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The early detection of tumors improves chances of decreased morbidity and prolonged survival. Serum biomarkers are convenient to use and have several advantages over other approaches, such as accuracy and straightforward protocols. Reliable biomarkers from easily accessible sources are warranted for the development of cost-effective assays for routine screening, particularly in veterinary medicine. Extracellular c-AMP-dependent protein kinase A (ECPKA) is a cytosolic leakage enzyme. The diagnostic accuracy of detecting autoantibodies against ECPKA was found to be higher than that of ECPKA activity from enzymatic assays, which use a complicated method. Here, we investigated the diagnostic significance of measuring serum ECPKA autoantibody levels using an in-house kit (AniScan cancer detection kit; Biattic, Anyang, Korea). We used sera from 550 dogs, including healthy dogs and those with malignant and benign tumors. Serum ECPKA and immunoglobulin G were determined using the AniScan cancer detection kit. ECPKA autoantibody levels were significantly higher (p < 0.01) in malignant tumors than in benign tumors, non-tumor diseases, and healthy controls. On the basis of sensitivity and specificity values, AniScan ECPKA is a rapid and easy-to-use assay that can be applied to screen malignant tumors from benign tumors or other diseases in dogs.

A heave motion of the offshore crane system with load is affected by unpredictable external factors. Therefore the offshore crane must satisfy rigorous requirements in terms of safety and efficiency. This paper intends to reduce the heave displacement of load position which is produced by rope extension and sea wave disturbance in vertical motion. In this system, the load position is compensated by the winch actuator control. The rope is modeled as a mass-damper-spring system, and a controller is designed by the input-output linearization method. The model system and the proposed control method are evaluated on the simulation results.

// Bang-Jin Kim 1,7 , Yong-Hee Kim 1 , Yong-An Lee 2 , Sang-Eun Jung 1 , Yeong Ho Hong 1 , Eun-Ju Lee 3 , Byung-Gak Kim 4 , Seongsoo Hwang 5 , Jeong Tae Do 6 , Myung-Geol Pang 1 and Buom-Yong Ryu 1 1 Department of Animal Science & Technology, Chung-Ang University, Anseong, Republic of Korea 2 Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore 3 Department of Internal medicine, Seoul National University, Seoul, Republic of Korea 4 Bio Environment Technology Research Institute, Chung-Ang University, Anseong, Republic of Korea 5 Animal Biotechnology Division, National Institute of Animal Science, Jeollabuk-do, Republic of Korea 6 Department of Stem Cell and Regenerative Biology, College of Animal Bioscience and Technology, Konkuk University, Seoul, Republic of Korea 7 Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America Correspondence to: Buom-Yong Ryu, email: // Keywords : germ-line stem cells; multipotent; testis; cardiac progenitor; differentiation Received : September 24, 2016 Accepted : February 28, 2017 Published : March 31, 2017 Abstract Cardiac cell therapy has the potential to revolutionize treatment of heart diseases, but its success hinders on the development of a stem cell therapy capable of efficiently producing functionally differentiated cardiomyocytes. A key to unlocking the therapeutic application of stem cells lies in understanding the molecular mechanisms that govern the differentiation process. Here we report that a population of platelet-derived growth factor receptor alpha (PDGFRA) cells derived from mouse multipotent germline stem cells (mGSCs) were capable of undergoing cardiomyogenesis in vitro . Cells derived in vitro from PDGFRA positive mGSCs express significantly higher levels of cardiac marker proteins compared to PDGFRA negative mGSCs. Using Pdgfra shRNAs to investigate the dependence of Pdgfra on cardiomyocyte differentiation, we observed that Pdgfra silencing inhibited cardiac differentiation. In a rat myocardial infarction (MI) model, transplantation of a PDGFRAenriched cell population into the rat heart readily underwent functional differentiation into cardiomyocytes and reduced areas of fibrosis associated with MI injury. Together, these results suggest that mGSCs may provide a unique source of cardiac stem/progenitor cells for future regenerative therapy of damaged heart tissue.

Objective Early and proper diagnosis of cancer is the most critical factor for the survival and treatment of veterinary cancer patients. In this study, we evaluated extracellular cyclic AMP-dependent protein kinase A (ECPKA) level in serum as a useful cancer biomarker in dogs. Methods ECPKA levels were detected in sera from dogs with cancers (n = 48), benign tumours (n = 18), and non-tumour diseases (n = 102) as well as healthy control dogs (n = 54) utilizing enzyme-linked immunosorbent assay (ELISA). Results Sera from dogs bearing various types of cancer exhibited markedly increased levels of ECPKA by up to 7.1-, 8.8-, and 10.9-fold compared with those from dogs harbouring benign tumours, dogs with non-tumour diseases, and healthy control dogs, respectively (P

The biological effects of a light-emitting diode (LED) light therapy device are determined by irradiation parameters, mainly wavelength and power density. However, using a battery to provide power causes a problem in the variation of LED power density during battery discharge. As a result, maintaining a stable LED power density, along with extending battery life and operating time, are the primary concerns in designing a LED light therapy device. The present study aims to introduce a LED light therapy device design with different LED color power density control. A Fuzzy logic, based on the relationship between LED power density and operating time, was proposed to control constant power density in this design. The experimental results demonstrate that by using the designed controller, the LED light therapy device's power density (40 mW/cm2, 50 mW/cm2, 60 mW/cm2 for red, blue, and green light, respectively) can be controlled. The newly designed LED light therapy device could be considered an advanced version with energy savings and stabilized LED power emitting property under a broad range voltage variation.

This paper presents the design of an Electrocardiogram (ECG) signal acquisition system based on 32-bit microprocessor platform. In hardware design, we present a complete design similar to the wearable devices used to monitor heart rates over the long term. The firmware is applied by an adaptive data compression method to increase storage space and reduces data transmission time between hardware devices and personal computer (PC). This adaptive algorithm is a combination of Joint Photographic Experts Group (JPEG) compression. The ECG signal was applied to the nearly-linear infinite impulse response (IIR) filter to eliminate noise from ECG signals. There are advantages of nearly-linear IIR filter over traditional IIR that the signal pass through filter is not distorted. Improvements in the JPEG compression algorithm make them suitable for one dimensional signals with a resolution greater than 8 bits and optimize the operation of the Huffman compression as well as the microcontroller unit (MCU) system with low processing speed. Placing the IIR filter at the end of the system promotes the ability to filter while reducing signal distortion after the decompression process. We tested the system with 8 records from the MIT/BIH cardiac arrhythmic database before and after we applied the compression algorithm to the firmware and software in the system that shows the effectiveness of these algorithms. The test results show that the system reached a maximum compression ratio of 8,8. The system deployed on the 32bit MCU (PIC32MZ2048), ECG analog front-end ADS1293 and operation time of ECG device up to 110 h (4.5 days).

Two isoforms of the 70-kDa ribosomal protein S6 kinase, S6K1 and S6K2, have been identified and are considered key downstream effectors of the mTOR signaling pathway, which is involved in tumor growth and progression. However, their biological roles in the tumor microenvironment are poorly understood. In this study, utilizing tumor xenograft models in S6k1−/− and S6k2−/− mice, we show that loss of S6K1 but not S6K2 in the tumor stroma suppresses tumor growth, accompanied by attenuated tumor angiogenesis. We found that while S6K1 depletion had no effect on the proangiogenic phenotype of endothelial cells, the growth and angiogenesis of tumor xenografts were significantly reduced in wild-type mice upon reconstitution with S6K1-deficient bone marrow cells. Furthermore, upon S6K1 loss, induction of both mRNA and protein levels of Hif-1α and those of the downstream target, Vegf, was compromised in bone marrow–derived macrophages stimulated with lactate. These findings indicate that S6K1 but not S6K2 contributes to establishing a microenvironment that favors tumor growth through mediating angiogenesis, and suggest that attenuated tumor angiogenesis upon loss of S6K1 in the tumor stroma is, at least in part, attributable to impaired upregulation of Vegf in tumor-associated macrophages.

Protein kinase A, a cyclic adenosine monophosphate (AMP)-dependent enzyme, normally exists within mammalian cells; however, in cancer cells, it can leak out and be found in the serum. Extracellular cyclic AMP-dependent protein kinase A (ECPKA) has been determined to increase in the serum of cancer-bearing dogs. However, there have been no reports in the veterinary literature on serum ECPKA autoantibody (ECPKA-Ab) expression in dogs with cancer. The aim of this study was to evaluate ECPKA-Ab and C-reactive protein (CRP) as serum biomarkers for cancer in dogs. ECPKA-Ab and CRP levels were detected by an enzyme-linked immunosorbent assay in serum samples from dogs with malignant tumours (n = 167), benign tumours (n = 42), or non-tumour disease (n = 155) and from healthy control dogs (n = 123). ECPKA-Ab and CRP levels were significantly higher in the dogs with malignant tumours than in those with benign tumours or non-tumour diseases, as well as in the healthy controls (P < 0.001, Kruskal-Wallis test). There was a significant positive correlation between the neoplastic index, which was developed using ECPKA-Ab and CRP levels, and the presence of cancer in dogs (P < 0.001); the area under the receiver-operating characteristic curve was estimated to be >0.85 (P < 0.001). In conclusion, ECPKA-Ab is a potential serum biomarker for a broad spectrum of cancers. Combined measurement of CRP and ECPKA-Ab levels in serum improves the sensitivity and accuracy of a diagnosis of cancer in dogs.

The objective of this study was to investigate the expression of bovine luteum expressed sequence tags (ESTs), vascular endothelial growth factor (VEGF), and tumor necrosis factor receptor 1 (TNFR1) and the presence of functional ESTs in the bovine corpus luteum (CL) during different stages of the estrus cycle. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a difference in the expression of ESTs during the CL stage. Concentration of ESTs in the CL tissue increased significantly from the mid-luteal stage and decreased thereafter. RT-PCR analysis showed higher levels of the EST genes in the CL of the mid-luteal stage than in other stages, and the same level of expression of VEGF. Immunohistochemistry analysis of the tissue from CL formation to regression showed low cytosol and aggregation of the nucleus. And activity caspase 3 (apoptosis detector) was most strongly detected in the CL1 stage of bovine. During the estrous cycle, the cytosol was magnified and differentiation of the nucleus was clearly manifested. The ESTs affected the CL, and the relationship between VEGF and TNFR1 played a pivotal role for CL development and activation, dependent on the stage of CL. These results suggest local production of ESTs, the presence of functional ESTs in the bovine CL, and that ESTs play a role in regulating the function of cell death in bovine CL.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

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.

Spermatogenesis is a complex developmental process of continuous cell division involving a phase of germ cell expansion, meiotic division and cytodifferentiation that ultimately generates mature gametes. The object of this study was the development of optimal culture system in order to induce spermatogenesis in vitro and for its establishment. We collected immature testicular tissues from different developmental-stage of mice and rats which were subsequently cultured on the agarose gel with serum-free culture media. After appropriate culture period, we counted multiple layered seminiferous tubules in the cultured testis tissue to find out whether any differentiation occured following spermatogenesis. Between our experimental mouse strains ICR and C57, as a result, we observed significant increased spermatogenic tubules in case of C57 mouse strain in neonatal stage. In our optimized culture condition, we detected the lectin PNA positive spermatid resulted from spermatogenesis in cultured testis tissue after 6 weeks of incubation. We also injected the lentiviral transduced rat spermatogonial stem cells (SSCs) into recipient testis to generate the transgenic gametes. The testicular fragments were then cultured based on our optimized culture condition. After culturing 8 weeks, we observed expression of GFP in differentiated germ cells including post meiotic germ cells, which suggested the induction of donor-derived spermatogenesis. Also, in order to optimize the in vitro culture system for rat testis, we have tested the additional effect of hormones and growth factors, but could not observe the significant increased effect of hormone and growth factors treated groups than the control. In conclusion, our in vitro tissue culture methods could be applicable for production of in vitro transgenic gametes in various kinds of mammalian species including domestic animal.

Spermatogonial stem cells (SSCs) are adult stem cells that transmit genetic information from the parent to the next generation (progeny) in males, and thus, SSCs have used in germline-modification for generating transgenic animals. In this study, we demonstrated the feasibility of transgenic sperm production by employing an effective busulfan treatment method to prepare recipient pigs for the transplantation of genetically modified donor porcine SSCs. We purified SSCs from pig testis cells by sequentially employing Laminin-coated dishes and culture dishes. The purified cells were transduced with lentivirus expressing enhanced green fluorescent protein (eGFP) at a multiplicity of infection (MOI) of 6 for 9 h. eGFP transduced pig SSCs were then transplanted into the seminiferous tubules of 12 to 16-week-old recipients born to busulfan-treated sows. We obtained six recipient pigs after transplantation and maintained them for more than 6 months. The collected viable spermatozoa from 2 out of 6 recipients were positive for eGFP gene expression in polymerase chain reaction. eGFP-expressing spermatozoa appeared morphologically normal under the microscope. When spermatozoa from these recipients were used for intra cytoplasmic sperm injection, eGFP expression could be detected in the embryos. Furthermore, eGFP colonies were derived from donor-transduced SSCs observed in the recipients’ testes. In summary, we demonstrated the successful production of functional-transgenic spermatozoa by transplantation of porcine SSCs where the transgenic was transduced by employing the lentiviral vector system.

Primordial germ cells (PGCs) as well as fetal germ line stem cells (GSCs) are pluripotent cells. Their differentiation potential is similar to that of embryonic stem cells (ESCs), suggesting that germ line lineage may retain the potential to form pluripotent cells. Here we report successful establishment of multipotent germ line stem cells (mGSCs) from cells obtained from adult mouse testis. We obtained testes from Pou5f1 (Oct4)-GFP transgenic mice and demonstrate that the germ cells present in testes can be converted to pluripotent stem cell-like cells. The newly established mGSCs had similar morphology and growth characteristics as that of ESCs and expressed markers of pluripotency. To assess the differentiation potential of mGSCs in vivo and in vitro, we performed a teratoma formation assay and in vitro differentiation via embryoid body (EB) formation. Multipotent GSC-derived teratomas contained derivatives of all the three embryonic germ layers and differentiated into multiple lineages in vitro, including cardiomyocytes and neural cells. Using real-time qPCR analysis, we compared the gene expression pattern in the newly established mGSCs with those of pluripotent stem cells, germ cells, and testicular cells. The differentiation potential of the newly established mGSCs was comparable to that of ESCs and induced pluripotent stem cells (iPSCs). Multipotent GSCs derived from adult testes can be used for personalized cell-based therapies without the ethical and immunological problems.

Endometrial cancer is the most common malignancy of the female genital tract. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4-resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, we generated a mouse model in which we specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of signal transducers and activators of transcription 3 (STAT3) and proliferation of the endometrial epithelial cells. The results from our immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 via protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr-positive cells (Pgrcre/+Mig-6f/f). However, Sprr2fcre+Mig-6f/f mice were P4-responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. We showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells, and the antitumor effects of P4 are mediated by the endometrial stroma. These data help to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer.

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.

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.

Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.

Endometrial cancer is the most frequently diagnosed malignancy of the female genital tract. Hysterectomy is typically the first line therapeutic strategy for endometrial cancer. However, there is an increasing demand for nonsurgical approaches for endometrial cancer, especially for women of reproductive age with endometrial cancer who wish to preserve their fertility. Most endometrial cancers are characterized by actively proliferating epithelial cells, increased AKT signaling and association with prolonged, unopposed E2 exposure. Mitogen-inducible gene 6 (Mig-6) is an important mediator of progesterone receptor action in the murine uterus. As P4 achieves the inhibition of proliferation by coordinating stromal-epithelial cross-talk, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f) to understand the role of epithelial Mig-6 in the uterus. Sprr2fcre+Mig-6f/f mice displayed endometrial hyperplasia upon 10 weeks of age and develop endometrial cancer by E2 treatment for 3 months. The levels of epithelial proliferation by Ki67 staining were significantly increased in epithelial cells of Sprr2fcre+Mig-6f/f mice at 10 weeks of age. The levels of phospho-AKT and phospho-S6, downstream of AKT, were remarkably higher in Sprr2fcre+Mig-6f/f mice. Interestingly, the hyperplasia exhibited by Sprr2fcre+Mig-6f/f mice was prevented by P4 treatment for 1 week in morphological and histological analysis. Aberrant activation of proliferation as well as AKT signaling was decreased in the epithelium of Sprr2fcre+Mig-6f/f mice by P4 treatment. Furthermore, we identified that MIG-6 inhibits AKT phosphorylation in human endometrial cancer cells. These data suggest that stromal P4 signaling including Mig-6 is critical in regulation of epithelial cell proliferation via regulating AKT phosphorylation during endometrial cancer development and progression. (This work was supported by American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Tae Hoon Kim, Jung-Yoon Yoo, Jae Hee Lee, Byung Gak Kim, Russell R. Broaddus, Jae-Wook Jeong. MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3674.

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.

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.

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.