**3. Attempts to differentiate spermatozoa outside the niche (***in vitro***,**  *in vivo,* **and** *ex vivo***)**

Understanding the components of SSCs niche and their interactions with each other are vital aspects in regeneration of spermatogenesis *in vitro*. The *in vivo* niche of mammalian SSCs is comprised of Sertoli cells, peritubular cells, and a complex array of matrix proteins. The normal SSC pool is maintained throughout adulthood, through signals provided by adhesion molecules and other cell surface receptors. SSCs are exposed to signals from both tubular lumen and the interstitial space sides of the basement membrane. Fate of SSCs is regulated mainly by Sertoli cells, inter-tubular blood vessels, and surrounding Leydig cells also have a role [51]. Steady state of germ cell niche can be disturbed by physiological changes of individual components by intrinsic or external factors. Regeneration of sperm has been difficult due to incomplete understanding of complex interactions within the niche environment, and germ cell-specific events such as, meiosis, chromatin re-modeling/repackaging, flagellum development and transcriptional reprogramming, etc. [52]. However, some recent advancements in sciences, such as construction of "omics" databases involving genomics, proteomics, and metabolomics have immensely contributed to rapid development of germ cell biology. Studies have shown that thousands of genes are successively expressed or suppressed leading to changes in biochemical composition in germ cells along the spermatogenesis pathway. Epigenetic reprogramming of genes and post transcriptional modifications of proteins are further favored this process. In addition, miRNAs play a significant role in regulating germ cells differentiation [37]. Based on the available data scientists have developed *in vitro* culture systems to induce male germ cells development from different types of stem cells. The improved culture systems facilitate to study the distinct pattern of gene expression in germ cells at various developmental stages.

Differential plating is the simplest technique isolating germ cells from digested testicular tissues. However, a highly purified SSCs population is expected only from sorting of cells using combination of surface markers. The array of potential markers reported for isolating SSCs are *GPR125, ITGA6, CD9,* or *GFRα1* [51]. Culturing of SSCs isolated using differential plating technique from testicular tissue of cancer patients, in laminin or gelatin coated wells (feeder free conditions) and serum free culture media supplemented with human GDNF, bFGF, EGF

adult SSCs were able to culture in laminin coated plates, up to 28 weeks with 18,000-fold increased in number in the presence of LIF, bFGF, GDNF, and EGF [60]. In contrast, SSCs underwent massive apoptosis in feeder free conditions, but testicular somatic cells together

Maintaining the spatial arrangement of testicular cells seems to be important in the process of regulation and completion of spermatogenesis. The goal may be achieved by arranging germ and somatic cells in three-dimensional (3D) culture systems by formation of embryoid bodies (EBs) or culturing the cells in soft agar or methyl cellulose [61]. Two culture systems are depicted in **Figures 3** and **4**. The studies have been highlighted that, low temperature (equal to testicular temperature), endocrine factors, and supporting somatic cells are prerequisites to be considered in *in vitro* spermatogenesis. Supportive mechanisms provided by somatic cells to develop germ cells are controversial. Presence of somatic cells, but not necessarily the direct contact is suggested for *in vitro* proliferation of male germ cells in one study [61] In contrast, significance of direct cell to cell contact between Sertoli cells and stem cells has been emphasized for successful germ cells formation from Warton's jelly-derived mesenchymal stem cells [62]. Reasons of such kind of variations, whether due to the source of cells used for differentiation of germ cells or specific stage of supporting along the differentiation process,

Transmeiotic differentiation is one of the critical step in the spermatogenesis pathway, and it is inducible employing bio-mechanical or chemical methods such as, simulated gravity, KL,

meiosis even in the absence of exogenous supplements or Sertoli cells. Microgravity may act as an inducer or accelerator in the progression of meiosis [43]. Co-culture with testicular somatic cells, induction with RA and BMP4 are other well documented methods for meiotic initiation of SSCs. Mouse embryonic stem cells (ESCs) are reported to enter early meiosis when co-cultured with Sertoli cells compared to culture provided with RA. Sertoli cells provide RA for germ cells in two ways; by direct delivery of RA and delivery of retinol via membrane receptor *STRA6* [63]. Rate of germ cells formation and meiotic entry may also vary in

spermatogonia cultured under simulated microgravity for 48 h entered into

with GDNF supported the propagation of SSCs more than 1 year [51].

cells from 2 to 70% [59]. In another study, human

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 35

and LIF resulted in the increase of GPR125<sup>+</sup>

should further be investigated.

**Figure 3.** Schematic of soft agar 3D culture system.

or RA. *c-KIT+*

Recapitulation of spermatogenesis completely or as in part, outside its niche is essential to understand the series of biological events associated with this complex process. The techniques can be utilized to study the germ cell biology (mitosis, meiosis, morphogenesis, initiation of motility, etc.) toxicological studies, fertility preservation, production of transgenic sperm, and have the potential for new therapeutic approaches in male infertility [53]. Continuous attempts have been made using pre-existing immature germ cells or various sources of stem or somatic cells as the starting source for *in vitro* derived gametes with satisfactory results [54]. However, same weight can be given for the doubts still have to be clarified. The strategies are broadly categorized into three aspects; development of different culture systems, haploidization, and differentiation of germ or somatic cells, and autologous or xenologous transplantation of germ or putative germ cells.

Methods to isolate SSCs from testicular tissue and differentiate into haploid cells or further to sperm, with feeder or feeder free conditions have been explored in different studies. Enrichment of SSCs *in vitro* facilitates dissection of germ cells biology, because SSCs represent a very rare population of germ cells consisting about 0.03% (20,000–35,000) in adult mouse testes [5] or even lesser 2000–3000 [6]. Early attempt to germ cell culture (in which whole segment of seminiferous tubules were maintained in culture) goes back to 1964 [55]. The first human SSC culture was reported in 1998 using a crude extract of testicular tissue, and the efficacy has now been improved with varying techniques [56]. Digested testicular tissues from obstructive azoospermic men cultured with high concentrations of follicle-stimulating hormone (FSH) and testosterone continued the *in vitro* reduction of germ cell ploidy with rapid morphological changes toward spermiogenesis. Two different mechanisms are possibly involved in endocrine regulation of the above process, and they would be, prevention of Sertoli cells apoptosis by testosterone and stimulation of the spermiogenesis by FSH [57]. Following a simple two enzymatic digestion protocol *OCT4+* SSCs were isolated from human testis with 87% purity. SSCs colonies were able to culture for around 1 month on a Sertoli cells feeder layer [40]. In another study, *THY1+* mouse SSCs have been cultured in the presence of Sertoli cells, hormones (FSH/testosterone) and vitamins (RA/vit.E/C), either with a mix of three components or with individual components. After 7 days of culture spermatidlike cells expressing post-meiotic markers were prominent in mixed supplement group compared to individual supplements [47]. Addition of FSH to bovine SSCs culture has proven the increased colonization capacity of spermatogonia [58].

Differential plating is the simplest technique isolating germ cells from digested testicular tissues. However, a highly purified SSCs population is expected only from sorting of cells using combination of surface markers. The array of potential markers reported for isolating SSCs are *GPR125, ITGA6, CD9,* or *GFRα1* [51]. Culturing of SSCs isolated using differential plating technique from testicular tissue of cancer patients, in laminin or gelatin coated wells (feeder free conditions) and serum free culture media supplemented with human GDNF, bFGF, EGF and LIF resulted in the increase of GPR125<sup>+</sup> cells from 2 to 70% [59]. In another study, human adult SSCs were able to culture in laminin coated plates, up to 28 weeks with 18,000-fold increased in number in the presence of LIF, bFGF, GDNF, and EGF [60]. In contrast, SSCs underwent massive apoptosis in feeder free conditions, but testicular somatic cells together with GDNF supported the propagation of SSCs more than 1 year [51].

Maintaining the spatial arrangement of testicular cells seems to be important in the process of regulation and completion of spermatogenesis. The goal may be achieved by arranging germ and somatic cells in three-dimensional (3D) culture systems by formation of embryoid bodies (EBs) or culturing the cells in soft agar or methyl cellulose [61]. Two culture systems are depicted in **Figures 3** and **4**. The studies have been highlighted that, low temperature (equal to testicular temperature), endocrine factors, and supporting somatic cells are prerequisites to be considered in *in vitro* spermatogenesis. Supportive mechanisms provided by somatic cells to develop germ cells are controversial. Presence of somatic cells, but not necessarily the direct contact is suggested for *in vitro* proliferation of male germ cells in one study [61] In contrast, significance of direct cell to cell contact between Sertoli cells and stem cells has been emphasized for successful germ cells formation from Warton's jelly-derived mesenchymal stem cells [62]. Reasons of such kind of variations, whether due to the source of cells used for differentiation of germ cells or specific stage of supporting along the differentiation process, should further be investigated.

Transmeiotic differentiation is one of the critical step in the spermatogenesis pathway, and it is inducible employing bio-mechanical or chemical methods such as, simulated gravity, KL, or RA. *c-KIT+* spermatogonia cultured under simulated microgravity for 48 h entered into meiosis even in the absence of exogenous supplements or Sertoli cells. Microgravity may act as an inducer or accelerator in the progression of meiosis [43]. Co-culture with testicular somatic cells, induction with RA and BMP4 are other well documented methods for meiotic initiation of SSCs. Mouse embryonic stem cells (ESCs) are reported to enter early meiosis when co-cultured with Sertoli cells compared to culture provided with RA. Sertoli cells provide RA for germ cells in two ways; by direct delivery of RA and delivery of retinol via membrane receptor *STRA6* [63]. Rate of germ cells formation and meiotic entry may also vary in

**Figure 3.** Schematic of soft agar 3D culture system.

flagellum development and transcriptional reprogramming, etc. [52]. However, some recent advancements in sciences, such as construction of "omics" databases involving genomics, proteomics, and metabolomics have immensely contributed to rapid development of germ cell biology. Studies have shown that thousands of genes are successively expressed or suppressed leading to changes in biochemical composition in germ cells along the spermatogenesis pathway. Epigenetic reprogramming of genes and post transcriptional modifications of proteins are further favored this process. In addition, miRNAs play a significant role in regulating germ cells differentiation [37]. Based on the available data scientists have developed *in vitro* culture systems to induce male germ cells development from different types of stem cells. The improved culture systems facilitate to study the distinct pattern of gene expression

Recapitulation of spermatogenesis completely or as in part, outside its niche is essential to understand the series of biological events associated with this complex process. The techniques can be utilized to study the germ cell biology (mitosis, meiosis, morphogenesis, initiation of motility, etc.) toxicological studies, fertility preservation, production of transgenic sperm, and have the potential for new therapeutic approaches in male infertility [53]. Continuous attempts have been made using pre-existing immature germ cells or various sources of stem or somatic cells as the starting source for *in vitro* derived gametes with satisfactory results [54]. However, same weight can be given for the doubts still have to be clarified. The strategies are broadly categorized into three aspects; development of different culture systems, haploidization, and differentiation of germ or somatic cells, and autologous or xenologous

Methods to isolate SSCs from testicular tissue and differentiate into haploid cells or further to sperm, with feeder or feeder free conditions have been explored in different studies. Enrichment of SSCs *in vitro* facilitates dissection of germ cells biology, because SSCs represent a very rare population of germ cells consisting about 0.03% (20,000–35,000) in adult mouse testes [5] or even lesser 2000–3000 [6]. Early attempt to germ cell culture (in which whole segment of seminiferous tubules were maintained in culture) goes back to 1964 [55]. The first human SSC culture was reported in 1998 using a crude extract of testicular tissue, and the efficacy has now been improved with varying techniques [56]. Digested testicular tissues from obstructive azoospermic men cultured with high concentrations of follicle-stimulating hormone (FSH) and testosterone continued the *in vitro* reduction of germ cell ploidy with rapid morphological changes toward spermiogenesis. Two different mechanisms are possibly involved in endocrine regulation of the above process, and they would be, prevention of Sertoli cells apoptosis by testosterone and stimulation of the spermiogenesis by FSH [57].

testis with 87% purity. SSCs colonies were able to culture for around 1 month on a Sertoli

ence of Sertoli cells, hormones (FSH/testosterone) and vitamins (RA/vit.E/C), either with a mix of three components or with individual components. After 7 days of culture spermatidlike cells expressing post-meiotic markers were prominent in mixed supplement group compared to individual supplements [47]. Addition of FSH to bovine SSCs culture has proven the

SSCs were isolated from human

mouse SSCs have been cultured in the pres-

in germ cells at various developmental stages.

34 Spermatozoa - Facts and Perspectives

transplantation of germ or putative germ cells.

Following a simple two enzymatic digestion protocol *OCT4+*

cells feeder layer [40]. In another study, *THY1+*

increased colonization capacity of spermatogonia [58].

affected on some cell lines, but not on others. Batch to batch variations of serum and positive or negative impact of unknown factors added by both feeder cells and serum are also concern [56]. Many authors emphasize the requirement of refined culture system eliminating serum and feeder cells to better understanding of individual cellular and molecular interactions.

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 37

The *in vitro* maturation of available germ cells has a little value considering the therapeutic aspects of infertile men especially for men with non-obstructive azoospermia. To overcome this situation, ESCs derived gametes were tested as a first line remedial step by scientific community. Initial attempts were least successful due to unresponsiveness of cells for specific media or culture conditions [74]. However, encouraging models have been described later on by different authors, not only for ESCs but also for other multipotent cell types including iPSCs and somatic stem cells. Two main approaches for this are (a) direct differentiation of stem cells into germ cell lineage using exogenous factors; (b) transfection of stem cells with marked or fluorescent proteins linked to specific gene promoters, such as *STRA8* and *PRM1* [75]. These methods can be employed as monolayer adherent cell culture or threedimensional embryoid bodies, with or without feeder cells. Optimum time point for obtaining germ-like cells from human pluripotent stem cells (PSCs) was recorded day 10, while day 7 cultures yielded lower numbers and day 15 not indicated a significant increase [67]. It is reported that germ cell formation in EB culture system seems to faster than in monolayer culture system [76]. Given the priority for direct differentiation method is more acceptable as gene transfection method disqualifies in clinical applications. However, the gene transfection and iPSCs systems may provide the necessary information on the behavior of related genes in germ cell development. Whatever the method employed, the imprinting regulation of gametes obtained from concerned methods has to be further validated, if ever they are to be used

The possibility of using PGCs and germ line stem cells (GSCs) in transplantation studies to restore fertility has been studied with varying degrees of success [65]. Grafting or transplantation of gonadal fragments, germ cells or genetically modified germ cells and transmeiotic pluripotent stem cells onto immune compromised animals is an alternative strategy to investigate germ cell development. Successful autologous-transplantation of spermatogonial stem cells has achieved in a wide range of species so far [77]. Autologous cryopreserved testicular tissue grafting is an option for preserving genetic materials in endangered species and immature cancer patients. Success of homing ability of grafts may depend on various factors such as, age of collecting graft (immature is the better), low GnRH level (suppressed spermatogenesis with more primitive cells), method of cryopreservation, etc. [78]. Homing ability of SSCs from different species including human, to basement membrane of seminiferous tubules of nude mice has been proven by many authors [60]. The niche for spermatogonial proliferation appears to be generally similar among different species, because proliferation is undisturbed between cross-species after xenotransplantation of spermatogonia. However, the niche for spermatogonial differentiation is thought to work through a

The most advanced progress in meiosis and qualified male gametes may be obtained following transplantation of *in vitro* derived PGCs or GSCs into the testis. The ability to develop more mature germ cells from PGCs like cells derived from mouse iPSCs has shown after

for clinical applications [73].

species-specific mechanism [78].

**Figure 4.** SSCs co-cultured with Sertoli cells in embryoid bodies culture system. A—initial culture (day 5) showing spermatocytes and spermatids like stages. B—late phase of culture (day 14), few sperms with normal morphology are observed (arrow heads).

different culture systems, and with the source of cells used. For example, 3% of bone marrow mesenchymal stem cells (MSCs) were differentiated into germ cells when treated with RA [64]. Another study achieved around 20% of germ cells after co-culturing human ESCs with fetal gonadal stromal cells [65]. Mouse-induced pluripotent stem cells (iPSCs) treated with BMP4 led to formation of 41% primordial germ cells like cells [66]. However, very limited or pseudoentry of meiosis was noted by many studies. Around 3–5% of haploid cells were present in FACS sorted cells after induction of mouse SSCs in differentiation medium [67]. Similarly, 1–5% of post-meiotic cells were emerged when hESCs derived embryoid bodies (EBs) were treated with mouse testes conditioned medium supplemented with RA and BMP4 [68]. In contrast, 20% of haploid cells were observed in BMP4-induced human IPCs culture, and around 70% was positive for acrosin after sorting for 1 N cells [69]. Cell organization in EBs may reflect more of the arrangement of embryonic gonadal ridge [64], and correct erasure of imprinting genes was observed with EBs culture system [70]. It has been reported that, testosterone causes to increase in *STRA8* mRNA levels (pre-meiotic marker) when cells were treated with both RA and testosterone [71]. The spontaneous differentiation of stem cells into haploid state may also be possible in appropriate culture conditions with a very low efficiency, amounting around 2% in human iPSCs culture [69]. Spontaneous differentiation may increase with prolongation of culture, and due to inducing factors contained in culture medium. For example, media supplemented with 10% fetal bovine serum contain approximately 3.6 × 10−8 M of RA [72]. However, spontaneous differentiation of ESCs into germ line is generally low and inefficient with majority of germ cells undergoing degeneration [71].

Most of data have produced from very short period of cultures (2–30 days), indicating germ cell differentiation proceed an unusual speed *in vitro*. It is not clear how the timing went shorten, and it has been suggested that in the absence of environmental cues, germ cells may develop according to an intrinsic clock [73]. However, establishing a standard culture system of SSCs is difficult due to many reasons. Inherent variability between cells of different species leads to inconclusive results. For example, feeder cells and serum in culture may positively be affected on some cell lines, but not on others. Batch to batch variations of serum and positive or negative impact of unknown factors added by both feeder cells and serum are also concern [56]. Many authors emphasize the requirement of refined culture system eliminating serum and feeder cells to better understanding of individual cellular and molecular interactions.

The *in vitro* maturation of available germ cells has a little value considering the therapeutic aspects of infertile men especially for men with non-obstructive azoospermia. To overcome this situation, ESCs derived gametes were tested as a first line remedial step by scientific community. Initial attempts were least successful due to unresponsiveness of cells for specific media or culture conditions [74]. However, encouraging models have been described later on by different authors, not only for ESCs but also for other multipotent cell types including iPSCs and somatic stem cells. Two main approaches for this are (a) direct differentiation of stem cells into germ cell lineage using exogenous factors; (b) transfection of stem cells with marked or fluorescent proteins linked to specific gene promoters, such as *STRA8* and *PRM1* [75]. These methods can be employed as monolayer adherent cell culture or threedimensional embryoid bodies, with or without feeder cells. Optimum time point for obtaining germ-like cells from human pluripotent stem cells (PSCs) was recorded day 10, while day 7 cultures yielded lower numbers and day 15 not indicated a significant increase [67]. It is reported that germ cell formation in EB culture system seems to faster than in monolayer culture system [76]. Given the priority for direct differentiation method is more acceptable as gene transfection method disqualifies in clinical applications. However, the gene transfection and iPSCs systems may provide the necessary information on the behavior of related genes in germ cell development. Whatever the method employed, the imprinting regulation of gametes obtained from concerned methods has to be further validated, if ever they are to be used for clinical applications [73].

different culture systems, and with the source of cells used. For example, 3% of bone marrow mesenchymal stem cells (MSCs) were differentiated into germ cells when treated with RA [64]. Another study achieved around 20% of germ cells after co-culturing human ESCs with fetal gonadal stromal cells [65]. Mouse-induced pluripotent stem cells (iPSCs) treated with BMP4 led to formation of 41% primordial germ cells like cells [66]. However, very limited or pseudoentry of meiosis was noted by many studies. Around 3–5% of haploid cells were present in FACS sorted cells after induction of mouse SSCs in differentiation medium [67]. Similarly, 1–5% of post-meiotic cells were emerged when hESCs derived embryoid bodies (EBs) were treated with mouse testes conditioned medium supplemented with RA and BMP4 [68]. In contrast, 20% of haploid cells were observed in BMP4-induced human IPCs culture, and around 70% was positive for acrosin after sorting for 1 N cells [69]. Cell organization in EBs may reflect more of the arrangement of embryonic gonadal ridge [64], and correct erasure of imprinting genes was observed with EBs culture system [70]. It has been reported that, testosterone causes to increase in *STRA8* mRNA levels (pre-meiotic marker) when cells were treated with both RA and testosterone [71]. The spontaneous differentiation of stem cells into haploid state may also be possible in appropriate culture conditions with a very low efficiency, amounting around 2% in human iPSCs culture [69]. Spontaneous differentiation may increase with prolongation of culture, and due to inducing factors contained in culture medium. For example, media supplemented with 10% fetal bovine serum contain approximately 3.6 × 10−8 M of RA [72]. However, spontaneous differentiation of ESCs into germ line is generally low and inefficient with majority of germ cells undergoing degenera-

**Figure 4.** SSCs co-cultured with Sertoli cells in embryoid bodies culture system. A—initial culture (day 5) showing spermatocytes and spermatids like stages. B—late phase of culture (day 14), few sperms with normal morphology are

Most of data have produced from very short period of cultures (2–30 days), indicating germ cell differentiation proceed an unusual speed *in vitro*. It is not clear how the timing went shorten, and it has been suggested that in the absence of environmental cues, germ cells may develop according to an intrinsic clock [73]. However, establishing a standard culture system of SSCs is difficult due to many reasons. Inherent variability between cells of different species leads to inconclusive results. For example, feeder cells and serum in culture may positively be

tion [71].

observed (arrow heads).

36 Spermatozoa - Facts and Perspectives

The possibility of using PGCs and germ line stem cells (GSCs) in transplantation studies to restore fertility has been studied with varying degrees of success [65]. Grafting or transplantation of gonadal fragments, germ cells or genetically modified germ cells and transmeiotic pluripotent stem cells onto immune compromised animals is an alternative strategy to investigate germ cell development. Successful autologous-transplantation of spermatogonial stem cells has achieved in a wide range of species so far [77]. Autologous cryopreserved testicular tissue grafting is an option for preserving genetic materials in endangered species and immature cancer patients. Success of homing ability of grafts may depend on various factors such as, age of collecting graft (immature is the better), low GnRH level (suppressed spermatogenesis with more primitive cells), method of cryopreservation, etc. [78]. Homing ability of SSCs from different species including human, to basement membrane of seminiferous tubules of nude mice has been proven by many authors [60]. The niche for spermatogonial proliferation appears to be generally similar among different species, because proliferation is undisturbed between cross-species after xenotransplantation of spermatogonia. However, the niche for spermatogonial differentiation is thought to work through a species-specific mechanism [78].

The most advanced progress in meiosis and qualified male gametes may be obtained following transplantation of *in vitro* derived PGCs or GSCs into the testis. The ability to develop more mature germ cells from PGCs like cells derived from mouse iPSCs has shown after


**Source of cells Method used Observations References**

Cells in both cultures, predominantly in EBs were differentiated, into primordial germ cells with correct gene expression patterns. Correct pattern of parental imprint erasure was confirmed (*PEG3 and* 

Large, round PGCs like cells with 0.25% positive for alkaline phosphatase (AP). Cells were positive for *c-KIT, OCT4, VASA, STELLA, DAZL.* 99% of CpG sites were unmethylated in *DMR1* of

AP & *CXCR4, c-KIT* positive primordial germ cells arose. Electron microscopic pictures showed large round nucleus with numerous mitochondria

spermatogenesis were observed with increasing meiotic and post-meiotic markers with time. Average 15 spermatozoa per well of 24 well plate were

Cultured SSCs effectively regenerated spermatogenesis in testes of busulfan-treated

More round and elongating spermatids emerged at day 12 of culture. *PRM2* + ve cells and haploid cells were increased

Mature sperm after 7 months of transplantation in recipient mice testes. Performance of ICSI confirmed the oocytes activating capacity of these sperm

Progression of meiosis up to day 5. Correct gene expression pattern (*SCP3* and *CREST* and haploid cells were obtained

Different stages of

present

recipient rats

with time

Wei et al. [70]

39

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505

Linher et al. [86]

Bucay et al. [87]

Elhija et al. [88]

Wu et al. [89]

Lee et al. [90]

Nayernia et al. [91]

Riboldi et al. [92]

*IGF2R*)

PGCs

ES cells Cells were cultured in Ham's

fluid for 50 days

systems

F12/IMDM medium with BMP4 as adherent or EBs culture

Induced with porcine follicular

50–250 cell colonies were cultured on mouse primary embryonic fibroblast in ESCs growth medium for 7d. The cells were co-cultured with putative Sertoli cells

3D agar culture system consisting 0.5 agar and 25% FCS in lower layer and 0.37 agar and 20% FCS in upper layer. Culture continued for 30d with PRMI

Cells were cultured in gelatin coated wells with a serum free formulated medium for 120 days. Other supplements were

3D culture in a collagen gel matrix with somatic cells. The media supplemented with RA

TC cells were transfected with *Stra8-EGFP* fusion construct and positive cells were induced

FACS sorted germ-like cells were transplanted.

Cells were cultured with Sertoli cells and culture media containing RA, GDNF, FSH & testosterone for 15 days

media

GDNF and FGF

and rFSH

with RA.

Stella-GFP+

Porcine skin-derived somatic stem cells

Human ESCs (HSF-6

Mouse SSCs from 7d

Laminin binding spermatogonia from Sprague-Dawley rats

*in vitro* generated germ cell line from teratocarcinoma cells

TESE dissociated *CD49f* positive cells from azoospermic men

Immature spermatogenic cells isolated from nonobstructive azoospermic

men

(F9)

& H-9)

old male


**Source of cells Method used Observations References**

3% cells differentiated into male germ cells assessed by *OCT4*, *Fragilis*, *STELLA*, *MVH*, *RNF17, DAZLl, c-KIT, PIWIL2, RBM, STRA8, TEX 18*. But arrested at

Nyernia et al. [64]

Park et al. [65]

Easley et al. [67]

Panula et al. [69]

Miryounesi et al. [63]

Aflatoonian et al. [68]

Stukenborg et al. [61]

Jia et al. [35]

pre-meiotic stage

observed.

*IGF2*

20% germ cells with triple positive markers (*cKIT, SSEA1* & *PLAP or VASA*). Repression of *HOX* genes and imprint erasure by day 7. No report of meiosis

*UTF1, PLZF* and *CDH* positive spermatogonia, *HIWIi* and *HILL*positive spermatocytes, and *ACR, TP1* and *PRM1* expressing haploid cells (3.9–4.5%) were

Unimpaired uniparental genomic imprints on two loci: *H19* and

Increased number of meiotic cells in *DAZL* overexpressed cells

*DAZL*, *SYCP3*, *PRM* expression in both groups after 12 days of induction. Number of colonies and positive cells were high in

Progressive elevation of both spermatogenesis and oogenesis markers. Effect was prominent with RA treatment. 1–5% postmeiotic cells & few with the beginning of flagellum formation

Pre-meiosis, meiosis and postmeiosis gene markers were expressed. Few cells exhibited spherical morphology with tail tike structure. But the cells were diploid indicating arrest at pre-

Morphologically normal but immotile spermatozoa

Increased *Vasa* and *DAZL* expression. 4–6% GFP positive

cells within 14 days.

Sertoli cell group

meiotic stage

Induced with RA (10 μm) for 10 days. and EGFP positive cells were sorted using FACS

Co-cultured with human fetal gonadal stromal cells for 14

Cultured in mouse SSCs differentiation medium for 10 days and haploid cells were confirmed after sorting by FACS

Induced with BMP-4, 7, and 8b for 14 days in feeder free

Transduced with *VASA-GFP*

EBs were induced with RA, Bmp4 and neonatal mouse testis conditioned medium for

*STRA8*-positive C2C12 myoblasts were treated with 10 μM all-trans-RA for 8 days

Cells were cultured in gel matrices (soft agar or methyl cellulose) with the support of somatic cells and gonadotropins

for 40 days

Co-cultured with Sertoli cells or RA (on gelatin coated plates)

conditions

reporter

14 days

days

BM stem cells from *STRA8-EGFP* transgenic

38 Spermatozoa - Facts and Perspectives

hESCs (HSF1, HSF6 and

hESCs (H1) and iPSCs

iPSCs (iHUF4/IMR90) hESCs (H9/HSF1)

*STRA8-EGFP* transfected Mouse ESC line (C57BL6)

& H7)

cells

mouse

hESC lines (Shef1–6

Mouse C2C12 myoblast

Pre-meiotic male germ cells from immature

mouse line

H9) and hIPS

(HFF1)


Adult somatic cells induction (SCI) and sperm cloning (male genome cloning) are recent advancements and future directions for generating clinically applicable germ cells from stem cells. In SCI technique, somatic cell nucleus is injected into enucleated oocyte and cells are cultured further to produce two separated chromosome sets. Thus, the immature oocyte helps somatic cell to become haploid. The resultant cells are genetically identical and immune-compatible with the donor of the somatic cells. In case of severe oligozoospermia, a single viable sperm from testicular biopsy sample can be used for replicating its genome. Here, a single sperm is injected into enucleated oocyte and allows it to become a haploid embryo dividing through parthenogenesis process. Resulting blastomeric cells may be used for further *in vitro* maturation or direct injection into oocyte. These new techniques still remain in experimented animal models with low efficiency [85]. The contribution by various authors for development of this field is summarized in **Table 1**.

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 41

Among the different models used in *in vitro* spermatogenesis better results have been achieved through 3D culture systems. Formation of EBs using germ and Sertoli cells seems to be more efficient and resemble the testicular niche environment compared to soft agar culture system. Although this system is well supported for the proliferation and differentiation of putative germ cells obtained from somatic stem cells up to the spermatids state there is no firm evidence to conclude the support for an efficient spermiogenesis process. Complete spermatogenesis has been achieved by transplanting stem cells or putative germ cells into testes in few studies, but with lower efficiency. Clinical applications of xeno or autologous transplantation studies among humans, and between humans and animals are still far away due to ethical and safety related issues. Similarly, use of autologous stem cells for differentiation of germ cells and infertility treatment may have little value for the patients with cancer or genetic diseases, as there is a possibility to re-infuse cancer cells or passing genetic abnormalities to offspring. However, *in vitro* maturation of germ cells may be immensely helpful for men with

maturation arrest or azoospermia due to non-genetic causes.

**4. Conclusion**

**Abbreviations**

AP alkaline phosphatase

BM bone marrow

ART assisted reproductive technologies

DMEM Dulbecco's modified eagle medium

bFGF basic fibroblast growth factor

DSP daily sperm production

EGF epidermal growth factor

EBs embryoid bodies

**Table 1.** A summary of selected studies designed to differentiate male germ cells from different sources of stem cells.

transplantation into testis of infertile mice [66]. Nayernia et al. reported 300–500-fold increase in spermatogonial population after transplantation of mouse bone marrow (BM) derived SSCs into germ cells depleted recipient mice, but the cells were arrested at pre-meiotic stage [64]. A similar study has shown that transplanted green fluorescence protein positive (GFP<sup>+</sup> ) mouse BM cells can differentiate into both somatic and germ cell lineages in a favorable testicular niche [79]. Transplanted adipose tissue-derived MSCs could induce spermatogenesis in busulfan-treated recipient rats in another study [80]. Toyooka et al. cultured *Mvh* knockin GFP or *Lac-Z* mouse embryoid bodies with *BMP 4,8* expressing embryonic trophoblast cells, and they could achieve morphologically normal sperm after transplantation of *MVH* overexpressing cells under the testis capsules of nude mice [81]. Use of combination of gene transfection and subsequent germ cells transplantation techniques could results live sperm (with reduced motility) and offspring after intracytoplasmic sperm injection (ICSI) in mice. Though the newborns died within few months due to imprinting defects, this finding paves the way to promising *in vitro*- and/or *ex vivo*-derived functional gametes in the future [82]. Additional treatment with hormones (hCG) after stem cell transplantation may be a powerful tool for increasing the efficiency of transplantation [83]. It is suggested that FSH and testosterone favor the survival of germ cells by regulating both intrinsic and the extrinsic apoptotic pathways. Furthermore, FSH is needed to initiation of meiosis and androgen is necessary for the completion of meiosis and spermiogenesis [84].

Adult somatic cells induction (SCI) and sperm cloning (male genome cloning) are recent advancements and future directions for generating clinically applicable germ cells from stem cells. In SCI technique, somatic cell nucleus is injected into enucleated oocyte and cells are cultured further to produce two separated chromosome sets. Thus, the immature oocyte helps somatic cell to become haploid. The resultant cells are genetically identical and immune-compatible with the donor of the somatic cells. In case of severe oligozoospermia, a single viable sperm from testicular biopsy sample can be used for replicating its genome. Here, a single sperm is injected into enucleated oocyte and allows it to become a haploid embryo dividing through parthenogenesis process. Resulting blastomeric cells may be used for further *in vitro* maturation or direct injection into oocyte. These new techniques still remain in experimented animal models with low efficiency [85]. The contribution by various authors for development of this field is summarized in **Table 1**.
