Homologous recombination in rat germline stem cells

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.     

Retrovirus-mediated modification of male germline stem cells in rats

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.     

精原干细胞的生物学特性

精原干细胞(spermatogonialstemcells,SSCs)是雄性生殖系干细胞,位于睾丸曲细精管基膜上,既具有自我更新潜能,又具有定向分化潜能,是自然状态下出生后动物体内在整个生命期间进行自我更新并能将基因传递至子代的唯一成体干细胞。自SSCs移植技术建立以来,有关其分离、鉴定、培养、冻存、转基因操作及移植等方面均已取得长足进步,使人们对其生物学特性有了更深入的了解。根据最近的相关进展,系统评述了SSCs的相关生物学特性,以期为该领域及其他干细胞研究提供借鉴。     

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.     

Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells

To study self‐renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA‐1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty‐five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (×10⁷/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (×10⁷/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples. Mol. Reprod. Dev. 77: 340–347, 2010. © 2010 Wiley‐Liss, Inc.     

The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation

Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self‐renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long‐term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X‐linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26‐Cre males. We obtained 96 live offspring from six long‐term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3‐conKO males as recipients for testis transplantation of SSC lines.     

精原干细胞移植及受体制备技术研究进展

精原干细胞移植为研究精子发生、雄性生殖能力及新型转基因技术奠定了基础.尽管已利用小鼠建立了较成熟的移植技术体系,白消安受体制备法和曲细精管及睾丸网移植法已得到广泛应用,但白消安可导致动物较高的死亡率,局部射线照射和无内源性精子发生受体动物的制备费用较昂贵,热处理受体制备法应用范围较窄且效果不稳定;三种移植方法均对操作有较高的技术要求,曲细精管、睾丸输出管移植需要显微注射装置,而睾丸网移植需要超声仪的辅助.而且,移植效果在不同实验间、物种间差异较大,移植效率有待提高,对移植排斥反应的认识也有待进一步深入.对睾丸结构和精原干细胞生物学特性的深入研究,将有助于建立更简单高效的受体制备和移植的方法.     

Is there a clinical future for spermatogonial stem cells?

Like every other adult stem cell in the human body, spermatogonial stem cells (SSCs) have the capacity to either renew themselves or to start the differentiation process, namely, spermatogenesis. Due to the continuation of the stem cell population in the testis, several possible options for preservation and re-establishment of the reproductive potential exist. Currently, spermatogonial stem cell transplantation (SSCT) is considered the most promising tool for fertility restoration in young cancer patients. This technique involves the injection of a testicular cell suspension from a fertile donor into the testis of an infertile recipient. Although, SSCT could prove important for fertility preservation, this technique is not without any risk. Testicular cell suspensions from cancer patients may be contaminated with cancerous cells. It is obvious that reintroduction of malignant cells into an otherwise cured patient must be omitted. Decontamination strategies to solve this problem are discussed. Another alternative to preserve male fertility could be in-vitro culture of SSCs. This approach may be applied to generate spermatozoa in-vitro from cultured spermatogonial stem cells, which, in turn, could be used for intracytoplasmic sperm injection. Xenogeneic transplantation and xenografting are two other hypothetical methods to preserve fertility. However, because of the ethical and biological concerns inherent to these approaches, xenogeneic transplantation and xenografting should be limited to research. When SSCT or SSC culture becomes available for clinical use, efficient protocols for the cryopreservation of SSCs and testicular tissue will be of great benefit. The search for an optimal freezing protocol is discussed. Apart from fertility preservation, SSC studies are useful for other applications as well, such as transgenerational gene therapy and cell-based organ regeneration therapy.     

Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice

Stem cells in the male germ line (spermatogonial stem cells [SSCs]) are an important target for male fertility restoration and germ line gene modification. To establish a model system to study the biology and the applications of SSCs in mice, I used a sequential transplantation strategy to analyze the process by which SSCs colonize the stem cell niche after transplantation and to determine the efficiency of the process (homing efficiency). I further analyzed the proliferation kinetics of SSCs after colonization. The number of SSCs gradually decreased during the homing process, and only 12% of SSCs successfully colonized the niche on Day 7 after transplantation, but the number of SSCs increased by Day 14. Thus, homing efficiency of adult mouse SSCs is 12%. These results indicate that SSCs are rapidly lost upon transplantation and require approximately 1 wk to settle into their niches before initiating expansion. Using this SSC homing efficiency, I calculated that approximately 3000 SSCs exist in one normal adult testis, representing approximately 0.01% of total testis cells. Between 7 days and 1 mo after transplantation, SSCs proliferated 7.5-fold. However, they did not significantly proliferate thereafter until 2 mo, and only 8 SSCs supported one colony of donor-derived spermatogenesis from 1 to 2 mo. These results suggest that self-renewal and differentiation of SSCs are strictly regulated in coordination with the progress of an entire unit of regenerating spermatogenesis.     

哺乳动物精原干细胞的研究进展

精原干细胞是雄性体内可以永久维持的成体干细胞,它具有自我更新和分化的能力,保证了雄性个体生命过程中精子发生的持续进行,从而实现将遗传信息传递给下一代。精原千细胞不仅可在体外实现长期培养或诱导分化为各级生精细胞,并且可在特定条件下将其诱导去分化成为多能性干细胞。同样,这种多能性干细胞如同胚胎干细胞,可被诱导形成造血细胞、神经元细胞、肌细胞等多种类型细胞。鉴于其独具的生物学特性,精原干细胞在揭示精子的发生机制、治疗雄性不育和转基因动物等研究中具有重要价值。该文对精原干细胞在生物学特性、纯化培养、移植、体外诱导分化及其相关调控方面的各项研究进行了小结,综述了近年来的研究历程和最新研究成果。     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

哺乳动物精原干细胞的操作技术及应用

精原干细胞(spermatogonial stem cells,SSCs)具有高度的自我更新能力和分化潜能。精原干细胞移植技术作为精原干细胞研究的重要手段,已成为一种新兴的动物繁殖技术,能够提高雄性动物的生殖能力。该技术是从适龄雄性供体动物中采集精原干细胞,注射入受体动物的生精小管中使其产生精子。通过对精原干细胞的体外培养、遗传修饰及移植等操作,可以为探讨精子的发生机制、重建不育个体的精子发生、生产转基因动物提供新的途径;同时为提高优良品种家畜的生产效率、保护野生动物资源及不育症的治疗提供了一种新的方法;在医学、生物学及动物科学方面有着广泛地应用。通过对培养体系的不断完善,筛选、移植方法的不断改进,可获得更高的移植成功率。本文将从利用精原干细胞法生产转基因动物的优势,精原干细胞的形态特性和增殖分化特性,精原干细胞的移植技术和影响移植效率的关键因素,精原干细胞的体外培养,以及相关操作技术的应用与前景展望等方面做一概述。     

Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells

Spermatogenesis originates from a small population of spermatogonial stem cells. These cells are believed to divide infinitely and support spermatogenesis throughout life in the male. In this investigation, we examined the possibility of deriving transgenic offspring from single spermatogonial stem cells. Spermatogonial stem cells were transfected in vitro with a plasmid vector containing a drug resistant gene. Stably transfected stem cell clones were isolated by in vitro drug selection; these clones were expanded and used to produce transgenic progeny following spermatogonial transplantation into infertile recipients. An average of 49% of the offspring carried the transgene, and the recipient mice continued to produce monoclonal transgenic progeny a year after transplantation. Thus, a somatic cell-based genetic approach can be used to modify and select clones of spermatogonial stem cells in a manner similar to embryonic stem cells. The feasibility of genetic selection using postnatal spermatogonial stem cells demonstrates their extensive proliferative potential and provides the opportunity to develop new methods for generating stable animal transgenics or for germline gene therapy.     

Male germ line stem cells have an altered potential to proliferate and differentiate during postnatal development in mice

Spermatogonial stem cells (SSCs) continuously support spermatogenesis after puberty. However, accumulating evidence suggests that SSCs differ functionally during postnatal development. For example, mutant mice exist in which SSCs support spermatogenesis in the first wave after birth but cease to do so thereafter, resulting in infertility in adults. Studies using a retroviral vector have shown that the vector transduces pup SSCs more efficiently than adult SSCs, which suggests that pup SSCs divide more frequently. Thus, it is hypothesized that the SSCs in pup and adult testes have different characteristics. As an approach to testing this hypothesis in the present study, we investigated the proliferation kinetics of pup SSCs (6-9 days old) and their self-renewal/differentiation patterns for the first 2 mo after transplantation, and compared them to those of adult SSCs. Using serial transplantation, we found that the number of pup SSCs declined over the first week after transplantation. Thereafter, it increased ~4-fold by 1 mo and ~9-fold by 2 mo after transplantation, which indicates that pup SSCs continuously proliferate from 1 wk to 2 mo after transplantation. Compared to the proliferation of SSCs derived from adult intact testes, that of pup SSCs was lower at 1 mo but similar at 2 mo, indicating the delayed proliferation of pup SSCs. However, the pup SSCs regenerated spermatogenic colonies at 1 mo that were similar in length to those of SSCs from adult intact testes. Therefore, these results suggest that some functional differences exist in SSCs during postnatal development, and that these differences may affect the abilities of SSCs to self-renew and differentiate.     

不同年龄小鼠精原干细胞系的建立

精原干细胞（spennatogonial stem cells,SSCs）是雄性动物体内能进行终生自我更新并能将亲代基因遗传给予子代的一类细胞。不同年龄段的小鼠有不同的建系方法。6-7d幼鼠,可以用差异贴壁或直接贴壁法;5-6周成年鼠,一般采用差异贴壁法;31周老年鼠,最好种于饲养层细胞上。通过对精原干细胞系的甲基化和特异基因分析以及睾丸体内移植验证分析,成功建立了具有功能的不同年龄段的小鼠精原干细胞系。     

Expression of the human alpha1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis.

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell-based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human alpha1, 2-fucosyltransferase (H-transferase, HT). H-transferase expression modifies the cell surface carbohydrate phenotype of the xenogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galalpha1,3-Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine-inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galalpha1,3-Gal epitope, reduced XNA reactivity, and an increased resistance to human serum-mediated cytolysis. Transgenic pigs that express H-transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.     

Spermatogonial stem cell transplantation and testicular function

Spermatogonial stem cells (SSCs) are responsible for the continual production of spermatozoa throughout adult life. Interactions between SSCs and the surrounding cells in the seminiferous tubules regulate the biological activity of these cells. Factors involved in the regulation of SSCs are beginning to be defined by animal models and the culture of SSCs in defined media. A critical development in the characterization of SSCs has been the development of the germ cell transplantation technique, which provides the only assay for the presence of SSCs in a population of cells, and which allows the determination of whether SSCs are proliferating or differentiating in culture. This approach has accelerated SSC-focused research and promises to provide a better understanding of the factors and mechanisms that regulate these cells. The knowledge provided by this work is also critical to an appraisal of the components of the SSC niche in the seminiferous epithelium. Thus, many aspects of testicular function can be defined by the investigation of SSCs and the factors, cells, and environment that regulate SSCs, thereby leading to a more comprehensive understanding of spermatogenesis.     

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.