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PubMed2006

Contribution of human embryonic stem cells to mouse blastocysts.

In addition to their potential for cell-based therapies in the treatment of disease and injury, the broad developmental capacity of human embryonic stem cells (hESCs) offers potential for studying the origins of all human cell types. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or in teratomas, yet these methods have stopped short of demonstrating the ESC potential exhibited in the mouse (mESCs), which can give rise to every cell type when combined with blastocysts. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. Our results show that hESCs can engraft into mouse blastocysts, where they proliferate and differentiate in vitro and persist in mouse/human embryonic chimeras that implant and develop in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation.

Animals↗

PubMed2005

Synthetic neomycin-kanamycin phosphotransferase, type II coding sequence for gene targeting in mammalian cells.

The bacterial neomycin-kanamycin phosphotransferase, type II enzyme is encoded by the neo gene and confers resistance to aminoglycoside drugs such as neomycin and kanamycin-bacterial selection and G418-eukaryotic cell selection. Although widely used in gene targeting in mouse embryonic stem cells, the neo coding sequence contains numerous cryptic splice sites and has a high CpG content. At least the former can cause unwanted effects in cis at the targeted locus. We describe a synthetic sequence, sneo, which encodes the same protein as that encoded by neo. This synthetic sequence has no predicted splice sites in either strand, low CpG content, and increased mammalian codon usage. In mouse embryonic stem cells sneo expressability is similar to neo. The use of sneo in gene targeting experiments should substantially reduce the probability of unwanted effects in cis due to splicing, and perhaps CpG methylation, within the coding sequence of the selectable marker.

Animals↗

PubMed2004

Hedgehog signaling is required for the differentiation of ES cells into neurectoderm.

Mouse embryonic stem cells can differentiate in vitro into cells of the nervous system, neurons and glia. This differentiation mimics stages observed in vivo, including the generation of primitive ectoderm and neurectoderm in embryoid body culture. We demonstrate here that embryonic stem cell lines mutant for components of the Hedgehog signaling cascade are deficient at generating neurectoderm-containing embryoid bodies. The embryoid bodies derived from mutant cells are also unable to respond to retinoic acid treatment by producing nestin-positive neural stem cells, a response observed in cultures of heterozygous cells, and contain cores apparently arrested at the primitive ectoderm stage. The mutant cultures are also deficient in their capacity to differentiate into mature neurons and glia. These data are consistent with a role for Hedgehog signaling in generating neurectoderm capable of producing the appropriate neuronal and glial progenitors in ES cell culture.

Animals↗

PubMed2006

Use of differentiating embryonic stem cells in pharmacological studies.

Adipocytes and osteoblasts are derived from a common precursor cell. It has been proposed that the bone loss commonly seen during aging or in the pathology of osteoporosis might be partly caused by a deregulation of the normal balance between osteoblast and adipocyte differentiation. In vitro differentiation of mouse embryonic stem cells toward the adipogenic and the osteogenic lineages provides a powerful system for testing effects of compounds on the developmental switch between adipogenesis and osteogenesis and identification of pharmacological targets. In this chapter, an improved protocol to commit mouse embryonic stem cells into both lineages at a correct rate is detailed.

Adipocytes↗

PubMed2005

Magnetic resonance tracking of implanted adult and embryonic stem cells in injured brain and spinal cord.

Stem cells are a promising tool for treating brain and spinal cord injury. Magnetic resonance imaging (MRI) provides a noninvasive method to study the fate of transplanted cells in vivo. We studied implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with iron-oxide nanoparticles (Endorem) and human CD34+ cells labeled with magnetic MicroBeads (Miltenyi) in rats with a cortical or spinal cord lesion. Cells were grafted intracerebrally, contralaterally to a cortical photochemical lesion, or injected intravenously. During the first week post transplantation, transplanted cells migrated to the lesion. About 3% of MSCs and ESCs differentiated into neurons, while no MSCs, but 75% of ESCs differentiated into astrocytes. Labeled MSCs, ESCs, and CD34+ cells were visible in the lesion on MR images as a hypointensive signal, persisting for more than 50 days. In rats with a balloon-induced spinal cord compression lesion, intravenously injected MSCs migrated to the lesion, leading to a hypointensive MRI signal. In plantar and Basso-Beattie-Bresnehan (BBB) tests, grafted animals scored better than lesioned animals injected with saline solution. Histologic studies confirmed a decrease in lesion size. We also used 3-D polymer constructs seeded with MSCs to bridge a spinal cord lesion. Our studies demonstrate that grafted adult as well as embryonic stem cells labeled with iron-oxide nanoparticles migrate into a lesion site in brain as well as in spinal cord.

Animals↗

PubMedDate not supplied

Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants.

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-beta leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.

Animals↗

PubMed2004

Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord.

Nuclear magnetic resonance (MR) imaging provides a noninvasive method for studying the fate of transplanted cells in vivo. We studied, in animals with a cortical photochemical lesion or with a balloon-induced spinal cord compression lesion, the fate of implanted rat bone marrow stromal cells (MSCs) and mouse embryonic stem cells (ESCs) labeled with superparamagnetic iron oxide nanoparticles (Endorem). MSCs were colabeled with bromodeoxyuridine (BrdU), and ESCs were transfected with pEGFP-C1 (eGFP ESCs). Cells were either grafted intracerebrally into the contralateral hemisphere of the adult rat brain or injected intravenously. In vivo MR imaging was used to track their fate; Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells. During the first week postimplantation, grafted cells migrated to the lesion site and populated the border zone of the lesion. Less than 3% of MSCs differentiated into neurons and none into astrocytes; 5% of eGFP ESCs differentiated into neurons, whereas 70% of eGFP ESCs became astrocytes. The implanted cells were visible on MR images as a hypointense area at the injection site, in the corpus callosum and in the lesion. The hypointense signal persisted for more than 50 days. The presence of GFP-positive or BrdU-positive and nanoparticle-labeled cells was confirmed by histological staining. Our study demonstrates that both grafted MSCs and eGFP ESCs labeled with a contrast agent based on iron oxide nanoparticles migrate into the injured CNS. Iron oxide nanoparticles can therefore be used as a marker for the long-term noninvasive MR tracking of implanted stem cells.

Animals↗

PubMed2000

The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells.

Differentiation of the human embryonal carcinoma cell line NTERA-2 is characterized by changes in morphology, altered patterns of gene expression, reduced proliferative potential, and a loss of tumorigenicity. The cellular repressor of E1A-stimulated genes, CREG, was previously shown to antagonize transcriptional activation and cellular transformation by the Adenovirus E1A oncoprotein. These properties suggested that CREG may function to inhibit cell growth and/or promote differentiation. Here we show that CREG is a secreted glycoprotein which enhances differentiation of NTERA-2 cells. Northern blot analysis reveals that, although CREG mRNA is widely expressed in adult tissues, CREG mRNA is not significantly expressed in pluripotent mouse embryonic stem cells or NTERA-2 embryonal carcinoma cells. CREG mRNA is rapidly induced upon in vitro differentiation of both mouse embryonic stem cells and human NTERA-2 cells. We show that constitutive expression of CREG in NTERA-2 cells enhances neuronal differentiation upon treatment with retinoic acid. Media enriched in CREG was also found to promote NTERA-2 differentiation in the absence of an inducer such as retinoic acid. These studies suggest that secreted CREG protein participates in a signaling cascade important for differentiation of pluripotent stem cells such as those found in teratocarcinomas.

Animals↗

PubMed2002

Embryonic stem cells and mouse models of human syndromes: examples from the T-box gene family.

Synergism between the fields of reproduction and development has long been an important factor for advances in each field, and the development of gene modification technology in mammals has progressed hand in hand with advances in reproductive technology. The discovery and exploitation of embryonic stem cells for producing targeted gene mutations in the mouse, combined with advances in genome analysis, has provided a means of producing mouse models of human genetic diseases, including congenital defects. This review presents examples of how gene targeting and reproductive biology techniques are being applied to the production of mouse models for specific human developmental syndromes caused by mutations in genes of the T-box transcription factor gene family. The ulnar-mammary syndrome and the DiGeorge syndrome are two developmental syndromes that are currently being explored in this way.

Animals↗

PubMed2003

Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube.

OBJECTIVE: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and quantitative analyses of cardiogenesis in vivo and in vitro. METHODS: Gene expression profiles were made by in situ hybridisation and real-time PCR and electrophysiological profiles by patch clamp analyses of cardiomyocytes derived from time series of differentiating HM1 mouse embryonic stem cells and from embryonic and adult mouse hearts. RESULTS: In embryoid bodies the in situ patterns of expression of alpha-myosin heavy chain, myosin light chain 2a and sarcoendoplasmic reticulum calcium ATPase 2a were similar to that of the heart muscle-specific marker gene cardiac troponin I. Myosin light chain 2v was expressed in part of the cardiac troponin I-expressing area, indicating heterogeneity within the cardiac cell population. Atrial natriuretic factor expression, indicative of the chamber-type program, could only very occasionally be detected by in situ hybridisation. Quantitative reverse transcriptase PCR showed that all cardiac genes, most notably atrial natriuretic factor, were expressed at relatively low levels, similar to those in embryonic hearts at embryonic day 8.75-9. Analysis of the electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes showed an increase of the upstroke velocity and a shorter duration of the action potential during prolonged differentiation in vitro. When embryonic mouse heart compartments of embryonic day 12.5 were used as a reference, the electrophysiological characteristics of a substantial part of the embryonic stem cell-derived cardiomyocytes were most reminiscent to those observed in the embryonic outflow tract. CONCLUSION: Together, these data suggest that most cardiomyocytes acquired by differentiation of embryonic stem cells maintain a phenotype reminiscent of that of the cardiomyocytes of the primary heart tube, and hardly any myocytes develop a chamber myocardial phenotype.

Action Potentials↗

PubMed2003

TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells.

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5-days postcoitum embryos. TGF-beta and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-beta/activin signals play important roles in regulating vascular growth and permeability.

Activins↗

PubMed2005

X-ray-induced deletion complexes in embryonic stem cells on mouse chromosome 15.

Chromosomal deletions have long been used as genetic tools in dissecting the functions of complex genomes, and new methodologies are still being developed to achieve the maximum coverage. In the mouse, where the chromosomal deletion coverage is far less extensive than that in Drosophila, substantial coverage of the genome with deletions is strongly desirable. This article reports the generation of three deletion complexes in the distal part of mouse Chromosome (Chr) 15. Chromosomal deletions were efficiently induced by X rays in embryonic stem (ES) cells around the Otoconin 90 (Oc 90), SRY-box-containing gene 10 (Sox 10), and carnitine palmitoyltransferase 1b (Cpt 1 b) loci. Deletions encompassing the Oc 90 and Sox 10 loci were transmitted to the offspring of the chimeric mice that were generated from deletion-bearing ES cells. Whereas deletion complexes encompassing the Sox 10 and the Cpt 1 b loci overlap each other, no overlap of the Oc 90 complex with the Sox 10 complex was found, possibly indicating the existence of a haploinsufficient gene located between Oc 90 and Sox 10. Deletion frequency and size induced by X rays depend on the selective locus, possibly reflecting the existence of haplolethal genes in the vicinity of these loci that yield fewer and smaller deletions. Deletions induced in ES cells by X rays vary in size and location of breakpoints, which makes them desirable for mapping and for functional genomics studies.

Animals↗

PubMed2004

Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation.

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.

Animals↗

PubMed2001

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation.

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.

Animals↗

PubMed2001

Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology.

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene "targeting"). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

Animals↗

PubMed2005

Generation of insulin-producing cells from stem cells.

Islet transplantation as a potential treatment for diabetes will always be limited mainly because of the difficulty in obtaining sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is the use of a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation. Therefore, these cells have the potential to proliferate and differentiate into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Moreover, these derived cells have proven to be useful in different animal models. In this regard, insulin-secreting cells derived from mouse embryonic stem cells normalize blood glucose when transplanted into streptozotocin-induced diabetic animals. Using a combination of several differentiation methods and a 'cell trapping' system, we have obtained insulin-secreting cells from undifferentiated embryonic stem cells. The construct used allows the expression of a neomycin selection system under the control of the regulatory regions of insulin gene and other beta cell genes, such as Nkx6.1. Transplanted animals correct hyperglycaemia within 1 week and restore body weight in four weeks. Graft removal rescued the diabetic condition. Glucose tolerance test (IPGTT) and blood glucose normalization after a challenge meal was similar in control and in transplanted animals. This approach opens new possibilities for tissue transplantation in the treatment of type 1 and 2 diabetes.

Animals↗

PubMed2002

Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling.

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).

Amino Acid Sequence↗

PubMed2025

Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells.

Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. Fibroblast growth factor (FGF), Wnt and mammalian target of rapamycin (mTOR) signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mouse embryonic stem cells (mESCs). Focusing on the Mediator subunit-encoding Med12 gene as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage but impairs pluripotency gene expression and slows down transitions between pluripotency states. These findings suggest that Med12 helps pluripotent cells to efficiently execute transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.

Animals↗