精原干细胞自我更新与分化的调控

精原干细胞(SSCs)是精子发生过程的起源,有独特的复制方式:一次分裂可形成两个分化细胞,也可形成一个分化细胞和一个干细胞,这种自我更新和分化受到严密的调控,包括微环境、胶质细胞源性神经营养因子(GDNF)、多种信号通路等。本文综述SSCs的自我更新与分化及其调节机制,对于深入了解精子发生及男性精子发生障碍性不育、探讨睾丸肿瘤发生和寻找新的潜在治疗靶点等诸多方面有理论意义。     

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.     

精原干细胞微环境的生物学特性

成体干细胞的自我更新和分化与其微环境关系密切。精原干细胞(spermatogonial stem cells,SSCs)是体内自然状态下唯一能将遗传信息传至子代的成体干细胞。探讨SSCs更新和分化的调控机制有助于精子发生机理的阐明,并为探究其他成体干细胞增殖分化的调节机制提供依据。因此,SSCs系统为成体干细胞微环境研究提供了理想模型。资料表明,SSCs的更新和分化受其微环境的调控。基于本室的工作,参考最新文献,本文主要从SSCs微环境的基本特性、构成及其产生的各种调控因子等角度,评述了SSCs微环境的生物学特性及其与SSCs更新和分化间的关系,以期为本领域研究及其他成体干细胞相关研究提供借鉴。     

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.     

成体干细胞在男性不育领域的应用及展望

干细胞是目前生物医学研究领域的热点,并由基础医学研究扩展到了临床应用。根据干细胞所处发育阶段和分化性质,通常可分为胚胎干细胞、诱导性多能干细胞(iPS)和成体干细胞。其中成体干细胞在诸多系统疾病中已经开展临床应用。目前,对成体干细胞范畴内的精原干细胞、间充质干细胞的基础研究走在生殖男科治疗研究的前列,应用于临床尚需时日。本文综述成体干细胞在男性不育领域的应用前景。     

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.     

精原干细胞在中药生精制剂中的生长特性

目的 观察精原干细胞在中药生精制剂中的生长特性,为探讨精原干细胞的多向分化能力提供实验依据.方法 取5～7d龄昆明系雄性小白鼠,脱颈椎处死后,无菌取出睾丸,进行精原干细胞分离、培养与鉴定.取培养3d的精原干细胞,分为对照组和实验组.对照组用DMEM/F12完全培养液(含10%FBS、4mmol/L谷氨酰胺、0.12%NaHCO3、0.5mmol/L丙酮酸钠、15mmol/L HEPES、50U/mL青霉素和50μg/mL链霉素)培养,实验组用条件培养液(DMEM/F12完全培养液中添加中药生精制剂生精胶囊浓缩液)培养.在倒置显微镜下观察细胞的形态变化,酶联吸收OD值,细胞DNA倍体分析.结果 实验组精原干细胞在中药生精制剂条件培养液中较对照组细胞生长快,条件培养3～7d后见细胞生长迅速,呈集落样增长,细胞立体感增强,但未见明显细胞间桥;诱导培养15d见细胞增殖达高峰,增殖细胞的胞质间桥仍然不明显.酶联吸收OD值两组之间有显著性差异(P<0.01).细胞DNA倍体分析,实验组DNA四倍体含量与对照组比较,两组之间有显著性差异(P<0.01).结论 小鼠精原干细胞在中药生精制剂培养条件下能较快速生长与增殖,在增殖过程中表现出与正常体内细胞增殖的某些特性,这为进一步研究精原干细胞多向分化潜能提供实验参考,也为中药制剂治疗少、弱精子症提供理论依据.     

精原干细胞增殖和分化相关因子的研究进展

临床上,非梗阻性无精子症患者由于睾丸中没有精子而无法进行辅助生殖治疗,精原干细胞方面的研究,可能为这部分患者带来希望。本文就近年来精原干细胞增殖分化的分子调控机制进行综述。     

Evaluation of the effects of cryopreservation on viability,proliferation and colony formation of human spermatogonial stem cells in vitro culture

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n = 8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen‐thawed SSCs were co‐cultured on fresh Sertoli cells (experimental group 1), and frozen‐thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co‐cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen‐thawed testicular cells after 2 weeks of culture had a significantly (P < 0.05) higher percentage of living cells compared to frozen‐thawed testicular cells at the beginning of culture (59.2 ± 7.05 and 46.3 ± 8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6 ± 2.8 and 8.33 ± 1.5, respectively, P < 0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P < 0.05) after 3 weeks of culture (269.7 ± 52.1, 204.34 ± 24.1 and 112.52 ± 23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer‐related illness and waiting for radiotherapy and/or chemotherapy.     

Human adipose‐derived stem cells: isolation,characterization and applications in surgery

The ideal stem cell for use in functional tissue engineering needs to be abundantly available, harvested with minimal morbidity, differentiated reliably down various pathways and able to be transplanted safely and efficaciously. Adult human adipose tissue contains a population of mesenchymal stem cells, termed ‘adipose‐derived stem cells’ (ASC), which seem to fulfil most, if not all, of these criteria. ASC can be harvested readily, safely, and in relative abundance by modern liposuction techniques. They are capable of differentiating into other mesenchymal tissue types, including adipocytes, chondrocytes, myocytes and osteoblasts. They also show angiogenic properties, with recent evidence of a potential role in healing radiotherapy‐damaged tissue, possibly due to their secretion of vascular endothelial growth factor. Similarly, they may have a role in healing chronic wounds, and as such are being investigated in phase 1 trials for their ability to aid healing of recurrent Crohn’s fistulae. Subsequently they have a wide range of potential clinical uses in all fields of surgery. This article reviews the current and potential clinical applications of ASC in relation to surgery, as well as methods for their isolation, differentiation and molecular characterization.     

人胚胎干细胞来源的成纤维细胞支持无动物源污染条件下的人精原干细胞生长

精原干细胞在人出生后可以连续分裂以支持精子形成,将遗传信息传递给下一代。在本文中,我们成功地从睾丸组织中分离并鉴别人精原干细胞的一种新方法,并在人胚胎干细胞来源的成纤维细胞上扩增人精原干细胞。在这种人胚胎干细胞来源的成纤维细胞上可维持大量的人精原干细胞并且在组合生长因子,特别是胶质细胞来源的神经生长因子存在的条件下扩增至少2个月。细胞表面标志分析显示这些精原干细胞具有高水平的碱性磷酸酶的表达并保持了高水平的胚胎特异阶段抗原SSEA-1,OCT4和CD49f的表达。用逆转录PCR反应检测出OCT4,SOX3,STRA8基因的表达。这些数据清晰地表明运用人胚胎干细胞来源的成纤维细胞来支持人精原干细胞的一个新的策略,并排除了异源物种的污染。这个系统提供了新的机会去了解干细胞巢对于控制精原干细胞自我更新的调节机制,并将对于精原干细胞潜在的临床应用提供有用的精原干细胞。     

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.     

Germline-competent stem cell in avian species and its application

Germ cells are the only cell type in the body that can transfer genetic information to the next generation. Germline-competent stem cells can self-renew and contribute to the germ cell lineage giving rise to pluripotent stem cells under specific conditions. Hence far, studies on germline-competent stem cells have contributed to the generation of avian model systems and the conservation of avian genetic resources. In this review, we focus on previous studies on germline-competent stem cells from avian species, mainly chicken germline-competent stem cells, which have been well established and characterized. We discuss different sources of germline-competent stem cells and recent advances for the future applications in birds.     

Morphological and ultrastructural studies of human spermatogonial stem cells from patients with maturation arrest

Destruction of spermatogonial stem cells (SSCs) along the chemotherapy and radiotherapy is one of the side effects of cancer treatments that lead to infertility. In vitro propagation of hSSCs is necessary to obtain an adequate number of cells for successful transplantation. In this study, hSSCs were isolated from testis biopsies of the patients with maturation arrest and proliferated in DMEM in the presence of LIF and bFGF for 5 weeks. The various types of human spermatogonia were identified in culture system and compared with testis tissue using morphological criteria at the ultrastructural level. The results showed that although many various types of spermatogonia were identified, but no remarkable difference was observed between spermatogonial cells in culture system and testis tissue. Electron and light microscopic studies of hSSC colonies did not show differentiated SSCs in the culture system. The results also showed that probably the suitable time for transplanting of SSCs in recipient testis is 2–3 weeks after culture. Because apoptosis which may affect the development of germ cells has not started in colony cells at this time and the population of apoptotic cells are low.     

人诱导性多潜能干细胞向表皮样干细胞分化的实验研究

目的 探讨人诱导性多潜能干细胞( iPSC)向表皮样干细胞分化的可能性.方法 (1)以经过灭活处理的小鼠胚胎Fb株作为滋养层,将人iPSC株接种于其上,加入胚胎干细胞完全培养液培养,Ⅳ型胶原酶消化法传代,倒置相差显微镜下观察人iPSC形态及生长状况并行碱性磷酸酶(AKP)染色.以胚胎干细胞不完全培养液悬浮培养iPSC,观察拟胚体形成能力.(2)将人iPSC接种于铺有人羊膜的6孔培养板培养,设为诱导组;另于未铺羊膜的6孔培养板上培养iPSC,作为对照组.观察2组iPSC形态,免疫细胞化学染色法检测整合素β1和细胞角蛋白19( CK19)表达.结果 (1)人iPSC在胚胎干细胞完全培养液中呈典型的干细胞克隆状生长,边界清楚,增殖旺盛;AKP染色阳性.iPSC在无滋养层条件下悬浮培养可形成拟胚体.(2)诱导组iPSC经培养4d后形成干细胞克隆,部分细胞整合素β1和CK19表达阳性.对照组细胞大量死亡,未见整合素β1和CK19表达.结论 人iPSC在羊膜诱导下可定向分化为表皮样干细胞,有望成为皮肤组织工程新的种子细胞.     

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.     

Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.     

Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation

This study investigated the effect of spermatogonial stem cell encapsulated in alginate hydrogel during cryopreservation, as cells were protected against damage during cryopreservation within the hydrogel. Spermatogonial stem cells were isolated from the testes of Balb/c mice pups (6 days old), purified in laminin‐coated dishes and CD90.1 microbeads, encapsulated in alginate hydrogel and then cryopreserved. After thawing, cell viability and Spermatogonial stem cell (SSC) colony diameter were evaluated. After RNA was isolated and cDNA was synthesised, the expression of stemness genes was considered using RT real‐time PCR. Finally, spermatogonial stem cells labelled with BrdU were transplanted to busulfan azoospermic mouse models. Lin28a and Sall4 genes were significantly upregulated after cryopreservation in alginate hydrogel. However, cell viability was significantly decreased. The diameter of colonies consisting of spermatogonial stem cells freeze‐thawed in alginate microbeads showed no significant difference with fresh spermatogonial stem cells and the control group. The injection of freeze‐thawed spermatogonial stem cells encapsulated in alginate hydrogel resulted in spermatogenesis recovery. Alginate mimics the extracellular matrices (ECM) for spermatogonial stem cells; therefore, it can support stemness potential during the cell cryopreservation process and restart spermatogenesis after transplantation.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.