Understanding Mammalian Germ Line Development with In Vitro Models.

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Stem Cells and Development Understanding mammalian germ line development with in vitro models (doi: 10.1089/scd.2015.0060) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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Understanding mammalian germ line development with in vitro models

1 Ana M. Martínez-Arroyo1#, Jose M. Míguez-Forján1#, Jose Remohí , Antonio Pellicer2

& Jose V. Medrano1,2*

1

Fundación Instituto Valenciano de Infertilidad (FIVI), Valencia University; INCLIVA;

Valencia, 46015; Spain 2

Fundación Instituto de Investigación Sanitaria La Fe; Valencia, 46015; Spain

Running title: In vitro models for mammalian germ line development

#

Equally contributing authors

*Correspondence and reprint requests should be addressed to: Jose Vicente Medrano at Fundación Instituto Valenciano de Infertilidad (FIVI), PARC CIENTIFIC UNIVERSITAT DE VALENCIA, Catedrático Agustín Escardino 9, 46980 Paterna (Valencia),

Spain;

Phone:

+34

963903305;

[email protected]

1

Fax:

+34

963902522;

E-mail:

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ABSTRACT Germ line development is crucial in organisms with sexual reproduction to complete their life cycle. In mammals, knowledge about germ line development is based mainly on the mouse model, in which genetic and epigenetic events are well described. However, little is known about how germ line development is orchestrated in humans, especially in the earliest stages. New findings derived from human in vitro models to obtain germ cells can shed light on these questions. This comprehensive review summarizes the current knowledge about mammalian germ line development, emphasizing the state of the art obtained from in vitro models for germ cell-like cells derivation. Current knowledge of the pluripotency cycle and germ cell specification has allowed different in vitro strategies to obtain germ cells with proven functionality in mouse models. Several reports during the last 10 years show that in vitro germ cell derivation with proven functionality to generate healthy offspring is possible in mouse. However, differences in the embryo development and pluripotency potential between human and mouse make it difficult to extrapolate these results. Further efforts on both human and mouse in vitro models to obtain germ cells from pluripotent stem cells may help to elucidate how human physiological events take place, so therapeutic strategies can also be considered.

Keywords: Germ line development; In vitro gamete generation; Pluripotency; Regenerative

medicine.

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1. INTRODUCTION Eggs and sperm are the mature and functional form of germ cells, a cellular lineage which is defined very early in development during embryonic stages. During their development, germ cells suffer different changes that prepare them to carry out meiosis in order to form gametes with half of the chromosomes [1,2]. Additionally, germ cells suffer an extensive and specific epigenetic reprogramming, including imprinting control regions, that allow the epigenetic contribution of both gametes to be unique and complementary [1]. At the end of these processes, mature gametes will be ready to give rise to a new individual after fertilization, closing the life-cycle and being responsible for the transmission of the genetic and epigenetic information through generations [3]. Infertility causes have been widely studied. However, there are infertility situations which cannot be overcome with current techniques. Thus, a better knowledge of the events that take place during human germ line development would help in the design of new strategies to treat infertility. In this report we will review the most important events during germ line development in mammals, with special focus in humans. Moreover, we will pay attention to how regenerative medicine can be a tool to study human germ line development and help us to understand its insights, deepening the current knowledge of how the pluripotency cycle is closed along development in vivo, and demonstrating its plasticity in vitro.

2. OVERVIEW OF GERM LINE DEVELOPMENT IN MAMMALS The differentiation between germ and somatic cells occurs very early in development with the escape of a group of mesodermic cells from their somatic fate during 3

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4 gastrulation, and is characterized by two fundamental facts: reacquisition of the pluripotency and extensive epigenetic remodeling. Next, after sexual determination, each one of the respective sexual features of future eggs and spermatozoa are acquired, during gametogenesis sensu stricto, as gonads are synchronically formed. In the mouse model, during gastrulation the founder population of primordial germ cells (PGCs) are specified by extrinsic signals driven by bone morphogenetic proteins (BMP4 and BMP8b), members of the TGFβ superfamily, from the adjacent extraembryonic ectoderm (ExE) at embryonic day 6.25 (E6.25) [4]. BMPs secreted by the ExE induce Prdm1 and Prdm14 expression in PGCs which, together with Tcfap2c, will act as master regulators over a very reduced group of epiblastic cells located close to the base of Allantois [5]. Thus, PGCs are specified from somatic precursors, in a process called epigenesis which has many implications. Firstly, at a signaling level, PGCs need to be specified by external signals which will turn on the germ cell program and turn off the somatic program. Opposite to this situation, in other model animals (e.g., Drosophila, C. elegans), germ line is specified thanks to inherited maternal determinants, called germ plasm, already present in the oocyte [3,6] in a process known as preformation. Secondly, mammals and other animals following epigenesis induction, endure a cellular reorganization locating PGC precursors outside the embryo in order to prevent their exposure to “somatizing signals” [6]. This reorganization is not needed in the preformation model since the “somatizing signals” are directly suppressed by the inherited maternal determinants. Therefore, PGC specification in response to extrinsic signals is revealed as the first crucial step in germ line development which determines future events, such as the need for pluripotency reacquisition and epigenetic reprogramming [7,8]. Thus, just after their specification, PGCs are characterized by the

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5 expression of several pluripotency markers such as Oct4 (also known as Pou5f1), Nanog and Sox2 [9], as well as other markers such as Fragilis and Stella [10,11]. Epigenetics comprises all the modifications of the DNA that do not imply changes in its sequence and includes DNA methylation [12] and histone modifications [13], both controlling gene expression pattern. At the moment of their specification, PGCs are epigenetically indistinguishable from surrounding epiblastic cells, and show inherited epigenetic modifications such as DNA methylation and X chromosome inactivation that represent epigenetic barriers to totipotency acquisition in somatic tissues. After their specification, PGCs start their migration at E8.5-9.5 through the posterior hindgut to the forming gonad [7,14] and during their migration, PGCs begin the first wave of a profound epigenetic remodeling in order to acquire the epigenetic state necessary to form functional gametes [15]. The first epigenetic changes in PGC reprogramming happen at the histone modification level, leading to a large chromatin remodeling. Only in the X chromosome, the inactive X chromosome of female PGCs show decreased levels of histone 3 lysine 27 trimethylation (H3K27me3), a repressive histone modification, as an indication of its reactivation, whereas in the somatic line H3K27me3 levels remain unchanged [15,16]. These changes in histone modifications occur in parallel to an overall reduction in DNA methylation during PGC migration [14,15]. Apparently, methylation levels do not change dramatically from PGC specification until their arrival to the genital ridges at E10.5, when a deeper reset of methylated DNA occurs in a second wave of epigenetic remodeling events [17]. This process involves a global erasure of DNA methylation patterns, including imprinted genes, and PGCs reach a basal epigenetic state by approximately E13.5, even though there are some sequences in the genome that are resistant to demethylation such as the

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6 intracisternal A particle (IAP) retrotransposon family and adjacent CpG islands (CGIs) [15,18], suggesting a possible epigenetic inheritance between generations. Recent studies suggest that hydroxymethylcytosine (hmC), a derivative of mC [19], is involved in PGC reprogramming. DNA methylation can be passively erased by rounds of replication not followed by the DNA-methylation maintenance system [20], but also actively removed by TET (Ten-Eleven Translocation) enzymes [19,20]. TET enzymes are 5mC-dioxygenases which catalyze the oxidation of mC into hmC, as well as into its other derivatives formylcytosine (fC) and carboxylcytosine (caC). The role that all these oxidated forms of mC play in the epigenome remains unclear, but some studies suggest that they not only act as intermediates preceding DNA-methylation erasure, but also have a structural function controlling gene expression [21]. Thus, in the PGC reprogramming context, the conversion of 5mC to 5hmC by TET enzymes provides a substrate for base excision repair (BER)-mediated active demethylation that could give rise to unmodified cytosine, hence actively erasing DNA methylation marks [22-24]. But not all methylated regions are prone to be demethylated in the first wave of demethylation, since imprinted loci or specific single copy genes maintain their methylation in CpG islands [17]. It is during the second wave of demethylation, which starts at E10.5, when these genomic regions with epigenetic memory are completely erased by E13.5 [17]. There is only one other epigenetic reprogramming event during the life cycle comparable to PGC reprogramming, and it takes place in the zygote just after fertilization and during pre-implantation development. However, the PGC reprogramming event is much deeper and more extensive than that which occurs in the zygote (in which parental imprints remain) and represents the most basal epigenetic state of the mammalian life cycle [15].

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7 When PGCs colonize the genital ridges, they start a process of proliferation and sex determination called gametogenesis that ends with the formation of sperm and eggs. Sex determination depends on the gonadal microenvironment regardless of sex chromosome [25,26] being synchronous with genital ridge formation and maturation. In the mouse, the bipotential genital ridges are formed on the ventral surface of the mesonephros, accompanied by proliferation of the coelomic epithelium around E9.5 [27]. During sex determination, PGCs must establish their sex-specific epigenetic patterns, which include paternal or maternal imprinted marks. The imprinting genes involve genomic sequences that exhibit differences in CpG methylation according to the parental origin, being one allele completely methylated and the other fully demethylated [28]. The establishment of these epigenetic patterns occurs before meiosis in sperm at E14.5-E16.5 [17], and after the first meiotic division in eggs after birth [29-33]. As a result of sex determination, spermatozoa are highly methylated (around 80%), while oocytes are about 30% methylated [17]. Thus, according to the somatic sex of the embryo, germ cells follow a different maturation pattern to finally give rise to mature and functional gametes. However, events that take place in the early phases of germ cell development have been demonstrated to be essential for future gametes to be able to restore totipotency after fertilization in the newly formed embryo. It is in this moment when the zygote endures epigenetic reprogramming to acquire totipotency, and in which some genomic regions escape demethylation, including centromeric repeats, IAP retrotransposons and differentially-methylated regions that are present in parentally imprinted genes [34]. Therefore, soon after fertilization, the paternal genome undergoes rapid and active DNA demethylation, while the maternal one is passively and slowly demethylated during segmentation. Subsequently in the blastocyst stage, the methylation profile 7

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8 characteristic of the new individual starts to be acquired as pluripotency potential is lost and first cellular fate decisions are taken, moving forward to a cycle that will be completed with the specification of PGCs of the new generation [35-37].

3. GERM LINE AND PLURIPOTENCY Fertilization of the oocyte gives rise a totipotent zygote, which is the only totipotent cell that can give rise to all cell types in an organism. Hence, this potential is hidden in a pluripotency cycle which is only completed and closed between generations with germ line development [1,7,38]. In the case of mammals, due to the epigenesis-specification mechanism of PGCs, this pluripotency cycle presents a gap between generations: postsegmentation embryos are composed entirely of somatic cells and germ line is specified by extrinsic signals that return early germ cells to a state of pluripotency. Opposite to epigenesis, in preformation mechanisms, the pluripotency cycle results are more obvious, as it is closed due to the fact that germ cells inherit the germ plasm that maintains their plasticity [7,39]. Consequently, germ cells harbor a latent pluripotent potential (FIGURE 1), although they have been traditionally considered unipotent. Therefore, germ cell specification via epigenesis requires a tight orchestration of signaling pathways that confers germ cell pluripotency reestablishment [7,40,41].

3.1.

SPECIFICATION

OF

PGCs

IN

TRIPARTITE

8

THE

MOUSE:

THE

GOLDEN

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9 As previously discussed, in the mouse model PGCs originate from the most proximal region of the post-implantation epiblast in response to external signals coming from the ExE [2]. These cells are developed from the ICM, where cells expressing Sox2, Oct4 and Nanog become the precursors from which PGCs will arise [42,43]. At E5.5, epiblast cells are able to respond to BMP4 which, in turn, activates the key PGC transcriptional regulator Prdm1 through SMAD1 and SMAD5 [44]. Thus, Prdm1 (also known as Blimp1) expression at E6.25 is the first component of a tripartite of transcription factors involved in mouse germ line specification and marks the onset of PGC specification, demarcating germ cells from the neighboring somatic cells through the repression of the incipient mesodermal program [45-47]. Also involved in this specification network is LIN28 [48], considered to be essential for PGC specification thanks to its role as negative regulator of Let-7 micro-RNA (miRNA) [49]. Because Let-7 is known to inhibit Blimp1 mRNA translation by binding to its 3′ UTR [50], it is proposed that LIN28 contributes to the activation of Blimp1 by blocking the maturation of functional Let-7 miRNA [49]. Together with Prdm1, Prdm14 expression is also enhanced in response to the activation signaling of BMP4. The onset of Prdm14 expression is not Prdm1-dependent, but Prdm1 is essential for Prdm14 expression maintenance in a dose-dependent manner, reinforcing its activity. [51,52]. Therefore, while Prdm1 repress the somatic program, Prdm14 acts to induce the epigenetic reprogramming in early germ cells. In fact, mutations in Prdm14 led to the formation of aberrant PGCs with a defective epigenetic programming by failure of global erasure of H3K9me2 histone modification at E8.5, losing PGCs by around E11.5 [20]. Tcfap2c, which encodes AP2γ, has also been described in mouse as a critical element that acts downstream of Prdm1, being the third component of the tripartite of 9

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10 transcription factors crucial for PGC specification. AP2γ might also be involved in the repression of somatic genes, including early mesodermal markers, such as Hoxb1 [11]. The induction of AP2γ by Prdm1 is crucial for the combinatorial role of Prdm14 and AP2γ in the induction of PGC-specific genes such as Dnd1, as well as Nanos. They also bind to the distal enhancer of Oct4, to maintain its expression in PGCs. Thus, Prdm1, Prdm14 and AP2γ together constitute the tripartite transcriptional network that is mutually interdependent for PGC specification in mouse [53]. Hence, this tripartite transcriptional network enables several programs to initiate in early PGCs, including the repression of somatic genes. For example, the migratory program, as well as the expression of PGC-specific genes, is executed mainly by Prdm14 and AP2γ, whereas Prdm14 and Prdm1 collaborate in both the induction and repression of epigenetic modifiers [54]. At an epigenetic level, the tripartite transcriptional network induces the global DNA demethylation characteristic of PGC reprogramming [17,19]. Prdm1 and Prdm14 promote DNA replication-coupled DNA demethylation by repressing the de novo DNA-methyltransferase Dnmt3b, as well as Uhrf1, a co-factor for the DNA-methylation maintenance system. Hydroxylation of 5-methyl-cytosine is mediated by TET1 and TET2, which are recruited to DNA target loci by Prdm14 [19,55]. The expression of Prdm1 prevents the somatic fate at the onset of PGC specification, initiating a reversion of some of these properties, including X-reactivation and re-expression of key pluripotency genes, such as Sox2 and Nanog [1]. Finally, the re-activation of the inactive X-chromosome in PGCs might also be facilitated by Prdm14 binding to intron 1 of Xist [54,56,57].

3.2. COMPARISON BETWEEN HUMAN AND MOUSE MODELS OF SPECIFICATION: UNSOLVED QUESTIONS 10

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11 Due to the limited access to the early stages of human development, current knowledge about very early germ line development in vivo is based in mouse model [58]. Interestingly, the timing and formation of the extra-embryonic tissues where early germ cells are specified, is significantly different in mouse and humans. This fact, hence, might have some implications for PGC specification and determination [59]. More specifically, before gastrulation, the mouse epiblast can be visualized as a thickwalled cup of tissue, which forms the entire embryo and some of the placental membranes. At this moment, the ExE arises from the polar trophectoderm underlying the epiblast. In humans, by contrast, the embryo consists of a bilaminar disc, the epiblast, and the primitive endoderm, or hypoblast. The epiblast cells will form the amnioblast and the amniotic cavity [59], whereas the polar trophectoderm over the epiblast differentiates towards the syncytiotrophoblast and the cytotrophoblast, which remains in contact with the epiblast (FIGURE 2). In other words, no equivalent structure to the mouse ExE, from which PGC specification is triggered, is apparently formed in the human embryo [59]. Thus, although mouse has been widely used as the mammalian model to define, at a molecular level, several signaling pathways in vivo, it does not seem to be the best model to compare PGC determination in humans. On the other hand, the use of other animal models with a discoidal epiblast may help to clarify some questions. Interestingly, in rabbits, PGCs arise in the primitive streak as well as in mouse, but BMP2 is expressed from the hypoblast and yolk sac epithelium at the boundary of embryonic disc, which is equivalent to the proximal and extraembryonic visceral endoderm in mice. Hence, BMP2 seems to play a more essential role in rabbit PGC specification than BMP4, since BMP4 is expressed after BMP2 and Blimp1-positive cells are identified close to BMP2 expression, following its same pattern [41,60]. In 11

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12 pigs, also with discoidal embryos, PGCs are not specified at an equivalent time to mouse embryos. In the proximal epiblast of pre-gastrulating pig embryos, few cells start to express OCT4 as the unique germ line marker. After that, and similar to other nonrodent mammals including humans, OCT4-positive cells move from the embryonic disc to the extraembryonic mesoderm of the yolk sac wall and become restricted to the ventral wall of the hindgut [61]. To date, there is no well-defined non-rodent animal model which enables us to identify where PGC specification signal(s) come from. In fact, precursors of human PGCs have been suggested to emerge from the epiblast around 10-11 days postcoitum (dpc) by the action of BMP signals from the hypoblast and cytotrophoblast, and probably from amnioblast or both, but this last point remains unclear. Two to four days after, these precursors migrate to the extraembryonic mesoderm and reach the region of the yolk sac where the allantois is formed at around 16 dpc [59]. Then, during the fourth week of gestation, PGCs are passively incorporated into the embryo with the yolk sac wall by folding the embryonic disc, and they start to migrate toward the gonadal ridges (FIGURE 2). In conclusion, anatomical differences between rodents and other mammals during early development are relevant. On the other hand, many legal and ethical limits imply that very little is known about early human embryo development and, particularly, germ line specification. Moreover, this first step in germ cell development reveals as crucial for proper reprogramming and pluripotency reversal in order for future germ cells to be able to generate a new organism. The role that the starting signaling pathway plays, as well as the putative function the specification tripartite has in humans, however, is not yet determined. In this sense, the pluripotent background that epiblastic cells possess

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13 might be different between mouse and human, conditioning the way that the first steps in PGC determination take place.

3.3. ARE THE ANATOMICAL DIFFERENCES BETWEEN HUMAN AND MOUSE EMBRYOS RELEVANT TO OBTAIN GAMETES IN VITRO? The anatomical differences between mouse and human embryos might have an effect on pluripotency re-acquisition mechanism. In fact, the consequences of this differential pattern should also be reflected in vitro (FIGURE 1). In mouse, the epiblast derives from the inner cell mass (ICM), as embryonic stem cells (ESCs) do in vitro [62-67]. Mouse ESCs (mESCs) pluripotency is defined by their ability to differentiate into representative cell types from the three germinal layers (ectoderm, mesoderm and endoderm) in vitro, and in vivo by the formation of teratomas after injection in immunosuppressed mice. Moreover, chimera formation [68], as well as tetraploid complementation both are the last and most consistent features of pluripotency (TABLE 1) [69]. In addition, pluripotent stem cells (PSCs) can also be derived from the epiblast of postimplantation mouse embryos. These cells are called mouse epiblastic stem cells (mEpiSCs), express pluripotency markers such as Oct4, Nanog and Sox2, and can be differentiated in vitro into the three germ layers and, also, into the germ line [70,71]. Furthermore, mEpiSCs are able to form teratomas in vivo, although they show a very low potential to contribute to chimera formation (TABLE 1) [72]. Interestingly, mEpiSC can be converted into ESC-like cells when cultured under conditions similar to mESCs with BMP4 and LIF/STAT3; likewise the ICM-derived mESC can convert to EpiSC-like cells when cultured under EpiSC conditions, i.e., under FGF and Activin signaling (FIGURE 1) [73,74]. 13

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14 The slightly different features that PSCs show, reveal the existence of several pluripotency states, distinguishing between naïve and primed pluripotent states [42]. Naïve pluripotency is defined as the initial ground state of pluripotency, in which the cells have not yet lost their ability to form every cell type in the organism, including germ cells. However, as cells become specified into a defined fate, they restrict their potential to a primed pluripotency state [7]. The mEpiSCs derived from postimplantation blastocysts exhibit primed characteristics, since they lose the expression of some pluripotency factors, such as Rex1 or Klf4, and are unable to form chimeras (TABLE 1) [75]. Interestingly, mouse PGCs from the post-implantation epiblast can be driven to become embryonic germ cells (EGCs) in vitro. EGCs are able to reverse this primed state toward a naïve pluripotent state as shown by their ability to form chimeras and differentiate into all cell types, including germ line (TABLE 1) [76]. Consequently, PGCs from the postimplantation embryo are considered to have a latent pluripotent potential [7]. First reports on germ line differentiation demonstrated that mESCs spontaneously differentiated in vitro towards germ cell lineages, forming oocyte-like cells (OLCs) [77], and sperm-like cells (SLCs) able to participate in spermatogenesis in vivo [78] and to form blastocysts after injection into oocytes by intra-cytoplasmic sperm injection (ICSI) [79]. These attempts, however, failed in overcoming meiotic checkpoints to give rise to healthy and fertile offspring, probably because earlier events such as establishment of primed pluripotency, were not properly reached (TABLE 2). Similar results from hESCs were later obtained by spontaneous differentiation, suggesting that hESCs consist of a heterogeneous population in which some cells are predisposed towards germ-cell-fate differentiation [80]. Moreover, hESCs show low potential to contribute to chimera formation although they are able to give rise to the 14

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15 three germ layers and forming teratomas in vitro, suggesting that they are more similar to mEpiSCs than to mESCs (73). Hence, hESCs represent a pluripotency state closer to primed pluripotency (72) from where they could directly respond to meiotic entry induction (82-85). In fact, acquisition of a primed pluripotent state has revealed as essential for functional mouse gamete generation in vitro [53,81,82], as discussed below (FIGURE 1).

4. IN VITRO MODELS TO EXPLORE HUMAN GERM LINE DEVELOPMENT Recent works highlighted the relevance of the specification signaling on PGC specification in vitro and hence, germ line development into functional gametes (TABLE 2) [53,81,82]. In these reports, mESCs cultured in 2i conditions to induce their naïve pluripotency state were first differentiated into Epiblastic-like cells (EpiLCs), evolving toward a primed stated by treating them with Activin A, Fibroblastic Growth Factor (bFGF) and 1% Knockout-Serum Replacement (KO-SR). Subsequently, EpiLCs were differentiated into PGC-like cells (PGCLCs) by two methods: adding BMP4/8b to the culture media, mimicking the BMP signal specification from the ExE in vivo, together with Stem Cell Factor (SCF), Leukemia Inhibitory Factor (LIF) and Epidermal Growth Factor (EGF) [81,82]; or alternatively, with the overexpression of Prdm1, Prdm14, and Tfap2C genes, which act downstream of the BMP signaling pathway [81]. The obtained PGCLCs showed imprinting patterns corresponding to their embryonic somatic sex and when transplanted into host mice, they went through both complete spermatogenesis [82,83] and oogenesis, and produced fertile offspring following ICSI [81]. With these experiments, the exclusive capability of epiblastic cells to respond to BMP signaling, as well as the important role of the BMP4/Prdm1 signaling pathway for PGC specification was demonstrated for first time in vitro. This has been, indeed, the 15

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16 only strategy able to generate viable gametes in vitro with proven healthy and fertile offspring. In the same way that these works contributed to a better understanding of the germ line specification process in mouse, in vitro models can also be useful for similar purposes on human germ cell development. Following this perspective, a recent publication highlighted some important differences in human germ line specification [84]. In this study, hESCs were cultured under 4i conditions to induce a distinct pluripotent state closer to the naïve state before pre-inducing them into an EpiLC state. In a similar way that previously reported in the mouse model [81,82], pre-induced hESCs differentiated into PGCLCs in suspension under the induction with BMPs, LIF SCF and EGF. Interestingly, resulting human germ-like cells displayed several differences compared to the ones obtained in mouse, such as null activation of SOX2, a delayed expression of PRDM14 and an up-regulation of SOX17 and GATA4. Due to the unexpected up regulation of SOX17, traditionally considered an endodermic transcription factor, its role in human PGC specification was investigated. In contrast with mouse PGC specification, SOX17 might play an essential role in human germ line specification by acting upstream and synergistically with BLIMP1 as the master regulators of PGC specification, at the same time that BLIMP1 acts as a repressor of the endodermic differentiation of PGCs [84]. Additionally, the first suggestions of germ line formation from hESCs in vitro were reported by spontaneous differentiation [80,85]. Undifferentiated hESCs expressed some early germ cell markers such as c-KIT and DAZL, but not late markers such as VASA or SYCP3 [86], a structural protein of the synaptonemal complex, suggesting that hESCs could be a heterogeneous pluripotent population in which some cells had a pre-disposition toward a germ cell fate. However, meiotic progression remained as one 16

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17 of the main obstacles. In fact, complete meiotic progression of in vitro derived human germ cells was achieved with the ectopic expression of the DAZ gene family members DAZ2, DAZL and BOULE not only in hESC but also in human induced PSCs (hiPSCs) lines subjected to spontaneous differentiation [87,88]. The expression of these highly conserved RNA-binding proteins lead the correct meiotic progression of human germ cells in vitro in the absence of a gonadal niche (TABLE 2). Along the same line, another report highlighted the possible role that RNA-binding proteins may have in meiotic entry control, showing how the ectopic expression of VASA also leads to meiotic induction of in vitro derived human germ cells [89]. In fact, recent reports demonstrate that DAZL limits pluripotency as well as differentiation to control apoptosis in PGCs through a robust interactive network. If this network is broken or DAZL expression is lost, the consequence is teratoma formation [90]. However, the current state of the art of germ line derivation from human pluripotent cells presents some limitations. Differences in the pluripotential status of mESCs (naïve-like state) and hESCs (primed state) make it difficult to compare their potential to fully develop into functional germ cells. Moreover ethical, restrictions make it difficult to carry out functional assays with the germ cells obtained from human pluripotent cells. Thus, xenotransplantation of human germ cell-like cells into seminiferous tubules of immunosuppressed mice to test their potential to colonize the seminiferous epithelium like human SSCs do, could be an important tool to assay the functionality of in vitro derived human germ cells. In this context, the role of RNAbinding proteins has also been tested, demonstrating that hiPSCs improved their ability to colonize the lumen of the seminiferous tubules of sterilized immunodeficient upon VASA overexpression in combination with pluripotency factors. These results highlight the role that VASA plays controlling the pluripotency-state in a combined in vitro/in 17

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18 vivo model [91]. This model also represents a very interesting analysis method from a clinical point of view, and has already been used to analyze the capability of hiPSCs derived from azoospermic men with different deletions in the Y chromosome, demonstrating how the genetic background affects their capability to differentiate into germ cells and colonize the seminiferous niche in vivo [92]. Finally, this same approach has been used recently to demonstrate that germ cell-like cells can be formed in vivo independently of X chromosomes geneticprofile [93]. In this case, hiPSCs from Turner syndrome patients were derived and xenotransplanted into seminiferous tubules, demonstrating that, regarding infertility in women with X chromosomes aneuploidies, two X chromosomes are not required for human germ cell formation but is for their maintenance until adulthood [93].

5. DISCUSSION AND CONCLUDING REMARKS The mammalian germ line development can be considered as a non-continuous pluripotency cycle between generations, due to the need of external signals for PGC specification during the embryonic development of each individual. Knowledge derived from germ line development is increasing since interest in this research field is getting bigger in part due to its possible clinical applications to treat infertility. Moreover, in vitro models give researchers the opportunity to improve the knowledge of human germ line development, a process which involves key milestones, mainly divided in two steps: pluripotency reacquisition (including epigenetic reprogramming) and gamete differentiation (including meiosis). According to the mouse model, the tripartite of transcription factors composed by Prdm1, Prdm14 and Tfap2C allows the post-implantation epiblast, a primed cell

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19 subpopulation, to be prone to reacquire the naïve pluripotency state. However, differences between rodents and other mammalian species are evident [84]. Rodents show an unusual method of early formation of egg cylinder epiblast and also form unique characteristic PSCs with a different pluripotency potential (i.e., mESCs and EpiSCs), probably due to the ability of rodent blastocysts to temporarily arrest their development depending on the niche (i.e., endometrium) conditions, known as diapause [94]. Mouse embryos arrested in diapause resume their development in response to LIF signaling, which is in concordance with the need of LIF/STAT3 pathway to derive mESCs [94]. Also, diapause capacity could also have a role in the explanation of why only mESCs and mEGCs, but not hESCs, respond to 2i conditions to acquire the true naïve pluripotent state [94]. Instead, other mammals, such as primates, show epiblast delamination, forming a simple flattened embryonic disc [60,95-99]. Both mESC and miPSCs show the capability to give rise to all cell types including germ cells and to form teratomas and chimeras, demonstrating a ground naïve pluripotent state. Otherwise, hESCs share characteristics with mEpiSC, which are derived from postimplantation epiblast, both showing limited capability to form chimeras corresponding to a primed pluripotency status. It is not known whether naïve pluripotency exists in the human embryo nor, if it does exist, our inability to recapitulate in vitro might be due to this status being only displayed in a very short window of time in vivo [94]. This important issue requires resolution. A more challenging approach is the study of human in vitro gamete generation. According to the complexity of the meiotic process, this seems to be the biggest obstacle in the in vitro gametogenesis reconstitution [100]. Firstly, since meiosis is a reductional division, the DNA content must be haploid (1N) for all chromosomes and must show an appropriate organization that could be analyzed by karyotyping and 19

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20 fluorescence in situ hybridization (FISH). Secondly, the recombination process must be assessed by immunolabeling of proteins involved in homologous recombination synapsis to demonstrate an appropriate localization [100]. Finally, the in vitro gametes must be able to form euploid and viable offspring and not only fertilization or early embryos. However, this last characteristic is the main handicap for human models due to its ethical controversy. Differences between mice and human embryology prevent the direct translation of knowledge obtained from mouse early germ line development to humans. Thus, in vitro models for germ line differentiation may be an important tool to fully understand this process in humans (TABLE 2) [84,91-93]. Herein further efforts must be addressed in the future to improve these models in order to them to complete our current knowledge on human germ line development, overcoming the main handicaps and, eventually, to allow improving human infertility treatments.

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ACKNOWLEDGEMENTS This work was supported by a research project grant PI13/00546 from ISCIII, cofounded by the European Regional Development Fund “A way to make Europe”, the KY Cha Award in Stem Cell Technology 2014 conceded by the American Society for Reproductive

Medicine

(ASRM),

a

VAL

I+D

grant

conceded

to

JMM

(ACIF/2014/077), a Sara Borrell grant conceded to JVM (CD12/00568) by the ISCIII and a FPU grant conceded to AMMA (AP-2009-4522) by the Spanish Ministry of Education.

CONFLICT OF INTERESTS The authors declare that they have no conflict of interest.

21

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FIGURE LEGENDS

27

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28

Figure 1: Pluripotency cycle. The blue inner circle represents pluripotent states in vivo; the grey circle represents in vitro-derived pluripotent states; and the white external circle represents the in vitro-induced pluripotent states. (A) After fertilization the embryo display the most naïve pluripotency state; the last cell population that keeps this feature is the PE, from where mESC can be derived; these cells also show a naïve pluripotency state. (B) At E7.25 the epiblast loses its naïve pluripotency state, exhibiting a primed pluripotent status. EpiSCs can be derived from PtE cells. EpiSCs can give rise to mESC in vitro (brown arrow). Alternatively, mESCs can be derived to EpiLCs (purplue arrow) which display features similar to PtE cells. (C) PGCs are specified from the PtE, and they show a latent pluripotent state. PGCs are derived in 28

Page 29 of 35


29 vitro to EGCs, which return the latent state to a naïve-like state. EGCs display features very similar to mESCs, although no direct conversion has been described to date (blue dotted arrow). Moreover PGCs and their derivatives sperm and eggs are the only cells able to recapitulate the naïve pluripotency in vivo after fertilization. ICM, inner cell mass; PE, Pre-implantation Epiblast; ExE, Extrambryonic Ectoderm; PtE, Postimplantation Epiblast; PGCs, Primordial Germ Cells; EpiSCs, Epiblast Stem Cells; EpiLCs, Epiblast-Like Cells; PGCLCs, PGC-Like Cells. EGCs: Embryonic Germ Cells.

29


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30

30

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31

Figure 2: Schematic anatomy models of mouse and human embryo. (A) Mouse embryo can be visualized as a thick-walled cup of tissue at E6.25-6.5. Polarizing signals determine an anterior-posterior and a proximal-distal axis, and then the epiblast region where pPGCs are located. At E7-7.25 gastrulation starts and PGCs arises in the base of allantois by BMP signaling from the ExE. Then the PGCs migrate and at approximately E10.0 reach the gonadal ridges. (B) In humans at 9dpc the embryo consists of a bilaminar disc, the Epiblast and Hypoblast. Epiblast form the amnios but no equivalent structure to the mouse ExE is apparently formed in the human embryo. BMP signals coming from the hypoblast and cytotrophoblast and probably from amnioblast or both, and pPGCs have been suggested to emerge from the proximal epiblast near the position of the primitive streak (17dpc). Similarly to mouse, the PGCs reach the gonad around

31

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32 33 dpc, after migration. ExE: Extraembryonic Ectoderm. PGCs: Primordial Germ Cells. pPGCs: Primordial Germ Cells precursors. PEc: Proximal Ectoderm. AVE:

Cell type

mESCs

Core pluripotency factors

Naïve pluripotency factors

(Oct4, Sox2 and Nanog)

(Klf4, Rex1, Klf2, Stella, Esrrb)

Yes

Yes

Derivation origin

Blastomere, ICM, pre-implantation epiblast

hESCs

Yes

No

Blastomere, ICM

mEpiSCs

Yes

No

Post-implantation epiblast

mEGCs

Yes

Yes

mPGCs

mPGCs

Yes

Yes (no Klf4)

Epiblast (in vivo)

Somatic Cells

No

No

Differentiated tissues

Anterior Visceral Endoderm. PPEc: Posterior Proximal Ectoderm. VE: Visceral Endoderm. DEc: Distal Ectoderm.

Table 1. Pluripotency features

32

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33

mESC, mouse embryonic stem cells; hESC, human embryonic stem cells; mEpiSCs, mouse epiblastic stem cells; mEGCs, mouse embryonic germ cells; mPG

Table 2. Hallmarks of germ cell-like cells in vitro derivation

Origin

Derivation conditions

In vivo assays

mESC

Spontaneous differentatio n in adherent cultures Differentiati on to EpiLCs

(XX, XY)

mESC and miPSC (XY)

(bFGF and activinA);

Epigenetic features

Germ cell markers

Meioti c marke rs

Haploid cell formatio n

Offspring obtaining

Not analyzed

Not analyzed

Not analyzed

SYCP3 , DMC1

Oocytelike cells

No (Parthenoge nic Blastocyst)

[78]

Transplantati on into neonatal mouse testis

H3K9me2 low/

DAZL

----

Spermlike cells

Healthy and fertile offspring (ICSI)

[86]

SYCP 3,

Oocytelike cells


[87]

Spermlike cells


[54]

PGCLC generation by BMP4

SSEA1 Integrinβ3

H3K27me3 high

Referen ce

mC low global levels no changes in Igf2r and Snrpn ICR; loss of methylation in H19 and Kcnq1ot1 ICR.

mESC and miPSC (XX)

Differentiati on to EpiLCs (bFGF and activinA);

Transplantati on into mouse ovarian bursa

(XY)

Differentiati on to EpiLCs (bFGF and activinA); PGCLC generation by Prdm1, Prdm14, Tcfap2C

SSEA1 Integrinβ3

XaXa

meiotic spindle

H19, Igf2r and Kcnq1ot1 ICR hypomethylati on

PGCLC generation by BMP4 mESC and miPSC

H3K27me3 high

Transplantati on into neonatal mouse testis

H3K9me2 low/ H3K27me3 high.

Not analyzed

H19 and Snrpn ICR demethylation

33

----

Page 34 of 35


34 overexpressi on.

hESC

Adherent culture;

Not analyzed

mC low global levels.

(XY, XX)

PRDM1 VASA

BMP4, BMP8b;

H19, PEG1, SNRPN and KCNQ ICR hypomethylati on.

DAZ2, DAZL, BOULE overexpressi on.

γH2A X, SYCP3

DAZL

Germ cell-like cells

Not analyzed

[106]

Not analyzed

[84]

(1N, TEKT1, ACROSI N)

STELLA

RA exposure. hiPSC- fetal (XX) and adult

Adherent culture;

Not analyzed

Not analyzed

PRDM1 A

BMP4 and BMP8b;

VASA

(XY)

STELLA R FITM1 PELOTA GCNF

RA exposure. Adherent culture;

Not analyzed

H19 ICR demethylation

(XX, XY) and hiPSC (XX)

Germ cell-like cells (1N, ACROSI N)

DAZL DAZ2, DAZL, BOULE overexpressi on.

hESC

SYCP3 , CENPA

PRDM1 VASA

BMP4 and BMP8b;

SYCP3 , CENPA

Germ cell-like cells

Not analyzed

[85]

SYCP3

Induced PGCs (2N)

Not analyzed

[107]

DAZL

VASA overexpressi on.

IFITM1

RA exposure.

GDF

GCN

cKIT PELOTA GDF9 ZP4 hiPSC (XX, XY)

BMP4 and BMP8b; VASA overexpressi on.

Transplantati on into mouse testis

KCNQ1OT1, PEG1, H19, and H19 ICR hypomethylati on. Global demethylation and 5mC to 5hmC conversion

34

PRDM14 VASA STELLA NANOS2 /3

Page 35 of 35


35 DAZ2

hiPSC

BMP4 and BMP8b

(XY, microdelecti ons in Y chr.)


Global DNA demethylation.

PRDM1 PRDM14

Not analyze d

Induced PGCs (2N)

Not analyzed

[108]

VASA DAZ STELLA IFITM3 NANOS3 PLZF UTF1 SALL4

hiPSC

BMP4 and BMP8b


??

??

Not analyze d

Induced PGCs (2N)

Not analyzed

[96]

Cell culture in bFGF and 4i medium. Derivation by BMP2 and BMP4 with SCF, LIF, EGF and ROCK inhibitor.

Not analyzed

Upregulation TET1 and TET2. 5mC to 5hmC conversion. Nucleus translocation of PRMT5. Downregulatio n of UHRF1, DNMT3A and DNMT3B.

BLIMP1, TFAP2C, DND1, KIT, SOX17 and CD38.

Not analyze d

Induced hPGCLC s (2N)

Not analyzed

[89]

(X0) hESCs and iPSCs (XX, XY)

mESC, mouse embryonic stem cell; miPSC, mouse induce pluripotent stem cell; hESC, human embryonic stem cell; hiPSC, human induce pluripotent stem cell; EpiLCs, epiblast like cells; BMP, bone morphogenetic protein; bFGF, basic fibroblast growth; SCF, stem cell factor; LIF, leukemia inhibitory factor; EGF, epidermal growth factor; ROCK, Rho-kinase; ICR, imprinted control region; Xa, X chromosome activation; ICSI, intra-cytoplasmatic sperm inyection; RA, retinoic acid; mC, 5-methylcytosine; hmC, 5-hydroximethylcytosine.

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Poised chromatin in the mammalian germ line.

How computational models contribute to our understanding of the germ line.

Origin and development of the germ line in sea stars.

Germ line development: lessons learned from pluripotent stem cells.

Perspectives of germ cell development in vitro in mammals.

Beyond the mouse monopoly: studying the male germ line in domestic animal models.

Sex determination in mammalian germ cells.

Mammalian pancreas development: regeneration and differentiation in vitro.

Germ-line and somatic recombination induced by in vitro modified P elements in Drosophila melanogaster.

Germ-line sex determination in Drosophila melanogaster.

Dynamic changes in DNA modification states during late gestation male germ line development in the rat.

In Vitro Cell Models for Ophthalmic Drug Development Applications.

Human germ line editing-roles and responsibilities.

Regulation of germ line stem cell homeostasis.

Germ-line gene therapy: back to basics.

Germ-line therapy: evolutionary and moral considerations.

A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency.

Don't edit the human germ line.

Genetic mosaics and the germ line lineage.

The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development.

Kif18a is specifically required for mitotic progression during germ line development.

Insights into female germ cell biology: from in vivo development to in vitro derivations.

Kremen1 regulates mechanosensory hair cell development in the mammalian cochlea and the zebrafish lateral line.

Models of germ cell development and their application for toxicity studies.