Around 10%–15% of couples are infertile worldwide, and male infertility contributes to about 50% of cases.1 The Global Burden of Disease Survey reveals that the age-standardized prevalence of infertility was increased annually by 0.291% in men between 1990 and 2017.2 It has been estimated that there are 50 million of patients with infertility in China, and male factors account for half of these patients. Notably, there are significant decreases in the newborn population by 7.82 million in 2020 (10.04 million) and by 6.07 million in 2019 (11.79 million) compared to 2016 (17.86 million) in China. Continuous production of functional sperm in the testis is the key to male reproduction, and the progression of spermatogenesis starts with the mitotic division of spermatogonial stem cells (SSCs) in puberty and continues throughout adulthood. Problems in any stages of the spermatogenesis, including the mitosis, meiosis, and spermiogenesis, can lead to male infertility. According to the testicular biopsy, male infertility can be divided into two categories, namely obstructive azoospermia (OA) and nonobstructive azoospermia (NOA).3 Patients with OA have normal spermatogenesis in the testes, and the sperm cannot be detected in the semen due to the obstruction of the sperm transport pipeline. Abnormal spermatogenesis without spermatids exists in the NOA testes. The pathogenesis of NOA is complex, including genetic mutations, chemicals, and endocrine disorders. In addition, many patients with NOA are idiopathic, and their pathogenesis remains largely unclear. Therefore, it is of great significance to explore genetic and epigenetic mechanisms underlying male germ cell development to fully understand the etiology of spermatogenesis failure.
Assisted reproductive technology (ART) has achieved great progress recently, which helps many couples realize their dream of having children. Nevertheless, ART is useful only for men who have mature spermatozoa. In addition, approximately 30% of cancer patients are permanently infertile due to the damage of testis by the chemotherapy or radiotherapy.4 Moreover, about 46% of male childhood cancer survivors have been reported to be sterile. The most extensively used method for fertility preservation is sperm cryopreservation, but it is invalid for the prepubertal boys who are unable to produce spermatozoa. It is thus essential to develop new strategies of fertility preservation for the patients without active sperm.
With the development of stem cell technology, it becomes feasible to derive male germ cells in vitro from various kinds of stem cells. Stem cells, including the embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), SSCs, and mesenchymal stem cells (MSCs), have the capacities of both self-renewal and differentiation. In 2003, Toyooka et al.5 have demonstrated that ESCs can be coaxed to differentiate into primordial germ cells (PGCs), and eventually, they give rise to the spermatids in vitro. PGCs can be derived from embryoid bodies, and they generate the functional spermatids that develop into blastocyst when injected into oocytes.6 These two studies illustrate that the production of sperm in vitro might pave the way to an analysis of the mechanism involved in spermatogenesis and sperm maturation and that it offers a novel approach of obtaining male gametes for fertility.
DERIVATION OF MALE GERM CELLS FROM THE SSCS
The PGCs are the precursors that can give rise to the SSCs (also known as male germline stem cells), and they migrate to the mesentery and finally form the testis together with the mesoderm cells of the genital ridge.7,8 PGCs stay mitotic arrest upon reaching the genital ridges until day 5 postpartum,9,10 while some of these cells differentiate into SSCs. Spermatogenesis in mammals occurs in the seminiferous epithelium of the testis, and it includes three main stages: the mitosis of SSCs, meiosis of spermatocytes, and maturation of spermatids. As the initial cells of spermatogenesis, SSCs are responsible for maintaining stem cell pool and differentiating into spermatocytes which further give rise to spermatids.
In 1992, Matsui et al.11 and Resnick et al.12 have reported that mouse PGCs can be proliferated and expanded at least 20 passages in vitro by the addition of basic fibroblast growth factor (bFGF) and LIF, and octamer-binding protein 4 (Oct4), bone morphogenetic protein 4 (Bmp4), and retinoic acid (RA) are essential for PGC survival.13–15 However, little is known about the progress of human PGC development. Tilgner et al.16 have enriched SSEA1-positive cells from human ESCs, and SOX17 has been shown to be a key regulator of human PGC fate.17 It has been reported that mouse PGCs can give rise to functional gametes when they are transplanted with somatic cells under the kidney capsule of adult animals, and these gametes are fertilized to produce normal offspring.18 Nevertheless, there are few studies showing the differentiation of PGCs into male sperm in vitro. Nakatsuji and Chuma19 developed a two-dimensional culture system with mixing PGCs with endogenous somatic cells and making KL-producing cell monolayer as feeder cells, which induced the differentiation of PGCs into meiotic prophase I cells. Recently, Yuan et al.20 have established a new culture system for human testicle organogenesis in vitro from the fetal gonadal ridge. The gonad tissue fragments were cultured on small agarose gels semi-submerged in the medium using a standard gas–liquid interphase method.20 Notably, complete process of spermatogenesis, including the mitosis of spermatogonia, meiosis of spermatocytes, and spermiogenesis of spermatids to form functional sperm,20 has been detected in human testicle organogenesis in vitro. Significantly, the spermatids produced by this testicle organogenesis possess the normal methylation status of imprinted genes, which implicates the abilities of fertilization and early embryonic development in vitro20 (Figure 1).
Compared with PGCs, different culture systems of SSCs have been established for their survival and the generation of the differentiated male germ cells. Kubota et al.21 have demonstrated that mouse SSCs can be cultured with serum-free medium containing glial cell line-derived neurotrophic factor (GDNF), bFGF, and GFRA1, which promotes SSCs to proliferate and survive for over 6 months. Long-term culture of human SSCs has been achieved by Sadri-Ardekani and his colleagues.22 We have revealed that a number of epigenetic and genetic factors stimulate the proliferation of human SSCs and inhibit their apoptosis. We have demonstrated that several microRNAs, including miR-1908-3p, miR-663a, miR-31-5p, and miR-122-5p, mediate the proliferation and apoptosis of human SSCs.23–26 We have also shown that PAK1 and FGF5 genes are involved in the regulation of human SSC development.27,28 These studies indicate the possibility for the expansion and differentiation of SSCs into sperm in vitro (Figure 1).
Several induction systems of SSC differentiation have been established. In 2002, Feng et al.29 established an immortalized cell line using the undifferentiated type A spermatogonia from 6-day-old mice by the overexpression of TERT, and the mouse spermatogonial cell line can be induced by stem cell factor (SCF) to differentiate into spermatocytes and the haploid cells. Wang and his colleagues isolated and purified murine SSCs, and RA was used to induce the differentiation of murine SSCs into haploid germ cells.30 A two-dimensional culture system has been developed for the proliferation and differentiation of mouse SSCs, and RA promotes the production of haploid germ cells derived from SSCs in vitro.31 Nevertheless, the fertilization ability of the sperm derived from mouse SSCs remains unknown. In 2011, Sato’s team has transplanted the mouse SSCs into the seminiferous tubules, and they cultured the host testis fragments in 1.5% agarose gel and α-MEM supplemented with 10% knockout serum replacement. After 43 days of culture, round spermatids can be collected, and these haploid cells can produce the pups after ROSI with oocytes.32 Notably, we have established a three-dimensional-induced system that can coax human SSCs to differentiate into functional sperm in vitro.33 Specifically, we cocultured human SSCs with the inactivated human Sertoli cells in a ratio of 1:3, and these cells were cultured in matrigel with the conditioned medium.33 Compared with the two-dimensional culture system, the percentages of haploid cells are obviously higher in three-dimensional-induced system.33 The in vitro differentiation of SSCs into functional sperm in rodents has been developed, and healthy offspring and second generation can be delivered by the in vitro-derived germ cells. Despite the generation of sperm from human SSCs, there are certain issues to be defined. For example, the number of human primary SSCs is very limited, and thus, the expansion of human SSCs in vitro is required to obtain sufficient cells. In addition, the concerns regarding the differentiation conditions in vitro, the efficiency, the safety of in vitro-produced spermatids, and the ethical issues remain to be resolved.
DERIVATION OF MALE GERM CELLS FROM THE ESCS
ESCs are derived from early embryos or primitive gonads. The main characteristics of ESCs are their pluripotency and differentiation into all cell types in the body.34,35 As such, it would be feasible to induce ESCs to differentiate into functional spermatids (Table 1). In 2003, ESCs have been shown to differentiate into PGCs by coculturing with trophoblast cells or the BMP4-producing cells. Eight weeks after the transplantation of ESCs-derived PGCs and gonadal cells under a host testis capsule, testicular tubules and spermatids are observed.5 Furthermore, Geijsen et al.6 have selected SSEA1+ cells from embryoid bodies (EBs) and induced the EB differentiation into round spermatids that are able to fertilize with oocytes and develop into the two-cell embryo and blastocyst. These studies suggest that ESCs can form functional haploid male gametes. Subsequently, Clark et al.36 reveal the transcriptional and translational profiles during ESC differentiation into spermatids. Low glucose medium suppresses germ cell formation from ESCs.37 The Stra8-EGFP and Prm1-DsRed promoters have been utilized to isolate spermatocytes and spermatids from the ESCs in vitro,38 respectively, and notably, these spermatids can fertilize with mouse oocytes to produce healthy offspring.38 Monkey ESCs can also be induced to differentiate into male germ cells by detecting the germ cell markers,39 while mouse ESCs give rise to male germ cells by the overexpression of DAZL.40 Complete meiosis of ESCs has been achieved by coculturing ESCs-derived PGCs with postnatal testicular cells and the addition of Activin A, BMPs, and RA,41 or by the overexpression of Eif2s3y.42 Human ESCs have been shown to be coaxed by the conditioned medium with the addition of RA and BMP4 to generate spermatids in vitro.43 Interestingly, coculturing human ESCs with mitomycin-C inactivated porcine ovarian fibroblasts has been found to be an appropriate condition for the differentiation of human ESCs into male germ cells in vitro,44 while Activin A promotes the differentiation potential of human ESCs into germ cells.45
Many factors affect the efficiency of male germ cells from ESCs. KIT ligand and BMP signaling enhance the differentiation of human ESCs into germ cells.46 The loss of KITL causes a significant decrease in the enrichment of human germ cells.46 BMP4 increases the levels of germ cell markers and appears to promote the differentiation of human ESCs into germ cells.46 MiR-34c has been shown to mediate mouse ESC differentiation through RARg,47 and hedgehog and JAK-STAT signaling pathways are involved in the regulation of chicken ESC differentiation into male germ cells.48,49 Nevertheless, the molecular mechanisms underlying human ESC differentiation into male germ cells remain largely unclear, and the effective methods to generate male germ cells from human ESCs need to be further explored.
DERIVATION OF MALE GERM CELLS FROM THE IPSCS
In 2006, Takahashi and Yamanaka50 have for the first time generated the iPSCs from mouse fibroblasts via the overexpression of four transcription factors, namely Oct3/4, SRY-Box transcription factor 2 (Sox2), c-Myc and Kruppel-like factor 4 (Klf4), as evidenced the findings that these cells are similar to ESCs in morphology, gene and protein expression profiles, epigenetic modification status, cell proliferation ability, embryoid body, teratoma formation ability, and differentiation ability. In human, fibroblasts can be reprogrammed to become the iPSCs which are almost identical to ESCs by the four defined factors, e.g., Oct3/4, Sox2, Klf4, and c-Myc (method of Shinya Yamanaka laboratory) or Oct3/4, Sox2, NANOG, and LIN28 (method of Thompson laboratory), respectively.51,52 Significantly, the iPSCs can be induced to differentiate into cell lineages of the three germ layers, including male germ cells (Table 2). The pluripotency of the iPSCs offers a novel application for generating male gametes in regenerative medicine without ethics issue compared to the human ESCs.
In 2010, the differentiation of iPSCs into male germ cells in vitro has been achieved via the coculture with M15-BMP4 cells transduced with GDNF and epidermal growth factor (EGF).53 In 2012, Zhu et al.54 induce the iPSCs into EBs using the LIF-free medium and differentiate the iPSCs-derived EBs into SSCs by RA, and EBs-derived SSCs undergo spermatogenesis and produce round spermatids when transplanted into mouse testes. The iPSC-derived EBs are grafted with testicular cells into the dorsal skin of mice and reconstitute the seminiferous tubules.55 Subsequently, several groups have demonstrated the feasibility of inducing mouse iPSCs to differentiate into spermatids in vitro,56–59 while RA and 17β-estradiol contribute to the generation of male germ cells from mouse iPSCs.60,61 In addition, porcine iPSCs can be produced from embryonic fibroblasts, and they could be induced to differentiate into SSCs.62
For human iPSCs, Eguizabal et al.63 have obtained the postmeiotic cells from human iPSCs via the RA-conditioned medium, and spermatogonia, spermatocytes, and haploid spermatids can be generated in the presence of LIF, bFGF, forskolin (FRSK), and CYP26 inhibitor.63 Notably, 2.3% of haploid cells can be obtained from human iPSCs. In addition, three-dimensional decellularized amnion membrane scaffold has been shown to enhance the differentiation efficiency with 3.95% of human iPSCs into haploid spermatids compared to the 2.22% of haploid cell formation by the two-dimensional culture system.64 On the other hand, not all iPSCs have the potential to produce male germ cells, and the function of spermatids derived from the iPSCs requires further studies. Compared with ESCs, the iPSCs have the advantage of overcoming the ethical restriction for their application in reproductive medicine. Another constrain is the genetic heterogeneity of the iPSCs and the variability in the differentiated cell phenotype and gene expression.
DERIVATION OF MALE GERM CELLS FROM THE MSCS
MSCs are multipotent stem cells with high self-renewal and differentiation abilities, and they can be obtained from the umbilical cord, bone marrow, peripheral blood vessels, and fat. Like the ESCs and other adult stem cells, they can be cultured and expanded in vitro and can also differentiate into a number of cell types, including nerve cells, osteoblasts, chondrocytes, muscle cells, and fats cells, under the specific conditions. One important property of MSCs is the immune suppressive, which is helpful for the application of MSCs in regenerative medicine and reproductive medicine. Here, we summarized the ability of MSCs with differentiating into germ cells in vitro (Table 3).
Human Wharton’s Jelly-derived MSCs have been shown to become germ cells in vitro when they are cultured with testicular cells-conditioned medium with the addition of RA and testosterone,65 as evidenced by the expression of germ cell markers, including Kit, CD49f, Stella, and Vasa.65 Human adipose-derived MSCs can also be induced into germ-like cells by RA.66 RA is an important regulator of meiosis in gametogenesis, and it induces the expression of meiosis markers during spermatogenesis.67 Growth factors, e.g., LIF, GDNF, and EGF, contribute to the long-term culture of MSCs, whereas RA promotes germ cell differentiation of MSCs.68,69
Sertoli cells are the only somatic cells within the seminiferous tubules, and they provide an essential microenvironment or the niche for germ cell development. Human MSCs are cocultured with mouse Sertoli cells to induce their differentiation into germ cells since STELLA, VASA, and DAZL are detected in these cells.70 This finding is consistent with the observations by Segunda et al.71 that coculturing bovine fetal MSC with Sertoli cells leads to male germ cell formation. Coculture with mouse testicular cells promotes the differentiation of human MSCs from the umbilical cord into germ-like cells.72 These studies illustrate that Sertoli cells and the testicular cells may provide the niche, which facilitates the differentiation of MSCs into male germ cells. The combination of RA with Sertoli cells-conditioned medium is employed as the stimulator to evaluate the MSC differentiation ability,73–76 while RA and testosterone have been shown to enhance male germ-like cell formation from adipose-derived MSCs when cocultured with Sertoli cells in vitro.77
Other efforts have been made to induce MSCs to differentiate into male germ cells in vitro. BMPs have been shown to be important for stem cell differentiation.78–80 BMP4 and bone morphogenetic protein 8b (BMP8b) promote the expression of PGCs-specific genes in bone marrow MSCs (BM-MSCs),79 and transforming growth factor beta1 (TGFβ1) induces the generation of the SSCs and spermatogonia from BM-MSCs.80 Moreover, canine adipose MSCs can be differentiated into male germ-like cells by BMP4 treatment.81 BMP4 increases Stra8 gene expression significantly in mouse BM-MSCs when cocultured with testicular cells.82DAZL and BOULE are members of the DAZ gene family, and they play a key role in mediating normal spermatogenesis. Overexpression of BOULE, DAZL, and STRA8 promotes the differentiation of goat BM-MSCs to germ cell-like cells.83,84 Notably, we have recently demonstrated that human Sertoli cells can be reprogrammed via overexpression of DAZ family genes to become SSCs with the self-renewal and differentiation potentials into spermatocytes and spermatids in vitro.85
SUMMARY AND PERSPECTIVES
The approaches, including the overexpression of male germ cell-related genes, the addition of growth factors and cytokines, coculture with testicular somatic cells, the two-dimensional (2D)- or three-dimensional (3D)-culture system, are useful and efficient for the differentiation of several kinds of stem cells into male germ cells in vitro (Figure 1 and 2), which might offer male gametes for the treatment of male infertility. Functional spermatids can be induced from human SSCs and ESCs, but the efficiency and safety need to be further improved. For the iPSCs and MSCs, they can be induced to produce male germ cells. Nevertheless, the function of male germ cells remains unknown.
With the development of next-generation sequencing, more information will be available about the regulation of the SSC fate determinations and the process of spermatogenesis.86,87 It is feasible to establish human SSC lines that can be differentiated in vitro and to establish culture systems which coax the efficient differentiation of stem cells into functional spermatids in vitro. Overexpression or knockout of the key genes in the MSCs or the iPSCs may reprogram them to develop male gametes with fertility and developmental capacities. These stem cells-derived male gametes might have significant application in reproductive medicine.
YHC and WC wrote the manuscript. SW and CLW helped the drawing of figures. ZH wrote and revised the manuscript. All authors read and approved the final manuscript.
All authors declared no competing interests.
This work was supported by the grants from the National Nature Science Foundation of China (32170862 and 31872845), Major Scientific and Technological Projects for Collaborative Prevention and Control of Birth Defect in Hunan Province (2019SK1012), Key Grant of Research and Development in Hunan Province (2020DK2002), High-Level Talent Gathering Project in Hunan Province (2018RS3066), and Natural Science Foundation of Hunan Province of China (2020JJ5380 and 2020JJ5383).
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