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Ronak Shabani,1,2 Mohsen Ashjari,3 Khadijeh Ashtari,1,4 Fariborz Izadyar,5 Babak Behnam,1,6,7 Samideh Khoei,8 Mohamad Asghari-Jafarabadi,9 Morteza Koruji1,2 1Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; 2Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 3Department of Chemical Engineering, Faculty of Engineering, University of Kashan, Kashan, Iran; 4Department of Medical Nanotechnology and Faculty of Advanced Technology in Medicine, Iran University of Medical Sciences, Tehran, Iran; 5Prime Gen Biotech LLC, Santa Ana, CA, USA; 6Department of Medical Genetics and Molecular Biology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 7NIH Undiagnosed Diseases Program, Common Fund, NHGRI, National Institutes of Health, Bethesda, MD, USA; 8Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 9Road Traffic Injury Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Background: Some male survivors of childhood cancer are suffering from azoospermia. In addition, spermatogonial stem cells (SSCs) are necessary for the improvement of spermatogenesis subsequent to exposure to cytotoxic agents such as cisplatin. Objective: The aim of this study was to evaluate the anticancer activity of cisplatin-loaded folic acid-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on mouse malignant cell line (EL4) and SSCs in vitro. Methods: SSCs were co-cultured with mouse malignant cell line (EL4) cells and divided into four culture groups: 1) control (cells were co-cultured in the culture medium), 2) co-cultured cells were treated with cisplatin (10 μg/mL), 3) co-cultured cells were treated with cisplatin-loaded folic acid-conjugated PLGA NPs, and 4) co-cultures were treated with folic acid-conjugated PLGA for 48 hours. The NPs were prepared, characterized, and targeted with folate. In vitro release characteristics, loading efficiency, and scanning electron microscopy and transmission electron microscopy images were studied. Cancer cells were assayed after treatment using flow cytometry and TUNEL assay. The co-cultures of SSCs and EL4 cells were injected into seminiferous tubules of the testes after treating with cis-diaminedichloroplatinum/PLGA NPs. Results: The mean diameter of PLGA NPs ranged between 150 and 250 nm. The number of TUNEL-positive cells increased, and the expression of Bax and caspase-3 were upregulated in EL4 cells in Group 4 compared with Group 2. There was no pathological tumor in testes after transplantation with treated co-cultured cells. Conclusion: The PLGA NPs appeared to act as a promising carrier for cisplatin administration, which was consistent with a higher activation of apoptosis than free drug. Keywords: spermatogonial cells, cancer cells, cisplatin, PLGA nanoparticles, drug delivery, folic acid

Introduction: Testicular cancer is the most common cancer affecting men of reproductive age. Cisplatin is one of the majority helpful chemotherapeutic agents for treatment of this cancer. In addition, spermatogonial stem cells (SSCs) are necessary for the improvement of spermatogenesis subsequent of exposure to cytotoxic agents such as cisplatin. The aim of this study was to evaluate the anticancer activity of cisplatin-loaded PLGA nanoparticles on mouse malignant testicular germ cell line (EL-4) and spermatogonial stem cells in vitro. Materials and Methods: The isolated spermatogonial cells were co-cultured with EL-4 cells. Then, cells were divided into six culture groups: Control (culture in DMEM/ F12 containing 1% FCS, 10 ng/mL GDNF and 20 ng/ml bFGF), Sham (basic media with 0.1% DMSO) and Experimental groups. Co-cultured cells in experimental groups were treated with different doses of cisplatin (5 μg/ml, 10 μg/ml, 15 μg/ml) and cisplatinloaded PLGA nanoparticles by effective dose for 12, 24, 48 and 72 hr and then, the cells culturing in media continued for 2 weeks with basic media. The nanoparticles were prepared by W/O/W double emulsion-solvent evaporation technique. After characterized, they were targeted with foliate. In vitro release characteristics, stability, drug loading and loading efficiency were studied. DLS data showed that the mean diameter of PLGA nanoparticles were ranging between 100-150 nm. The particles were investigated by SEM and TEM to observe surface topography and the morphology. Percentage of Cells was assayed after treatment using Flow cytometry assay. Results: This result was associated with a higher activation of apoptosis in EL-4 cells especially in experimental groups were treated with cisplatin-loaded PLGA nanoparticles. Conclusion: The PLGA nanoparticles seem to provide a promising carrier for cisplatin administration, which was consistent with a higher activation of apoptosis than free drug. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have emerged as a new therapy for various immune-mediated inflammatory diseases. In this study we perform the first double-blinded, placebo-controlled evaluation of the efficacy of adipose-derived allogenic canine MSCs for the treatment of canine atopic dermatitis (cAD). Enrolled canine patients were randomly divided into placebo (PBS saline), low-dose (5 × 10 5 cells/kg), and high-dose (5 × 10 6 cells/kg) treatment groups. Each patient received three subcutaneous MSCs treatments or PBS saline at four-week intervals with injections at five sites. Patients were monitored by physical exams, pruritus visual analog scales (PVAS) signed by the primary caretaker, canine atopic dermatitis extent and severity index-4 (CADESI-4) scores by two veterinarians, and complete blood count and serum chemistry analysis along with laboratory analysis for potential biomarkers. Patients were kept off any immune-modulating drugs during the study period, and oral antibiotics and topicals were used for managing pruritus and secondary infections. The PVAS scores and the serum miR-483 levels were significantly lower in the high dose group compared to the placebo group at day90 post first-treatment. The CADESI-4 scores of the high dose group also showed downward trends. No severe adverse effects were observed in any patient in this study. The high dose MSC treatment is efficacious in alleviating the clinical signs of cAD until 30 days after the last subcutaneous administration of MSCs, and miRNA-483 may be a reliable prognostic biomarker for cAD. The MSCs efficacy and potential biomarkers should be further explored by a larger scale clinical trial., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

This study was conducted to investigate the therapeutic effect of allogeneic adipose-derived MSCs on dogs with hip osteoarthritis (OA). Twenty dogs with bilateral osteoarthritis of the coxofemoral (hip) joint, diagnosed by a veterinarian through physical examination and radiographs were randomly allocated into four groups. Group 1 served as a placebo control and were injected with 0.9% sodium chloride (saline) ( n = 4). Group 2 were injected with a single dose of 5 million MSCs ( n = 5). Group 3 received a single dose of 25 million MSCs ( n = 6) and Group 4 received a single dose of 50 million MSCs ( n = 5). Intra-articular administration of allogeneic MSCs into multiple joints did not result in any serious adverse events. The average lameness score of the dogs in the placebo control group (-0.31) did not show improvement after 90 days of intra-articular saline administration. However, the average lameness score of the all MSC-treated dogs was improved 2.11 grade at this time point ( P < 0.001). Overall, sixty five percent (65%) of the dogs that received various doses of MSCs showed improvement in lameness scores 90 days after intra-articular MSC administration. Our results showed that intra-articular administration of allogeneic adipose derived MSCs was well-tolerated and improved lameness scores and reduced pain in dogs associated with hip OA. All doses of MSCs were effective. Subsequent studies with more animals per group are needed to make a conclusion about the dose response. The improved lameness effect was present up to 90 days post-injection. Serum interleukin 10 was increased in a majority of the dogs that received MSCs and that also had improved lameness., (Copyright © 2020 Maki, Beck, Wallis, Choo, Ramos, Tong, Borjesson and Izadyar.)

Canine Atopic Dermatitis (AD) is a common complex and multifactorial disease involving immune dysregulation, genetic predisposition, skin barrier defects, environmental factors and allergic sensitization. To date, diagnosis of canine AD relies on a combination of patient history, clinical examination, allergy testing and response to diet trials/therapies with no reliable biomarkers available to distinguish AD from other diseases with similar clinical presentations. A handful of studies to identify potential biomarkers in the peripheral blood of AD dogs and healthy controls have been performed with some showing inconsistent and contradictory results. In this study, we, for the first time, report statistically significant increases in expression of phosphodiesterase 4D (PDE4D) gene in peripheral blood mononuclear cells (PBMCs) and miR-203 in plasma from AD dogs compared to healthy controls. In addition, we report a statistically non-significant change of the CD4+/CD8+ ratio, a dramatic decrease of three gene markers (PIAS1, RORA and SH2B1) as well as a panel of differential expression of cytokines in AD dogs in comparison to the healthy controls. Our study provides important insight into the complexities of canine AD, and further studies to verify the specificity of these findings for canine AD at a larger-scale are warranted., Competing Interests: This work was supported by the intramural grant of Western University of Health Sciences to JH and the grant from VetCell Therapeutics for JH and GK. CM, TR, and FI are employees of VetCell Therapeutics and received funding in the form of salaries. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The recent discovery of ovarian stem cells in postnatal mammalian ovaries, also referred to as putative stem cells (PSCs), and their roles in mammalian fertility has challenged the long-existing theory that women are endowed with a certain number of germ cells. The rare amount of PSCs is the major limitation for utilizing them through different applications. Therefore, this study was conducted in six phases to find a way to increase the number of Fragilis- and mouse vasa homolog (MVH)-positive sorted cells from 14-day-old NMRI strain mice. Results showed that there is a population of Fragilis- and MVH-positive cells with pluripotent stem cell characteristics, which can be isolated and expanded for months in vitro. PSCs increase their proliferation capacity under the influence of some mitogenic agents, and our results showed that different doses of stem cell factor (SCF) induce PSC proliferation with the maximum increase observed at 50 ng/mL. SCF was also able to increase the number of Fragilis- and MVH-positive cells after sorting by magnetic-activated cell sorting and enhance colony formation efficiency in sorted cells. Differentiation capacity assay indicated that there is a basic level of spontaneous differentiation toward oocyte-like cells during 3 days of culture. However, relative gene expression was significantly higher in the follicle-stimulating hormone-treated groups, especially in the Fragilis- sorted PSCs. We suggest that higher number of PSCs provides us either a greater source of energy that can be injected into energy-impaired oocytes in women with a history of repeat IVF failure or a good source for research.

Cryopreservation of spermatogonial stem cells is considered as a useful procedure for preserving fertility in children with testis cancer. SSCs were isolated from testes mice, and then antioxidant was added to the freezing medium. The Bax expression level in antioxidant groups was significantly (P ≤ 0.05) lower than the control group and a significant rise of Bcl2 expression was detected in the antioxidant groups. ROS production with antioxidant was significantly lower compared with the control group. Cryopreservation with the addition of the antioxidants can help increase the number of SSCs and improve the quality and viability of these cells after cryopreservation.

Background: Spermatogonial stem cells (SSCs) are unique in mammals because they can transmit genetic information from generation to generation and it is of significant importance. In testes, Sertoli cells, peritubular myoid cells, Leydig cells and other interstitial cells contribute to the spermatogonial stem cell "niche". So, creation of niche in an in vitro condition that mimics the in vivo environment is essential to maintain functional characteristic of SSCs., Objective: In this review, we describe the impact of nanofiber scaffolds on the culture of SSCs derived from human-to-mouse., Results: Nanofiber Matrices mimic the architecture and size scale of the natural extracellular matrix (ECM). The scaffold provides more three-dimensional (3D), biomimicking and topographical signals to the cells and results in a more physiologically relevant cellular phenotype. Several investigators use different nanofiber scaffold-like carbon nanotubes (CNTs) scaffold, poly-L-lactic acid (PLLA) nanofiber scaffold, 3D soft agar culture system, human serum albumin (HSA)/tri calcium phosphate nanoparticles (TCPNPs) and electrospun polyamide nanofiber for proliferation and maintenance of self-renewal activity of the SSCs., Conclusion: Application of nanofiber scaffolds for in vitro culture of the SSCs may produce spermatogonial stem cells that can be used in regenerative medicine, tissue engineering, assisted reproductive technology and in the treatment of infertility in pre-pubertal cancer patients., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Cryopreservation of spermatogonial stem cells (SSCs) is an applicable method for young males seeking fertility preservation before starting a treatment. It increases reactive oxygen species (ROS) formation and oxidative stress, which damages cellular structures. In this study, we added two antioxidants, catalase and α-tocopherol (α-TCP), to the basic freezing medium to evaluate their effects on the efficiency of SSCs. SSCs were isolated from testes of 3- to 6-day-old male mice using enzymatic digestion. The enrichment of isolated cells was evaluated by flow cytometry and Stra8 antibody. Catalase (40 μg/mL), or α-TCP (200 μg/mL) was added to the basic freezing medium. The cell viability was evaluated by the methylthiazoltetrazolium (MTT) assay. After thawing, cells were cultured for 1 month, and the expression pattern of specific genes of SSCs and the ability of the cells to restore spermatogenesis were used to determine the efficiency of the cryopreservation method. The survival rate of the frozen cells in the presence of catalase or α-TCP was significantly higher than the control group (p < 0.05). The number of colonies and their diameter measured after 1 month were significantly higher in the antioxidant groups than in the control group (p < 0.05). Gene expression and resumption of spermatogenesis also followed the same pattern. Thus, adding antioxidants to the basic freezing medium can be helpful in increasing the quality and viability of SSCs after cryopreservation. This new approach to stem cells cryopreservation can also be a promising strategy for fertility preservation in patients who suffer from malignancy.

Introduction: An increasing body of evidence has emerged regarding the existence and function of spermatogonial stem cells (SSCs); however, their female counterparts are the subject of extensive debate. Theoretically, ovarian germ stem cells (GSCs) have to reside in the murine ovary to support and replenish the follicle pool during the reproductive life span. Recently, various methods have been recruited to isolate and describe aspects of ovarian GSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate GSCs., Material and Methods: A cell suspension of mouse neonatal ovaries was cultured. Colonies of GSCs were harvested mechanically and cultivated on mouse embryonic fibroblasts (MEF). Alkaline phosphatase activity was assessed to verify stemness features of cells in colonies. Expression of germ and stem cell specific genes (Oct-4, Nanog, Fragilis, C-kit, Dazl, and Mvh) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunofluorescence of Oct4, Dazl, Mvh, and SSEA-1 was also performed., Results: Small colonies without a clear border appeared during the first 4 days of culture, and the size of colonies increased rapidly. Cells in colonies were positive for alkaline phosphatase activity. Reverse transcription-polymerase chain reaction showed that Oct-4, Fragilis, C-kit, Nanog, Mvh, and Dazl were expressed in colony-forming cells. Immunofluorescence revealed a positive signal for Oct4, Dazl, Mvh, and SSEA-1 in colonies as well., Conclusions: The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economical than other techniques. The availability of ovarian stem cells can motivate further studies in development of oocyte and cell-based therapies.

Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients.

Background: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes., Methods: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers., Results: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics., Conclusions: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics., (Copyright 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Adipose tissue has become a reliable source of adult stem cells, which appear to possess a yet-undetermined degree of plasticity. With the difficulties associated with harvesting adult bone marrow stem cells, adipose tissue may represent a valuable and easily acquired source of stem cells. Stem cells have been identified using the DNA binding dye Hoechst 33342 and flow cytometry in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population stem cells in adult adipose tissues. Flow cytometric identification and isolation of this subpopulation of stem cells revealed that in the mouse there are 2.5% of adipose SP cells within the stromal vascular fraction of adipose tissue. In culture, mouse adipose SP cells showed the capacity to undergo in vitro differentiation into osteogenic, chondrogenic and adipogenic lineages. In NOD/SCID mice, freshly sorted mouse adipose SP cells were able to engraft and assist in wound healing. This animal model study showed that adipose SP cells were able to regenerate epithelial layers and connective tissue with minor scar formation. The ability of this novel cell population within adipose tissue to undergo directional differentiation in vitro and to regenerate skin in vivo has potential impact for uses in surgical dermal applications.

Background: Knowledge about the identity and characteristics of spermatogonial stem cells (SSCs) in human is very limited. Here, Rhesus monkey was used as an animal model to investigate molecular and phenotypic characteristics of SSCs in the adult testes., Methods: A variety of immunohistological, molecular biological and functional assays were used to study different populations of SSCs in the adult testes., Results: In adult primate testes, there are distinct populations of CD90+ CD49f+ CD117- (Triple Stained) cells and a small population of stage-specific embryonic antigen-4 (SSEA-4)+ cells which both localized at the basement membrane of seminiferous tubules. Both SSEA-4+ and Triple Stained cells express germ cell and SSC-specific markers and show high telomerase activity; however, only adult Rhesus monkey SSEA-4+ testis cells appear to contain functional and actively dividing SSCs that can repopulate recipient mouse testes following spermatogonial transplantation. DNA analysis of these populations showed that SSEA-4+ cells contain a DNA profile similar to the actively dividing cells, whereas Triple Stained cells showed an accumulated number of cells arrested in the S phase of the cell cycle. SSEA-4+ cells also showed significantly higher proliferation activity, as shown by proliferating cell nuclear antigen staining, than Triple Stained cells (P < 0.01). Interestingly, SSEA-4+ cells expressed a significantly higher level of promyelocytic leukemia zinc finger, a factor required for SSC self-renewal, than Triple Stained cells (P < 0.001)., Conclusions: Our data indicate that Triple Stained cells may represent a quiescent population of SSCs, whereas SSEA-4 might be expressed on a subpopulation of actively dividing SSCs.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.

The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM) supplemented with 1% bovine serum albumin (BSA) in a final concentration of 6 x 10(6) per mL, and the effects of different cryoprotectants and freezing protocols were tested. Cells frozen/thawed in medium containing 10% fetal calf serum (FCS) and 1.4 M glycerol or dimethyl sulfoxide (DMSO) had a significantly (P <.05) higher percentage of living cells compared to medium with only FCS, whereas DMSO gave a significantly better cell survival rate than glycerol did. An increase in the concentration of FCS in the DMSO-based medium to 20% had no effect on survival after freezing and thawing. Furthermore, inclusion of 0.07, 0.14, or 0.21 M sucrose in DMSO-based medium resulted in a significant improvement of cell survival, cell proliferation in culture, and colonization efficiency in recipient testes. A controlled slow-freezing rate (1 degrees C/min) resulted in significantly (P <.05) more viable cells than fast (5 degrees C/min) freezing. However, noncontrolled-rate freezing, with a comparably low cooling rate, gave even better results than the controlled-rate slow freezing. Cryopreservation in MEM-based medium containing 10% FCS, 10% DMSO, and 0.07 M sucrose using a non-controlled-rate freezing protocol appeared to be a simple and effective way to preserve type A spermatogonia, with a high yield of almost 70% living cells after thawing. Frozen/thawed spermatogonia survived in culture and retained the ability to proliferate as determined by colorimetric and bromodeoxyuridine incorporation assays. To test whether the stem cells among the A spermatogonia retained their ability to colonize the testis of a recipient mouse, bovine spermatogonia were transplanted. This resulted in colonization 2-3 months after transplantation. In conclusion, for the first time, a method specific for cryopreservation of type A spermatogonia, including spermatogonial stem cells was developed, which allows long-term preservation of these cells without apparent harmful effects to their function.