RETRACTED ARTICLE: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment. Electronic supplementary material The online version of this article (doi:10.1007/s00018-015-2020-1) contains supplementary material, which is available to authorized users.

Bovine iPSCs and testicular cells have been successfully used as in vitro models to study the toxicity of phthalate esters. We found that bovine iPSCs were more resistant to androgen receptor (AR)-dependent apoptosis than testicular cells, most likely attributed to regulation of the AR-p21 Cip1 cascade via p53, which showed significantly enhanced expression. Phthalate esters significantly reduced AR expression n bovine iPSCs. Collectively, these studies indicate that iPSCs may be useful for screening for adverse effects from endocrine disruptor [36,37]. This screening system has also promise as a useful model for studying the effects of environmental factors on human germ cell development.  Table 2. Basic fibroblast growth factor (bFGF) and feeder cells increased the expression of PGC marker genes such as VASA (DDX4), DAZL, and OCT4 in human germ-like cells differentiated from hESCs [85]. Tilgner et al. [20] reported the enrichment of putative PGCs from hESCs that had been sorted using an antibody specific for stage specific embryonic antigen-1 (SSEA-1). Gelatin-bound monolayers are obviously a robust system for generating large number of differentiated cells. However, these cells do not enter meiosis.

ESCs and iPSCs
Transplantation of ESC-derived somatic cells or tissues is promising for curing many human diseases. However, derivation of gametes from unrelated ESCs is associated with incompatibilities of the immune systems. Well-characterized iPSCs may be a good option for obtaining sufficient numbers of autologous cells. HiPSCs could be successfully differentiated to post-meiotic cells However, round spermatids could not fertilize human oocytes. The feasibility and safety of the culture systems will need to be established in animal models.
Mouse ESCs and iPSCs can be induced to form epiblast-like cells that, in turn, develop into PGC-like cells when the culture medium is supplemented with BMP4 [17] (Fig. 2). The resulting PGC-like cells were then transferred to the testes of infertile mice and produced sperm that were used for ICSI; transfer of the resulting embryos into recipient females gave rise to viable offspring. This is the most advanced protocol for the deviation of functional gametes from PSCs until now.
Further experiments are required before this system could be used for therapeutic treatments in human patients because some of the offspring showed malignant tumors in the neck area [17].
Human iPSC-derived cells should be monitored carefully to eliminate mutations, specifically in tumor suppressor genes [83,84].

Epigenetic control of germ cell development
A bimodal pattern of DNA methylation has been detected during the specification and maturation of mouse male germ cells (Fig. 3). PGCs derived from the epiblast at E6.5-E7.5 are stimulated by BMP4, then migrate from the epiblast to the hindgut at E7.5-E9, and finally to the genital ridge at   (Table 3).

MiR-34c expression is up-regulated in spermatocytes and round spermatids trigger apoptosis
[101]. This process is at least partially mediated by targeting transcription factor ATF-1 [102]. Thus, miR34c is critical for germ cell development. MiR-469 has been shown to target transition protein 2 (TP2) and protamine mRNAs to be repressed in pachytene spermatocytes and round spermatids Collectively, miRNAs play essential roles by regulating each step of male germ cell development, including mitosis, meiosis, and spermatogenesis in rodents. Nevertheless, it remains to be defined which miRNAs are required for the three major stages of spermatogenesis in humans, including spermatogonia, pachytene spermatocytes, and round spermatids [106]. A better understanding these processes may provide new targets for the treatment of male infertility.

In vitro gametogenesis from bovine iPSCs and production of genetically modified (GM) cattle from transgenic iPSCs
Bovine iPSCs established in our laboratory exhibited characteristics similar to those of mESCs and iPSCs with regard to gene expression, transcription factor dependency, and active signaling molecules [36,37]. Expression of pluripotency markers, including OCT4, NANOG, SOX2, STAT3, c-MYC, KLF4, TERT, and DNMT3A, is maintained in bovine iPSCs (Table 3). Mouse ESCs and iPSCs expressed SSEA-1, but not SSEA-4, whereas human ESCs and iPSCs expressed SSEA-4, but not SSEA-1 (Table 3) Spermatozoa may be useful as vectors for producing GM animals [113][114][115][116]. It could be a valuable option in the cattle industry to use spermatids differentiated from genetically modified iPSCs to produce transgenic animals by transplantation into the testes of recipient bull calves or by injecting them into bovine oocytes. We propose to produce transgenic animals by using sperm-like cells differentiated from transgenic iPSCs via in vitro fertilization or ICSI. Bovine SSCs could successfully be propagated in the presence of LIF, epidermal growth factor or fibroblast growth factor 2; however, no full spermatogenesis was established from SSCs transplanted into recipient mouse testis [117]. Complete spermatogenesis has been obtained from autologous transfer of bovine SSCs [47,48,118]. Thus, the methodologies described above need significant improvements, and cell-based approaches in livestock reproduction are a challenging task. The derivation of PSCs in livestock is promising for the development of novel disease-resistance strategy, cell or organ therapies, drug screening, and human disease models. It is also important for increasing the efficiency of the livestock industry. For example, dairy manufacturers could derive protein-rich milk from GM cows and thereby reduce the cost of cheese production.
The rapidly emerging DNA nucleases such as ZFNs, TALEN, and CRISPER/Cas may provide additional new options for producing livestock species with targeted genetic modifications with novel traits useful for application in agriculture and biomedicine [119]. There is no doubt that the application of genetic modifications and PSC techniques will improve our understanding of the dynamics of gametogenesis and reproductive biology in general, and will play an important role in the development of novel therapeutic treatments in humans and other mammalian species.

Conclusions
Over the past decade, revolutionary progress has been made in the derivation and characterization

Conflict of interest
The authors have declared that they have no conflict of interest.