The generation of artificial gametes will not only provide therapeutic advantages for those people wishing to have genetically related progeny and for whom donor gametes is their only current chance to have babies, but it will also provide new knowledge in the field of germ cell biology. The in vitro generation of human gametes will help to understand the biological process of germ cell specification and maturation. Nowadays, developmental studies regarding human germ cell development are performed by using animal models due to the lack of available human samples. However, it has been widely shown that gametogenesis is species-specific, and although the general developmental process is conserved among mammals, there are some differences that hampers the direct translation of the knowledge acquired in mouse into human [13,14]. The main interests on artificial gametes production are focused on the establishment of a reproducible method to generate fully committed haploid sperm and oocytes that will offer large amounts of cells at different stages of germ cell formation and that will be the basis for further experiments. This is of outstanding interest for the understanding of the different molecular and cellular events that take place during normal human gametogenesis and subsequent study of human disease models related to germ cell defects or other diseases producing infertility and lack of functional gametes. If obtaining artificial gametes from disease patients, the mechanisms of some kinds of infertility could be unraveled and thus potential treatments could be developed for those patients devoid of functional gametes. Also, by combining artificial gamete production from stem cells with new gene editing techniques, the correction of diseases could be potentially carried out in those patients suffering from inherited genetic disorders, so that their offspring would not suffer, nor transmit the disease to their progeny. On the other hand, the information obtained from in vitro gametogenesis models could be used to avoid the maturation of germ cells, leading to new and effective contraceptives.

As an example of the scientific strength of artificial gamete production from pluripotent cells, Dominguez and collaborators have recently published a study in which induced pluripotent stem cells from Turner syndrome individuals were differentiated in vitro into germ cell-like cells (GCLC) and compared to GCLC from control individuals [15]. The results of this work showed that aneuploidies in X chromosome do not impair germ cell formation but the correct dose of X chromosome is critical for the maintenance or functionality of those GCLC until adulthood, uncovering the origin of infertility in Turner syndrome patients.

There are numerous adult tissues, if not all, that have the ability to regenerate themselves due to the existence of a specific population of adult stem cells with capacity for self-renewal and specification within their cell lineages [30,31,32]. The existence of this type of stem cells in the testis, the so-called spermatogonial stem cells (SSCs ), has been widely demonstrated [33]. They are located in the basal lamina of the seminiferous tubule and are responsible for the continued production of sperm during the life of males. SSCs start to asymmetrically divide when sexual maturity is reached, giving rise to mitotically active spermatogonia, whilst also self-renewing [34,35,36,37], always under the control of a very specific microenvironment directed by Sertoli cells by building the balance between self-renewal and differentiation. It is possible to identify and purify human SSC by using different membrane markers such as α6-integrin (CD49f) and β1-integrin, two laminin receptors [38], CD9, a basal membrane cell attachment protein [39] and other markers [37,40,41,42,43,44]. SSC culture is challenging due to the need of blocking differentiation, maintenance of long-term survival, and promoting of proliferation. Attempts of in vitro spermatogenesis demonstrate promising results in humans, where SSCs isolated from human biopsies and co-cultured with Sertoli cells have shown expression of markers for meiotic progression and haploid cell formation [45]. However they have not fared better, due in part to the need for a co-culture system that mimics the microenvironment of testicular niche. Perhaps for this reason neither fertilization nor healthy offspring have been reported to date with gametes generated by this mechanism (Table 1) [13,14,15,45,46,47,48,49,50,51,52,53,54,55,56,57,58].

In the female counterpart, it is generally accepted that human ovaries contain a fixed number of non-growing follicles established before birth that decreases with female age and is depleted in menopause [59,60,61,62,63,64,65,66,67,68]. Some researchers very early on struggled against the growing dogma about the finite number of follicles and oocytes at birth [69]. In the mouse model, the existence of ovarian stem cells leading to viable offspring was first reported by Zou et al. [70] in 2009. In spite of the fact that there is no evidence of persistence of oogonia in adult human ovaries, a new idea about germinal (oogonial) stem cells in adult ovaries has been developed [71,72,73,74]. The existence of stem cells in the human ovary has been surrounded by controversy. Some works in mouse and human have described a population of rare ovarian stem cells able to generate oocytes in vitro [55,75], representing an invaluable promise in regenerative medicine to treat infertile women. However, these works have been widely refuted, and some other authors consider that the presence of ovarian stem cells has been overestimated [76,77,78,79]. The ovarian stem cells are the most interesting population of cells for potential autologous de novo oogenesis and regeneration of non-functional ovaries in infertile women. In the human adult ovaries, putative ovarian stem cells (OSC) can be isolated by Fluorescence-activated cell sorting (FACS) as small rounded VASA positive-cells from the ovarian surface epithelium. These cells can grow and develop into oocyte-like cells in vivo and in vitro [55,80,81,82]. Interestingly there appears to be more positive results for the in vitro generation of female gametes rather to sperm (Table 1). This may be due to the greater ease to perform co-culture of OSC with the surrounding somatic cells; because follicle-like structure is easier to reproduce in vitro [55]. However, this starting material is limited or even absent in infertile patients so they do not seem to be the best option for regenerative medicine purposes on female infertile patients. For this reason pluripotent stem cells (PSCs) seem to be a better option, according to its greater availability in those kind of patients.

First evidences of in vitro germ line formation from pluripotent stem cells in humans were reported by spontaneous differentiation [83,84] of hESCs embryoid bodies [51,54]. Undifferentiated hESCs expressed some early germ cell markers such as c-KIT and DAZL, but not later markers such as VASA (a RNA helicase involved in germ cell maturation in both sexes) or SYCP3 (a structural protein of the synaptonemal complex critical for meiosis) [85], suggesting that hESCs could be a heterogeneous pluripotent population in which some cells had a pre-disposition towards a germ cell fate. Addition of several molecules have been shown to improve the differentiation efficiency of pluripotent cells to germ cells, this is the case of BMP cytokines [51,52,56], and retinoic acid (RA) that has been demonstrated to induce meiosis [15,50,86]. However, meiotic progression remained as one of the main obstacles for artificial gametes derivation [87]; most of the works published to date report spontaneous differentiation towards putative PGCs or PGC-like cells (PGCLC) with diploid (2N) content of DNA (Table 1), but the completion of meiosis to give haploid (1N) cells remains a challenge when spontaneous differentiation is used.

In fact, complete meiotic progression of in vitro derived human germ cells was achieved by inducing the ectopic expression of the DAZ gene family members (DAZ2, DAZL, and BOULE) not only in hESC but also in human iPSCs (hiPSCs) lines subjected to spontaneous differentiation [46,47]. 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. Also RNA-binding proteins, like VASA, could have a possible role in meiotic entry control [48]. A recent study have provided a more accurate process, in which pluripotent stem cells were first cultured in 4i conditions to induce the naïve pluripotent state, this step make cells prone to response to specification signals [88]. Then, the derivation process was performed in culture media supplemented with BMPs, leukemia inhibitory factor (LIF), Rho kinase (ROCK) inhibitor and Knockout serum replacement, to obtain the PGC precursors and then PGCLCs [13,14]. This system allows for the generation of large numbers of cells for use in research procedures. In summary, human artificial meiotic or sperm-like cells had been generated from both ESCs and iPSCs, assessed by the expression of meiotic and post-meiotic markers and DNA ploidy (Table 1).

On the female side, less studies have been performed with hESC and hiPSC for oocytes generation, partially due to the difficulties derived from the complexity of the own oocyte cell. Nonetheless previous data has shown that oocyte-like cells, expressing oocyte-specific markers, can be developed from ESCs and iPSCs [53,89,90,91,92,93]. These oocyte-like cells were developed from stem cells that expressed at least a degree of pluripotency, and the resulting oocyte-like cells expressed some germinal markers like the structural meiotic protein SYCP3 [90] as well as c-KIT, ZP1, ZP2, ZP3, VASA, and DMC1 [81] but were still far from being real and competent oocytes. Finally, the most challenged goal in the artificial gamete generation seems to be the production of sperm and eggs from the opposite sex. This would be of great importance to gay couples who wish to have genetically related offspring. However, little has been achieved in this direction and only a couple of papers reporting generation of sperm from female cells exist, albeit this sperm was not fully functional and presented incorrect or unknown epigenetic status [94,95].

Determining functionality of derived gametes will, therefore, require establishing their capacity for fertilization and early embryogenesis. In order to consider artificial sperm for clinical purposes, they should be tested to be able to produce the main biological proceses leading to fertilization as the initiation of oocyte activation, nuclear remodelling and subsequent fusion to the oocyte nucleus, and loss of the own nuclear envelop [96]. On the other side, when artificially derived eggs needs to be assessed for clinical use they should be able to escape from meiotic arrest, undergo pronuclear formation, initiate embryonic gene activation while recapitulating nuclear maturation events (i.e., germinal vesicle breakdown, chromosomal segregation, polar body extrusion) and cytoplasmic reorganization (i.e., adenosine triphosphate (ATP) supply by mitochondria, protein and mRNA storage) [97]. Finally, to fully address the generation of functional artificial gametes they need to be proved to successfully give a healthy embryo upon fecundation, showing correct genetic and epigenetic reprogramming, cleavage divisions and zygotic genome activation [98].

Ethical restrictions make difficult to carry out functional assays in humans with germ cells obtained from stem cells. Moreover, differences in the pluripotent status of mESCs (naïve-like state) and hESCs (primed state) make difficult to compare their potential to fully develop into functional germ cells [99]. The main obstacle to develop fully committed sperm and eggs from stem cells is the lack of knowledge regarding molecules and factors, which are important in the gamete maturation process in the gonad niche and are not present when the immature gametes (OSC or SSC) are exposed to in vitro culture conditions. For all these reasons, the functionality and safety of the obtained GCLC needs to be first tested in animal models.

The most similar to functional assays are xenotransplantation of human germ cell-like cells into seminiferous tubules of immunosuppressed mice, in the male counterpart. These animal models are very helpful to test the ability of human germ cell-like cells to behave like human germ cell progenitors. In this context, the role of RNA-binding proteins has also been tested, demonstrating that hiPSCs improved their ability to colonize the lumen of the seminiferous tubules of sterilized immunodeficient mice upon VASA overexpression in combination with pluripotency factors. These results highlight the role of VASA in making cells competent to form germ cell development and in controlling the pluripotency-state in a combined in vitro/in vivo model [49].

This murine/human heterologous model represents a very interesting analysis method from a clinical point of view, and has already been used to analyze the ability of hiPSCs-GCLC derived from azoospermic men with different deletions in the Y chromosome to colonize the spermatogonial niche, demonstrating how the genetic background affects their capability to differentiate into germ cells and colonize the seminiferous niche in vivo [49]. Finally, this same approach has been recently used to demonstrate that GCLC can be formed in vivo independently of X chromosome dosage [15]. In this case, hiPSCs from Turner syndrome patients were obtained, differentiated to GCLC and xenotransplanted into the seminiferous tubules of sterilized immunodeficient mice, demonstrating that the two X chromosomes are not required for human germ cell formation but for their maintenance until adulthood [15].

Also in vitro maturation of human oocytes is one of the most difficult tasks in the IVF programs. It has been proposed that transplantation of stem cells or stem cell-derived "oocytes" into the ovary or other organs would be of great advantage because it would provide a natural niche for maturation of oocytes and bypass several obstacles related to oocyte in vitro maturation [100]. In the mouse model it was confirmed that maturation of ESC-derived oocytes ultimately fails in vitro, and to overcome this obstacle the transplantation of ESC-derived oocyte-germ cells into an ovarian niche has been proposed to direct their natural and functional maturation. To demonstrate this, ESC-derived germ cells were enclosed into ovarian follicles, and some of them reached the primordial or primary stage of development after the two-step protocol by performing in vitro specification from mESCs and subsequent maturation in an in vivo model [100].

In 2006, the birth of murine offspring obtained by fertilization of murine oocytes with artificial mESCs-derived sperm was achieved [101] proving for the first time that stem cells could be developed in vitro to obtain autologous germ cells for reproductive treatments. However, the offspring presented phenotypic abnormalities as well as reduced life-span, mainly because of epigentic defects accumulated during germline in vitro specification and maturation [101]. Later on, Hayashi and collaborators reported the generation of healthy and fertile pups by fertilization of mouse oocytes with artificial sperm obtained by two-step differentiation of mESC through an intermediate epiblastic-like cells [102]. In this work, the authors assessed not only the cellular events and transcriptomics along the generation of gametes, but also their epigenetic profiles, showing that the in vitro-derived germ cells closely resemble the physiological process [102], revealing the importance of the acquisition of a new epigenetic program in parallel to transcriptomic changes during differentiation from stem to germ cells. It has been also reported that erroneous epigentic imprinting can produce diseases in humans [103] enphasizing the relevance of epigenetics in the developmental process from germ cells to adult life.

As discussed before, one of the crucial facts in PGC specification is an extensive epigenetic remodeling. This epigenetic remodeling is mainly characterized by an overall reduction in DNA methylation during migration of PGCs to the gonadal ridge [22]. DNA methylation is a repressive mark that mainly targets cytosines within CpG islands (palindromic CpG dinucleotides in the genome) [24] producing a complete erase of the methylation patterns and thus the acquisition of totipotency in gametes of both sexes [104]. Then, during sex determination, PGCs must establish their sex-specific epigenetic patterns, which include paternal or maternal imprinted marks in order to acquire the epigenetic state necessary to form functional gametes [21]. The imprinting genes involve genomic sequences that exhibit differences in CpG methylation according to the parental origin. These differentially methylated regions (DMRs) can influence the allele-specific expression of one or more genes [21]. After fertilization, a second wave of epigenetic reprogramming is produced in all the genome but in the imprinted regions, which in turn regulates fetal and placental growth, differentiation, development and other important functions after birth [105].

It has been recently suggested that in vitro manipulation of cells could lead to epigenetic alterations affecting either stem cells or the obtained artificial gametes [106,107,108,109]. Not all the studies performed in the field have analyzed the epigenetic status of generated cells, but when authors assesed epigenetic features of these cells, they found similar patterns than their in vivo counterparts, with a sharp decrease in global methylation levels, and methylation patterns in DMRs of imprinting genes according to their fate [46,48]. Nonetheless, the correct epigenetic state of artificial gametes is an important issue that will determine the successful development of the offspring. Especial attention should be paid to the epigenetic analysis on DNA metyhlation and histone modification to ensure the complete imprimting of artificial gametes and avoid partially reprogrammed artifacts [53].

Finally, the long-term safety of the obtained artificial gametes will be a critical issue to assess the quality and applicability of derived artificial gametes into the clinical practice. Observational and biochemical tests can be performed to demostrate the safety of artificial sperm or eggs, but these assays are insufficient to judge whether the cells would support normal development in humans upon fertilization until the adult life. The most robust and stringent studies reporting human PCGs generated in vitro have shown that these cells present normal germ cell genetic and epigenetic profiles and are able to colonize [14] and proliferate when xenotransplanted into mice testes [15,49]. However, some other works have reported incomplete imprinting of the generated germ-like cells [53]. These findings highlight the need for more consistent research that should be focused to the following main directions: (1) tumorigenicity of the cells as in vitro culture of stem cells for long periods as well as reprogramming of patient-specific somatic cells to hiPSCs have been shown to constitute risks factors for accumulation of chromosomal aneuploidies [110,111]; (2) immunogenicity of in vitro generated gametes, which remains a challenge for their use as potential progenitors to be transplanted into the host as they could produce immunological rejection problems even if they are used in autologous therapy, as it has been reported for some hiPSCs cell lines [112]; (3) the future use of artificial gametes in regenerative medicine programs would require the obtention of therapeutic grade sperm and eggs attending to strict production conditions that guarantee that these cells do not present additional risks acquired during their manipulation.