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 vitrodifferentiation and in vivotransplantation. 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 StraSand 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 GFRal, 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.

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） ceils 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.

Transplantation of Goat Bone Marrow Mesenchymal Stem Cells (gMSCs) Help Restore Spermatogenesis in Endogenous Germ Cells-Depleted Mouse Models

Mesenchymal stem cells （MSCs） derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs （gMSCs） and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these ceils were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid （RA）, as analysed by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture;however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

Revolutionize livestock breeding in the future: an animal embryo-stem cell breeding system in a dish

Meat and milk production needs to increase ～ 70–80% relative to its current levels for satisfying the human needs in 2050.However,it is impossible to achieve such genetic gain by conventional animal breeding systems.Based on recent advances with regard to in vitro induction of germ cell from pluripotent stem cells,herein we propose a novel embryo-stem cell breeding system.Distinct from the conventional breeding system in farm animals that involves selecting and mating individuals,the novel breeding system completes breeding cycles from parental to offspring embryos directly by selecting and mating embryos in a dish.In comparison to the conventional dairy breeding scheme,this system can rapidly achieve 30–40 times more genetic gain by significantly shortening generation interval and enhancing selection intensity.However,several major obstacles must be overcome before we can fully use this system in livestock breeding,which include derivation and mantaince of pluripotent stem cells in domestic animals,as well as in vitro induction of primordial germ cells,and subsequent haploid gametes.Thus,we also discuss the potential efforts needed in solving the obstacles for application this novel system,and elaborate on their groundbreaking potential in livestock breeding.This novel system would provide a revolutionary animal breeding system by offering an unprecedented opportunity for meeting the fast-growing meat and milk demand of humans.

High dose chemotherapy with stem cell support in the treatment of testicular cancer

Testicular germ cell cancer(TGCC) is rare form of malignant disease that occurs mostly in young man between age 15 and 40. The worldwide incidence of TGCC is 1.5 per 100000 man with the highest rates in North Europe. After discovery of cisplatin cure rates of TGCC are very favorable between 90%-95% and unlike most solid tumors, cure rate for metastatic TGCC is around 80%. Metastatic TGCC is usually treated with 3-4 cycles of bleomycin, etoposide, cisplatinum chemotherapy with or without retroperitoneal surgery and cure rates with this approach are between 41% in poor risk group and 92% in good risk group of patients. Cure rates are lower in relapsed and refractory patients and many of them will die from the disease if not cured with first line chemotherapy. High dose chemotherapy(HDCT) approach was used for the first time during the 1980 s. Progress in hematology allowed the possibility to keep autologous haematopoietic stem cells alive ex-vivo at very low temperatures and use them to repopulate the bone marrow after sub-lethal dose of intesive myeloablative chemotherapy. Despite the fact that there is no positive randomized study to prove HDCT concept, cure rates in relapsed TGCC are higher after high dose therapy then in historical controls in studies with conventional treatment. Here we review clinical studies in HDCT for TGCC, possibilities of mobilising sufficient number of stem cells and future directions in the treatment of this disease.

Reprogramming of germ cells into pluripotency

Primordial germ cells(PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

Types of Human Stem Cells and Their Therapeutic Applications

The present review examines in the first place various kinds of naturally occurring stem cells, including germ cells and embryonic stem cells (ES cells), as well as haemopoietic stem cells, which are historically the first to be used for medical treatment. Attention is also given to cancer stem cells, as a source of perseverant malignant disease. The main interest is now represented by the variety of somatic cells, which can be re-programmed to different types of differentiated cells, the so-called induced pluripotent stem cells (IPSC’s). Focus is now directed not only to the factors which make such events possible like de-differentiation and reconversion but also to the stages involved in this process. It is actually postulated that the transition from differentiated cells to pluripotent cells follows a definite sequence with evidence of two waves of gene regulations. Main applications of stem cell therapy are reviewed, from the established use of haemopoietic stem cells for clinical transplantation in a variety of haematological disorders to the initial attempts to employ stem cells for the treatment of other disparate conditions. Problems related to stem cell treatment with both ES and IPS cells, like the necessity of a large in vitro expansion to provide sufficient amounts of cells and the related risk of genomic abnormalities are illustrated. The necessity of safe procedures for the development of this venture is also outlined.

Stem cells of the reproductive tract of women

Research in stem cells is one of the most rapidly evolving fields of investigation in medicine today. Stem cells are defined as cells that have the capacity to both generate daughter cells identical to the cell of origin (self-renewal) and to produce progeny with more restricted, specialized potential (differentiated cells). This dual ability to self-renew and differentiate offers great promise for expanding our understanding of organ systems, elucidating disease pathophysiology, and creating therapeutic approaches to difficult diseases. The goal of this review is to offer an overview of the different types of stem cells and to provide an introduction to the applications of stem cells to the field of obstetrics and gynecology.

Developmental potency of mouse primitive ectoderm cells, embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.

鱼类原始生殖细胞发育与生殖操作技术研究进展

原始生殖细胞是胚胎发育过程中最早建立的一群生殖干细胞,是有性生殖动物生殖发育的基础。鱼类原始生殖细胞特化遵循“先成论”的模式,早期胚胎发育过程中获得母源生殖质组分的细胞特化为原始生殖细胞,特化形成的原始生殖细胞需要维持其生殖干细胞命运,并经过长距离迁移最终到达性腺原基的位置。原始生殖细胞特化、迁移和命运维持过程受到多种基因和信号通路的综合调控。研究鱼类原始生殖细胞发育不仅有助于深入理解脊椎动物细胞特化、迁移和命运维持等基本生物学过程的调控机理,而且是开发新的养殖鱼类生殖控制和生殖干细胞移植技术的重要基础。本文概述了鱼类原始生殖细胞发育的基础理论以及生殖操作技术研究进展,并展望了未来的发展趋势,以期为开发重要养殖鱼类优良种质创制新技术提供参考。

间充质干细胞在小型猪重组牙胚同种异体移植中的免疫调节作用

目的 探索在小型猪重组牙胚同种异体移植实现颌骨内牙再生过程中,间充质干细胞BMMSCs的免疫调节作用。方法 将胚胎小型猪帽状期牙胚解离重组后,移植至成年小型猪拔牙区,BMMSCs经分离鉴定后,单独或与他克莫司FK506联合应用,进行同种异体移植免疫干预;评估受体小型猪的免疫状况和牙再生小型猪百分比。结果 BMMSC组受体小型猪CD3^(+)细胞和CD3^(+)CD8^(+)细胞比例均显著下降(P<0.05),Tregs细胞比例显著上调(P<0.05),有牙再生的小型猪百分比为75%;而BMMSC^(+)FK506组CD3^(+)和CD8^(+)T细胞比例均显著下调(P<0.05),但Tregs比例与空白对照组无显著差异,该组25%小型猪有牙再生;空白对照组的免疫排斥相关指标上调,未发现牙再生。结论 相对于与FK506合用,单独使用BMMSCs更有利于同种异体重组牙胚移植。

小鼠诱导多能干细胞向雄性生殖细胞样细胞诱导分化的研究

目的:对小鼠诱导多能干细胞向原始生殖细胞样细胞进行诱导培养,并对获得的原始生殖细胞样细胞进行鉴定。方法:在诱导干细胞培养基中对小鼠诱导多能干细胞进行培养,用蛋白质印迹法(Western blot)对Oct-4及C-kit蛋白表达水平进行鉴定,用定时定量PCR对Mvh,Fragilis,Stella的mRNA水平进行鉴定。结果:由小鼠诱导多能干细胞体外诱导分化获得的原始生殖细胞样细胞特异性表达Oct-4蛋白、C-kit蛋白以及Mv hmRNA,Fragilis RNA,Stella RNA。结论:由小鼠诱导多能干细胞成功诱导培养出原始生殖细胞样细胞,为进一步研究其向精子细胞的转化奠定了较好的基础。

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.

Study on Transplanting Germ Like Cells Differentiated from Embryonic Stem Cell into Mouse Seminiferous Tubules

Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, stella, fragilis, Tex14, Sry, Hsp90-a, Stra8 and integrin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body （EB） derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1＋ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-a, Stra8 and integrin a6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.

Reprogramming of the pig primordial germ cells into pluripotent stem cells: a brief review

Primordial germ cells(PGCs) are regarded as unipotent cells that can produce only either spermatogonia or oocytes. However, PGCs can be converted into the pluripotent state by ?rst dedifferentiation to embryonic germ cells and then by reprogramming to induce them to become pluripotent stem cells(iPSCs). These two stages can be completely implemented with mouse cells. However, authentic porcine iPSCs have not been established.Here, we discuss recent advances in the stem cell ?eld for obtaining iPSCs from PGCs. This knowledge will provide some clues which will contribute to the regulation of reprogramming to pluripotency in farm species.

牛原始生殖细胞的分离与培养

从4～15周龄的牛胎儿生殖嵴中分离得到牛原始生殖细胞,以STO(一种建系的小鼠胎儿成纤维细胞)及MEF(小鼠胎儿成纤维细胞)为饲养层抑制分化培养,其中1个细胞系传至4代。研究发现,STO较MEF更有利于牛类EG细胞的分离与培养,体长小于5cm的胎儿适合牛EG细胞分离与克隆。同时观察到这些细胞在体外进行自发性分化,可形成上皮样细胞、神经样细胞和成纤维样细胞。

影响牛胚胎干细胞分离克隆因素的研究

采用与同源胎儿成纤维细胞共同培养及传统饲养层培养方式 ,以高糖DMEM ,添加 0 .1mM2 -巯基乙醇、犊牛血清、细胞因子为培养基 ,以 4～ 13周龄屠宰牛胎儿为实验材料 ,探讨影响牛原始生殖细胞分离克隆胚胎干细胞的相关因素。结果发现 :当犊牛血清为 15 %时效果最好 ;细胞因子添加与否对胚胎干细胞的分离及同源牛胎儿成纤维细胞的贴壁与生长影响并不显著 ,而在传代过程中有一定影响 ;以 0 .2 %胰酶 +0 .0 4 %EDTA为细胞消化液效果最佳 ;以同源胎儿成纤维细胞共培养的方式分离克隆牛胚胎干细胞 。