Byung-Gak Kim

Enrichment of Testicular Gonocytes and Genetic Modification Using Lentiviral Transduction in Pigs

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Stage-specific embryonic antigen-1 expression by undifferentiated spermatogonia in the prepubertal boar testis

The objective of this study was to use fluorescence-activated cell sorting (FACS) and spermatogonial stem cell (SSC) xenotransplantation to identify cell surface markers of putative porcine SSC. Analysis of porcine testis cells enriched for spermatogonia using FACS indicated that nearly half of stage-specific embryonic antigen-1 (SSEA-1) expressing testis cells expressed the undifferentiated spermatogonia marker protein gene product 9.5 (PGP 9.5) whereas significantly fewer (P < 0.05) cells selected for thymus cell antigen-1 (Thy-1), also known as cluster of differentiation 90 (CD90), cluster of differentiation 9 (CD9), or other SSC markers expressed PGP 9.5. Immunocytochemical analysis indicated that promyelocytic leukemia zinc finger (PLZF) protein and germ cell lineage marker VASA homolog (VASA), also known as DEAD box protein 4 (DDX4), were expressed by SSEA-1 expressing germ cells. Spermatogonial stem cell xenotransplantation of testis cell populations enriched for cells expressing SSEA-1 generated significantly (P < 0.05; greater than 15-fold) more colonies of donor derived germ cells than unselected testis cells. In conclusion, these data indicate that SSC markers identified in rodents are likely not entirely conserved in pigs and that SSEA-1 is a marker for porcine undifferentiated spermatogonia including SSC in prepubertal boars and its expression may serve as a target for the further study of porcine germ cells.

Cryopreservation of porcine spermatogonial stem cells by slow-freezing testis tissue in trehalose.

Spermatogonial stem cells provide the foundation for continued adult spermatogenesis and their manipulation can facilitate assisted reproductive technologies or the development of transgenic animals. Because the pig is an important agricultural and biomedical research animal, the development of practical application techniques to manipulate the pig Spermatogonial stem cell is needed. The ability to preserve porcine Spermatogonial stem cell or testis tissue long term is one of these fundamental techniques. The objective of this study was to optimize methods to cryopreserve porcine Spermatogonial stem cell when freezing testis cells or testis tissue. To identify the most efficient cryopreservation technique, porcine testis cells (cell freezing) or testis tissue (tissue freezing) were frozen in medium containing dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) or DMSO, FBS, and various concentrations of trehalose (50, 100, or 200 mM). After thawing, undifferentiated germ cells were enriched and treatments were evaluated for cryopreservation efficiency. The tissue freezing method resulted in significantly greater germ cell recovery (P = 0.041) and proliferation capacity (P < 0.001) compared to the cell freezing treatment. Regardless of freezing method (cell vs. tissue), addition of 200 mM trehalose to freezing medium increased germ cell recovery and proliferation capacity compared to cells frozen using the same freezing method without trehalose. Interestingly, addition of trehalose to the tissue freezing medium significantly increased germ cell recovery (P = 0.012) and proliferation capacity (P = 0.004) compared to the cell freezing treatment supplemented with trehalose. To confirm that cryopreservation in trehalose improves the survival of Spermatogonial stem cell, testis cells enriched for undifferentiated germ cells were xenotransplanted into recipient mouse testes. Germ cells recovered from tissue frozen with 200 mM trehalose generated significantly more (P < 0.001) donor derived colonies than tissue frozen without trehalose. Regardless of cryopreservation medium or freezing method, testis cell recovery, viability, and proliferation capacity of germ cells after thawing were significantly lower compared to those of untreated fresh control. Nevertheless, these data demonstrate that undifferentiated porcine germ cells can be efficiently cryopreserved in the presence of 200 mM trehalose.

Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells.

OBJECTIVE To study the influence of sugars and establish a serum-free freezing method for the cryopreservation of spermatogonial stem cells (SSCs). DESIGN Animal study. SETTING University laboratory. ANIMAL(S) C57BL/6-TgEGFP, C57BL/6 mice. INTERVENTION(S) Germ cells enriched from testis cells were frozen using standard freezing medium containing sugars, including monosaccharides, disaccharides, and trisaccharides at 50, 100, and 200 mM, respectively. To study the feasibility of establishing a serum-free freezing method, fetal bovine serum was substituted with knockout serum replacement. MAIN OUTCOME MEASURE(S) Freeze-thawed germ cells were evaluated for recovery rate, proliferation capacity, and stem cell activity after transplantation to recipient testes. RESULT(S) Supplementation of freezing medium with 200 mM disaccharide is an effective method for cryopreservation of SSCs. Trehalose is the most effective cryoprotectant among all the sugars tested and only lactose was comparable to trehalose. Our proliferation and transplantation data show that serum-free freezing can be achieved in freezing medium supplemented with 200 mM trehalose, 10% knockout serum replacement, and 10% dimethyl sulfoxide (DMSO) for cryopreservation of SSCs. CONCLUSION(S) These findings raise the possibility of effectively banking frozen SSCs from various species, including humans, in a traditional serum-free medium for germ cell research and male infertility treatments.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Lentiviral modification of enriched populations of bovine male gonocytes.

Undifferentiated germ cells have the capacity to develop into sperm capable of fertilizing oocytes and contributing genetic material to subsequent generations. The most primitive prepubertal undifferentiated germ cells include gonocytes and undifferentiated spermatogonia, including spermatogonial stem cells (SSC). Gonocytes, present in the testis at birth, differentiate into SSC, which maintain spermatogenesis for the remainder of the males life. Because of their capacity to contribute to lifelong spermatogenesis, undifferentiated germ cells are attractive targets for genetic modification to produce transgenic animals, including cattle. To maximize the efficiency of genetic modification of bovine gonocytes and SSC, effective enrichment techniques need to be developed. Selection of bovine gonocytes using differential plating was improved 8-fold (P < 0.001) when using a combination of extracellular matrix proteins, including laminin, fibronectin, collagen type IV, and gelatin, compared to using laminin and gelatin alone. Selected cells labeled with PKH26 formed colonies of donor-derived germ cells after transplantation into recipient mouse testes, indicating putative stem cell function. Significantly more colonies (P < 0.001) per 1 × 10(5) viable transplanted cells were formed from isolated nonadherent cells (203 ± 23.2) compared to adherent (20 ± 2.7) or Percoll (45.5 ± 4.5) selected cells. After selection, some gonocytes were transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Collectively, these data demonstrate effective methods for the selection and genetic modification of bovine undifferentiated germ cells.

Efficient enhancement of lentiviral transduction efficiency in murine spermatogonial stem cells

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male’s lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

In vitro spermatogenesis using bovine testis tissue culture techniques

Spermatogenesis is a complex process initiated by spermatogonial stem cells (SSCs) that have the ability to differentiate into mature spermatozoa or to self-renew to maintain the SSC population and long-term fertility. However, a technique for complete spermatogenesis in vitro using cell culture has not yet been developed. In the present study, we developed in vitro spermatogenesis techniques using bovine testis tissue culture. The effects of specific temperatures and different media on maintaining tubule and germ cell competency were investigated. We found that the optimal temperature and media were 37°C and mouse serum-free medium (mSFM), respectively. In addition, the efficacy of various hormones and growth factors on spermatogenesis in bovine testis tissues maintained in vitro was evaluated. We found that the addition of triiodothyronine (T3) and stem cell factor (SCF) induced spermatogenesis of bovine SSCs in vitro. Therefore, tissue fragments were cultured in the presence of T3 and SCF for three months to induce spermatogenesis in vitro. Overall, in vitro spermatogenesis was enhanced 2.4- to 2.7-fold. Our tissue culture technique may serve as a model system that leads to a more comprehensive understanding of the biology of SSCs as well as the factors that regulate male fertility. Furthermore, the results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.