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Spermatogonial stem cells (SSCs) are unique with an important role in the transmission of genetic information to the next generation. Thus, they play an important role for the production of interspecies germ line chimeras. Therefore, the objective of this study was to produce chimera through the intraperitoneal transplantation of Caspian brown trout SSCs into newly-hatched rainbow trout. Spermatogonial cell were isolated from the testes of 8-month-old Caspian brown trout through enzymatic digestion. The spermatogonial cell suspension was enriched using differential plating technique to remove testicular somatic cells. After culturing for 48 h in L15 supplemented with 10% serum, suspended cells were collected and stained with the fluorescent membrane dye PKH26. The stained cells were intraperitoneally transplanted into triploid rainbow trout hatchlings. At 15 and 30 days after transplantation, the recipients were investigated under a fluorescent microscope. The gonads of recipients were dissected for molecular analysis at 180 days after transplantation. Transplanted spermatogonial cells migrated toward and incorporated into recipient genital ridges. The presence of the Caspian brown trout genetic material was confirmed by PCR in 41.4% of the rainbow trout testes. These results demonstrated for the first time that the interspecies spermatogonial transplantation was successful in rainbow trout and that the somatic microenvironment of the rainbow trout gonad can support the colonization and survival of intraperitoneally transplanted cells derived from a fish species belonging to a different genus. Therefore, the SSCs transplantation can be used as a tool for conservation of Caspian brown trout genetic resources.

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Objective: It is hypothesized that stem cells have the capability to form tumors after transplantation. Spermatogonial stem cells have proliferation potency and colonization ability related to express pluripotency genes such as c-Myc. The primary aim of this study is to investigate tumorigenicity ability of these cells after in vitro cultivation and inoculation in athymic animals. Methods: Spermatogonial stem cells from 3-5 day-old neonatal mice testes (NMRI) were cultured following two-step enzymatic digestion. After one month of culturing the spermatogonial stem cells, the obtained colonies were identified by Oct4 and PLZF markers. Expressions of Nanog, Oct4 and c-Myc pluripotency genes were subsequently studied. We subcutaneously inoculated 5 x 10⁶ cells into athymic mice and assessed tumor formation after 8 weeks. Mouse embryonic stem cells (CCE line) were used as the positive control. Generated tumors were measured by a caliper. Results: The colonies expressed Oct4 and PLZF proteins. Ratio of pluripotency gene expressions in these cells compared to embryonic stem cells significantly decreased (P≤0.05). Mouse embryonic stem cells formed tumors however the spermatogonial colonies did not form any tumors. Conclusion: Mouse spermatogonial stem cells in comparison with embryonic stem cells are not capable of forming tumors in vivo. We have observed that the tumorigenic ability of these cells decreased significantly with down regulation of pluripotency gene expressions, particularly c-Myc. However, this study should be reassessed by using human tissue samples.

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Objective: This study aims to investigate the testes cultures of patients with previous histories of maturation arrest in spermatogenesis and find the appropriate methods to overcome this problem.
Methods: We divided spermatogonial stem cells (SSCs) isolated from testes biopsies into 3 groups: 1) culture of SSCs without feeder layer; 2) co-culture of SSCs with patient-derived Sertoli cells; and 3) co-culture of SSCs on Sertoli cell feeder layer derived from healthy donors. We calculated the numbers and diameters of stem cell-derived colonies and the percentage of cell viability in the different groups. The presence of SSCs at different culture times was determined by immunochemistry, alkaline phosphatase, and xenotransplantation of SSCs into an azoospermic mouse model.
Results: The microenvironment of the feeder layer derived from the patient’s own Sertoli cells produced numerous (36.1±4) large colonies (213.2±17 µm) after 3 weeks of culture. However, the ratio of germ cell-specific expressions of Stra8 (2.3) and Vasa (2.2) was more than the pluripotency gene, Nanog (0.45) in SSCs cultured on the Sertoli cell layer of a healthy person. After xenotransplantation of human SSCs into the testis of an azoospermic mouse model, we observed that the cells grow on basement membrane of seminiferous tubules, which confirmed their nature.
Conclusion: SSCs could be co-cultured with Sertoli cells derived from healthy donors in order to overcome the arrest of spermatogenesis observed in the co-culture of SSCs with patient-derived Sertoli cells. The results of the present study indicated that spermatogenesis could possibly be resumed in cancer patients previously treated by chemotherapy and∕or radiotherapy.

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Spermatogonial stem cells are foundation of the male reproductive system. These cells are the only conduit capable of transferring genetic traits from one generation to the next. Isolation and long-term preservation of spermatogonial stem cells for use in inducing spermatogenesis is one technique to preserve fertility in male patients who need chemotherapy. In vitro spermatogenesis is an alternative to achieve this goal. The use of an optimal model of human spermatogenesis is a major step in understanding the physiology and genetic pathways in the male reproductive system. In vitro spermatogenesis is crucial to reducing a complex process into smaller parts for experimentation, manipulation, and deriving cellular and molecular level knowledge. Is it possible to manipulate the paracrine environment and separately evaluate the effects of growth factors. Different in vitro culture systems are used to explore alternatives to spermatogenesis and obtain mature, functional spermatozoa for ultimate use in infertility treatment. In order to present a useful and practical method, this study provides an overview of different methods for the long-term preservation of spermatogonial stem cells and in vitro culture systems used in spermatogenesis.

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Objective: Pluripotent stem cells derived from testis are a new, unlimited source for cell therapy in regenerative medicine. Recently, studies show that spermatogonial stem cells can form embryonic stem-like cells (ES-like cells) in vitro. New procedures such as low intensity ultrasound (LIUS) can have positive effects on cell growth and differentiation. However, the effect of LIUS stimulation on ES-like cells has not been explored. In this study we investigate the effects of LIUS on colonization of ES-like cells.
Methods: Initially, we isolated SSCs from neonatal mice. The spermatogonial and Sertoli cells were cultured together in DMEM/F12 supplemented with 15% fetal bovine serum (FBS) and leukemia inhibitory factor (LIF). ES-like cells were stimulated by LIUS at intensity doses of 200 millwatt/square centimeter (mW/cm²) over 5 days. Characteristics of the isolated cells were confirmed by immunocytochemistry with Sox2 and SSEA-1 protein for ES-like cells. We also investigated colonization features in the ES-like cells.
Results: After 21 days, we observed there was a significant increase in diameter and number of colonies in the 200 mW/cm² group compared to the control group (p≤0.05). Pluripotency proteins, ES-like cell marker Sox2, and SSEA-1 expressed in the ES-like cells.
Conclusion: LIUS treatment can be an efficient, cost-effective method to improve colonization of ES-like cells during culture.

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Introduction:
Spermatogonial stem cells (SSCs) because of its ability to be reprogrammed into embryonic-like stem cells (ELSCs) can be a new source of pluripotent stem cells which can play a promising role in regenerative medicine. In this study, SSCs were transdifferentiated into neuron-like cells (NLCs) using two-step differentiation protocol. pluripotency and germ cells markers were analyzed in SSCs and ELSCs. Also neural markers were analyzed in ELSCs and NLCs.
Methods:
Neonatal rat testes were mechanically dissected and digested then was cultured in DMEM supplemented with 15% FBS. The medium was replaced with DMEM containing LIF, mercaptoethanol, EGF, bFGF, and GDNF. After 5 weeks, ELSCs colonies appeared. SSCs and ELSCs were evaluated by Stra8, plzf (germ cells markers) Oct4, and sox2 (pluripotency markers) using qRT-PCR. The ELSCs colonies were isolated and cultured in DMEM containing 0.5 mM lithium chloride. In day 5, ELSCs transdifferentiated to NLC. They were evaluated using neural marker including Neurofilament 200 (NF-200), choline acetyltransferase (CAT), synaptophysin (Syp), Nestin (Nes), Neurogenin1 (NG1), Neurod1 (Nd1), and Neurofilament 68 (NF-68)gene expression.
Results:
Result showed increasing expression of Oct4 and sox2 genes and low level of Stra8 and plzf expression in ELSCs than SSCs. After neural transdifferentiation by lithium chloride induction, neural markers were examined by RT-PCR in ELSCs and NLCs. The result showed expression of NF-200, CAT, Syp, Nes, NG1, Nd1 and NF-68 in NLCs opposed to ELSCs.
Conclusion:
This study indicates lithium chloride can promote ELSCs to transdifferentiate into NLCs.