山羊精原干细胞体外培养分化

探索山羊精原干细胞体外培养体系。收集2月龄关中奶山羊睾丸,一步酶法消化分离曲细精管细胞,台盼兰检测平均存活率82.7%,以1×106个/ml接种含15%胎牛血清DMEM/F12培养瓶,37℃、5%CO2和饱和湿度条件下培养,4周后FBS逐渐降至10%。原代培养以多突起和片状的睾丸体细胞铺壁生长为主,10天左右精原干细胞数量增加,可见二联体和四联体,3周左右有鸟巢状和山脉状集落形成,碱性磷酸酶染色阳性,培养30天集落数不断增加,散在分布有贴壁和漂浮精子,换液后精子丢失。挑取单集落重新接种铺壁的曲细精管体细胞饲养层后陆续有精子细胞及精子形成,主要分布于集落周围。     

哺乳动物精原干细胞体外分离培养研究进展

精原干细胞(spermatogonial stem cells,SSCs)作为成体干细胞的一种,具有高度自我更新和分化潜能。近几年研究发现,SSCs的体外培养是表观遗传基础研究、精子发生机制深入探索以及治疗雄性不育的基础条件。本文根据国内外SSCs相关研究,对哺乳动物SSCs的生物学特性、体外分离培养和鉴定等作一综述,以期为哺乳动物SSCs和其他干细胞的长期体外培养以及建系提供一定的借鉴。     

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。     

体外培养条件下SCF、LIF与bFGF对昆明白小鼠精原干细胞增殖的影响

生长因子作为细胞体外培养和在体细胞生长及增殖必需的调节因子 ,一直被广泛的关注。业已证明干细胞因子(ＳｔｅｍＣｅｌｌＦａｃｔｏｒ,ＳＣＦ)、白血病抑制因子 (ｌｅｕｋａｅｍｉａｉｎｈｉｂｉｔｏｒｙｆａｃｔｏｒ,ＬＩＦ)和碱性成纤维细胞生长因子 (ＢｓａｉｃＦｉｂｒｏｂｌａｓｔＧｒｏｗｔｈＦａｃｔｏｒ,ｂＦＧＦ)具有刺激细胞增殖的作用[1～ 4] ,但大都是对单一因子进行研究。本实验探讨用这三种生长因子的不同组合观察对小鼠精原干细胞增殖的作用 ,以期定性和定量的探讨出体外培养初期三种因子对小鼠精原干细胞生长的影响 ,为小…     

美国成功培养小鼠精原干细胞

美国宾夕法尼亚州大学兽医学院通过改进培养介质成功地在实验室中培养了小鼠的精源干细胞，并将这种细胞移植给不育小鼠，结果这些受体小鼠不但能够产生精子，而且产生了具有捐献小鼠遗传特征的后代。该研究成果发表在Proceedingc 0f the National Academy 0f Sciences网络版上。     

小鼠精原干细胞在三种培养基中的生长行为

目的：建立小鼠精原干细胞（SSCs）的体外长期培养体系。方法：用分别添加了等量的胶质细胞源神经营养因子（GDNF）、可溶性GFRα1和hFGF的DMEM／F12、KSR和StemPro-34 SFM三种无血清培养基和MEF饲养层分别培养经差异贴壁分选富集的小鼠SSCs,通过形态观察、标志基因的RT-PCR和免疫细胞化学分析检测其SSCs本原。结果：DMEM／F12与KSR可支持小鼠SSCs在体外存活6-7d,而StemPro-34 SFM能能维持SSCs体外增值一个月。结论：StemPro-34 SFM支持小鼠SSCs的体外增殖。     

以支持细胞为饲养层培养小鼠精原干细胞

为探索精原干细胞(Spermatogonialstemcells,SSCs)体外自增殖的条件以及SSCs体外快速扩增的方法,以6-8日龄昆明乳鼠为材料,分离小鼠睾丸细胞,采用Percoll梯度离心法富集SSCs;以经丝裂霉素C处理的Sertoli细胞作饲养层,以DMEM为基本培养基,加入5%胎牛血清和103u/ml的白血病抑制因子(Leukemiainhibitoryfactor,LIF),体外培养SSCs;运用免疫荧光技术,以SSCs特异性表面分子Thy1为标志,对原代培养20d和传代培养14d的细胞进行鉴定。该培养体系下,SSCs贴壁时间为6h-9h,48h后可见细胞分裂,迅速增殖出现在接种12d以后。接种后第20d形成数十至上百个细胞的细胞团,细胞总数比接种时增加了45-245倍,100倍显微镜下观察可见,单位视野内细胞团数为26±4个。传代后细胞增殖较快。原代培养20d和传代培养14d的细胞均为Thy1阳性;而传代20d后,细胞周缘不整,有伪足出现,呈现出死亡迹象。该培养条比较适合SSCs短期快速增殖。     

精原干细胞技术研究进展

精原干细胞(spermatogonial stem cells,SSCs)是位于睾丸曲细精管基膜上能自我更新和连续分化产生精子的最原始精原细胞,是雄性体内唯一能将遗传信息自然传至子代并可终生复制的双倍体细胞,对复杂的精子发生过程有着至关重要的作用。作为一个未分化细胞群体,SSCs在精子生成和物种进化所必需的基因传递中发挥作用。基于课题组多年的研究,该文较系统地评述了SSCs的生物学特性、分离富集、体外培养影响因素和移植技术等方面的进展,以期对雄性辅助生殖、细胞再生治疗、畜牧业生产等研究应用提供借鉴。     

大鼠精原干细胞的微滴培养法

精原干细胞（spermatogonial stem cells,SSCs）作为成体干细胞的一类,既具有自我更新和分化的潜能,又可向子代传递遗传信息。阐明其增殖过程及分化特性对SSCs的进一步应用具有重要意义。小鼠SSCs的微滴培养研究显示,微滴培养技术与常规培养方法相比具有独特的优势。然而其他物种的SSCs能否实现微滴培养尚有待证实。该研究旨在利用微滴培养法建立大鼠SSCs体外培养技术。5、8、10、20、40个大鼠SSCs分别置于20此微滴中培养,用丝裂霉素处理的STO细胞作为滋养层。倒置显微镜观察记录大鼠SSCs的增殖状态。一个月后,对微滴培养的SSCs进行免疫荧光双标记染色鉴定。结果显示,一个微滴内接种5个SSCs就能实现扩增培养;培养一个月后,SSC仍然表达其特异的标记基因分子如CDH1、OCT4、PLZF、Thy1和Gfra1。体外诱导分析显示,微滴培养的大鼠SSCs具有分化为精母细胞的能力。大鼠SSCs微滴培养法的建立,为其他物种SSCs的培养提供了借鉴,也为再生医学和生命科学相关领域的研究提供了技术平台。     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.     

Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells

Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I(-) Thy-1(+) alpha 6-integrin(+) alpha v-integrin(-/dim) throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.     

Radioprotective effect of misoprostol on mouse spermatogonial stem cells

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH x 101/F1 hybrid mice (3H1) were analysed by establishing dose--response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1.7 and 3.6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3.6 to 5.0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose--response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.     

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.     

Nonrandom distribution of mouse spermatogonial stem cells surviving fission neutron irradiation

Colony formation by surviving spermatogonial stem cells was investigated by mapping pieces of whole mounted tubuli at intervals of 6 and 10 days after doses of 0.75 and 1.50 Gy of fission neutron irradiation. Colony sizes, expressed in numbers of spermatogonia per colony, varied greatly. However, the mean colony size found in different animals was relatively constant. The mitotic indices in large and small colonies and in colonies in different epithelial stages did not differ significantly. This finding suggests that size differences in these spermatogenic colonies are not caused by differences in growth rate. Apparently, surviving stem cells start to form colonies at variable times after irradiation. The number of colonies per unit area varied with the epithelial stages. Many more colonies were found in areas that during irradiation were in stages IX-III (IX-IIIirr) than in those that were in stages IV-VII (IV-VIIirr). After a dose of 1.50 Gy, 90% of all colonies were found in areas IX-IIIirr. It is concluded that the previously found difference in repopulation after irradiation between areas VIII-IIIirr and III-VIIIirr can be explained not by differences in colony sizes and/or growth rates of the colonies in these areas but by a difference in the number of surviving stem cells in both areas. In area XII-IIIirr three times more colonies were found after a dose of 0.75 Gy than after a dose of 1.50 Gy. In area IV-VIIirr the numbers of colonies differed by a factor of six after both doses. This finding indicates that spermatogonial stem cells are more sensitive to irradiation in epithelial stages IV-VII than in stages XII-III. In control material, spermatogonia with a nuclear area of 70-110 micron2 are rare. However, especially 6 days after irradiation, single cells of these dimensions are rather common. These cells were found to lie at random over the tubular basement membrane with no preference for areas with colonies. It is concluded that the great majority of these cells were not or do not derive from surviving stem cells. These enlarged cells most likely represent lethally injured cells that will die or become giant cells (nuclear area greater than 110 micron2).     

Sertoli cells dictate spermatogonial stem cell niches in the mouse testis

Sustained spermatogenesis in adult males relies on the activity of spermatogonial stem cells (SSCs). In general, tissue-specific stem cell populations such as SSCs are influenced by contributions of support cells that form niche microenvironments. Previous studies have provided indirect evidence that several somatic cell populations and the interstitial vasculature influence SSC functions, but an individual orchestrator of niches has not been described. In this study, functional transplantation of SSCs, in combination with experimental alteration of Sertoli cell content by polythiouracil (PTU)-induced transient hypothyroidism, was used to explore the relationship of Sertoli cells with SSCs in testes of adult mice. Transplantation of SSCs from PTU-treated donor mice into seminiferous tubules of normal recipient mice revealed a greater than 3-fold increase in SSCs compared to those from testes of non-PTU-treated donors. In addition, use of PTU-treated mice as recipients for transplantation of SSCs from normal donors revealed a greater than 3-fold increase of accessible niches compared to those of testes of non-PTU treated recipient mice with normal numbers of Sertoli cells. Importantly, the area of seminiferous tubules bordered by interstitial tissue and percentage of seminiferous tubules associated with blood vessels was found to be no different in testes of PTU-treated mice compared to controls, indicating that neither the vasculature nor interstitial support cell populations influenced the alteration of niche number. Collectively, these results provide direct evidence that Sertoli cells are the key somatic cell population dictating the number of SSCs and niches in mammalian testes.     

Establishment of a short-term in vitro assay for mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.     

小鼠精原干细胞与胚胎干细胞样细胞

近年来,通过培养小鼠精原干细胞(spermatogonial stem cells,SSCs)获得了胚胎干细胞样细胞(,embryonic stem cell-like cells,ES样细胞).这些研究表明小鼠精原干细胞不仅具备特异分化为精子的干细胞潜能,而且具备胚胎干细胞(embryonic stem cell,ES)分化为三胚层的多向分化潜能.因此.这将有助于研究干细胞的分化调控机制,并且这些研究成果延伸至人类精原干细胞,也将为再生医学获取特殊的胚胎干细胞样细胞或特异分化的精子细胞开辟了蹊径.