Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.     

Targeted mutation of Ncam to produce a secreted molecule results in a dominant embryonic lethality.

The neural cell adhesion molecule (NCAM) is a membrane-associated member of the immunoglobulin superfamily capable of both homophilic and heterophilic binding. To investigate the significance of this binding, a gene targeting strategy in embryonic stem (ES) cells was used to replace the membrane-associated forms of NCAM with a soluble, secreted form of its extracellular domain. Although the heterozygous mutant ES cells were able to generate low coat color chimeric mice, only the wild-type allele was transmitted, suggesting the possibility of dominant lethality. Analysis of chimeric embryos with high level of ES cell contribution revealed severe growth retardation and morphological defects by E8.5-E9.5. The second allele was also targeted, and embryos derived almost entirely from the homozygous mutant ES cells exhibited the same lethal phenotype as observed with heterozygous chimeras. Together, these results indicate that dominant lethality associated with the secreted NCAM does not require the presence of membrane-associated NCAM. Furthermore, the data indicate that potent bioactive cues or signals can be generated by NCAM.     

PGD-derived human embryonic stem cell lines as a powerful tool for the study of human genetic disorders

Human embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocyst embryos. They are established from spare embryos that have been obtained by in vitro fertilization (IVF) and donated for research purposes. The ICM-derived cell lines have two unique properties, they can be propagated indefinitely in culture and have the potential to develop into practically any cell type in vitro and in vivo. Human embryonic stem (hES) cells carrying specific mutations can be used as a valuable tool for studying genetic disorders in human. One favorable approach to obtain such mutant ES cell lines is their derivation from affected preimplantation genetic diagnosed (PGD) embryos. This review focuses on the importance of deriving human ES cell lines from genetically abnormal embryos, especially in cases where no good cellular and/or animal models exist.     

Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos

The first differentiation event of the mammalian embryo is thought to occur during blastulation and results in two populations of cells, the inner cell mass (ICM) and the trophectoderm. Most embryonic stem (ES) cell lines have been derived from the ICM or a further subset of ICM cells known as the epiblast. There appears to be a limited period of embryonic development during which pluripotent ES cells can be adapted from the cells of the blastocyst to culture. A method is presented here that allows ES cell lines to be isolated from preblastocyst mouse embryos. These lines were derived from 129S2/SvHsd mouse morulae and earlier cleavage stages with high efficiency. The lines expressed genes and antigens characteristic of pluripotent ES cells. XY cell lines remained karyotypically stable through extensive passaging and produced germ-line-competent chimeras upon blastocyst injection. These results suggest that true ES cells can be derived from embryos explanted at any stage of preimplantation development in the mouse. This finding raises the interesting question of whether ES cell lines derived from embryos at different stages of preimplantation development possess the same potential.     

RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.

We describe a system to evaluate the function of lymphocyte-specific and generally expressed genes in the differentiation and/or function of lymphocytes. RAG-2 (recombination-activating gene 2)-deficient mice have no mature B and T lymphocytes due to the inability to initiate VDJ recombination. Blastocysts from RAG-2-deficient mice generate animals with no mature B and T cells following implantation into foster mothers. However, injection of normal ES cells into RAG-2-deficient blastocysts leads to the generation of somatic chimeras with mature B and T cells all of which derive from the injected ES cells (referred to as RAG-2-deficient blastocyst complementation). Complementation of RAG-2-deficient blastocysts with mutant ES cells heterozygous for a targeted mutation that deletes all immunoglobulin heavy-chain joining (JH) gene segments (JH+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (JH-/-) generates animals with normal T cells but no B cells, due to a block in B-cell development at a very early stage. Transfection of a functionally assembled mu heavy-chain gene into the JH-/- ES cells prior to blastocyst injection rescues the JH-/- mutation and allows the generation of both mature T and mature B cells. The rescued B cells express IgM but not IgD and respond normally to bacterial lipopolysaccharide stimulation by proliferating and by secreting IgM.     

Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.

We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.     

Long-term reconstitution of the mouse hematopoietic system by embryonic stem cell-derived fetal liver.

Murine embryonic stem (ES) cells are permanent blastocyst-derived cell lines capable of contributing to a wide variety of tissues, including the germ line, after injection into host blastocysts. Recently, we have shown that ES cells can produce all of the cells of the developing fetus after aggregation with developmentally compromised tetraploid embryos. Completely ES cell-derived embryos die perinatally, but the liver of these embryos is a source of entirely ES cell-derived hematopoietic progenitors. We have taken 14- to 15-day fetal liver cells from ES cell-tetraploid chimeras and reconstituted the hematopoietic system of lethally irradiated adult recipient mice. ES cell-derived hematopoietic stem cells were capable of long-term (greater than 6 months) repopulation of irradiated recipients, and all hematopoietic cell lineages analyzed (erythrocytes, T cells, mast cells, and macrophages) were derived exclusively from ES cells in such recipients. Thus, ES cells retain the capacity to differentiate into all hematopoietic cell types after prolonged passage in culture. This approach should provide a direct route to the production of mice whose hematopoietic cells carry genetic alterations that would be lethal if passed through the germ line.     

Functional abrogation of p53 is required for T-Ag induced proliferation in cardiomyocytes.

Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death.     

Transplantation of embryonic stem cells into the infarcted mouse heart: formation of multiple cell types

Initial studies have suggested that transplantation of embryonic stem (ES) cells following myocardial infarction (MI) in animal models is beneficial; however, the mechanism of benefit is largely unknown. The present study investigated the fate of mouse ES cells transplanted post-MI to determine if the ES cells give rise to the range of major cell types present in the native myocardium. MI was produced by coronary artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing eGFP and beta-galactosidase, respectively, were tested. Post-MI intramyocardial injection of 3 x 10(4) ES cells was compared to injection of media alone. Histochemistry and immunofluorescence were used to track the transplanted ES cells and identify the resulting cell types. Echocardiography assessed the cardiac size and function in a blinded fashion. Two weeks post-MI, engraftment of the transplanted ES cells was demonstrated by eGFP or beta-galactosidase-positive cells in the infarct region without evidence for tumor formation. Co-immunolabeling demonstrated that the transplanted ES cells had become cardiomyocytes, vascular smooth muscle, and endothelial cells. Echocardiographic analysis showed that ES cell transplantation resulted in reduced post-MI remodeling of the heart and improved cardiac function. In conclusion, transplanted mouse ES cells can regenerate infarcted myocardium in part by becoming cardiomyocytes, vascular smooth muscle, and endothelial cells that result in an improvement in cardiac structure and function. Therefore, ES cells hold promise for myocardial cellular therapy.     

Developmental and adult phenotyping directly from mutant embryonic stem cells

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.     

Improved experimental procedures for achieving efficient germ line transmission of nonobese diabetic (NOD)-derived embryonic stem cells

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g., Idd locus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBS Lett., 455, 101-104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a "pure" NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.     

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.     

CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.

To study the role of B-cell antigen CD40 in immune responses, mouse embryonic stem (ES) cells in which both copies of the gene encoding CD40 had been disrupted by homologous recombination were injected in RAG-2 (recombination-activating gene-2)-deficient blastocysts to generate chimeras in which all mature lymphocytes are derived from the CD40-deficient ES cells. T- and B-cell number and phenotype were normal in the CD40-/- chimeras. However, B cells failed to proliferate and undergo isotype switching in vitro in response to soluble CD40 ligand (sCD40L) with interleukin 4 (IL-4) but responded normally to lipopolysaccharide (LPS) with IL-4. CD40-/- chimeras completely failed to mount an antigen-specific antibody response or to develop germinal centers following immunization with the T cell-dependent (TD) antigen keyhole limpet hemocyanin. In contrast, CD40-/- mutant mice responded normally to the T cell-independent (TI) antigens, 2,4,6-trinitrophenyl (TNP)-LPS and TNP-Ficoll. The most noticeable alteration in the serum immunoglobulin levels of young (6-8 weeks old) CD40-/- animals was absence of IgE and severe decrease of IgG1 and IgG2a. These results confirm the essential role of CD40- CD40L interactions in the antibody response to TD antigens and in isotype switching.     

Functional analysis of mammalian members of the transforming growth factor-beta superfamily.

Analysis of growth factor function has come from studies both in vitro and in vivo. However, the generation of mice deficient in a specific growth factor via gene targeting (for example, "knockout") strategies in embryonic stem (ES) cells will often evaluate the essential roles of the protein in vivo and, in many cases, discover new functions. In this review, studies to date are discussed on the generation and analysis of mice deficient in members of the transforming growth factor (TGF-beta) superfamily. Among the genes targeted via ES cell strategies are the TGF-beta1, Müllerian-inhibiting substance (MIS), inhibin alpha, activin betaA, and activin betaB genes. In addition, the mouse short ear and brachypodism mutants and insertional mutant 413-d have been identified as mutations in the BMP-5, GDF-5, and nodal loci, respectively. These studies have led to critical insights into the functions of these gene products and have further emphasized the importance of members of the TGF-beta superfamily in mammalian development, reproduction, and oncogenesis.     

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.     

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice.     

Red blood cell production from immortalized progenitor cell line

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and have established many immortalized erythroid progenitor cell lines so far. Although their precise characteristics varied among cell lines, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. Considering the number of human ES cell lines that have been established so far and the number of induced pluripotent stem cell lines that will be established in future, the intensive testing of a number of these lines for establishing immortalized erythroid progenitor cell lines may allow the establishment of such cell lines similar to the mouse erythroid progenitor cell lines.     

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.