Plasticity of male germline stem cells and their applications in reproductive and regenerative medicine

Spermatogonial stem cells (SSCs), also known as male germline stem cells, are a small subpopulation of type A spermatogonia with the potential of self-renewal to maintain stem cell pool and differentiation into spermatids in mammalian testis. SSCs are previously regarded as the unipotent stem cells since they can only give rise to sperm within the seminiferous tubules. However, this concept has recently been challenged because numerous studies have demonstrated that SSCs cultured with growth factors can acquire pluripotency to become embryonic stem-like cells. The in vivo and in vitro studies from peers and us have clearly revealed that SSCs can directly transdifferentiate into morphologic, phenotypic, and functional cells of other lineages. Direct conversion to the cells of other tissues has important significance for regenerative medicine. SSCs from azoospermia patients could be induced to differentiate into spermatids with fertilization and developmental potentials. As such, SSCs could have significant applications in both reproductive and regenerative medicine due to their unique and great potentials. In this review, we address the important plasticity of SSCs, with focuses on their self-renewal, differentiation, dedifferentiation, transdifferentiation, and translational medicine studies.     

Plasticity of spermatogonial stem cells

There have been significant breakthroughs over the past decade in the development and use of pluripotent stem cells as a potential source of cells for applications in regenerative medicine. It is likely that this methodology will begin to play an important role in human clinical medicine in the years to come. This review describes the plasticity of one type of pluripotent cell, spermatogonial stem cells (SSCs), and their potential therapeutic applications in regenerative medicine and male infertility. Normally, SSCs give rise to sperm when in the testis. However, both human and murine SSCs can give rise to cells with embryonic stem (ES) cell-like characteristics that can be directed to differentiate into tissues of all three embryonic germ layers when placed in an appropriate inductive microenvironment, which is in contrast to other postnatal stem cells. Previous studies have reported that SSCs expressed an intermediate pluripotent phenotype before differentiating into a specific cell type and that extended culture was necessary for this to occur. However, recent studies from our group using a tissue recombination model demonstrated that SSCs differentiated rapidly into another tissue, in this case, prostatic epithelium, without expression of pluripotent ES cell markers before differentiation. These results suggest that SSCs are capable of directly differentiating into other cell types without going through an intermediate ES cell-like stage. Because SSCs do not require reprogramming to achieve a pluripotent state, they are an attractive source of pluripotent cells for use in regenerative medicine.     

诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

Spermatogonial stem cells: progress and prospects

Twenty years ago, the transplantation of spermatogonial stem cells (SSCs) from a mouse to other recipient mice was shown to be feasible, which clearly demonstrated the functional identity of SSCs. Since then, several important new findings and other technical developments have followed, which included a new hypothesis on their cell kinetics and spermatogonial hierarchy in the testis, a culture method allowing their self-renewal and proliferation, a testis tissue organ culture method, which induced their complete differentiation up to sperm, and the in vitro induction of germ cells from embryonic stem cells and induced pluripotent stem cells. These advancements reinforced or advanced our understanding of this unique cell. Nonetheless, there are many unresolved questions in the study of spermatogonial stem cells and a long road remains until these cells can be used clinically in reproductive medicine.     

诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究

目的：探讨诱导的多潜能干细胞（induced pluri potent stem cells,iPS cells）通过类胚体长期分化后残留未分化细胞的特性。方法：小鼠iPS细胞株,体外类胚体分化20天后消化打散,重新给予i PS细胞常规培养液培养。观察扩增的残留细胞形态;流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力。将残留细胞扩增后注射入裸鼠背部皮下,6周后注射部位取材进行大体和组织学检查。结果：分化20天的类胚体中存在残留未分化的细胞,呈克隆样生长,高度表达SSEA-1、CD-9和OCT-4等多潜能性标志。残留细胞能反复传代,并可在体外再次分化和残留。残留细胞注射部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织。结论：iPS细胞分化为类胚体后残留部分未分化细胞,残留细胞在体内、外可再次分化,并能在体外分化中再次残留。     

Renal differentiation from adult spermatogonial stem cells

Abstract

There is considerable interest in the use of multi-potent stem cells in kidney tissue regeneration. We studied if spermatogonial stem cells have the ability to undergo kidney differentiation. Spermatogonial stem cell differentiation was induced using in vitro and ex vivo co-culture techniques. Conditioned media from human kidney fibroblasts induced the expression of epithelial and endothelial lineages in spermatogonial stem cells, consistent with nephrogenesis. Furthermore, we showed that these cells up-regulated renal tubular-specific markers alkaline phosphatase, mineralocorticoid receptor, renal epithelial sodium channel and sodium-glucose transporter-2 (p?<?0.05). GFP-labeled spermatogonial stem cells were engrafted into metanephric kidney organ cultures harvested from E12.5 mouse embryos. After 5 days of organ culture, focal anti-GFP staining was detectable in all inoculated kidneys demonstrating integration of spermatogonial stem cells into the developing kidney (p?<?0.01). Histological assessment showed early nephron-like architecture. In summary, we show that spermatogonial stem cells have the potential to generate renal tissue and lay the foundations for further investigations into a novel therapeutic approach for renal insufficiency.     

Recent advances in isolation,identification,and culture of mammalian spermatogonial stem cells

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells(SSCs).SSCs,the only male reproductive stem cells that tr...     

Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.     

诱导小鼠骨髓间充质干细胞向雄性生殖细胞的定向分化

目的探讨小鼠骨髓间充质干细胞是否能够在体外被诱导发生向雄性生殖细胞方向的分化。方法从雄性小鼠骨髓中分离能够长期贴壁生长的细胞,并鉴定其是否为间充质干细胞。对分离的细胞进行生殖细胞特异性报告基因标记(stra-8-GFP)。采用视黄酸诱导标记的细胞发生向生殖细胞方向的分化。通过观察报告基因表达和生殖细胞相关基因mRNA表达情况确定是否发生了分化。结果从小鼠骨髓中分离到的贴壁生长的细胞表达间充质干细胞的表面标志CD90、CD44、CD105和Sca-1;细胞在体外可以被诱导分化为成骨、成软骨及成脂肪细胞。报告基因标记的间充质干细胞在被视黄酸诱导2d后开始表达绿色荧光蛋白和生殖细胞相关基因Mvh、Fragilis和Stella的mRNA。未经视黄酸诱导的细胞不表达绿色荧光蛋白和生殖细胞相关基因。结论小鼠骨髓间充质干细胞在体外可以被视黄酸诱导发生向雄性生殖细胞方向的分化。     

胶质细胞源性神经营养因子对哺乳动物精原干细胞增殖及分化的影响

胶质细胞源性神经营养因子(GDNF)是转化因子β(TGF-β)超家族的一个相关成员,在哺乳动物睾丸中由支持细胞分泌,对精原干细胞(SSCs)的增殖和分化具有非常重要的作用。体外培养SSCs时培养液中添加适量的GDNF能够促进SSCs的增殖。GDNF介导多种信号通路调节SSCs的自我更新和分化。本文就GDNF对哺乳动物精原干细胞增殖与分化的作用及其所介导的信号通路进行了综述。     

Mouse undifferentiated spermatogonial stem cells cultured as aggregates under simulated microgravity

Dynamic simulated microgravity (SMG) culture systems provide environments that stimulate stem cell proliferation and differentiation. However, the effect of SMG on spermatogonial stem cells (SSCs) remains unclear. Here, we used a rotating cell culture system (RCCS) to determine its effect on mouse SSC proliferation and differentiation. SSCs were enriched from mouse pub testis and cocultured with Sertoli cell feeders pre‐treated with mitomycin C on fibrin scaffolds in a rotary bioreactor for 14 days. Our results show that mouse SSCs cultured in a rotary bioreactor exhibited enhanced proliferation surpassing those cultured in static conditions, although SSC cultures in SMG underwent a growth lag at initial 3 days. After 14 days, mouse SSCs and feeders grew into cell aggregates with average diameters of 242.63 ± 16.53 μm compared with those in conventional static culture (49.51 ± 15.64 μm). Related detection revealed that proliferating SSCs in SMG remained undifferentiated, maintained clone‐forming capacity and were capable of differentiation into round spermatids with flagella. The growth characteristics of mouse SSCs in RCCS suggest that the resulting aggregates are similar to native in vivo cells. Rotary bioreactors that create SMG environments may be an alternative to conventional systems for the clinical application of SSCs.     

BALB/c小鼠精原干细胞长期培养和移植的实验研究

目的 建立小鼠精原干细胞(SSC)长期培养体系,探讨SSC体外增殖分化的关键因子.方法 收集出生4～6 d BALB/c绿色荧光小鼠睾丸,采用改良两步消化法获得细胞悬液,3次差速贴壁去除体细胞获得富集的精原细胞,采用添加生长因子的无血清基础培养液重悬,种植到小鼠胚胎成纤维细胞饲养层上培养.基础培养液为StemPro-34 SFM干细胞培养基并补充15种添加成分;生长因子为10 ng/ml碱性成纤维细胞因子、20 ng/ml胶质细胞源性神经营养因子和200 ng/mlGDNF家族受体a1.取4～5周龄BALB/c雄性小鼠15只,腹腔注射40 mg/kg的白消安建立受体模型,采用三维显微注射系统将培养的SSC移植到受体左侧睾丸精曲小管内,右侧睾丸作为自身对照;分别采用体视荧光显微镜观察和HE染色检测细胞移植后睾丸生精功能恢复情况.结果 改良消化富集法消化后细胞活性>98%,SSC富集约18.5倍.饲养层培养1～2 d后细胞成对称或线形排列,细胞间可见明显的胞质桥连接.3～4 d后精原细胞增殖形成典型的克隆,为边缘不清楚的团块;小鼠SSC能在该培养体系中稳定培养、传代3个月.移植后2个月,体视荧光显微镜下受体睾丸内可见明显绿色阳性克隆,HE染色证实移植的SSC在受体睾丸内克隆增殖并分化产生成熟的精子.结论 成功建立了BALB/c小鼠SSC培养体系,为研究SSC增殖分化调控机制及SSC移植治疗男性不育提供了实验依据.     

Derivation of motor neuron‐like cells from neonatal mouse testis in a simple culture condition

Embryonic stem cell (ESC) therapy is an exciting way to treat neurodegenerative disease and central nervous system injury. However, many ethical and immunological problems surround the use of embryonic stem cells. Finding an alternative source of stem cells is therefore pertinent. In this study, spermatogonia stem cells (SSCs) were used to generate mature motor neurons. SSCs were extracted from neonatal testes and cultured in DMED/F12 medium for 3 weeks. Characterisation of SSC‐derived ESC‐like cells was confirmed by RT‐qPCR, immunostaining, alkaline phosphatase activity and their ability to form embryoid bodies (EBs). The EBs were induced by retinoic acid and Sonic hedgehog and trypsinised to obtain single induced cells. The single cells were cultured in neural medium for 18 days. Characterisation of neural precursors and motor neuron‐like cells was confirmed by RT‐qPCR and immunocytochemical analysis at the 7th day (early stage) and 18th day (late stage), respectively, of culturing. The neural precursors were found to be positive for nestin and Sox2, and a small fraction of cells expressed β‐tubulin III. Upon further differentiation, multipolar neurons were detected that expressed β‐tubulin III and MAP2 markers. Moreover, the expression levels of Olig2 and PAX6 were significantly lower, while HB9, Isl1 and Isl2 expression levels were higher at the late stage when compared to the early stage. These results show that SSCs have the potential to differentiate to motor neuron‐like cells and express markers specific for mature motor neurons. However, the functional ability of these cells remains to be evaluated in future studies.     

Coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system

Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-Agar-Culture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfralpha-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.     

Amniotic fluid stem cell-based models to study the effects of gene mutations and toxicants on male germ cell formation

Male infertility is a major public health issue predominantly caused by defects in germ cell development. In the past, studies on the genetic regulation of spermatogenesis as well as on negative environmental impacts have been hampered by the fact that human germ cell development is intractable to direct analysis in vivo. Compared with model organisms including mice, there are fundamental differences in the molecular processes of human germ cell development. Therefore, an in vitro model mimicking human sperm formation would be an extremely valuable research tool. In the recent past, both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been reported to harbour the potential to differentiate into primordial germ cells and gametes. We here discuss the possibility to use human amniotic fluid stem (AFS) cells as a biological model. Since their discovery in 2003, AFS cells have been characterized to differentiate into cells of all three germ layers, to be genomically stable, to have a high proliferative potential and to be non-tumourigenic. In addition, AFS cells are not subject of ethical concerns. In contrast to iPS cells, AFSs cells do not need ectopic induction of pluripotency, which is often associated with only imperfectly cleared epigenetic memory of the source cells. Since AFS cells can be derived from amniocentesis with disease-causing mutations and can be transfected with high efficiency, they could be used in probing gene functions for spermatogenesis and in screening for male reproductive toxicity.     

Differentiation of embryonic stem cells after transplantation into peritoneal cavity of irradiated mice and expression of specific germ cell genes in pluripotent cells

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.     

Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas

Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs.     

Extrinsic and intrinsic factors controlling spermatogonial stem cell self-renewal and differentiation

Spermatogonial stem cells (SSCs), the stem cells responsible for male fertility, are one of a small number of cells with the abilities of both self-renewal and generation of large numbers of haploid cells. Technology improvements, most importantly, transplantation assays and in vitro culture systems have greatly expanded our understanding of SSC self-renewal and differentiation. Many important molecules crucial for the balance between self-renewal and differentiation have been recently identified although the exact mechanism(s) remain largely undefined. In this review, we give a brief introduction to SSCs, and then focus on extrinsic and intrinsic factors controlling SSCs self-renewal and differentiation.     

Xenotransplantation assessment: morphometric study of human spermatogonial stem cells in recipient mouse testes

The purpose of this study was (i) To establish in vitro propagation of human spermatogonial stem cells (hSSCs) from small testicular biopsies to obtain a high number of cells; (ii) to evaluate the presence of functional hSSCs in culture system by RT‐PCR using DAZL, α6‐Integrin, β1‐Integrin genes; and (iii) to evaluate the effects of cell concentration on successful xenotransplantation of hSSCs in mice testis. Donor hSSCs were obtained from men with maturation arrest of spermatogenesis duration 1 year ago. These cells were propagated in DMEM containing 1 ng ml^?1 bFGF (basic fibroblast grow factor) and 1500 U ml LIF (leucaemia inhibitory factor) for 5 weeks. Different concentrations of hSSCs transplanted into seminiferous tubules of busulfan‐treated immunodeficient mice and analysed up to 8 weeks after transplantation. The results showed that expression of DAZL and α6‐Integrin mRNA was increased as well as the colony formation of SSCs in vtro culture during 5 weeks. Proliferation occurred about 4 weeks after transplantation, but meiotic differentiation was not observed in recipient testis after 8 weeks. The difference in donor cells concentration had effect on homing spermatogenesis in recipient testis. Homologous transplantation of proliferated SSCs to seminiferous tubules of that patient individually may allow successful differentiation of transplanted cells.     

环磷酰胺干扰精原干细胞功能的初步研究

目的:初步探讨环磷酰胺对精原干细胞分化功能的干扰作用。方法:根据W istar大鼠生精细胞发育不同阶段分为1、3、9周龄组,每组24只,随机均分为实验组和对照组,实验组腹腔内注射环磷酰胺100 mg/(kg.体重),对照组注射等量生理盐水,24 h后用原位缺口末端标记法检测生精细胞凋亡。另取仅有精原干细胞的1周龄W istar雄性大鼠60只,随机均分为实验组和对照组(给药方法同上),在给药后24 h、3周、9周分别用免疫组化法检测c-K it蛋白,流式细胞仪分析细胞周期。结果:环磷酰胺干预后,1周龄实验组与对照组大鼠生精细胞凋亡差异无显著性(P>0.05),其余各周龄组实验组生精细胞凋亡均显著增高(P<0.01)。1周龄实验组大鼠睾丸在环磷酰胺处理后不同时间点的细胞周期S期细胞比例、精原细胞c-K it蛋白表达水平均明显低于对照组(P<0.01)。结论:环磷酰胺诱导精原干细胞凋亡不明显,可能更重要的是干扰精原干细胞的增殖分化功能。