF2 is sufficient to ‘reboot’ other components of the pluripotency network, and to reset primed hESCs towards a naı¨ve pluripotent state (Takashima et al., 2014). Remarkably, the resulting (reset) hESCs could be cultured in defined medium without serum products or growth factors. It was shown that the ‘reset’ pluripotent state is characterized by the same transcription factor circuitry and DNA methylation pattern as (naı¨ve) mESC. Moreover, the metabolism of these (reset) hESCs was altered, resulting in an increased mitochondrial respiration. To date the direct derivation to naı¨ve hESCs from preimplantation human embryos has been achieved with very low efficiency: 1 naı¨ve hESC line out of 128 blastocysts (Ware et al., 2014); 4 naı¨ve hESC lines out of an unstated number of blastocysts (Gafni et al., 2013); and an unknown number of lines from an unstated number of blastocysts (Theunissen et al., 2014). By culturing the ICM to PICMI in the most suitable ‘naı¨ve medium’ or taking the PICMI as the starting point to culture further in naı¨ve conditions may prove a more efficient way to obtain gold standard naı¨ve hESCs instead of the primed state in the future. Concluding, the growing number of studies reporting new culture conditions, successful for the conversion from primed to naı¨ve hESCs, implies that multiple routes can lead to the induction of naı¨ve pluripotency in hESCs, both at molecular and epigenetic level. However, as the factors and inhibitors are acting on different signalling pathways, all of the converted naı¨ve hESCs probably represent intermediate stages between the naı¨ve and primed state of pluripotency. Remarkably, the golden combination of 2i together with hLIF and/or bFGF seems to be indispensable for promoting the naı¨ve state in hESCs.

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research groups by adapting the culture conditions and overexpressing several transcription factors (reviewed in Festuccia et al., 2013). Differences between these two pluripotency states (naı¨ve and primed) in mouse, in contrast to human, are relatively straight forward to assess in female lines, because mESCs are characterized by having two active XX chromosomes while mEpiSCs undergo X chromosome inactivation. The appearance of the silent X is easily recognizable by the characteristic perinuclear spot, corresponding to the inactive X chromosome (Nichols and Smith 2009, 2011). In addition, the promoter sequences in mEpiSCs are in general more methylated than promoter sequences in mESCs (Veillard et al., 2014). Recently, it was demonstrated that mESCs and mEpiSCs use different enhancers to regulate transcription (Buecker et al., 2014; Factor et al., 2014). One of the best-studied examples is the differential usage (regulated by DNA methylation) of the distal and proximal enhancer to regulate the expression of Oct4 in mESCs (as well as in the ICM and primordial germ cells) and mEpiSCs (and in the egg cylinder or epiblast, respectively) (Yeom et al., 1996; Hattori et al., 2004).

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Treatment

Hanna et al. (2010)a

Gu et al. (2012)b

Gafni et al. (2013)c

Chan et al. (2013)d

Ware et al. (2014)e

Valamehr et al. (2014)f

Theunissen et al. (2014)g

Takashima et al. (2014)h

Duggal et al. (2015b)i

.......................................................................................................................................................................................................................................................... MEKi

×

×

×

×

×

×

×

×

×

GSK3i

×

×

×

×

×

×

×

×

×

JNKi

×

P38i

×

PKCi

×

ROCKi

×

× ×

×

×

×

×

BMPi TGFbi

×

Ascorbic acid

×

× × ×

BRAFi

×

SRCi PKA agonist

×

Transgene overexpression

OCT4, KLF2/4

× NANOG, KLF2 ×

bFGF TGFb/ActA

×

×

NANOG, KLF2

×

TGFb

× ActA

hLIF

×

×

×

×

×

×

×

×

Base medium

N2B27

N2B27 + 20% KOSR

DMEM + Albumax + N2

TeSR1

DMEM/F12 + 20% KOSR

DMEM/F12 + 20% KOSR

N2B27

N2B27

KODMEM + 20% KOSR

Oxygen levels

20%

20%

20%

?

5%

20%

5%

5%

5%

Conversion hESCs

×

×

×

×

×

×

×

×

×

×

×

Derivation hESCs

Role of post-inner cell mass intermediate in pluripotency

Table I Different culture media used to generate human naive pluripotent stem cells.

Conversion hiPSCs

×

×

×

×

×

Derivation hiPSCs

×

×

×

×

×

MEKi, MAPK/Erk kinase inhibitor; GSK3i, Glycogen synthase kinase 3 inhibitor; JNKi, Jun N-terminal kinase inhibitor; p38i, p38 mitogen-activated kinase inhibitor; PKCi, Protein kinase C inhibitor; ROCKi, Rho-associated coiled-coil kinase inhibitor; BMPi, Bone morphogenetic protein inhibitor; TGFbi, Transforming growth factorb inhibitor; BRAFi, B-Raf inhibitor; SRCi, Src (sarcoma) inhibitor; PKA, Protein kinase A; bFGF, basic fibroblast growth factor; ActA, ActivinA; hLIF, human leukemia inhibitory factor; OCT4, Octamer-binding transcription factor 4; KLF2/4, Kru¨ppel-like factor 2/4; NANOG, Nanog homeobox. a First conversion of hESC towards naive, using hLIF (20 mg/l), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM), and ectopic gene expression of OCT4 and KLF2/4. The PKA (protein kinase A) agonist forskolin (10 mM) can substitute for the ectopic transgene expression (in red). b Mouse ESC-like hESC (mhESC) medium: hLIF (2000 U/ml), bFGF (16 ng/ml), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM), TGFbi (SB431542 2 mM), ascorbic acid (50 ng/ml). c Naive human stem cell medium (NHSM): hLIF (20 ng/ml), TFGb (1 ng/ml), bFGF (8 ng/ml), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM), JNKi (SP600125 10 mM), p38i (SB203580 10 mM). Optimizing components (in red) are PKCi (Go¨6983 5 mM) and ROCKi (Y27632 5 mM). d 3i/LIF medium : hLIF (10 ng/ml), MEKi (PD0325901 1 mM), GSK3i (BIO 2 mM), BMPi (dorsomorphin 2 mM). Optimizing component (in red) is ROCKi (thiazovivin 1 mM). e Reverse toggle protocol for conversion: pretreatment with HDACi (0.1 mM sodium butyrate and 50 nM SAHA), culture with bFGF (10 ng/ml), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM); for direct derivation: bFGF (10 ng/ml), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM). f Feeder-free two-step protocol (1) fate reprogramming medium (FRM): bFGF (100 ng/ml), hLIF (10 ng/ml), MEKi (PD0325901 0.4 mM), GSK3i (CHIR99021 1 mM), ROCKi (thiazovivin 5 mM) and TGFbi (SB431542 2 mM); (2) fate maintenance medium (FMM): FRM without SB431542. g 5i/LA medium : ectopic gene expression of NANOG and KLF2, followed by pretreatment of cells with ROCKi (Y27632 10 mM) and by trysinization and culture in 5i/LA (MEKi (PD0325901 1 mM), GSK3i (CHIR99021 0.3 –3 mM), BRAFi (SB590885 0.5 mM), SRCi (5WH-4-023 1 mM), ROCKi (Y27632 10 mM), hLIF (20 mg/l) and ActA (20 ng/ml). h Two-step protocol: pretreatment of cells with ROCKi (Y27632), followed by (1) ectopic gene expression NANOG and KLF2 and culture in 2iL/t2iL: hLIF (made in house), MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM in 2iL or 1 mM in t2iL); (2) t2iL+Go¨: t2iL with PKCi (Go¨6983 5 mM). i Induction of naive pluripotency in hESC, using MEKi (PD0325901 1 mM), GSK3i (CHIR99021 3 mM), ascorbic acid (50 ng/ml), PKA agonist (forskolin 10 mM), bFGF (12 ng/ml) and hLIF (1000 U/ml).

7

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8

Human regenerative medicine: which pluripotency state should be used?

compromised in the capacity to undergo efficient differentiation towards a mature phenotype (Honda et al., 2013). Although the naı¨ve hESCs have now been established, in-depth investigation is required to determine their homogenous lineage-specific differentiation potential. Most probably, naı¨ve stem cells would first have to be differentiated in a later developmental state mimicking the natural course of embryogenesis, in contrast to primed stem cells, which are already poised for differentiation (Mascetti and Pedersen, 2014). Still, apart from the in vivo chimera formation tests there is little evidence, especially in human, that the naı¨ve pluripotent stem cells are more competent for differentiation and more promising to be used for regenerative medicine purposes than their primed counterparts (Pera, 2014).

Application of human pluripotent stem cells towards stem cell-derived gametes Based on germ cell differentiation experiments, the answer to what may be the ultimate pluripotent state to use in regenerative medicine remains uncertain. In mouse, primed EpiSCs possess an increased propensity to give rise to PGCs-like cells in vitro. This was proven by differentiating transgenic lines with either a Blimp1 or Stella reporter construct in the presence of BMP4 (Hayashi and Surani, 2009). In any case, despite the predisposition, the efficiency of mEpiSCs to form PGC-like cells remains low, as the majority of the EpiSCs has lost competency towards a PGC fate (Hayashi and Surani, 2009). To circumvent this hurdle, naı¨ve mESCs were differentiated towards an intermediate epiblast-like cells (EpiLCs) stage in the presence of ActA. This transient stage resembled the early pluripotent epiblast that gives rise to PGCs in vivo (Hayashi et al., 2011, 2012). The in vitro differentiation of these mEpiLCs to PGC-like cells was more efficient, and in two landmark studies in mice functional oocytes and sperm could be obtained (Hayashi et al., 2011, 2012). This remarkable achievement was thus based on the careful choice of the starting population of stem cells. Not much is known about the best state of pluripotency for germ cell formation from human pluripotent stem cells. Primed state hESCs have been extensively used to generate precursor gametes, however, derivatives have not reached beyond the premeiotic stage without the need for genetic manipulation (Handel et al., 2014; Sun et al., 2014). Recently, it has been shown that naı¨ve-like hESCs also exhibit efficient differentiation towards an early germ cell stage (premeiotic) (Irie et al., 2015). Moreover, it was shown that the endodermal lineage marker SOX17 could contribute to specify the germ cell fate in humans (Irie et al., 2015), whereas in mice it has also been suggested that the endoderm plays a critical role in inducing germ cell fate (de Sousa Lopes et al., 2004). With the identification of the PICMI and the generation of novel culture conditions facilitating naı¨ve pluripotency in hESCs, it has been possible to derive hESC lines predisposed towards the germ cell lineage by exposing the PICMI to medium containing ActA (Duggal et al., 2013). Interestingly, similar to the role of ActA in facilitating human germ cell development in vivo (Coutts et al., 2008; Childs et al., 2010, 2011), ActA-derived hESCs displayed an inherent increased expression of the early PGC markers DPPA3 and KIT, compared with standard-derived hESCs in their undifferentiated status (Duggal et al., 2013). Inducing differentiation of ActA-derived hESCs towards the PGC-lineage with BMP4 resulted in significant up-regulation of the germline postmigratory marker DDX4 (also known as VASA) both at the mRNA and protein level compared with when standard-derived hESCs were used (Duggal et al., 2015a).

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These aforementioned findings open the debate about which pluripotency state is preferred for future clinical applications. As derivatives from primed hESCs are already being used in clinical trials (Schwartz et al., 2014), it is necessary to further investigate the ideal starting population for differentiation. Should different somatic cells be tested for reprogramming to specific cell types providing a tailored approach? Should we start from a naı¨ve or primed state of pluripotency? Considerable differences have been shown in the differentiation propensity of different primed hESC lines (Osafune et al., 2008), probably caused by the use of various derivation and culture methods, inherent individual genetic differences, and embryo stage or quality (Allegrucci and Young, 2007; O’Leary et al., 2011, 2012a). In fact, the majority of the in vitro differentiation protocols are plagued by a relatively low yield of differentiated cells and contamination with undesired cell types. Moreover, defined and predictable protocols for directed differentiation and methods of cell delivery have yet to be optimized. Most differentiation protocols using primed hESCs have been modified, since the protocols used for naı¨ve mESC differentiation were not working efficiently. For future regenerative applications, the shortcomings of primed hESCs, such as their intolerance to single-cell passaging which makes them less ideal for bulk production or genetic manipulation, as well as their heterogeneity during lineage-specific differentiation can now be encountered by the production of hESCs in a naı¨ve state of pluripotency. For example, naı¨ve hESCs supported more efficient homologous recombination and were able to grow as single-cell colonies, in contrast to their primed counterparts (Gafni et al., 2013). As previously discussed, different routes have been described to capture the naı¨ve state of pluripotency in human (Chan et al., 2013; Gafni et al., 2013; Takashima et al., 2014; Theunissen et al., 2014; Ware et al., 2014). The main questions that remain are what differences exist between these naı¨ve hESCs obtained by diverse culture conditions and how this affects their functionality, especially their differentiation potential and hence utility in regenerative medicine. The fundamental distinction between the naı¨ve and primed state of mESCs is that primed EpiSCs, derived from the post-implantation blastocyst, do not contribute efficiently to chimeras whereas naı¨ve mESCs do (Rossant, 2008). But, when EpiSCs are grafted into the post-implantation embryo (,E8.5) for whole mount culture, they could be traced back in tissues derived from the three germ layers and as primordial germ cells (PGCs) (Huang et al., 2012). Still, there is now a general consensus that primed mouse or human pluripotent stem cells display a more advanced stage of development, in comparison with naı¨ve pluripotent stem cells, which symbolize a more potent naı¨ve state of pluripotency (Pera, 2014). Consequently, the naı¨ve epigenetic state should be more amenable for (efficient) directed differentiation into various multiple cell lineages. Interestingly, neural stem cells derived from mESCs were more similar to bona fide neural stem cells compared with those originating from EpiSCs, suggesting the superior differentiation potential of naı¨ve stem cells (Jang et al., 2014). Similarly, hiPSCs derived from human fibroblasts versus cord blood possess biased propensities for differentiation due to the resilient epigenetic memory of the somatic cells, but this was eliminated by first converting the hiPSCs to a more naı¨ve state (Lee et al., 2014a, b). Also, it was shown that naı¨ve rabbit iPSCs could robustly differentiate towards oligodendrocytes unlike their primed state, which was

Van der Jeught et al.

9

Role of post-inner cell mass intermediate in pluripotency

Conclusion

Conflict of interest

The generation and cryopreservation of PICMIs from embryos cryopreserved but no longer needed for reproductive purposes, as well as from the fresh PQEs in cycle cohorts, could be beneficial to ART patients. Similar to cord blood banking for the potential treatment of diseases, cryopreserved PICMIs could be an important resource for the regenerative treatment of born siblings. Routinely extending embryo culture beyond the blastocyst stage for PQEs and thawed blastocysts would require optimized culture conditions and increased efficiency, similar to the early trials of ART. As progress is made, it is conceivable to initiate ART cycles with the goal of producing PICMIs and hESC lines rather than the controversial ‘saviour siblings’ to help treat affected siblings. In any event, IVF centres could play an increasingly important role in the future application of stem cells in regenerative medicine. Until now, the PICMI has only been linked to the derivation of (primed) hESCs. Additional studies may prove that the PICMI represents the human peri-implantation embryo, a unique stage during human development that is under studied. Therefore, by understanding the PICMI’s metabolism and genome-wide transcriptional profile, we could gain precious insight into the mechanism of implantation in humans. The majority of cultured blastocysts during IVF do not develop into viable pregnancies and the efficiency of PICMI/hESC generation is similarly low. Could the mechanisms responsible for the blastocyst’s transition into a PICMI be comparable or related to its potential to implant? By studying the interaction between cultured human endometrial cells with both normal and aneuploid PICMI structures, our understanding of the events responsible for human embryo implantation would increase substantially. Implantation failure is thought to be a major cause for the low efficiency of human fecundity. The development of new experimental systems to better understand implantation in humans would undoubtedly benefit a large number of ART patients.

None declared.

We would like to thank the medical illustrator of Anatomy/LUMC Bas Blankevoort for preparing Fig. 1.

Authors’ roles M.V.d.J., T.O., G.D., S.C.d.S.L., P.D.S. and B.H. were responsible for the literature search, making the figures and tables, writing and approval of the final manuscript.

Funding P.D.S. is holder of a fundamental clinical research mandate by the Flemish Foundation of Scientific Research (FWO Vlaanderen), Belgium. S.C.d.S.L. is supported by the Netherlands Organization of Scientific Research (NOW) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). We would like to thank Ferring Company (Aalst, Belgium) for financial support. G.D. and this research is supported by the Flemish Foundation for Scientific Research (FWO-Vlaanderen, grant number FWO-3G062910) and by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).

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