全反式视黄酸影响小鼠胚胎干细胞向原始生殖细胞分化的相关基因表达

目的 探讨全反式视黄酸(all-trans retinoic acid,atRA)诱导小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向原始生殖细胞(Primordial germ cellS,PGCs)分化的相关基因表达改变及其机制.方法 mESCs分化形成拟胚体(embryoid bodies,Ebs),不同浓度atRA(1 μM,2 μM,5 μM)持续诱导Ebs 16h、2d和5d,观察Ebs形态变化,实时PCR和Western blot检测PGCs分化相关基因和蛋白表达变化.分析基因肩动子中RA反应元件,以Stra8(Stimulated by Retinoic Acid Gene 8,Stra8)基因为阳性对照,确定各基因对atRA刺激的原发性反应和继发性反应.结果 不同浓度atRA诱导Ebs 16h和2d后形态无明显变化.诱导后Ebs的PGCs分化相天基因mRNA表达分4种情况:16h内(Stra8)表达量达最大,然后下降;2d内(Scp3)表达量达最大,然后下降;5d达到最大量(Mvh);表达量无明显变化(Fragilis,Blimp-1和Prm1).Mvh表达符合继发反应特点;Stra8和Scp3表达符合原发反应特点;Blimp1,Prm1和Fragilis表达可能与atRA调节无关.Stra8蛋白变化与mRNA变化相一致,而Mvh则不一致.结论 atRA诱导mESCs向PGCs分化,各阶段标志基因表达变化均在相对短的时间内完成(2～5d),atRA诱导浓度可选用1 μM.根据基因表达模式初步得出,atRA调控的靶基因为Mvh,具备继发反应特点,与特异性分化有关,而Blimp-1、Fragilis和Prm1调控PGCs分化可能与atRA无关.     

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。     

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.     

脐带间充质干细胞增强无精子症模型睾丸特异性基因表达

在生殖男科领域，探索人脐带间充质干细胞（HUCMSCs）对无精子症的治疗作用。HUCMSCs具有潜在的免疫抑制功能，并能够分泌多种细胞因子和生长因子，因此具有潜在的临床应用价值。作为探索，我们移植HUCMSCs进入无精子症小鼠睾丸间质，检测是否能够促进精子发生过程。从不同来源的脐带中分离HUCMSCs，移植进入白消安处理的小鼠睾丸间质中，采用注射生理盐水和HEK293细胞作为对照，对侧睾丸不注射。三周之后，RT-PCR检测10个生殖细胞特异性表达的基因，并检N3个特异性蛋白的表达。结果表明，注射人脐带间充质干细胞之后的表达明显高于对照组，证实生殖细胞特异性基因的表达上调，从而表明HUCMSCs对睾丸生精功能的恢复具有促进作用，为治疗无精子症探索一条新的途径。     

Quantification of germ cells and seminiferous tubules by stereological examination of testicles from 50 boys who suffered from sudden death

A stereological study of the development of testicular volume, the germ cell population and the seminiferous tubules was carried out on testes from 50 boys between 0 and 18 years of age who had suffered from sudden unexpected death. Semi-thin sections (0.5 μm) of testicular tissue were prepared and examined by point and profile counting using a projection microscope. Testicular weight was recorded in 48 of the cases. Median volume of pairs of testes increased from 1.1 (range 0.3–1.9) to 3.0 cm³ (range 2.8–3.4) during the 0–10 year period, while the median of the total number of germ ceils per boy was augmented from 13 times 10⁶ (range 4.4–36 times 10⁶) to 83 times 10⁶ (range 32–122 times 10⁶) during the same period. The median length of the seminiferous tubules per boy was 181 m (range 27–361) in the 0–1 year period and 411 m (range 277–660) in the group 5–10 years. Mean tubular diameter was unchanged during the first 10 years of life, whereas the mean nuclear diameter of the germ cells showed significant negative correlation to age in boys less than 10 years. After the age of 10 years a more marked growth of testicular volume, an increase in the number of germ cells and the diameter as well as the length of the seminiferous tubules were observed. Thus, the study showed that testicular size, the number of germ cells and the total length of the seminiferous tubules per boy increased not only in puberty but also during the first ten years of life.     

Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow

Development of novel therapeutic approaches to repair fracture non‐unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)‐derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non‐union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM‐MSCs) or a no cell control group (n = 10–12 per group). Preliminary studies demonstrated that both for hESC‐derived MSCs and hBM‐MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM‐MSC as compared to the control group that received no cells; values for these parameters in the hESC‐derived MSC group were intermediate between the hBM‐MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X‐ray in the hESC‐derived MSC group. Our results thus indicate that while hESC‐derived MSCs may have potential to induce fracture healing in non‐unions, hBM‐MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1804–1811, 2011     

Apoptosis precedes detachment of germ cells from the seminiferous epithelium after hormone suppression by short-term oestradiol treatment of rats

Spermatogenesis is a highly synchronized process in which FSH and testosterone are considered the major regulators. Nevertheless, the mechanism by which these hormones act on germ cells is unclear. Cell adhesion has been proved to play an essential role in the regulation of programmed cell death in epithelial cells and it is now known that FSH and testosterone withdrawal results in the triggering of apoptosis as well as germ cell detachment from the seminiferous epithelium. Therefore, it seemed important to investigate whether the triggering of apoptosis in germ cells by experimental hormone suppression occurred as a result of their previous detachment from the epithelium. To achieve this goal, adult male rats were injected with 50 μg oestradiol benzoate for 1, 2, 3, 4, 5 or 10 days to suppress gonadotrophin secretion and thus intratesticular levels of testosterone. Germ cell apoptosis was assessed in testes from these animals by in situ 3' end-labelling of DNA fragments and quantified in seminiferous tubule sections at stages VII–VIII. Serial sections throughout the epididymides from these animals were analysed to search for immature germ cells detached from the epithelium. These cells were scored and quantified in non-consecutive randomly selected epididymal sections. Our data indicate that the triggering of apoptosis in germ cells precedes germ cell detachment, suggesting that detachment of germ cells from the epithelium, occurring after hormone suppression, is not necessary for germ cell apoptosis.