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ScienceDirect Germ line development: lessons learned from pluripotent stem cells Ana M Martı´nez-Arroyo1,2,5, Jose V Medrano1,3,5, Jose´ Remohı´1,3 and Carlos Simo´n1,2,4 Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. Addresses 1 Fundacio´n Instituto Valenciano de Infertilidad (FIVI), Dept. Obst. & Gynec., Valencia University, Valencia, Spain 2 INCLIVA Biomedical Research Institute, Valencia 46015, Spain 3 Fundacio´n Instituto de Investigacio´n Sanitaria La Fe, Valencia 46026, Spain 4 Department of Obstetrics and Gynecology, Stanford University, Stanford, CA 94305, USA Corresponding author: Simo´n, Carlos ([email protected], [email protected]) 5 These authors contributed equally to this work.
Current Opinion in Genetics & Development 2014, 28:64–70 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Jose´ CR Silva and Renee A Reijo Pera For a complete overview see the Issue and the Editorial
‘switch on’ the germ line program so that these cells can acquire pluripotency properties [1]. Thus, there is a latent pluripotent cycle in germ cells that is maintained by a network that controls the non-restrictive PGC epigenome [2,3,4]. In this review we will focus on the main genetic and epigenetic events in germ line development examining evidence from in vivo studies that is supported by in vitro models (Figure 1).
Genetic regulation of germ line development PGCs can first be identified in mice on embryonic day 6.25 (E6.25), when some cells in the proximal epiblast start to express Prdm1 [5] in response to the BMP4 and BMP8b signals secreted by extraembryonic ectoderm [6]. Specification by BMP4 and BMP8b induce Prdm1 and Prdm14 expression in a cooperative dose-specific way to ensure sufficient exposure to epiblast BMP signaling in order to specify germ lineage fate [6,7]. The transcriptional repressor Prdm1 is essential for suppressing the somatic program [5,8]; subsequently Prdm14 promotes epigenetic reprogramming so that a pluripotent status can be reacquired. Prdm14 expression is initially independent of Prdm1 however its maintenance is strictly dependent on it [9,10]. Together with these two transcription factors, Tcfap2C has also been described as a critical element which acts downstream of Prdm1, preventing mesodermal differentiation and enhancing the germ cell program [11].
Available online 14th October 2014 http://dx.doi.org/10.1016/j.gde.2014.09.011 0959-437X/# 2014 Elsevier Ltd. All rights reserved.
Pluripotency and the germ cell cycle In organisms with sexual reproduction, germ cells allow the life-cycle to continue through generations. In the case of mammals and other vertebrates, there is a clear separation of the germ line from the somatic lineages at the early developmental stages of the embryo. In the murine model, the germ line is formed at gastrulation from a small number of founder cells called primordial germ cells (PGCs) which escape their somatic fate and acquire a germinal fate. The moment of PGC specification in mammals involves global epigenetic reprogramming mechanisms that ‘turn off’ the somatic program and Current Opinion in Genetics & Development 2014, 28:64–70
The BMP4/Prdm1 signaling pathway which initiates at the moment of PGC specification seems to be crucial for future PGCs to acquire the proper epigenetic and transcriptional background which would enable them to be correctly reprogrammed and develop into functional gametes. In fact, studies based on mouse models describe the derivation of live offspring by recapitulating the germ cell-specification pathway in a two-step in vitro model. Since only cells from the epiblast are able to respond to BMP signaling, mouse pluripotent stem cells (PSCs) were first differentiated into epiblast-like cells (EpiLCs), which represent a cellular state very close to epiblast cells before gastrulation. Subsequently, EpiLCs were differentiated into PGC-like cells (PGCLCs) by two methods: adding BMP4 to the culture media, mimicking the BMP4 signal specification which epiblastic cells receive from the adjacent ectoderm in vivo [12,13]; or alternatively the overexpression of Prdm1, Prdm14, and Tfap2C genes, which act on the PSCs downstream of the www.sciencedirect.com
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Figure 1
Fertilization
E3.5 Oct4, Nanog
Meiosis Oct4, Nanog
Oct4, Nanog
Spc I SSC Spg
E6.5
E7.5
E8.5
E9.5
PGC determination
E11.5
PGC migration
Spg
E13.5 Sex determination
Gametogenesis
Mvh
Prdm1 Prdm14
Daz
Tcfap2c
Boule
Current Opinion in Genetics & Development
Primordial germ cell specification and migration in the post-implantation embryo. Primordial germ cell (PGC) precursors and PGCs are shown in pink during determination at embryonic day 6.5 (E6.5), E7.5, E8.5, during migration at E9.5 and E11.5, and in sex determination at E13.5. Extraembryonic ectoderm from which BMP signaling specifies PGC precursors is shown in purple. Only male gametogenesis is shown. The gene expression timing pattern is indicated with black arrows. Dotted line indicates loss of gene expression. SSC, spermatogonial stem cell; Spg, spermatogonia; Spc I, spermatocyte I.
BMP signaling pathway [13]. The PGCLCs obtained by these approaches showed imprinting patterns according to their embryonic somatic sex and when transplanted into host mice, they went through both complete spermatogenesis [12,14] and oogenesis, producing fertile offspring following intracytoplasmic sperm injection (ICSI)$ [13]. In summary, germ line development in the murine model requires somatic program suppression, then the re-acquisition of pluripotency, and finally reprogramming to a unique and non-restrictive epigenome [6,15]. The signaling for this process is triggered by BMP and the Prdm1/Prdm14/Tfap2c pathways. From their specification at the proximal epiblast region at E6.25, the germ line development process occurs sequentially during their migration from the posterior hindgut to the forming gonad at around E8.5–E9.5 [16,17]. Once they reach the gonads, PGCs start to migrate bilaterally at the gonadal ridges at E10.5–E11.5. From this point, germ cells change their genetic expression program and epigenetic profiles, and start to express key genes for survival and maturation like DAZL [18] and VASA [19] (Figure 1). In humans, other genes from the deleted in azoospermia (DAZ) family, such as DAZ2 or BOLL, have been demonstrated to be necessary for proper male germ line development [19–21]. After gonadal colonization, PGCs initiate their sexual determination guided by the www.sciencedirect.com
surrounding microenvironment of the gonadal niche (i.e. sertoli or granulosa cells) and acquire a sex-specific epigenetic status that involves DNA methylation and, in females, X chromosome inactivation. Thus, as gametogenesis goes forward pluripotency decreases and the germ cell specific expression program becomes activated. In contrast to the somatic tissues, the germ line is characterized by a set of specific features among which the most important is their ability to reduce their chromosomal charge during meiosis. The importance of RNA-binding proteins on meiotic progression has been elucidated using in vitro models in several studies that describe how the ectopic overexpression of DAZ family genes (i.e. DAZ2, DAZL, BOULE, etc.) enables proper meiotic progression in both hESCs and human induced pluripotent stem cell (hiPSC)-derived germ cell-like cells [22,23]. It has been proposed that germ line related RNA-binding proteins, including VASA, NANOS, DAZL, and PIWI, that are highly conserved at different stages of germ line development [24] may act as chaperones, mediating proper protein folding [25], as well as acting as post-transcriptional meiosis-related protein regulators. In humans, the DAZ family is composed of the autosomal genes BOULE and DAZL, and the DAZ gene cluster which is encoded on the Y chromosome. In mouse, Current Opinion in Genetics & Development 2014, 28:64–70
66 Cell reprogramming, regeneration and repair
Daz acts as a master regulator of spermatogenesis, whilst Dazl is critical for meiosis [26]. In humans, evidence suggests that loss of BOULE gene function may result in azoospermia with primary defects in meiotic transition [27]. Similarly, ectopic expression of VASA, another highly conserved RNA-binding protein has shown a similar effect on the induction of meiosis [28]. Interestingly, mouse knock-out models for the Mouse vasa homolog (Mvh) show male infertility, while the Drosophila phenotype shows female sterility [21].
methylated (around 80%), while in females, oocytes are about 30% methylated [1].
Epigenetic regulation during germ line development
The spermatogonial stem cell (SSCs) subpopulation leads to male germ cell propagation through adult life [31], and are considered to be the male germ line stem cells [32]. In humans, SSCs are present at the basal membrane of the seminiferous tubules from the moment of birth while in mice the gonocyte-to-spermatogonia transition takes place 1–4 days postpartum [33]. Once sexual maturity is reached, quiescent spermatogonia start to asymmetrically divide giving rise to originating mitotically active spermatogonia, to advance into spermatogenesis, but also self-renew in order to maintain the adult testicular stem cell population [34–37]. Interestingly, although pluripotency markers seem to be lost after sexual determination, SSCs still express some of them [15].
Throughout the mammalian life cycle there are two reprogramming events that involve massive DNA demethylation. The first, after fertilization at the zygote stage, erases the epigenetic signature inherited from gametes (except for imprinted marks) and allows totipotency to be reacquired. The second, during PGC development, restores the epigenetic signature and erases imprinted marks. Just after specification, PGCs have a stable epigenetic status inherited from their epiblastic origin involving DNA methylation, and in females, X chromosome inactivation, which establishes an epigenetic barrier for the acquisition of totipotency. When PGCs enter the gonadal ridges they undergo global epigenetic methylation mark erasure and chromatin remodeling, a process that ends at around E13.5 when a basal epigenetic status (the most naı¨ve in mammals) is reached [2]. This germ line-specific epigenetic reprogramming process initiates at E8.0–E8.5. According to the most recent studies, it takes place in two phases: first, at an early phase, during migration, where active methylation is maintained in some specific regions; second, after gonadal ridge colonization, which affects sequences with epigenetic memory (imprinted regions, chromosome X CpG islands [CGIs], germ cell-related CGI promoters, etc.). However, there are other repetitive regions such as intracisternal A particles (IAPs) that are not affected by this epigenetic erasure. The mechanisms by which 5-methylcytosine (5mC) is lost is mediated by both 5-hydroxymethylcytosine (5hmC) conversion [1,29] and passive loss of 5mC marks over rounds of cellular divisions giving rise to a hypomethylated status at E13.5 [1]. After PGC demethylation, the genome must be remethylated de novo to acquire the sex-specific methylation profile of mature gametes. In male PGCs, de novo methylation occurs several days after the end of the erasure period at E14.5–E16.5, and continues up to the spermatogonia stage, while in female PGCs, the methylation pattern is established in the growing oocyte after birth. The end result is the mature methylation pattern characteristic of gametes, with their respective somatic sex imprinting marks [1]: in males, spermatozoa are highly Current Opinion in Genetics & Development 2014, 28:64–70
Closing the pluripotency cycle: the male side Gamete maturation is different depending on the sex. In females, germ cells go into meiotic arrest until puberty [30]. In males, germ cells remain in mitotic arrest, and when puberty arrives, they resume cellular divisions and differentiate in order to start meiosis giving rise to mature spermatozoa.
Box 1 Signaling mechanisms of primordial germ cell specification in mouse and humans. In mice, during early gastrulation (E5.0–E6.5), the cells located in the proximal epiblast are in direct contact with the signaling pathway from the extra-embryonic ectoderm (ExE) and acquire a germ cell fate when they become competent to respond to BMP4 through anteroposterior-axis signals, mainly NODAL and WNT3. Concomitantly, a BMP8b signals from the ExE prevents anti-posteriorization events, blocking inhibitory signals against germ line specification from the anterior visceral endoderm. BMP4, also from ExE, then induces Prdm1 and Prdm14 expression in a dose-specific manner through the Alk3 complex and BMP type II receptors (mainly BmprII) via SMAD1 and SMAD5 in the subpopulation of epiblast cells which will become specified to PGCs [6]. In humans, however, before gastrulation at day 8 of development, some pluripotent cells from the epiblast form the amnioblast, resulting in the formation of the amniotic cavity that separates the epiblastic cells from the trophoblastic layer. This means that in humans an equivalent structure to the murine ExE does not exist; the first evidence of PGCs in human embryos is at the end of the third week of gestation which can be recognized by their alkaline phosphatase (AP) activity detected at the wall of the yolk sac close to the base of the allantoides [10]. These differences between mice and humans in early postimplantation embryo biology suggest the existence of different mechanisms for PGC specification that correlates with the differing success in germ line cell derivation from PSCs from mouse or human origin. In mouse, embryonic stem cells (mESCs) show features similar to pre-implantation epiblastic cells and only have postimplantation-like characteristics when culture conditions are modified, thus allowing them to become epiblastic stem cell (EpiSCs)-like cells. Moreover, only mouse EpiSCs, but not PSCs can be differentiated in vitro into PGCs [3], while in humans PSCs respond directly to BMP4 signaling [23].
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The role of RNA-binding proteins in in vivo differentiation of human-derived germ cells has also recently been investigated. Germ-cell like cells derived from hiPSCs were able to colonize the lumen of the seminiferous tubules of sterilized immunodeficient mice better when VASA was added to the cocktail of reprogramming factors (OCT4, SOX2, KLF4, and c-MYC). These results highlight the role VASA plays in making cells competent to properly enter into meiosis and in controlling the pluripotency-state in a mixed in vitro/in vivo model [38]. This same model has been used to analyze the capability of human iPSCs derived from azoospermic men with different deletions in the Y chromosome, demonstrating how the
genetic background affects in vivo spermatogonial marker expression efficiency in these cells [39].
In vitro models for germ line generation from pluripotent stem cells Although pre-implantation embryo development is similar between mice and humans, there are some relevant differences during the early gastrulation events (Box 1). These differences are translated into the fact that there are morphological divergences between the mouse egg-cylinder and the human discoidal embryo; therefore it is not certain which specific area of the epiblast acquires a germinal fate in humans or where these induction
Table 1 In vitro germ cell-like derivation from pluripotent stem cells Type of pluripotent stem cell
Derivation method
Germ cell-like formation
Reported conclusions Human DAZL functions in PGC formation, whereas DAZ and BOULE promote later stages of meiosis and the development of haploid gametes in vitro Proper meiotic progression with extensive synaptonemal complex and post-meiotic haploid cell formation with an Acrosin staining pattern similar to humans spermatids in vitro Better efficiencies in meiotic progression and haploid cell formation in vitro with VASA overexpression Complete spermatogenesis in host mice and fertile offspring after ICSI Complete oogenesis in host mice and fertile offspring after ICSI
Human
ESC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of DAZ2, DAZL, BOULE. RA exposure.
Germ cell-like-cells
Human
iPSC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of DAZ2, DAZL, BOULE. RA exposure.
Germ cell-like-cells
Human
ESC and iPSC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of VASA. RA exposure.
Germ cell-like-cells
Mouse
ESC and iPSC
Sperm-like-cells
Mouse
ESC and iPSC
Mouse
ESC and iPSC
Human
iPSC
Human (deletions in the Y chromosome)
iPSC
Differentiation to EpiLCs with bFGF and activin A and then PGCLC generation by BMP4. Transplantation into neonatal mouse testis. Differentiation to EpiLCs with bFGF and activin A and then PGCLC generation by BMP4. Transplantation into mouse ovarian bursa Differentiation to EpiSCs with bFGF and activin A and then PGCLC generation by ectopic overexpression of Prdm1, Prdm14 and TFAP2 C Culture media supplementation with human BMP4, and BMP8a; ectopic expression of VASA; and transplantation into murine seminiferous tubules Culture media supplementation with human BMP4, and BMP8a; and transplantation into murine seminiferous tubules
Oocyte-like-cells
Sperm-like-cells
Induced PGCs
Induced PGCs
Complete spermatogenesis in host mice and fertile offspring after ICSI Morphologically and immunohistochemically recognizable human germ cells in vivo The efficiency of in vivo differentiation of induced PGCs depends on their genetic background
Reference [23]
[22]
[28]
[12]
[13]
[14]
[38]
[39]
ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; RA, retinoic acid; PGC, primordial germ cell; ICSI, intracytoplasmic sperm injection.
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68 Cell reprogramming, regeneration and repair
Figure 2
NAÏVE PLURIPOTENCY
(a)
PRIMED PLURIPOTENCY
Epiblast-like status induction
BMP4 signaling
GERM CELL DIFFERENTIATION In vivo microenvironment
Mouse preimplantation embryo
Mouse somatic cell
ICSI
mPSCs (mESCs & miPSCs)
mEpi-like
(b)
PGC-like
BMP4 signaling
Human preimplantation embryo
Functional male and female gametes
RNA-binding proteins
Fertile offspring
RA In vitro meiotic progression
Spg SSC
Human somatic cell
hPSCs (hESCs & hiPSCs)
PGC-like
Spg
In vivo microenvironment Spermatogonial markers
Current Opinion in Genetics & Development
Model for germ cell derivation in vitro. (a) Mouse germ cell derivation studies from [12,13,14]. Mouse embryonic stem cells (mESCs) or mouse induced pluripotent stem cells (miPSCs), in general PSCs, must be induced into an epiblastic-like (mEpi-like) status in which they are able to respond to the signaling pathway started by BMP4. A primordial germ cell (PGC)-like state is reached and these cells, in an appropriate in vivo microenvironment (i.e. transplantation into neonatal mouse testis or ovarian bursa) become functional spermatocytes or oocytes. After intracytoplasmic sperm injection (ICSI) these gametes generate fertile and healthy offspring of both sexes. (b) Human germ cell derivation studies from [22,23,28,38,39]. Human pluripotent stem cells (hPSCs) either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) present a primed pluripotency state, more similar to a mEpi-like state, and so they can directly respond to BMP4 signaling to attain a PGC-like status. PGC-like cells seem to need the presence of different RNA-binding proteins, to progress through meiosis and form haploid cells in vitro under the induction of retinoic acid (RA) and to express the correct spermatogonial markers when subjected to in vivo microenvironment control after xenotransplantation in immunosuppressed mouse testes. SSC, spermatogonial stem cell; Spg, spermatogonia.
signals originate [10]. Due to moral, ethical, technical, and legal difficulties regarding the use of post-implantation human embryos at the time when most of the main germ line specification events take place (gestational week 2 and 3), very little is known about how this process occurs in humans [40]. Therefore human embryonic stem cells (hESCs) and hiPSCs, generally referred to as pluripotent stem cells (PSCs) which are capable of differentiating not only into the three germinal layers but also into germ cells [22,27,28,38,41–44] have been used as a model to understand the genetic and epigenetic basis of germ cell specification and development in humans [45], and thus the in vitro induction of a germ-cell-like stage from a pluripotent base line has been demonstrated (Table 1). More recent and relevant assays demonstrate that two main events are required for PGC formation, again revealing the differences between mouse and human development. hESCs are more similar to mouse epiblast stem cells (EpiSCs) than to mouse embryonic stem cells (mESCs)$ [46,47]; in fact two different pluripotency Current Opinion in Genetics & Development 2014, 28:64–70
states are represented by these cells: a naı¨ve state, characteristic of mESCs, and a primed pluripotent state characteristic of EpiSCs and hESCs in which cells are more likely to differentiate towards different cell types [48]. Therefore, in mouse, the key event required to obtain a properly primed pluripotency state is the induction of an epiblast-like state before germ cell derivation while in humans, this major obstacle seems to be correct entry into meiosis led by RNA-binding proteins (Figure 2). In this review we have shown key examples of how in vitro models can be used as a tool to understand the main genetic and epigenetic steps for germ cell specification and posterior development, not only in mouse but also in humans, which explains why interest in in vitro gamete derivation research is increasing [40]. The networks related to pluripotency in PSCs and PGCs, the strong correlation between germ line specification and maintenance of pluripotency [3,15,49], and paradoxically, the fact that in vivo PGCs must overcome epigenetic pluripotency barriers may explain why, to date, gamete derivation in vitro has only been described from a PSC origin, and the reason for the differences between mouse and www.sciencedirect.com
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human in vitro models. A remaining question is whether epigenetic pluripotency barriers must be completely overcome or instead, direct derivation of germ cell-like cells through meiotic stages can be achieved from either mouse or human somatic cells.
12. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M: Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011, 146:519-532. PGCLC derivation in vitro from both ESCs and PSCs via an Epi-LC state caused by BMP4 induction. These PGCLCs recapitulated the spermatogenic program when injected into neonatal mouse testis, giving rise to healthy and fertile offspring. In the following year authors from this group obtained offspring from oocytes derived with the same technology.
Acknowledgements
13. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M: Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012, 338:971-975.
Research in CS lab is supported by a research project grant PI13/00546 from ISCIII, co-founded by the European Regional Development Fund ‘A way to make Europe’, a FPU grant conceded to AMMA (AP-2009-4522) by the Spanish Ministry of Education, and a Sara Borrell grant conceded to JVM (CD12/00568) by the ISCIII.
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Current Opinion in Genetics & Development 2014, 28:64–70
39. Ramathal C, Durruthy-Durruthy J, Sukhwani M, Arakaki JE, Turek PJ, Orwig KE, Reijo Pera RA: Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to murine seminiferous tubules. Cell Rep 2014. iPSCs reprogrammed from patients with azoospermia can be differentiated into germ cell-like cells in vitro and in vivo after xenotransplantation into germ-cell depleted seminiferous tubules from immunodeficient mice. 40. Medrano JV, Pera RA, Simon C: Germ cell differentiation from pluripotent cells. Semin Reprod Med 2013, 31:14-23. 41. Clark AT, Bodnar MS, Fox MS, Rodriquez RT, Abeyta MJ, Firpo MT, Reijo Pera RA: Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Genet 2004, 13:727-739. 42. Park TS, Galic Z, Conway AE, Lindgren A, van Handel BJ, Magnusson M, Richter L, Teitell MA, Mikkola HK, Lowry WE et al.: Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells. Stem Cells 2009, 27:783-795. 43. Bucay N, Yebra M, Cirulli V, Afrikanova I, Kaido T, Hayek A, Montgomery A: A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells. Stem Cells 2009, 27:68-77. 44. Tilgner K, Atkinson S, Golebiewska A, Stojkovic M, Lako M, Armstrong L: Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 2008, 26:3075-3085. 45. Marques-Mari AI, Lacham-Kaplan O, Medrano JV, Pellicer A, Simon C: Differentiation of germ cells and gametes from stem cells. Hum Reprod Update 2009, 15:379-390. 46. Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RD: New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007, 448:196-199. 47. Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, Kubota Y, Ogawa T: In vitro production of functional sperm in cultured neonatal mouse testes. Nature 2011, 471:504-507. 48. Nichols J, Smith A: Naive and primed pluripotent states. Cell Stem Cell 2009, 4:487-492. 49. Hayashi K, Saitou M: Perspectives of germ cell development in vitro in mammals. Anim Sci J 2014.
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ScienceDirect Germ line development: lessons learned from pluripotent stem cells Ana M Martı´nez-Arroyo1,2,5, Jose V Medrano1,3,5, Jose´ Remohı´1,3 and Carlos Simo´n1,2,4 Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. Addresses 1 Fundacio´n Instituto Valenciano de Infertilidad (FIVI), Dept. Obst. & Gynec., Valencia University, Valencia, Spain 2 INCLIVA Biomedical Research Institute, Valencia 46015, Spain 3 Fundacio´n Instituto de Investigacio´n Sanitaria La Fe, Valencia 46026, Spain 4 Department of Obstetrics and Gynecology, Stanford University, Stanford, CA 94305, USA Corresponding author: Simo´n, Carlos ([email protected], [email protected]) 5 These authors contributed equally to this work.
Current Opinion in Genetics & Development 2014, 28:64–70 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Jose´ CR Silva and Renee A Reijo Pera For a complete overview see the Issue and the Editorial
‘switch on’ the germ line program so that these cells can acquire pluripotency properties [1]. Thus, there is a latent pluripotent cycle in germ cells that is maintained by a network that controls the non-restrictive PGC epigenome [2,3,4]. In this review we will focus on the main genetic and epigenetic events in germ line development examining evidence from in vivo studies that is supported by in vitro models (Figure 1).
Genetic regulation of germ line development PGCs can first be identified in mice on embryonic day 6.25 (E6.25), when some cells in the proximal epiblast start to express Prdm1 [5] in response to the BMP4 and BMP8b signals secreted by extraembryonic ectoderm [6]. Specification by BMP4 and BMP8b induce Prdm1 and Prdm14 expression in a cooperative dose-specific way to ensure sufficient exposure to epiblast BMP signaling in order to specify germ lineage fate [6,7]. The transcriptional repressor Prdm1 is essential for suppressing the somatic program [5,8]; subsequently Prdm14 promotes epigenetic reprogramming so that a pluripotent status can be reacquired. Prdm14 expression is initially independent of Prdm1 however its maintenance is strictly dependent on it [9,10]. Together with these two transcription factors, Tcfap2C has also been described as a critical element which acts downstream of Prdm1, preventing mesodermal differentiation and enhancing the germ cell program [11].
Available online 14th October 2014 http://dx.doi.org/10.1016/j.gde.2014.09.011 0959-437X/# 2014 Elsevier Ltd. All rights reserved.
Pluripotency and the germ cell cycle In organisms with sexual reproduction, germ cells allow the life-cycle to continue through generations. In the case of mammals and other vertebrates, there is a clear separation of the germ line from the somatic lineages at the early developmental stages of the embryo. In the murine model, the germ line is formed at gastrulation from a small number of founder cells called primordial germ cells (PGCs) which escape their somatic fate and acquire a germinal fate. The moment of PGC specification in mammals involves global epigenetic reprogramming mechanisms that ‘turn off’ the somatic program and Current Opinion in Genetics & Development 2014, 28:64–70
The BMP4/Prdm1 signaling pathway which initiates at the moment of PGC specification seems to be crucial for future PGCs to acquire the proper epigenetic and transcriptional background which would enable them to be correctly reprogrammed and develop into functional gametes. In fact, studies based on mouse models describe the derivation of live offspring by recapitulating the germ cell-specification pathway in a two-step in vitro model. Since only cells from the epiblast are able to respond to BMP signaling, mouse pluripotent stem cells (PSCs) were first differentiated into epiblast-like cells (EpiLCs), which represent a cellular state very close to epiblast cells before gastrulation. Subsequently, EpiLCs were differentiated into PGC-like cells (PGCLCs) by two methods: adding BMP4 to the culture media, mimicking the BMP4 signal specification which epiblastic cells receive from the adjacent ectoderm in vivo [12,13]; or alternatively the overexpression of Prdm1, Prdm14, and Tfap2C genes, which act on the PSCs downstream of the www.sciencedirect.com
Germ line development Martı´nez-Arroyo et al. 65
Figure 1
Fertilization
E3.5 Oct4, Nanog
Meiosis Oct4, Nanog
Oct4, Nanog
Spc I SSC Spg
E6.5
E7.5
E8.5
E9.5
PGC determination
E11.5
PGC migration
Spg
E13.5 Sex determination
Gametogenesis
Mvh
Prdm1 Prdm14
Daz
Tcfap2c
Boule
Current Opinion in Genetics & Development
Primordial germ cell specification and migration in the post-implantation embryo. Primordial germ cell (PGC) precursors and PGCs are shown in pink during determination at embryonic day 6.5 (E6.5), E7.5, E8.5, during migration at E9.5 and E11.5, and in sex determination at E13.5. Extraembryonic ectoderm from which BMP signaling specifies PGC precursors is shown in purple. Only male gametogenesis is shown. The gene expression timing pattern is indicated with black arrows. Dotted line indicates loss of gene expression. SSC, spermatogonial stem cell; Spg, spermatogonia; Spc I, spermatocyte I.
BMP signaling pathway [13]. The PGCLCs obtained by these approaches showed imprinting patterns according to their embryonic somatic sex and when transplanted into host mice, they went through both complete spermatogenesis [12,14] and oogenesis, producing fertile offspring following intracytoplasmic sperm injection (ICSI)$ [13]. In summary, germ line development in the murine model requires somatic program suppression, then the re-acquisition of pluripotency, and finally reprogramming to a unique and non-restrictive epigenome [6,15]. The signaling for this process is triggered by BMP and the Prdm1/Prdm14/Tfap2c pathways. From their specification at the proximal epiblast region at E6.25, the germ line development process occurs sequentially during their migration from the posterior hindgut to the forming gonad at around E8.5–E9.5 [16,17]. Once they reach the gonads, PGCs start to migrate bilaterally at the gonadal ridges at E10.5–E11.5. From this point, germ cells change their genetic expression program and epigenetic profiles, and start to express key genes for survival and maturation like DAZL [18] and VASA [19] (Figure 1). In humans, other genes from the deleted in azoospermia (DAZ) family, such as DAZ2 or BOLL, have been demonstrated to be necessary for proper male germ line development [19–21]. After gonadal colonization, PGCs initiate their sexual determination guided by the www.sciencedirect.com
surrounding microenvironment of the gonadal niche (i.e. sertoli or granulosa cells) and acquire a sex-specific epigenetic status that involves DNA methylation and, in females, X chromosome inactivation. Thus, as gametogenesis goes forward pluripotency decreases and the germ cell specific expression program becomes activated. In contrast to the somatic tissues, the germ line is characterized by a set of specific features among which the most important is their ability to reduce their chromosomal charge during meiosis. The importance of RNA-binding proteins on meiotic progression has been elucidated using in vitro models in several studies that describe how the ectopic overexpression of DAZ family genes (i.e. DAZ2, DAZL, BOULE, etc.) enables proper meiotic progression in both hESCs and human induced pluripotent stem cell (hiPSC)-derived germ cell-like cells [22,23]. It has been proposed that germ line related RNA-binding proteins, including VASA, NANOS, DAZL, and PIWI, that are highly conserved at different stages of germ line development [24] may act as chaperones, mediating proper protein folding [25], as well as acting as post-transcriptional meiosis-related protein regulators. In humans, the DAZ family is composed of the autosomal genes BOULE and DAZL, and the DAZ gene cluster which is encoded on the Y chromosome. In mouse, Current Opinion in Genetics & Development 2014, 28:64–70
66 Cell reprogramming, regeneration and repair
Daz acts as a master regulator of spermatogenesis, whilst Dazl is critical for meiosis [26]. In humans, evidence suggests that loss of BOULE gene function may result in azoospermia with primary defects in meiotic transition [27]. Similarly, ectopic expression of VASA, another highly conserved RNA-binding protein has shown a similar effect on the induction of meiosis [28]. Interestingly, mouse knock-out models for the Mouse vasa homolog (Mvh) show male infertility, while the Drosophila phenotype shows female sterility [21].
methylated (around 80%), while in females, oocytes are about 30% methylated [1].
Epigenetic regulation during germ line development
The spermatogonial stem cell (SSCs) subpopulation leads to male germ cell propagation through adult life [31], and are considered to be the male germ line stem cells [32]. In humans, SSCs are present at the basal membrane of the seminiferous tubules from the moment of birth while in mice the gonocyte-to-spermatogonia transition takes place 1–4 days postpartum [33]. Once sexual maturity is reached, quiescent spermatogonia start to asymmetrically divide giving rise to originating mitotically active spermatogonia, to advance into spermatogenesis, but also self-renew in order to maintain the adult testicular stem cell population [34–37]. Interestingly, although pluripotency markers seem to be lost after sexual determination, SSCs still express some of them [15].
Throughout the mammalian life cycle there are two reprogramming events that involve massive DNA demethylation. The first, after fertilization at the zygote stage, erases the epigenetic signature inherited from gametes (except for imprinted marks) and allows totipotency to be reacquired. The second, during PGC development, restores the epigenetic signature and erases imprinted marks. Just after specification, PGCs have a stable epigenetic status inherited from their epiblastic origin involving DNA methylation, and in females, X chromosome inactivation, which establishes an epigenetic barrier for the acquisition of totipotency. When PGCs enter the gonadal ridges they undergo global epigenetic methylation mark erasure and chromatin remodeling, a process that ends at around E13.5 when a basal epigenetic status (the most naı¨ve in mammals) is reached [2]. This germ line-specific epigenetic reprogramming process initiates at E8.0–E8.5. According to the most recent studies, it takes place in two phases: first, at an early phase, during migration, where active methylation is maintained in some specific regions; second, after gonadal ridge colonization, which affects sequences with epigenetic memory (imprinted regions, chromosome X CpG islands [CGIs], germ cell-related CGI promoters, etc.). However, there are other repetitive regions such as intracisternal A particles (IAPs) that are not affected by this epigenetic erasure. The mechanisms by which 5-methylcytosine (5mC) is lost is mediated by both 5-hydroxymethylcytosine (5hmC) conversion [1,29] and passive loss of 5mC marks over rounds of cellular divisions giving rise to a hypomethylated status at E13.5 [1]. After PGC demethylation, the genome must be remethylated de novo to acquire the sex-specific methylation profile of mature gametes. In male PGCs, de novo methylation occurs several days after the end of the erasure period at E14.5–E16.5, and continues up to the spermatogonia stage, while in female PGCs, the methylation pattern is established in the growing oocyte after birth. The end result is the mature methylation pattern characteristic of gametes, with their respective somatic sex imprinting marks [1]: in males, spermatozoa are highly Current Opinion in Genetics & Development 2014, 28:64–70
Closing the pluripotency cycle: the male side Gamete maturation is different depending on the sex. In females, germ cells go into meiotic arrest until puberty [30]. In males, germ cells remain in mitotic arrest, and when puberty arrives, they resume cellular divisions and differentiate in order to start meiosis giving rise to mature spermatozoa.
Box 1 Signaling mechanisms of primordial germ cell specification in mouse and humans. In mice, during early gastrulation (E5.0–E6.5), the cells located in the proximal epiblast are in direct contact with the signaling pathway from the extra-embryonic ectoderm (ExE) and acquire a germ cell fate when they become competent to respond to BMP4 through anteroposterior-axis signals, mainly NODAL and WNT3. Concomitantly, a BMP8b signals from the ExE prevents anti-posteriorization events, blocking inhibitory signals against germ line specification from the anterior visceral endoderm. BMP4, also from ExE, then induces Prdm1 and Prdm14 expression in a dose-specific manner through the Alk3 complex and BMP type II receptors (mainly BmprII) via SMAD1 and SMAD5 in the subpopulation of epiblast cells which will become specified to PGCs [6]. In humans, however, before gastrulation at day 8 of development, some pluripotent cells from the epiblast form the amnioblast, resulting in the formation of the amniotic cavity that separates the epiblastic cells from the trophoblastic layer. This means that in humans an equivalent structure to the murine ExE does not exist; the first evidence of PGCs in human embryos is at the end of the third week of gestation which can be recognized by their alkaline phosphatase (AP) activity detected at the wall of the yolk sac close to the base of the allantoides [10]. These differences between mice and humans in early postimplantation embryo biology suggest the existence of different mechanisms for PGC specification that correlates with the differing success in germ line cell derivation from PSCs from mouse or human origin. In mouse, embryonic stem cells (mESCs) show features similar to pre-implantation epiblastic cells and only have postimplantation-like characteristics when culture conditions are modified, thus allowing them to become epiblastic stem cell (EpiSCs)-like cells. Moreover, only mouse EpiSCs, but not PSCs can be differentiated in vitro into PGCs [3], while in humans PSCs respond directly to BMP4 signaling [23].
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The role of RNA-binding proteins in in vivo differentiation of human-derived germ cells has also recently been investigated. Germ-cell like cells derived from hiPSCs were able to colonize the lumen of the seminiferous tubules of sterilized immunodeficient mice better when VASA was added to the cocktail of reprogramming factors (OCT4, SOX2, KLF4, and c-MYC). These results highlight the role VASA plays in making cells competent to properly enter into meiosis and in controlling the pluripotency-state in a mixed in vitro/in vivo model [38]. This same model has been used to analyze the capability of human iPSCs derived from azoospermic men with different deletions in the Y chromosome, demonstrating how the
genetic background affects in vivo spermatogonial marker expression efficiency in these cells [39].
In vitro models for germ line generation from pluripotent stem cells Although pre-implantation embryo development is similar between mice and humans, there are some relevant differences during the early gastrulation events (Box 1). These differences are translated into the fact that there are morphological divergences between the mouse egg-cylinder and the human discoidal embryo; therefore it is not certain which specific area of the epiblast acquires a germinal fate in humans or where these induction
Table 1 In vitro germ cell-like derivation from pluripotent stem cells Type of pluripotent stem cell
Derivation method
Germ cell-like formation
Reported conclusions Human DAZL functions in PGC formation, whereas DAZ and BOULE promote later stages of meiosis and the development of haploid gametes in vitro Proper meiotic progression with extensive synaptonemal complex and post-meiotic haploid cell formation with an Acrosin staining pattern similar to humans spermatids in vitro Better efficiencies in meiotic progression and haploid cell formation in vitro with VASA overexpression Complete spermatogenesis in host mice and fertile offspring after ICSI Complete oogenesis in host mice and fertile offspring after ICSI
Human
ESC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of DAZ2, DAZL, BOULE. RA exposure.
Germ cell-like-cells
Human
iPSC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of DAZ2, DAZL, BOULE. RA exposure.
Germ cell-like-cells
Human
ESC and iPSC
Culture media supplementation with human BMP4, and BMP8a; and ectopic expression of VASA. RA exposure.
Germ cell-like-cells
Mouse
ESC and iPSC
Sperm-like-cells
Mouse
ESC and iPSC
Mouse
ESC and iPSC
Human
iPSC
Human (deletions in the Y chromosome)
iPSC
Differentiation to EpiLCs with bFGF and activin A and then PGCLC generation by BMP4. Transplantation into neonatal mouse testis. Differentiation to EpiLCs with bFGF and activin A and then PGCLC generation by BMP4. Transplantation into mouse ovarian bursa Differentiation to EpiSCs with bFGF and activin A and then PGCLC generation by ectopic overexpression of Prdm1, Prdm14 and TFAP2 C Culture media supplementation with human BMP4, and BMP8a; ectopic expression of VASA; and transplantation into murine seminiferous tubules Culture media supplementation with human BMP4, and BMP8a; and transplantation into murine seminiferous tubules
Oocyte-like-cells
Sperm-like-cells
Induced PGCs
Induced PGCs
Complete spermatogenesis in host mice and fertile offspring after ICSI Morphologically and immunohistochemically recognizable human germ cells in vivo The efficiency of in vivo differentiation of induced PGCs depends on their genetic background
Reference [23]
[22]
[28]
[12]
[13]
[14]
[38]
[39]
ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; RA, retinoic acid; PGC, primordial germ cell; ICSI, intracytoplasmic sperm injection.
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68 Cell reprogramming, regeneration and repair
Figure 2
NAÏVE PLURIPOTENCY
(a)
PRIMED PLURIPOTENCY
Epiblast-like status induction
BMP4 signaling
GERM CELL DIFFERENTIATION In vivo microenvironment
Mouse preimplantation embryo
Mouse somatic cell
ICSI
mPSCs (mESCs & miPSCs)
mEpi-like
(b)
PGC-like
BMP4 signaling
Human preimplantation embryo
Functional male and female gametes
RNA-binding proteins
Fertile offspring
RA In vitro meiotic progression
Spg SSC
Human somatic cell
hPSCs (hESCs & hiPSCs)
PGC-like
Spg
In vivo microenvironment Spermatogonial markers
Current Opinion in Genetics & Development
Model for germ cell derivation in vitro. (a) Mouse germ cell derivation studies from [12,13,14]. Mouse embryonic stem cells (mESCs) or mouse induced pluripotent stem cells (miPSCs), in general PSCs, must be induced into an epiblastic-like (mEpi-like) status in which they are able to respond to the signaling pathway started by BMP4. A primordial germ cell (PGC)-like state is reached and these cells, in an appropriate in vivo microenvironment (i.e. transplantation into neonatal mouse testis or ovarian bursa) become functional spermatocytes or oocytes. After intracytoplasmic sperm injection (ICSI) these gametes generate fertile and healthy offspring of both sexes. (b) Human germ cell derivation studies from [22,23,28,38,39]. Human pluripotent stem cells (hPSCs) either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) present a primed pluripotency state, more similar to a mEpi-like state, and so they can directly respond to BMP4 signaling to attain a PGC-like status. PGC-like cells seem to need the presence of different RNA-binding proteins, to progress through meiosis and form haploid cells in vitro under the induction of retinoic acid (RA) and to express the correct spermatogonial markers when subjected to in vivo microenvironment control after xenotransplantation in immunosuppressed mouse testes. SSC, spermatogonial stem cell; Spg, spermatogonia.
signals originate [10]. Due to moral, ethical, technical, and legal difficulties regarding the use of post-implantation human embryos at the time when most of the main germ line specification events take place (gestational week 2 and 3), very little is known about how this process occurs in humans [40]. Therefore human embryonic stem cells (hESCs) and hiPSCs, generally referred to as pluripotent stem cells (PSCs) which are capable of differentiating not only into the three germinal layers but also into germ cells [22,27,28,38,41–44] have been used as a model to understand the genetic and epigenetic basis of germ cell specification and development in humans [45], and thus the in vitro induction of a germ-cell-like stage from a pluripotent base line has been demonstrated (Table 1). More recent and relevant assays demonstrate that two main events are required for PGC formation, again revealing the differences between mouse and human development. hESCs are more similar to mouse epiblast stem cells (EpiSCs) than to mouse embryonic stem cells (mESCs)$ [46,47]; in fact two different pluripotency Current Opinion in Genetics & Development 2014, 28:64–70
states are represented by these cells: a naı¨ve state, characteristic of mESCs, and a primed pluripotent state characteristic of EpiSCs and hESCs in which cells are more likely to differentiate towards different cell types [48]. Therefore, in mouse, the key event required to obtain a properly primed pluripotency state is the induction of an epiblast-like state before germ cell derivation while in humans, this major obstacle seems to be correct entry into meiosis led by RNA-binding proteins (Figure 2). In this review we have shown key examples of how in vitro models can be used as a tool to understand the main genetic and epigenetic steps for germ cell specification and posterior development, not only in mouse but also in humans, which explains why interest in in vitro gamete derivation research is increasing [40]. The networks related to pluripotency in PSCs and PGCs, the strong correlation between germ line specification and maintenance of pluripotency [3,15,49], and paradoxically, the fact that in vivo PGCs must overcome epigenetic pluripotency barriers may explain why, to date, gamete derivation in vitro has only been described from a PSC origin, and the reason for the differences between mouse and www.sciencedirect.com
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human in vitro models. A remaining question is whether epigenetic pluripotency barriers must be completely overcome or instead, direct derivation of germ cell-like cells through meiotic stages can be achieved from either mouse or human somatic cells.
12. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M: Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011, 146:519-532. PGCLC derivation in vitro from both ESCs and PSCs via an Epi-LC state caused by BMP4 induction. These PGCLCs recapitulated the spermatogenic program when injected into neonatal mouse testis, giving rise to healthy and fertile offspring. In the following year authors from this group obtained offspring from oocytes derived with the same technology.
Acknowledgements
13. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M: Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012, 338:971-975.
Research in CS lab is supported by a research project grant PI13/00546 from ISCIII, co-founded by the European Regional Development Fund ‘A way to make Europe’, a FPU grant conceded to AMMA (AP-2009-4522) by the Spanish Ministry of Education, and a Sara Borrell grant conceded to JVM (CD12/00568) by the ISCIII.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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