Conversion of female germline stem cells from neonatal and prepubertal mice into pluripotent stern cells

Pluripotent stem cells derived from neonatal or adult testes are a useful tool to examine the mechanisms of pluripotency and a resource for cell-based therapies. However, therapies usingthese cells will only benefit males but not females. Recently, female germline stem cells （FGSCs） were discovered in ovaries. Whether FGSCs can be converted into pluripotent stem cells, similar to spermatogonial stem cells, is unknown. Here, we demonstrate that female embryonic stem-like cells （fESLCs） can be generated within 1 month from the stably proliferating FGSCs cultured in embryonic stem cell （ESC） medium, fESLCs exhibit properties similar to those of ESCs in terms of marker expression and differentiation potential. Thus, our findings suggest that generation of patient-specific fESLCs is feasible and provides a foundation for personalized regenerative applications.     

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.     

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10⁰ TCID₅₀ MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10⁻⁵ TCID₅₀ MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.     

Epigenetic modifications and self-renewal regulation of mouse germline stem cells

Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

A versatile tool for tracking the differentiation of human embryonic stem cells

The ability of human embryonic stem cells (hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are powerful tools for tracking cell types of interest in differentiated cell populations.Here,we describc the construction of modular lentivectors containing different tissue-specific promoters(Tαl of α-tubulin:αP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla green fluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to bc traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.     

The transfection of embryonic stem cells with Tet-on system and its responsiveness to doxycycline

We transiently transfected pTet-on and pTRE2hyg-luciferase into the mouse embryonic stem cells (ESCs) using lipofectamine, and analyzed its inductive effect by adding serial concentrations of doxycycline (DOX). The results showed that in the transfected group, the luciferase activity of the cells was gradually increased along with the increasing concentration of DOX. While in the non-transfected group, the luciferase activity was not detectable even with DOX treatment. This indicated that the ESCs transfected with Tet-on system could response to DOX very well, and the regulation of target gene expression is dose dependent.     

Pooling and PCR as a method to combat low frequency gene targeting in mouse embryonic stem cells

The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.     

Bi-allelic gene targeting in mouse embryonic stem cells

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

胚胎干细胞诱导分化为雄性生殖细胞的研究进展

胚胎干细胞（embryonic stem cells,ES细胞）具有自我更新及无限分化潜能,理论上可以分化为生殖细胞。目前,在人及鼠中已有体外诱导ES细胞分化为成熟精子的报道。系统阐述影响ES细胞分化为雄性生殖细胞的内源性及外源性因素,并结合国内外最新研究进展总结其诱导分化方法,展望应用前景,期望为从事相关研究的学者提供参考。     

Genetic recombination pathways and their application for genome modification of human embryonic stem cells

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.