Spermatogonial Stem Cell Isolation, Storage, and Transplantation

The past 30 years have been marked by unparalleled accomplishments in the medical treatment of malignancy. Prior to advances in chemotherapeutic and radiation treatment, many oncologic conditions had dismal survival rates. Today, medical interventions have success rates that approach complete remission for many malignancies. An inadvertent complication of these therapies, however, has been the high rate of infertility following treatment. Male germinal tissue, like many malignancies, is mitotically active and therefore is particularly susceptible to the toxic effects of both chemotherapy and radiotherapy (Meistrich et al, 1982; Meistrich, 1993). Consequently, posttreatment patients often develop severe oligozoospermia or azoospermia (Wallace et al, 1991). Potential infertility complications can be anticipated, and adult male patients interested in future procreation are counseled to cryopreserve semen before instituting treatment. With present-day capabilities of in vitro fertilization, particularly intracytoplasmic injection, male patients can maintain posttreatment fertility. Pretreatment sperm banking, however, is not a viable option for prepubescent males. These individuals have not yet begun spermatogenesis and thus lack viable spermatozoa. It is estimated that, by the end of the decade, 1 in 250 young men will be childhood cancer survivors (Blatt, 1999). For these patients, infertility has often been an accepted consequence of their life-saving treatment. A great deal of interest has recently been shown in testicular autologous transplantation, an intervention that may provide a future therapeutic fertility option for these individuals. Having been successfully demonstrated in rodent models, investigators have now begun to explore the possibility of using testicular autotransplantation to restore fertility in humans. This paper will review the history of spermatogonia transplantation with an emphasis on the clinical pertinence of this field of investigation. Current innovations involving the isolation of spermatogonial stem cells (SSCs) and the present capabilities of in vitro proliferation will additionally be reviewed. Spermatogonia are male germinal progenitor cells and are composed of differentiated nonstem and stem cells. Stem cells are characterized by a capacity for self-renewal and an ability to produce differentiating cell lines (Loeffler and CS, 1997; van der Kooy and Weiss, 2000). Spermatogonia are diploid germ cells that originated from primordial germ cells (PGCs). These precursor cells originate from embryonal ectoderm. PGCs migrate to the genital ridge, where they become known as gonocytes. Gonocytes are surrounded by Sertoli precursor cells in what become the seminiferous cords. Tight junctions between adjacent Sertoli cells later become the basis of the blood testis barrier. The gonocytes undergo mitotic division, followed by arrest in the G0 phase of the cell cycle. They are mitotically inactive until after birth, when they become spermatogonia (Clermont and Perey, 1957; de Rooij and van Dissel-Emiliani, 1997). Understanding spermatogonial nomenclature, differentiation, and regulation is important for comprehending testicular transplantation. First, distinguishing SSCs from differentiating spermatogonia has been a challenge that has been met with limited success. The term undifferentiated spermatogonia refers to As, Apr, and Aal cell types. Undifferentiated spermatogonia are considered distinct from “differentiating spermatogonia.” The latter group, ordered in succession, consists of A1, A2, A3, A4, Ain, and then B spermatogonia. It has been speculated that only As spermatogonia are stem cells that may divide into 2 identical daughter As spermatogonia or into 2 Apr (paired) daughter cells that are functionally committed to differentiation (Huckins, 1971). Apr cells differentiate into 4, 8, or 16 Aal cells; there is no cell division from Aal to A1; however, there is a transformation while in the G0/G1 phase (de Rooij and van Dissel-Emiliani, 1997). Some controversy surrounds the As-based categorization of spermatogonia, and additional classification and nomenclature schemes exist. However, most concur with the As hypothesis of spermatogonia development (Russell et al, 1990). Throughout the remainder of this review, the As nomenclature will be used. Various techniques have been suggested to distinguish As spermatogonia from spermatogonia committed to differentiation. Morphology is inadequate for this purpose. It has been asserted that undifferentiated (As-Aal) spermatogonia can be distinguished from differentiating spermatogonia (A1–4, Ain, B) because the latter cells will be in the G2 or M phase, while undifferentiated cells will not divide synchronously (de Rooij and van Dissel-Emiliani, 1997). Additionally, Apr and further differentiated spermatogonia form cellular bridges, which allow the sharing of gene products and facilitate synchronized development (Weber and Russell, 1987; Braun et al, 1989). SSCs do not have intercellular bridges. However, definitively determining that a cell lacks a bridge is a profound challenge, limited by tissue sectioning. Additionally, the capacity to distinguish As spermatogonia on the basis of the absence of intercellular bridges is limited, since it is known that some gonocytes possess intercellular bridges. There are methods to help identify the spermatogonial stage on the basis of topographical criteria (Huckins, 1971; Oakberg, 1971). However, these methods provide neither an efficient nor an effective means by which to distinguish the As or SSCs from other spermatogonia. Lastly, spermatogonial density regulation takes place at the A2 through B spermatogonia (de Rooij and Lok, 1987). This phenomenon is influenced by programmed cell death and ensures that the number of differentiated germ cells does not exceed the organism's need (de Rooij and Grootegoed, 1998). When larger numbers of differentiated spermatogonia are present, degeneration occurs more frequently, thus reducing the cellular population. This apoptosis is similar to events occurring in somatic cells (Conlon and Raff, 1999). In 1994, Brinster and Zimmerman published their landmark findings in the field of testicular tissue transplantation. Using a mixed cellular solution obtained from dissociated testicular parenchyma, they infiltrated recipient mouse seminiferous tubules with the donor cells. Among the hallmark findings of this experiment was the discovery that donor spermatogonial cells could interact with the host environment, migrate from the adluminal compartment, and negotiate past Sertoli-Sertoli tight junctions to enter the basal compartment (Griswold, 2000). Brinster and Zimmerman (1994) demonstrated successful donor spermatogenesis from testicular tissue transplanted between different mouse subjects. They used donor testicular tissue harvested from postnatal mice between days 4 and 12 of life. The assumption was that immature mice would have the highest concentration of undifferentiated spermatozoal progenitor cells or gonocytes, thereby providing the largest quantity of viable cells for transplantation. Testicular tissue was mechanically and enzymatically dissociated into a cellular suspension. The suspension was microinjected into mice pretreated with busulfan to eliminate native spermatogenesis. Donor cells came from transgenic mice expressing the LacZ (Escherichia coli B-galactosidase) gene; these cells, when differentiated to the round spermatid phase, stained blue, distinguishing them from the recipient's native sperm cells. The authors identified restored spermatogenesis in the recipient mouse along with colonization and differentiation of the donor tissue (Brinster and Zimmermann, 1994). Brinster and Avarbock in 1994 reported successful spermatogenesis in a mouse allogenic spermatogonial cell transplantation experiment. They found that the donor-derived spermatogonia were responsible for generating offspring; transmission was confirmed by the presence of a donor haplotype in the resulting progeny (Brinster and Avarbock, 1994). Cryopreservation before transplantation was first described by Avarbock and colleagues (1996). They reported successful transplantation after freezing the donor tissue for up to 156 days. Clouthier and colleagues (1996) published the subsequent landmark investigation in testicular transplantation. In this investigation, rat testicular tissue was introduced into immunodeficient mouse testis. The transgenic rat tissue was identified in the mouse seminiferous tubules, and differentiated rat germinal tissue (including spermatozoa) was recovered from the mouse epididymis (Clouthier et al, 1996). Nagano and colleagues (1998) then demonstrated the capacity to culture spermatogonial cells in vitro, followed by testicular transplantation. Their study found that spermatogonia survived in culture for up to 4 months. Further investigations found that the intraluminal transplanted germ cells degenerated and disappeared by 1 month's time. The successfully transplanted spermatogonia localized at the basement membrane and began to show evidence of division by the first week after transplantation. Donor spermatogonia migrated to the basal compartment during the first month, and donor spermatozoa were noted by that time (Parreira et al, 1998; Nagano et al, 1999). The limits of spermatogonia transplantation were noted in more distant, xenogeneic transplantation experiments. Although limited colonization did occur with rabbit, monkey, bull, and human transplantation, no spermatozoa or postmeiotic germ cells were found after these transplantations. Schlatt and colleagues (1999a,b) transplanted germinal tissue in primates and found evidence of spermatogonial survival at 4 weeks. When bromodeoxyuridine (BrdU) was introduced into donor tissue before transplantation, immunostaining located cells in the interstitium and seminiferous tubules that were identified with the BrdU label in their nuclei at 4 weeks. Morphologic criteria indicated these were type B or differentiated spermatogonia (Schlatt et al, 1999a). In 2001, Nagano and colleagues demonstrated the transplantation of baboon testicular tissue into nude mice. In this investigation, the authors identified the survival and propagation of the transplanted cells for up to 6 months. They used a rabbit-produced anti-baboon antibody in conjunction with an anti-human antibody to identify the baboon cells. They noted that baboon cells had migrated to the basement membrane of the seminiferous tubules—indicating that mouse Sertoli cells had somehow interacted with the baboon spermatogonia and had allowed passage through the blood testis barrier. Despite this evidence of favorable interactions between the 2 tissue types, the baboon spermatogonia showed no signs of spermatogenesis (Nagano et al, 2001). To date, successful donor-derived spermatogenesis has been primarily limited to phylogenetically similar species. In addition to mouse-to-mouse transplants, spermatogenesis has been noted in rat-to-immunodeficient mouse transfers (Clouthier et al, 1996), hamster-to-immunodeficient mouse transfers (Ogawa et al, 1999a), and mouse-to-rat transfers (Ogawa et al, 1999b). It has been theorized that evolutionary distance is primarily responsible for the failure of more distant xenogolous transplantations. Mice and rats are thought to have diverged evolutionarily 10 to 11 million years ago, and hamster and mice are thought to have diverged 16 million years ago (Catzeflis et al, 1993). Transplants with animals separated by greater evolutionary have been This is to spermatogonia and Sertoli cell and other and colleagues found that hamster spermatogenesis in mice in hamster spermatozoa. This suggested that the recipient Sertoli cells had influenced the differentiation of the spermatozoa and that the in the in Despite the differentiated germinal transplantation between different has in of The described interactions between the host testicular and the donor germinal tissue these using human tissue have in have reported the survival of at some undifferentiated spermatogonia during distant et however, reported that there was no evidence of donor tissue survival following a mouse testicular tissue transplantation. These investigators were using the antibody to to identify successful transplantation. is a of differentiated human spermatogonia and and would not have transplanted cells that had survived or not the other et reported successful spermatogenesis from of human tissue into rat and mouse In Nagano and colleagues reported on the of anti-baboon antibody to identify the survival of human spermatogonia in mouse for up to 6 These investigators found no evidence of the donor The of following testicular tissue transplantation further investigation in this the basis of the findings from other it is that complete spermatogonial differentiation will not be The distance between will into between the host testicular and donor spermatogonia that complete spermatogenesis. of spermatogonia have great into the of spermatogenesis and different and colleagues found that mice demonstrated restored fertility when transplanted spermatogonia from stem cell mice. a found on differentiated spermatogonia, has been with a of that spermatogonia can in which as et al, and survival et al, et al, are Stem cell a of Sertoli cells, is the et al, 1999). by and colleagues demonstrated that infertility resulting from a was a germ cell The testicular in these the Sertoli cells, could facilitate spermatogenesis differentiated spermatogonia. Additionally, this study showed that the stem cell did not the differentiating capacity of spermatogonia when in a The that mice could spermatogenesis has The Sertoli cells in these mice had no to differentiated spermatogonia. However, they were of spermatogenesis from transplanted germ cells. This discovery to the of testicular transplantation to restore fertility in cancer and in a severe of germinal tissue in these of the the testicular of these posttreatment patients primarily of Sertoli cells. spermatogonia and differentiation in the it is that, after treatment, many cancer survivors could spermatogenesis viable spermatogonia were into their testis. Further spermatogonial transplantation have from investigations using mice. Testicular tissue from these transplanted into mice with did not in donor-derived spermatogenesis et al, 2000). This suggested that the differentiation a germ cell and not from a in the The determining the of spermatogenesis were further by transplantation in the Brinster mouse spermatogenesis takes place while rat spermatogenesis is to days. The spermatogonial showed that spermatogenesis of the rat donor germ cells, in a mouse testicular environment, followed the rat days before rat spermatozoa were et al, 1998). This showed that the of spermatogenesis is a by the testicular somatic environment, spermatogenesis is not by Testicular transplantation was used by and colleagues to the of gene on gene mice are known to be to a failure of spermatogenesis et al, 1996). When spermatogonia from mice were transplanted into donor-derived spermatozoa The of these spermatozoa were confirmed by the of progeny the donor This discovery demonstrated that was for spermatogonial differentiation because of on testicular cells, not germinal cells et al, 2001). The transplantation was as time to The that host colonization by the donor spermatogonia the the quantity of undifferentiated spermatogonia the transplanted the host environment, and in the transplantation 2 in testicular cells are SSCs (Meistrich and van 1993). most testicular transplantation cellular an estimated to stem cells and a to success rate in generating (Nagano et al, 1999). the of spermatogonia in the transplanted investigators have that a greater quantity of stem cell spermatogonia can be introduced into the the of SSCs in a transplanted solution the of colonization with the donor germinal the isolation of spermatogonia, particularly spermatogonia As or stem cells, has been an active Among the methods to more SSCs are the or of animals with spermatogonial failure germ cell the in vitro of spermatogonia, and the cellular isolation of stem cells on the basis of cell or other animals have seminiferous tubules with only undifferentiated spermatogonia. it is estimated that 1 in cells in a testis is an and 2000). An effective for the undifferentiated spermatogonial for donor transplantation has been the of tissue from mice. mice were after 2 to have only type A spermatogonia and and mice are of testis thought to of undifferentiated spermatogonia. The a in the of the mice spermatogonial As a their are by a of undifferentiated spermatogonia are of mice (Ogawa et al, 2000). However, investigations have into the quantity of stem cells in these and 2000). Using spermatogonial transplantation as a for the presence of spermatogonial cells, et reported that from in a in the number of spermatogonial was with donor cells from mice. Additionally, the of undifferentiated spermatogonia can be by medical mice and rats have only undifferentiated spermatogonia and may as for these cells et al, 1996). arrest at Aal can be with A et al, It is that A the differentiation of spermatogonia a with cells that are in stage of the cycle. A to synchronized differentiation et al, 1987; and 1987; van and de 1990). of the numbers of SSCs in the many that antibody or advances in cell will be to spermatogonial transplantation a therapeutic The is a that has been in to undifferentiated spermatogonia. antibody that undifferentiated spermatogonia are while differentiated spermatogonia are et al, et al, to SSCs have been the of these cells. cell is a cell that is on of cells and cell In a in a of spermatogonia stem cells, as by a subsequent transplantation In the described spermatogonia were on the basis of This was because cells with a of undifferentiated spermatogonia, could be by this These cells or had of and they were for 6 the presence of did not stem cell isolation et al, The most of the and of SSCs used a cell to identify SSCs as a in the adult testis et al, Spermatogonia do not and for in a of stem cells are for the and and the for testicular cells in the that and are for a that of the et al, this cell is for other or used to for stem cells, and et al, The for the and of SSCs along with the transplantation a means of a of these cells, thereby the of a of stem cells. the for mouse SSCs are and and are not present in cells with the cell is that may be used to spermatogonia. and colleagues reported the isolation of spermatogonia a cell A solution of testicular cells was to was then using with A of to of the cells were on the basis of and and were used by and colleagues to an of cells to type A spermatogonia. Using to the isolation of SSCs has been noted to to et al, 1999). and colleagues (1996) used in conjunction with to an of type spermatogonia. cell may be a for the isolation of spermatogonia on the basis of and density et al, germ cells in the adult only 2 are stem cells (Meistrich and van 1993). It has been asserted that the numbers of spermatogonia in would the isolation of spermatogonia future therapeutic by density and inadequate et al, 2000). As a treatment for before with autologous testicular transplantation, it will be to develop more effective The culture of male germinal cells has been a challenge, and have met with limited success. and reported on a Sertoli cell from mouse germ cells that demonstrated no of at days. et found that a culture demonstrated up to for spermatogonia at days. have demonstrated further culture success et al, Schlatt et al, et al, 2000). Nagano and colleagues (1998) demonstrated that, using testicular transplantation as a spermatogonial survival and propagation could be demonstrated after days of cells survived for 4 in a on a of cells Using their culture that was on cells, Nagano and colleagues were to SSCs with a They noted the rates by the number of transplanted the were from cells in cells with to the They that the successful rate was limited to 1 in Additionally, the of donor spermatogonia were noted in the recipient for up to 6 following transplantation (Nagano et al, and colleagues demonstrated in rat success was by donor-derived spermatogenesis the the it was estimated that only to of the SSCs survived for days or using and an The of the that SSCs and the ability to these to proliferation in culture have for these cells and using them for et demonstrated that SSCs can be for 4 to 12 when culture was with 2 and in addition to The number of donor-derived from SSCs with these as time from 4 to 12 weeks. In the culture of SSCs in a known to the proliferation and of cell and in an in the number of SSCs as time et al, The of of round cells that on the of the that to form on of cells after The number of SSCs for and the transplantation of the SSCs restored fertility to mice. An of this study was that the reported successful culture and donor-derived spermatogenesis of SSCs from mice not from the SSCs of mice with or using this et al, This there may be important in the culture conditions for germ cells from different and this may the conditions to culture of SSCs with cells no in the demonstrated that the stem cells and to et al, The SSCs in and days. SSCs could be these conditions for 6 and donor-derived spermatogenesis when transplanted into These the hypothesis that is important in the undifferentiated of spermatogonia in the mouse testis. mice that in the testis are known to undifferentiated spermatogonia et al, This to the of SSCs will of the the stem cell in the testis. and provide a for gene This could allow to be from the germ An effective for SSCs would allow a quantity of tissue from the to into a quantity for transplantation. an option would allow a before with effects to germinal this quantity of native spermatogonia would be The success of the intervention would be by the of the with germinal tissue, effective and fertility as by semen and rates. The investigation by Brinster and (1994) that the host pretreated with busulfan was more to transplanted spermatogonia the testis of animals with spermatogenesis. they found a success rate mice with an success rate mice with spermatogonia these may have been to methods for the of successful transplantation. severe of spermatogonia from the testis. of a to 1 or more of has been with a of by days (de Rooij et al, to the in cellular particularly of the more cells. in quantity the differentiated spermatogonia, while the As are the most der et al, has not been in the of recipient Among the is the greater with radiotherapy with this would the of this Using a and colleagues (1998) demonstrated colonization of recipient a of of the cells of the to from testicular cells. The testicular was found to be more of transplanted germinal tissue (Ogawa et al, 1998). were by the donor cellular solution through the investigators used into the seminiferous In larger host introduced the donor cellular solution into the testis. Schlatt and colleagues (1999a,b) reported on these techniques used on and They described the as more efficient in mouse recipient These investigators noted that into were limited in of They this to high In that were or immature had a greater of the seminiferous tubules during This was to a quantity of Sertoli cell marked by a seminiferous (Schlatt et al, 1999a). cell transplantation of testicular cells from donor transgenic for human into the of immature recipient in 2 of the recipient sperm for the The germ cells in a of were into the immature by an of of the recipient 1 that was for the human et al, To this the first time that donor-derived spermatogenesis in the transmission of a donor haplotype to the It that immature similar to rodent may the for donor-derived spermatogenesis in species. This may be to the of the seminiferous and the lack of Sertoli junctions before The most clinical of testicular transplantation will chemotherapy with These individuals could undergo a testicular The tissue could be used for isolation and or of treatment with no evidence of these patients could undergo an autotransplantation with their or spermatogonia. The subsequent of their with germinal tissue would in spermatogenesis and fertility. the may be or in the there are that be considered before clinical that are blood as and be considered a great for of into the the other as may not a for patients autologous et al, 2000). and colleagues reported on the transmission of rat testicular transplantation from These findings the profound of spermatogonial isolation and The investigators noted that as as cells introduced into the recipient testis were of into the et al, 2001). be with the of human xenogeneic as a clinical it is an In the have human cells et al, 1997). the possibility of a into human germ the of clinical investigations Among the further is the of human spermatogonial cells. there is no for human spermatogonia. The for used as the et al, 1996). There have been of using freezing to cryopreserve testicular tissue and However, the lack of investigation in the of is of success. It can be that future on the of SSCs will of and rate of during the freezing and This will in the more These as be and before and transplantation become a For these of are and the future and of these in the of in young male cancer