Tetraploid embryonic stem cells contribute to the inner cell mass of mouse blastocysts

The demonstration that mouse somatic cells can be reprogrammed following fusion with embryonic stem (ES) cells may provide an alternative to somatic cell nuclear transfer (therapeutic cloning) to generate autologous stem cells. In an attempt to produce cells with an increased pool of reprogramming factors, tetraploid ES cells were produced by polyethylene glycol mediated fusion of two ES cell lines transfected with plasmids carrying puromycin or neomycin resistance cassettes, respectively, followed by double antibiotic selection. Tetraploid ES cells retain properties characteristic of diploid ES cells, including the expression of pluripotent gene markers Oct4 and Rex1. On injection into the testis capsule of severe combined immunodeficient (SCID) mice, tetraploid ES cells are able to form teratomas containing cells representative of all three germ layers. Further, these cells demonstrated the ability to integrate into the inner cell mass of blastocysts. This study indicates that tetraploid ES cells are promising candidates as cytoplasm donors for reprogramming studies.     

Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences

The mechanisms governing the emergence of the earliest mammalian neural cells during development remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition; however, an instructive process has also been proposed. We used mouse embryonic stem (ES) cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals and what cellular fate will result. To assess this default state, ES cells were placed in conditions that minimize external influences. Individual ES cells were found to rapidly transition directly into neural cells, a process shown to be independent of suggested instructive factors (e.g., fibroblast growth factors). Further, we provide evidence that the default neural identity is that of a primitive neural stem cell (NSC). The exiguous conditions used to reveal the default state were found to present primitive NSCs with a survival challenge (limiting their persistence and proliferation), which could be mitigated by survival factors or genetic interference with apoptosis.     

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic stem cells induce pluripotency in somatic cell fusion through biphasic reprogramming

It is a long-held paradigm that cell fusion reprograms gene expression but the extent of reprogramming and whether it is affected by the cell types employed remain unknown. We recently showed that the silencing of somatic genes is attributable to either trans-acting cellular environment or cis-acting chromatin context. Here, we examine how trans- versus cis-silenced genes in a somatic cell type behave in fusions to another somatic cell type or to embryonic stem cells (ESCs). We demonstrate that while reprogramming of trans-silenced somatic genes occurs in both cases, reprogramming of cis-silenced somatic genes occurs only in somatic-ESC fusions. Importantly, ESCs reprogram the somatic genome in two distinct phases: trans-reprogramming occurs rapidly, independent of DNA replication, whereas cis-reprogramming occurs with slow kinetics requiring DNA replication. We also show that pluripotency genes Oct4 and Nanog are cis-silenced in somatic cells. We conclude that cis-reprogramming capacity is a fundamental feature distinguishing ESCs from somatic cells.     

Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras

Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76–80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11–13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells. A. A. Kruglova and E. A. Kizilova contributed equally to this work. This study was financially supported by grants from the Russian Academy of Sciences, Siberian Branch 5.2 and 14.0.     

Embryonic stem cells alone are able to support fetal development in the mouse

The developmental potential of embryonic stem (ES) cells versus 3.5 day inner cell mass (ICM) was compared after aggregation with normal diploid embryos and with developmentally compromised tetraploid embryos. ES cells were capable of colonizing somatic tissues in diploid aggregation chimeras but less efficiently than ICMs of the same genotype. When ICM in equilibrium with tetraploid and ES in equilibrium with tetraploid chimeras were made, the newborns were almost all completely ICM- or ES-derived, as judged by GPI isozyme analysis, but tetraploid cells were found in the yolk sac endoderm and trophectoderm lineage. Investigation of ES contribution in 13.5 day ES in equilibrium with tetraploid chimeras by DNA in situ hybridization confirmed the complete tetraploid origin of the placenta (except the fetal blood and blood vessels) and the yolk sac endoderm. However, the yolk sac mesoderm, amnion and fetus contained only ES-derived cells. ES-derived newborns failed to survive after birth, although they had normal birthweight and anatomically they appeared normal. This phenomenon remains unexplained at the moment. The present results prove that ES cells are able to support complete fetal development, resulting in ES-derived newborns, and suggest a useful route for studying the development of genetically manipulated ES cells in all fetal lineages.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Embryonic stem cells and cell replacement therapies in the nervous system

Embryonic stem (ES) cells are pluripotential cells derived from the pre-implantation embryo. They can proliferate indefinitely in vitro while retaining pluripotency. ES cells can also be made to differentiate into a large variety of cell types in vitro. This has paved the way to research aimed at using ES-derived cells for cell replacement therapies. Hence, mouse ES cells can efficiently differentiate into neural precursors which can further generate functional neurons, astrocytes, and oligodendrocytes. Methods have also been developed to coax mouse ES-derived neural stem cells to differentiate into either dopaminergic neurons or motoneurons. Mouse ES-derived neural stem cells, or their fully differentiated progeny, have been shown to survive, integrate, and to some extent, function following transplantation within appropriate rodent host tissue. Research on human ES cells is still in its infancy. Considerable work has to be done: (1) to master growth and genetic manipulation of human ES cells; (2) to master their differentiation into specific cell types; and (3) to demonstrate that they can provide long term therapeutical benefits upon grafting into damaged tissues in humans. From the ethical point of view, the establishment of appropriate primate model will be an obligatory prerequisite to clinical trials based on ES cells derivatives grafting.     

Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.     

The stem cells of a primordial germ cell-derived teratocarcinoma have the ability to form viable mouse chimeras

A euploid testicular teratocarcinoma line, STT-3, has been established from a tumor spontaneously occurring in the testis of a 129/Sv-ter male. Developmental ability of the STT-3 stem cells was tested by injecting these cells into mouse blastocysts. The frequency and the extent of chimerism were examined in mid-gestational fetuses and in live-born mice. STT-3 stem cells form viable chimeras at a high rate and differentiate into normal tissues. This is the first reported testicular teratocarcinoma-derived stem line with a proven capacity to form viable chimeric mice upon injection into the blastocysts.     

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。     

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.     

Systematic elimination of parthenogenetic cells in mouse chimeras

The developmental potential of primitive ectoderm cells lacking paternal chromosomes was investigated by examining the distribution of parthenogenetic cells in chimeras. Using GPI-1 allozymes as marker, parthenogenetic cells were detected in most organs and tissues in adult chimeras. However, these cells were under severe selective pressure compared with cells from normal fertilized embryos. In the majority of chimeras, parthenogenetic cells in individual animals were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. Nevertheless, parthenogenetic cells were detected more consistently in some organs, especially the brain, heart, kidney and spleen. In contrast, there was apparently a systematic selection against parthenogenetic cells in some tissues, most notably in skeletal muscle, liver and pancreas. These results suggest that paternally derived genes are probably required not only for the development of extraembryonic structures but also for subsequent development of embryonic tissues derived from the primitive ectoderm lineage.     

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.     

Nonmitogenic survival-enhancing autocrine factors including cyclophilin A contribute to density-dependent mouse embryonic stem cell growth

An improved understanding of the role of extracellular factors in controlling the embryonic stem cell (ESC) phenotype will aid the development of cell-based therapies. While the role of extracellular factors in controlling the pluripotency and differentiation of embryonic stem cells (ESCs) has been the subject of much investigation, the identity and role of extrinsic factors in modulating ESC growth under conditions supporting self-renewal remain largely unknown. We demonstrate that mouse ESC (mESC) growth is density dependent and that one of the mechanisms underlying this phenomenon is the action of survival-enhancing autocrine factors. Proteomic analysis of proteins secreted by mouse ESCs demonstrates significant levels of cyclophilin A which increases the growth rate of mouse ESCs in a dose-dependent manner. Additionally, inhibition of the cyclophilin A receptor CD147 decreases the growth rate of mESCs. These findings identify cyclophilin A as a novel survival-enhancing autocrine factor in mouse ESC cultures.     

Distribution of androgenetic cells in fetal mouse chimeras

To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.     

Extrahepatic cells contribute to the progenitor/stem cell response following reduced-size liver transplantation in mice

The extent to which extrahepatic cells participate in liver regeneration following transplantation is not known. Either full-size or reduced-size livers from wild-type mice were implanted into green fluorescent protein-positive (GFP(+)) transgenic recipient mice to determine whether regenerated liver contained host-derived GFP(+) hepatic cells. After reduced-size liver transplantation, GFP(+) cells were localized to the portal zone of the liver lobule. Interestingly, GFP(+) cells stained for CD117, a marker for progenitor cells, beginning 2 days after transplantation. A significant number of GFP(+) CD117(+) cells were identified in donor livers after 28 days. GFP(+) cells comprised nearly 9% of the donor liver 28 days after reduced-size liver transplant. Moreover, GFP(+) cells also expressed the hepatic progenitor cell marker A6 and novel marker hepatic-specific antigen (HSA), as well as stem cell antigen-1 (Sca-1). Interestingly, some GFP(-) cells also were stained for CD117 and A6, suggesting that both extrahepatic and intrahepatic stem cells were present and may have contributed to the regenerative response under these conditions. Reduced-size liver transplantation using GFP(+) transgenic mice supports the hypothesis that recipient-derived progenitor cells are present and may contribute to liver regeneration following transplantation.