Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

Background:NANOG is a core transcription factor(TF)in embryonic stem cells(ESCs)and primordial germ cells(PGCs).Regulation of the NANOG gene by TFs,epigenetic factors,and autoregulatory factors is well characterized in ESCs,and transcriptional regulation of NANOG is well established in these cells.Although NANOG plays a key role in germ cells,the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied.Therefore,we investigated the mechanism that regulates transcription of the chicken NANOG(cNANOG)gene in PGCs and ESCs.Results:We first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis.Then,we measured the promoter activity of various 5′flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay.cNANOG expression required transcriptional regulatory elements,which were positively regulated by POU5F3(OCT4)and SOX2 and negatively regulated by TP53 in PGCs.The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element(CCAAT/enhancer-binding protein(CEBP)-binding site)in ESCs.Furthermore,small interfering RNA-mediated knockdown demonstrated that POU5F3,SOX2,and CEBP played a role in cell type-specific transcription of cNANOG.Conclusions:We show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner.This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

Improved Isolation and Culture of Embryonic Germ Cells from Guanzhong Dairy Goat

A total of 219 embryonic-germ-cell-like （EG-like） clumps were derived from 15 selected goat fetuses. Isolation of primordial germ cells （PGCs） based on co-culture with primary goat embryonic fibroblast showed no difference from traditional feeder layer-based culture method used in mouse and human. The putative primary EG colonies were multilayer clumps of compact cells with unclear cell-cell boundaries. Three subculture methods of goat EG-like colony, traditional enzymatic digestion, mechanical cutting and combination of the both, were compared in this study. As a result, EG-like colonies traditionally disassociated with collagenase 1V could be subcultured for up to 4 passages. And the mechanically disaggregated EG-like colonies were successfully maintained 9-12 passages with or without enzymatic treatment. The pluripotency of the EG-like colonies was identified by their specific marker staining, spontaneous differentiation and embryoid bodies （EBs） formation in vitro. Most goat EG-like colonies （〉 80%） were AKP positive and immunocytochemically characterized with positive SSEA-1, Oct-4 and c-kit staining but SSEA-4. Under the condition of delaying passage, goat EG-like cells could differentiate into fibroblast-like, epithelium-like, and neuron-like cells. In addition, EBs could be obtained successfully in routine hanging drop culture. The serum free culture system （feeder layer-based） used in this study was suitable for keeping PGCs and EG-like cells in their undifferentiated condition, but failed to converse them to immortal cells. These results indicated that mechanical cutting is an effective method for passaging goat EG cell colonies. However, the microenvironment of conversing EG cells to immortal cells is still unclear.

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.

Primary mixed germ cell tumor of the liver with sarcomatous components

Germ cell tumor(GCT)of the liver is extremely rare. Here,we describe a case of hepatic mixed GCT with significant sarcomatous components and elevated serum α-fetoprotein(AFP)in a 34-year-old man.Histopathologically,the tumor was composed of two GCTs components:yolk sac tumor and immature teratoma.The predominant components of immature teratoma consisted of several types of tissue that represented different germinal layers(endoderm,mesoderm and ectoderm) and showed varying degrees of differentiation with significant sarcomatous components.The yolk sac component showed positivity for AFP and cytokeratin(AE1/ AE3).The immature teratoma components showed positivity for varying differentiation markers.Interphase cytogenetic analysis revealed that the yolk sac tumor and immature teratoma were positive for i(12p)and 12p over-representation.In particular,the rhabdomyoblastic components also showed typical i(12p)and 12p overrepresentation.This suggested that sarcomatous components may be associated with dedifferentiation or malignant transformation of certain mesenchymal components within teratoma.

Culture of Embryonic Stem Cell by Primordial Germ Cells of Down Producing Goat

[Objective] The paper was to establish embryonic stem cell system of goats. [Method] Numerous primordial germ cell colonies were derived from gonadal ridge and the surrounding tissues in 20 millimeter fetuses of down producing goat. Primordial germ cells and goats embryonic fibroblasts obtained from conceptus of equivaient gestational age were co-cultured. [Result] The colonies showed some characteristics of embryonic stem cells, such as the morphology of nest-like, they continued to be AKP positive and the ability to be continuously passed [Conclusion] These cells were pluripotent and ES-like cells.

Primary Embryonal Lung Carcinoma and Testicular Seminoma in the Same Patient Ten-Years Later: Case-Report and Literature Review

Primary germ cell tumors of lung are extremely rare. The prognosis is usually poor, with various symptoms seriously affecting quality of life. In this paper we describe the unique case of a patient affected by an embryonal carcinoma of lung and a testicular seminoma after ten years. We also report literature about pulmonary extragonadal germ cell tumors.

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.

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.

Diagnosis and treatment of primary seminoma of the prostate:A case report and review of literature

BACKGROUND Primary seminoma of the prostate(PSP)is a rare type of extragonadal germ cell tumour that is easily misdiagnosed,owing to the lack of specific clinical features.It is therefore necessary for clinicians to work toward improving the accuracy of PSP diagnosis.CASE SUMMARY A 59-year-old male patient presenting with acute urinary retention was admitted to a local hospital.A misdiagnosis of benign prostatic hyperplasia led to an improper prostatectomy.Histopathology revealed PSP invading the bladder neck and bilateral seminal vesicles.Further radiotherapy treatment for the local lesion was performed,and the patient had a disease-free survival period of 96 mo.This case was analysed along with 13 other cases of PSP identified from the literature.Only four of the cases(28.6%)were initially confirmed by prostate biopsy.In these cases,imaging examinations showed an enlarged prostate(range 6-11 cm)involving the bladder neck(13/14).Of the 14 total cases,11(78.6%)presented typical pure seminoma cell features,staining strongly positive for placental alkaline phosphatase,CD117,and OCT4.The median age at diagnosis was 51(range 27-59)years,and patients had a median progression-free survival time of 48(range 6-156)mo after treatment by cisplatin-based chemotherapy combined with surgery or radiotherapy.The remaining three were cases of mixed embryonal tumours with focal seminoma,which had clinical features similar to those of pure PSP,in addition that they also had elevated serum alpha fetoprotein,beta-human chorionic gonadotropin,and lactose dehydrogenase.CONCLUSION PSP should be considered in patients younger than 60 years with an enlarged prostate invading the bladder neck.Further prostate biopsies may aid in proper PSP diagnosis.Cisplatin-based chemotherapy is still the main primary therapy for PSP.

Real case of primitive embryonal duodenal carcinoma in a young man

We report here the case of a young man suffering from a rare germ cell tumour. The patient was a 25-year-old man who was referred to our centre for asthenia, stinging epigastric pain, and an iron deficiency anaemia. Gastroscopy revealed a circumferential vegetating lesion on the second portion of the duodenum. The lesion was indurated at the third portion of the duodenum, responsible for a tight stenosis. A computerized tomography-scan of the chest, abdomen and pelvis, and a pancreatic MRI showed a circumferential lesion with a bi-ductal dilatation (i.e., of the common bile duct and Wirsung’s duct) without metastatic localisation. The patient underwent a pancreaticoduodenectomy with lymph node dissection including all cellular adipose tissues of the hepatic pedicle from the hepatic common artery and of the retroportal lamina. Histological findings were suggestive of a duodenal embryonal carcinoma with pancreatic infiltration. This is the second published case highlighting the duodenal primitive localisation of an embryonal carcinoma with pancreatic infiltration.

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.

The role of sex chromosomes in mammalian germ cell differentiation： can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes？

The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

Derivation of Putative Porcine Embryonic Germ Cells and Analysis of Their Multi-Lineage Differentiation Potential

Embryonic germ （EG） cells are cultured pluripotent stem cells derived from the primordial germ cells （PGCs） that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22-30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24-28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase （AP）. These cells were able to form embryoid bodies （EBs） in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein （AFP）, s-smooth muscle actin （^-SMA）, and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunoftuorescence for neuronal class III [3-tubulin （Tuj 1）, glial fibrillary protein （GFAP） and galactosylceramidase （GALC）, respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.

Single-cell sequencing: New insights into pre-implantation development, pluripotency, and germ cells

Early mammalian embryonic development is a complex,dynamic,and heterogeneous process.Although numerous efforts have been made to delineate the mechanism underlying embryonic development,many questions still remain unanswered because of the scarcity of available human materials and the minute amounts of DNA and RNA.In recently years,technical developments in high-throughput single cell sequencing have revolutionized biology and provided a novel and powerful tool to investigate the dynamics of early embryo development in a quantitative and systematic manner.Gene expression networks,epigenetic modification(including DNA methylation and chromatin accessibility),and genetic variation can be recorded simultaneously in a single cell at single base resolution.The present review highlights how the single cell sequencing has provided new insights into mammalian pre-implantation development,embryonic stem cell biology,and germline development.

Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors

Testicular germ cell tumors(TGCTs)are a cancer pharmacology success story with a majority of patients cured even in the highly advanced and metastatic setting.Successful treatment of TGCTs is primarily due to the exquisite responsiveness of this solid tumor to cisplatin-based therapy.However,a significant percentage of patients are,or become,refractory to cisplatin and die from progressive disease.Mechanisms for both clinical hypersensitivity and resistance have largely remained a mystery despite the promise of applying lessons to the majority of solid tumors that are not curable in the metastatic setting.Recently,this promise has been heightened by the realization that distinct(and perhaps pharmacologically replicable)epigenetic states,rather than fixed genetic alterations,may play dominant roles in not only TGCT etiology and progression but also their curability with conventional chemotherapies.In this review,it discusses potential mechanisms of TGCT cisplatin sensitivity and resistance to conventional chemotherapeutics.

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。

人胚胎生殖干细胞的分离和体外培养

目的:体外培养人胚胎生殖干细胞(EG),在不添加细胞因子的培养条件下,观察细胞生长情况。方法:取5～10周人胚胎的生殖腺嵴和肠背系膜,进行组织块培养,采用组织化学及免疫细胞化学技术对培养的细胞进行鉴定。结果:培养4d后,在成纤维细胞的上面出现EG细胞集落;培养2周后,显示细胞呈圆形,胞质染成深蓝色;细胞染色体均为正常的二倍体核型;碱性磷酸酶活性强阳性;并检测到SSEA-1。结论:体外培养人胚胎生殖腺嵴,利用源于自身胚胎组织的成纤维细胞作为饲养层,可观察到EG细胞集落的形成;培养的细胞初步鉴定为人胚胎干细胞。

从原始生殖细胞分离克隆鸡胚胎生殖细胞的研究

从孵化 5 5天的鸡胚生殖腺中分离得到大量原始生殖细胞 (PGCs)集落 ,这些集落的细胞经多次克隆传代具有胚胎生殖细胞 (EG)的诸多特征 ,如有连续传代的能力 (传至第 9代 ) ,细胞集落有典型鸟巢状结构 ,PAS染色阳性 ,AKP染色阳性 ,在无饲养层无分化抑制因子LIF时可以自发分化成几种细胞类型 ,包括成纤维细胞、神经细胞、自律细胞等 ,悬浮培养时具有形成类胚体的能力。上述发现表明该细胞具EG细胞的诸多特性 。