Spermatogonial Stem Cell Technology and Regenerative Medicine

Germ cell development begins with the characteristics of the primordial germ cells (PGCs) early in human fetal life. PGCs arise from the proximal epiblast and will replace to the extra embryonic mesoderm during the fourth and fifth week of embryonic development. By the finish of the fifth week to early sixth week, PGCs will embark for a second immigration via the dorsal mesenterium of the gut to the gonadal ridge. During their immigration, PGCs proliferate but once they has arrived the gonadal ridge, they enter into a mitotic arrest while differentiating into gonocytes. Shortly after birth these gonocytes put on the basal membrane of the seminiferous tubules where mitosis is reinitiated. From then on, they are called spermatogonial stem cells or SSCs [1]. Spermatogonial stem cells (SSCs) are a subpopulation of unspecialized stem cells that lie along the basement membrane of the seminiferous tubules of the testis and give rise to the germ line lineage in males. In the adult testis, only 0.03% of all germ cells are spermatogonial stem cells [2]. Testicular SSCs are typically unipotent and are only capable of giving rise to the germ cell lineage and ultimately spermatozoa.


Spermatogonial stem cell
Germ cell development begins with the characteristics of the primordial germ cells (PGCs) early in human fetal life. PGCs arise from the proximal epiblast and will replace to the extra embryonic mesoderm during the fourth and fifth week of embryonic development. By the finish of the fifth week to early sixth week, PGCs will embark for a second immigration via the dorsal mesenterium of the gut to the gonadal ridge. During their immigration, PGCs proliferate but once they has arrived the gonadal ridge, they enter into a mitotic arrest while differentiating into gonocytes. Shortly after birth these gonocytes put on the basal membrane of the seminiferous tubules where mitosis is reinitiated. From then on, they are called spermatogonial stem cells or SSCs [1]. Spermatogonial stem cells (SSCs) are a subpopulation of unspecialized stem cells that lie along the basement membrane of the seminiferous tubules of the testis and give rise to the germ line lineage in males. In the adult testis, only 0.03% of all germ cells are spermatogonial stem cells [2]. Testicular SSCs are typically unipotent and are only capable of giving rise to the germ cell lineage and ultimately spermatozoa.
This ability of SSCs, the only adult stem cell population that transmits genetic information to the next generation [3][4][5], to generate spermatozoa is critically dependent on the micro environment or niche that surrounds these cells [6][7][8][9][10]. Daily, 45-207 million spermatids are generated in the normal adult testis. This coining of germ cells is not regulated by the stem cells themselves but by the microenvironment environs the stem cells, i.e. the stem cell ''niche''. The niche is described as ''the microenvironment around stem cells that provides support and generates signals regulating self-renewal and differentiation'' [11]. The niche can action on a stem cell through various mechanisms: niche cells can make straight contact with the stem cell, niche cells can disguise paracrine factors acting on the stem cell or intermediate cells can ''communicate'' among the niche and the stem cells. When removed from their normal stem cell niche and cultured in vitro, SSCs display a broader developmental potential than they normally manifest in vivo. This has led to interest in their potential suitability for human regenerative medicine.

Open Access Journal of Surgery
Klinefelter's syndrome or AZF deletion [16,17]. Because children do not have the feasibility to bank spermatozoa, the preservation and transplantation of SSCs may become an important and main strategy to treat reproductive stem cell loss disturbances. In the last decade, most of research has been done on human SSC protection and transplantation [18]. Spermatogonial stem cell transplantation was first presented in the mouse in 1994 by Brinster and Zimmermann [19]. Spermatogenesis could be reinitiated in infertile receiver mice after transplantation of testicular cell suspensions from fertile donors. Shortly later, this technology was done in other mammalian species, including primates [20][21][22][23][24]. Even the transplantation between various species was proven successful [25,26].
These results, particular those from primate studies, propose a possibility of banking and transplanting human spermatogonial stem cells to barricade sterility caused by SSC loss. In mice and rat, the efferent duct has been shown to be an affective site for reintroducing SSCs by injection. However, compared to mouse and rat testis, human testis are bigger than and more fibrous. Thus, the injection technique has to be rectified. By using excised human testis donated by orchidectomy patients, different injection sites have been checked: the seminiferous tubules, the rete testis, the epididymis and the deferent duct. Schlatt et al.
[21] have demonstrated that ultrasound-guided intratesticular rete testis injection was the better and least invasive injection technique with maximal infusion efficiency for larger testis. Tagged cells could be found in tubules near to the rete testis but not in tubules far away the rete testis. Brook et al.
[27] evaluated the performance of single and multiple injections through the rete testis of isolated human testis.
Nevertheless, the hole into the testis was not monitored and it was unclear whether the injected hue had been injected in the seminiferous tubules or in the interstitial tissue or both. If spermatogonial stem cell transplantation has been demonstrated to be a successful procedure to produce live offspring in a mouse model [28], genetic and epigenetic changes due to the procedure should be taken into attention. Goossens et al. [29,30] observed that after in vivo and in vitro conception, transplanted males generated smaller litter sizes compared to normal fertile control mouse. However, the offspring demonstrated normal karyotypes and methylation patterns [31,32]. In another study, a detailed analysis of the motility kinematics and attentions of spermatozoa acquired after transplantation was performed demonstrating a less sperm concentration and sperm motility after transplantation [33]. The latter results may explain the reduced litter size as watched in the in vivo mating studies.
Taking into account the significance of the spermatogonial stem niche in spermatogenesis, grafting testicular tissue may be another to obtain functional sperm that is able to fertilize oocytes. In rodents and rabbits, perfect spermatogenesis has been seen and fertile offspring could be acquired through assisted reproductive techniques after grafting immature testicular tissue [34,35]. Grafting testicular tissue of human, however, did not produce spermatozoa. In prepubertal xenografts, this period was even lengthy (9 months) [36]. Lately, differentiation up to primary spermatocytes and the attendance of a few secondary spermatocytes was showed in testicular tissue from a peripubertal boy after xenotransplantation to mouse [37].

Spermatogonial stem cells as pluripotent cells
Newly, the pluripotency of SSCs from neonatal and adult mouse testis has been reported by various research teams. Kanatsu-Shinohara et al. [38] demonstrated the derivation of ES-like cells from neonatal mouse testis in culture. In their researches, ES-like cells were shown to be phenotypically alike to embryonic stem cells and to have the ability to differentiate in vitro into different types of somatic cells and to generate teratomas after injection into nude mice. In addition to, these ES-like cells formed germ line chimeras when injected into blastocysts, which proves the pluripotency of the spermatogonial stem cells derived from neonatal testis.
Afterwards, Guan et al. [39] demonstrated that SSCs from adult mouse testis could produce multipotent cells in vitro that were able to differentiate into various cell types of all three germ layers and to produce teratoma in immune deficient mice.

Conclusion
Transplantation of SSCs may become a promising procedure to keep the fertility of prepubertal patients. Their testicular tissue could be removed and cryopreserved before treatment. After recuperation, the tissue/cells could be grafted or transplanted into the patient's own testis and fortunately spermatogenesis will be reinitiated. For cancer patients, additional methods would be essential for the isolation of SSCs and the removal of malignant cells. Other future choice would be in vitro maturation of SSCs after isolation and malignant cell removal. By this means, functional spermatozoa could be derived from mouse SSCs [41]. These in vitro generated spermatozoa could be used to fertilize oocytes by assisted reproductive techniques. These above indicated strategies could also be appropriate for adult patients with non-obstructive azoospermia. After seclusion and in vitro maturation, SSCs from these donors may have the capacity to generate functional spermatozoa. Although, before using these methods in patients, more research is required.

Open Access Journal of Surgery
The potential of human spermatogonial stem cells to differentiate into pluripotent cells or transdifferentiate into other cell types is interesting and may create an additional function for spermatogonial stem cell banking. Apart from fertility preservation, the banked cells could potentially be used as a provenance for stem cell therapy targeting other diseased organs. SSCs could be induced into pluripotent cells in vitro, by adding sure growth factors or inductive mesenchymes before their induction into the target cell type. Other strategy may be the straight injection into the human body at the location of injury or disease, e.g. bone marrow, myocardium. However, a number of problems abide before SSCs can be used as an alternative origin for pluripotent stem cells. The main challenge is to acquire sufficient numbers of purified SSCs from a small part of testicular biopsy.
A large part (about 1 g) of testicular biopsy includes less than 5 million cells with only 1500 being right stem cells. This is far from the number required for cell therapy besides mentioning the low de-and transdifferentiation rates. Therefore, an efficient procedure for SSC isolation and in vitro enlarging has to be developed. Several researches on the in vitro amplification of human adult SSCs has been reported [42]; an important challenge for any clinical application is to eschew the risk of teratoma formation after transplantation. The development of an effective induction protocol and the regulation of the recipient's immunology system may be essential to remove the risk of teratoma formation. Although there is not yet an obvious clinical prospect for SSC-based cell therapy in the clinic, SSC banking may become an encouraging and applicable strategy in the future.