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Results for “Mouse Embryonic Stem Cells”

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PubMed2006

Microinjection of targeted embryonic stem cells and establishment of knockout mouse lines for Fmo genes.

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.

Animals↗

PubMed2003

Stem cells in diabetes: what has been achieved.

Beta-cell replacement therapy via islet transplantation has received renewed interest due to the recent improved success. In order to make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must be readily available. The most promising sources are stem cells, with efforts of deriving new beta-cells from both embryonic and adult stem cells. Several groups have reported generating insulin-producing cells from mouse embryonic stem cells. The strategies in the first two acclaimed reports were very different. One strategy, used by Soria's group, is gene trapping in which an introduced antibiotic resistance under the control of the insulin promoter allowed the selection of insulin-expressing cells that had spontaneously differentiated within embryoid bodies. Another strategy, used by McKay's group, manipulated culture conditions in a multistep protocol used for generating neural cells but with changed final conditions. Since these reports, there have been modifications of the protocols in efforts to improve the yields and maturity of the resulting cells. While it is unclear if the insulin-producing cells in any of these studies are truly mature beta-cells, these studies show the clear potential of embryonic stem cells and support optimism that similar results will be possible with human embryonic stem cells. We know that new beta-cells are generated throughout adult life, but the identity of adult pancreatic stem cells has been elusive. The potential for expansion and differentiation of pluripotent adult stem cells, whether from bone marrow or as non-pancreas tissue resident SP cells, is being explored but has not yet yielded insulin-producing tissue. In contrast, insulin-producing cells have been generated in vitro from adult pancreatic tissues. We have been examining the hypothesis that the functional source for new beta-cells in the adult pancreas are mature duct epithelial cells that have regressed or lost their mature phenotype after replication. Others have isolated putative stem cells from islets and ducts. For adult cells the issue of expansion as well as of differentiation is a question. The field of generating new beta-cells from stem cells, either embryonic or adult, is still in its infancy. Each new report has been met with a mixture of excitement and skepticism. With continued efforts and rigorous assessments, hopefully the potential of generating enough new beta-cells from stem cells will be realized.

Animals↗

PubMed2005

[Expression patterns of PPARgamma2 in differentiation of mouse embryonic stem (ES) cells into adipocytes].

To investigate the expression patterns of peroxisome proliferator activated receptor2 (PPARgamma2) gene in the differentiation of mouse embryonic stem (ES) cells into adipocytes, mouse ES cells were transfected with the vector of pPPARgamma2-promoter-luciferase, and PPARgamma2 expressions were analyzed by detecting luciferase activities and by detecting the protein expressions using western blotting. The results showed that the gene PPARgamma2 did not express in undifferentiated mouse ES cells and in embryoid bodies (EBs) within the first 2 days of differentiation induction after EB formation, and began to express from the third day of differentiation induction after EB formation to the finish of the differentiation. The gene's expression in differentiated adipocytes was much stronger than that in differentiating preadipocytes. In Conclusion our results reported for the first time the five-step expression patterns of the gene PPARgamma2 during the whole differentiation procedures from mouse ES cells into adipocytes via preadipocytes, and supported the previous studies that PPARgamma2 is a fat-specific gene that expresses only in developed and developing adipose tissues.

Adipocytes↗

PubMed2006

Identification of Jmjd1a as a STAT3 downstream gene in mES cells.

Mouse embryonic stem (mES) cells can be maintained in undifferentiated state in the presence of a cytokine, leukemia inhibitory factor (LIF). Many investigators found that STAT3 activation is important for the maintenance of pluripotency by LIF. However, the downstream pathways of STAT3 activation are still unknown. To look for STAT3-downstream target genes, we performed DD-RT PCR in the presence or absence of LIF. Through further confirmation, we finally selected 8 genes whose expressions were significantly dependent upon the presence of LIF. Among them, Jmjd1a was down-regulated after LIF withdrawal, and it was selected for further investigation. Its expression started to decrease 1 day after the removal of LIF, and disappeared on day 3. It was also shown that STAT3 could bind to the promoter region of Jmjd1a gene. These data demonstrate that Jmjd1a might be a critical signaling molecule underlying the maintenance of pluripotency in mES cells.

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PubMedDate not supplied

[Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

Animals↗

PubMed2001

In-vitro differentiation of pancreatic beta-cells.

Stem cell biology is a new field that holds promise for in-vitro mass production of pancreatic beta-cells, which are responsible for insulin synthesis, storage, and release. Lack or defect of insulin produces diabetes mellitus, a devastating disease suffered by 150 million people in the world. Transplantation of insulin-producing cells could be a cure for type 1 and some cases of type 2 diabetes, however this procedure is limited by the scarcity of material. Obtaining pancreatic beta-cells from embryonic stem cells would overcome this problem. We have derived insulin-producing cells from mouse embryonic stem cells by a 3-step in-vitro differentiation method consisting of directed differentiation, cell-lineage selection, and maturation. These insulin-producing cells normalize blood glucose when transplanted into streptozotocin-diabetic mice. Strategies to increase islet precursor cells from embryonic stem cells include the expression of relevant transcription factors (Pdx1, Ngn3, Isl-1, etc), together with the use of extracellular factors. Once a high enough proportion of islet precursors has been obtained there is a need for cell-lineage selection in order to purify the desired cell population. For this purpose, we designed a cell-trapping method based on a chimeric gene that fuses the human insulin gene regulatory region with the structural gene that confers resistance to neomycin. When incorporated into embryonic stem cells, this fusion gene will generate neomycin resistance in those cells that initiate the synthesis of insulin. Not only embryonic, but also adult stem cells are potential sources for insulin-containing cells. Duct cells from the adult pancreas are committed to differentiate into the four islet cell types; other possibilities may include nestin-positive cells from islets and adult pluripotent stem cells from other origins. Whilst the former are committed to be islet cells but have a reduced capacity to expand, the latter are more pluripotent and more expandable, but a longer pathway separates them from the insulin-producing stage. The aim of this review is to discuss the different strategies that may be followed to in-vitro differentiate pancreatic beta-cells from stem cells.

Animals↗

PubMed2002

Validation successes: chemicals.

The ECVAM validation concept, which was defined at two validation workshops held in Amden (Switzerland) in 1990 and 1994, and which takes into account the essential elements of prevalidation and biostatistically defined prediction models, has been officially accepted by European Union (EU) Member States and by the Federal regulatory agencies of the USA and the OECD. The ECVAM validation concept was introduced into the ongoing ECVAM/COLIPA validation study of in vitro phototoxicity tests, which ended successfully in 1998. The 3T3 neutral red uptake in vitro phototoxicity test was the first experimentally validated in vitro toxicity test recommended for regulatory purposes by the ECVAM Scientific Advisory Committee (ESAC). It was accepted by the EU into the legislation for chemicals in the year 2000. From 1996 to 1998, two in vitro skin corrosivity tests were successfully validated by ECVAM, and they were also officially accepted into the EU regulations for chemicals in the year 2000. Meanwhile, in 2002, the OECD Test Guidelines Programme is considering the worldwide acceptance of the validated in vitro phototoxicity and corrosivity tests. Finally, from 1997 to 2000, an ECVAM validation study on three in vitro embryotoxicity tests was successfully completed. Therefore, the three in vitro embryotoxicity tests, the whole embryo culture (WEC) test on rat embryos, the micromass (MM) test on limb bud cells of mouse embryos, and the embryonic stem cell test (EST) including a permanent embryonic mouse stem cell line, are considered for routine use in laboratories of the European pharmaceutical and chemicals industries.

3T3 Cells↗

PubMed2006

Effects of hesperetin on vessel structure formation in mouse embryonic stem (mES) cells.

OBJECTIVE: The present study investigated the effects of hesperetin on vessel structure formation in mouse embryonic stem (mES) cells with regard to whether hesperetin acts as an antioxidant or pro-oxidant. Some flavonoids enhance antioxidant systems while increasing oxidative stress in the body. METHODS: After their differentiation into endothelial-like cells for 10 d, mES cells were treated with 1 to 100 muM of hesperetin for 24 h. RESULTS: Hesperetin efficiently inhibited the formation of vessel-like tubular structures consisting of platelet-endothelial cell adhesion molecule-1-immunoreactive cells and significantly (P < 0.05) increased the generation of reactive oxygen species in a concentration-dependent manner. Although glutathione (in its reduced and oxidized forms) in mES cells was not affected by hesperetin, the 8-iso-prostaglandin F2(alpha) content was decreased. In addition, cytotoxicity-induced hesperetin was not found; lactate dehydrogenase release and cell viability were determined as an index of cell damage. CONCLUSION: Taken together, the present study shows that hesperetin inhibits vessel formation by pro-oxidant means and suggests its potential as an antiangiogenic agent.

Animals↗

PubMed2006

Staining embryonic stem cells using monoclonal antibodies to stage-specific embryonic antigens.

Stage-specific embryonic antigens (SSEAs) are cell-surface molecules that exhibit lineage-restricted patterns of expression during development. These antigens provide useful markers for identifying embryonic stem cells and their differentiated derivatives. SSEA-1 provides a surface marker for mouse and human primordial germ cells and mouse embryonic stem cells. SSEA-3 and SSEA-4 provide surface markers for human embryonic stem cells. Here, we describe methods for staining embryonic stem cells in culture and suspension using specific primary antibodies and fluorescent dye- or enzyme-conjugated secondary antibodies. Methods for alkaline phosphatase staining and immunohistochemical localization of antigens in paraffin sections are also described. Monoclonal antibodies directed to SSEAs are valuable research tools that can be used to: (1) determine the percentage of stem cells in mixed cultures; (2) examine the spatial distribution of stem cells and differentiated derivatives in monolayer cultures and embryoid bodies; and (3) isolate populations of differentiated and undifferentiated cells by immunomagnetic or fluorescence-activated cell sorting for cellular and molecular analyses.

Alkaline Phosphatase↗

PubMed2004

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction.

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 microg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-microl aliquots of 10(7) SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long-term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction.

Animals↗

PubMed1995

Development of mouse embryonic stem (ES) cells: IV. Differentiation to mature T and B lymphocytes after implantation of embryoid bodies into nude mice.

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cells differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies "ES fetuses." In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed.

Animals↗

PubMed1998

Spontaneous calcium oscillations in embryonic stem cell-derived primitive endodermal cells.

In vitro differentiation of mouse embryonic stem cells within three-dimensional cell aggregates called embryoid bodies parallels the development of postimplantation embryos at the egg cylinder stage, where visceral and parietal endoderm diverge from the primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal laser-scanning microscopy in primitive endodermal cell layers of embryoid bodies during their differentiation to parietal and visceral endoderm. The frequency of [Ca2+]i oscillations increased from day 4 to day 19 of development, whereas their duration decreased from day 3 to days 16-17. Oscillations depended on both extracellular Ca2+ and Ca2+ release from intracellular stores as they were abolished in Ca(2+)-free solution and in the prescence of Ni2+ and thapsigargin. Signal transduction operated via the phospholipase C (PLC)-mediated inositol 1,4,5-triphosphate (InsP3) pathway with a negative feedback loop via protein kinase C (PKC) as U73,122, a blocker of PLC; bisindolylmaleimide 1, staurosporine, and H-7, blockers of PKC; and 10 mM caffeine totally inhibited [Ca2+]i spiking. Thimerosal, which hypersensitizes the InsP3 receptor, as well as vasopressin and bradykinin, which act via the InsP3 pathway, increased the frequency of [Ca2+]i spikes. In the prescence of brefeldin A (50 microM) or monensin (20 microM), which both inhibit endo/exocytotic vesicle pathways, an immediate transient increase in spiking activity was followed by a decline within 1 to 2 h. In the presence of brefeldin A or thapsigargin or in the absence of extracellular Ca2+, endocytotic vesicles were absent, suggesting that oscillating [Ca2+]i transients are involved in the exo/endocytotic vesicle shuttle.

Animals↗

PubMed2006

Epigenetic reprogramming of somatic genomes by electrofusion with embryonic stem cells.

Cell fusion is an approach for combining genetic and epigenetic information between two different types of cells. Electrofusion for generating hybrid cells between mouse embryonic stem cells and somatic cells, which is a type of nonchemically induced and nonvirus-mediated cell fusion, is introduced here as a highly effective, reproducible, and biomedically safe in vitro system. Under optimized electrofusion conditions, cells are ligned and form pearl chains between electrodes in response to AC pulse stimulation, and subsequently adjacent cytoplasmic membranes are fused by DC pulse stimulation. Hybrid cells survive as drug-resistant colonies in selection medium. Cell fusion is a technique that is applied widely in the life sciences. A recent topic of great interest in the field of stem cell research is the successful production of cloned animals via epigenetic reprogramming of somatic nuclei. Interestingly, nuclear reprogramming for conferring pluripotency on somatic nuclei also occurs via cell fusion between pluripotential stem cells and somatic cells. Furthermore, it has been shown that spontaneous cell fusion contributes to generating the intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of regenerative medicine and epigenetic reprogramming.

Animals↗

PubMed1995

The initiation of senescence and its relationship to embryonic cell differentiation.

Mouse embryonic stem cells have an unlimited lifespan in cultures if they are prevented from differentiating. After differentiating, they produce cells which divide only a limited number of times. These changes seen in cultures parallel events that occur in the developing embryo, where immortal embryonic cells differentiate and produce mortal somatic ones. The data strongly suggest that differentiation initiates senescence, but this view entails additional assumptions in order to explain how the highly differentiated sexual gametes manage to remain potentially immortal. Cells differentiate by blocking expression from large parts of their genome and it is suggested that losses or gains of genetic totipotency determine cellular lifespans. Cells destined to be somatic do not regain totipotency and senesce, while germ-line cells regain complete genome expression and immortality after meiosis and gamete fusions. Losses of genetic totipotency could induce senescence by lowering the levels of repair and maintenance enzymes.

Animals↗

PubMed2003

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

Cell Culture Techniques↗

PubMed1997

Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos.

Embryonic stem (ES) cells are pluripotent and capable of differentiating into somatic as well as germ cell lineages when conjoined with blastomeres of early mouse embryos. However, the developmental potential of single ES cells has not been fully investigated. We injected single murine ES cells (A3-1 cell line) of 129 origin into 8-cell mouse embryos (B6xBDF1) and examined the patterns of distribution of ES-cell-derived cells in the blastocysts as well as in the fully grown chimeric mice. The ES cells underwent 1-2 cycles of mitosis between the 8-cell and the blastocyst stage when they were introduced as single cells, whereas those introduced as groups of 2-5 cells did not proliferate during the same period of development. The ES cells and their daughter cells were predominantly incorporated into the ICM. From the 63 8-cell embryos which received single ES cells microinjected into the perivitelline space, 24 newborns were obtained, and 4 (2 fertile males, 1 sterile female and 1 hermaphrodite) of them (16.6%) were chimeric. The test breeding studies revealed that all the progeny of the two chimeric males were derived from spermatozoa of 129 genotype. The relative contribution of the host-derived and the ES-cell-derived cells in different tissues of the chimeric mice was assessed by PCR analyses of the microsatellite polymorphism of genomic DNA extracted from the tissues. In two male germ line chimeras, the testes, the kidneys and the dorsal skeletal muscles exhibited exceptionally high 129 contents. Our results demonstrated that single ES cells which maintain totipotency or pluripotency of high degree are present in a colony of ES cells, and that single ES cells conjoined with the blastomeres of 8-cell-stage embryos may colonize, if the circumstances allow, almost exclusively the germ cells and concomitantly the urogenital cell lineages. Possible correlation between the allocation of the germ line and the urogenital lineages is discussed.

Animals↗

PubMed2005

Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain.

alpha-Mannosidosis is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-D-mannosidase. A major feature of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment available. We have a guinea pig model of alpha-mannosidosis that models the human condition. This study investigates the feasibility of implanting differentiated mouse embryonic stem cells in the neonatal guinea pig brain in order to provide a source of alpha-mannosidase to the affected central nervous system. Cells implanted at a low dose (1.5 x 10(3)cells per hemisphere) at 1 week of age were found to survive in very low numbers in some immunosuppressed animals out to 8 weeks. Four weeks post-implantation, cells implanted in high numbers (10(5) cells per hemisphere) formed teratomas in the majority of the animals implanted. Although implanted cells were found to migrate extensively within the brain and differentiate into mature cells of neural (and other) lineages, the safety issue related to uncontrolled cell proliferation precluded the use of this cell type for longer-term implantation studies. We conclude that the pluripotent cell type used in this study is unsuitable for achieving safe engraftment in the guinea pig brain.

Animals↗

PubMed1992

Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene.

A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker--the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications.

Animals↗