The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.     

Regulation of hematopoiesis and the hematopoietic stem cell niche by Wnt signaling pathways

Hematopoietic stem cells （HSCs） are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal （in which the ability to function as HSCs is retained） and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. Wnt signaling pathways play a crucial role during embryogenesis and deregulation of these pathways has been implicated in the formation of solid tumors. Wnt signaling also plays a role in the regulation of stem cells from multiple tissues, such as embryonic, epidermal, and intestinal stem cells. However, the function of Wnt signaling in HSC biology is still controversial. In this review, we will discuss the basic characteristics of the adult HSC and its regulatory microenvironment, the ＂niche＂, focusing on the regulation of the HSC and its niche by the Wnt signaling pathways.     

The inhibitory effect of transthyretin gene on growth of human hepatoma cells

Transthyretin(TTR) gene was highly expressed in normal liver and it has been found to be deleted in part of DNA samples from human hepatic cancer.Its mRNA expression was suppressed in most hepatoma samples.In order to study the biological effect of TTR gene on the growth of hepatoma cells,a recombinant vector containing TTR cDNA was constructed by pCMV,then it was transfected into hepatoma cell lines SMMC-7721 and Q3.It has been demonstrated that the inhibition of growth rate of TTR cDNA transfected hepatoma cells was about 50% in strength compared with that of the control.This inhibition was further enhanced when the transfected hepatoma cells were treated with all-trans retinoic acid.Hepatoma cells of cell lines PLC/PRF/5,SMMC-7721 and Q3 as well as hepatoma cells SMMC-7721 transfected with pCMV or pCMV-TTR were analyzed for TTR expression by Northern hybridization.The low level of TTR expression was found in both hepatoma cell lines and in SMMC-7721 cells transfected with pCMV alone.However,a remarkable TTR mRNA expression was observed in hepatoma SMMV-7721 cells transfected with pCMV-TTR.It seems possible that TTR gene might be a candidate of cancer suppressor gene for human hepatic cancer.     

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique

Transgenic cell lines of loblolly pine （Pinus taeda L.） were analyzed by a compact laser-tweezers-Raman-spectroscopy （LTRS） system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

Getting the right stuff： controlling neural stem cell state and fate in vivo and in vitro with biomaterials

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem ceils are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell （NSC） therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.     

Advances in cellular technology in the hematology field: What have we learned so far?

Despite the advances in the hematology field, blood transfusion-related iatrogenesis is still a major issue to be considered during such procedures due to blood antigenic incompatibility. This places pluripotent stem cells as a possible ally in the production of more suitable blood products. The present review article aims to provide a comprehensive summary of the state-of-theart concerning the differentiation of both embryonic stem cells and induced pluripotent stem cells to hematopoietic cell lines. Here, we review the most recently published protocols to achieve the production of blood cells for future application in hemotherapy, cancer therapy and basic research.     

siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines

Glioma is one of the most highly angiogenic tumors, and glioma stem cells （GSCs） are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silen- cing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The suscep- tibUity of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a po- tential target in GSCs for glioma treatment.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

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), α-smooth muscle actin (α-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 immunofluorescence for neuronal class Ⅲ β-tubulin (Tuj1), 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.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

Transgenesis and nuclear transfer using porcine embryonic germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.     

人胚胎干细胞建系的研究现状与存在的问题

人胚胎干细胞系的建立,对人类胚胎发生和人类发育生物学研究、人类新基因的发现和功能研究以及基因治疗、细胞和组织的移植治疗等领域的突破性进展具有重大意义;回顾了人胚胎干细胞建系研究的历程,就建系的几种方案、路线、意义和可行性进行了探讨;详细系统地说明了迄今为止建立人胚胎干细胞系所需要的饲养层类型、培养基组成、添加细胞因子种类及其作用;分析了建立和维持人胚胎干细胞系所需消化酶的种类及其作用以及目前常用的几种传代方法;从若干方面总结了人胚胎干细胞系的鉴定方法,并对建立和维持人胚胎干细胞系中存在的若干问题进行了剖析,提出了目前急待解决的问题。     

Establishment of porcine transgenic embryonic germ cell lines expressing enhanced green fluorescent protein

The purpose of this study was to isolate porcine embryonic germ (EG) cells and establish transgenic EG cell lines. Plasmid DNA was the enhanced green fluorescent protein (EGFP) vector. Porcine EG cells in rapid proliferation (4th to 9th passage) were transfected with LipofecTamine 2000 and TransFast reagents. Porcine EG cells transfected with a complex of 1 microg of DNA and 2 microL of LipofecTamine 2000 reagent yielded four EG-EGFP cell lines, which emitted bright green fluorescence. EG-EGFP cells cultured for more than 2 weeks without passage gave rise to various differentiated phenotypes. In addition, to determine the degree to which EG cells become integrated into the inner cell mass of host embryos, 135 embryos were injected with porcine EG-EGFP cells; 110 embryos survived and developed into blastocysts (81.5%). Eighty-four chimeric embryos contained fluorescent cells after culture; 49 blastocysts contained EG-EGFP cells in the inner cell mass. Our results suggested that the chimeric rate would not be improved via using different stages of embryos for injection.     

Attempts to enhance production of porcine chimeras from embryonic germ cells and preimplantation embryos

Porcine embryonic germ (EG) cells share common features with porcine embryonic stem (ES) cells, including morphology, alkaline phosphatase activity and capacity for in vitro differentiation. Porcine EG cells are also capable of in vivo development by producing chimeras after blastocyst injection; however, the proportion of injected embryos that yield a chimera and the proportion of cells contributed by the cultured cells in each chimera are too low for practical use in genetic manipulation. Moreover, somatic, but not germ-line chimerism, has been reported from blastocyst injection using porcine ES or EG cells. To test whether efficiency of chimera production from blastocyst injection can be improved upon by changing the host embryo, we used as host embryos four groups according to developmental stage or length in culture: fresh 4-cell and 8-cell stage embryos subsequently cultured into blastocysts, fresh morulae, fresh blastocysts, and cultured blastocysts. Injection and embryo transfer of fresh and cultured blastocysts produced similar percentages of live piglets (17% versus 19%). Four piglets were judged to have a small degree of pigmentation chimerism, but microsatellite analysis failed to confirm chimerism in these or other piglets. Polymerase chain reaction analysis for detection of the porcine SRY gene in female piglets born from embryos injected with male EG cells identified six chimeras, at least one, but not more than two, from each treatment. Chimerism was confirmed in two putative pigmentation chimeras and in four piglets without overt signs of chimerism. The low percentage of injected embryos that yielded a chimera and the small contribution by EG cells to development of each confirmed chimera indicated that procedural changes in how EG cells were combined with host embryos were unsuccessful in increasing the likelihood that porcine EG cells will participate in embryonic development. Alternatively, our results suggested that improvements are needed in EG cell isolation and culture procedures to ensure in vitro maintenance of EG cell developmental capacity.     

Effects of growth factors and feeder cells on porcine primordial germ cells in vitro

As embryonic stem (ES) cells are not available in swine, embryonic germ (EG) cells derived from primordial germ cells (PGCs) are an alternate source of pluripotent embryonic cells for genetic modification through homologous recombination. Although morphological and biochemical characteristics are similar between ES and EG cells, culture conditions are quite different. To optimize the culture condition for the establishment of porcine EG cells, porcine PGCs were cultured in vitro with various combinations of growth factors (leukemia inhibitory factor [LIF], stem cell factor [SCF], and basic fibroblast growth factor [bFGF]) and on different kinds of feeder cells (STO, TM(4), Sl/Sl(4) m220, porcine embryonic fibroblasts, and COS-7 cells). Optimal results were obtained when all three growth factors (LIF, SCF, and bFGF) were present in the media. Also, feeder cells expressing membrane-bound SCF are required for survival and establishment of porcine EG cells. Therefore, a combination of growth factors and proper feeder cells are critical for the establishment of undifferentiated porcine EG cells.     

Derivation and characterization of pluripotent embryonic germ cells in chicken

Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.     

Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells

Human embryonic stem (ES) cells and embryonic germ (EG) cells are pluripotent and are invaluable material for in vitro studies of human embryogenesis and cell therapy. So far, only two groups have reported the establishment of human EG cell lines, whereas at least five human ES cell lines have been established. To see if human EG cell lines can be reproducibly established, we isolated primordial germ cells (PGCs) from gonadal ridges and mesenteries (9 weeks post-fertilization) and cultured them on mouse STO cells. As with mouse ES colonies, the PGC-derived cells have given rise to multilayered colonies without any differentiation over a year of continuous culture. They are karyotypically normal and express high levels of alkaline phosphatase, Oct-4, and several cell-surface markers. Histological and immunocytochemical analysis of embryoid bodies (EBs) formed from floating cultures of the PGC-derived cell colonies revealed ectodermal, endodermal, and mesodermal tissues. When the EBs were cultured in the presence of insulin, transferrin, sodium selenite, and fibronectin for 1 week, markers of primitive neuroectoderm were expressed in cells within the EBs as well as in cells growing out from the EBs. These observations indicate that our PGC-derived cells satisfy the criteria for pluripotent stem cells and hence may be EG cells.