胚胎干细胞移植到大鼠梗死心脏后的分化研究

目的研究胚胎下细胞在梗死心脏微环境下向心肌细胞、成纤维细胞的分化情况。方法将大鼠分为两组,梗死组为正常大鼠通过结扎左前降支(LAD)制备,对照组为正常大鼠。将4,6-二氨基(DAPI)标记的具有伞能分化能力的鼠胚胎干细胞(mESCs)注射人急性心肌梗死大鼠(18只)或对照绀大鼠(16只)的心脏,观察胚胎干细胞在休内的分化情况。结果 DAPI标记的移植mESCs在对照和梗死心脏均能成活并形成稳定的移植岛,同时在移植区有巨噬细胞浸润。mESCs移植2～4周后,心脏特异性肌钙蛋白T(cTnT)阳性的移植mESCs比例在正常心脏较梗死心脏高(2.67%±0.79%比1.06%±0.52%,P0.01),但4周后cTnT阳性的DAP1标记细胞在正常和梗死心脏的比例差异无统计学意义(1.17%±0.98%比1.07±1,02%,P0.05)。mESCs在埘照组和梗死组心脏都能分化为成纤维细胞。结论移植mESCs不仪能仔活,还可分化进入大鼠梗死心肌细胞。但是,梗死心脏的微环境不能选择性促进mESCs分化进入心肌细胞。     

A genomewide study identifies the Wnt signaling pathway as a major target of p53 in murine embryonic stem cells

Both p53 and the Wnt signaling pathway play important roles in regulating the differentiation of mouse embryonic stem cells (mESCs). However, it is not known whether they directly and/or functionally crosstalk in mESCs. Here we report a surprising antidifferentiation function of p53 in mESCs through directly regulating the Wnt signaling pathway. A chromatin-immunoprecipitation-based microarray (ChIP-chip) and gene expression microarray assays reveal that the Wnt signaling pathway is significantly (P value, 0.000048) overrepresented in p53-regulated genes in mESCs. The expression of five Wnt ligand genes is robustly induced by various genotoxic and nongenotoxic insults in a p53-dependent manner. Moreover, the induction of these Wnt genes is greatly attenuated in mouse embryonic fibroblast (MEF) cells and ESC-derived neural stem/progenitor cells, suggesting that the induction is mESC specific. It is established that the activation of the Wnt signaling pathway inhibits the differentiation of mESCs. Consistent with this notion, we detected an antidifferentiation activity from the conditioned medium (CM) collected from UV (UV)-treated mESCs. This antidifferentiation activity can be lowered by either the addition of Wnt antagonists into the CM or the reduction of p53 levels in UV-treated mESCs. Therefore, reminiscent of its dual functions on death and survival in somatic cells, p53 appears to regulate both prodifferentiation and antidifferentiation programs in mESCs. Our findings uncover a direct and functional connection between p53 and the Wnt signaling pathway, and expand the catalog of p53 regulated genes in mESCs.     

Poor response of Wv/Wx mice to a grafted neutrophilia-inducing, colony-stimulating-factor-producing tumor

Although mice possessing two mutant genes at the W locus have a defect in multipotential hematopoietic stem cells that form macroscopic colonies in the spleen of irradiated mice, the number of neutrophils in the blood of these mutant mice is normal or nearly normal. We investigated neutrophil production using the NFSA fibrosarcoma of C3H mouse origin, which induces neutrophilia accompanied by production of a neutrophil-macrophage colony-stimulating factor by the tumor. When the NFSA tumor was transplanted to (C57BL/6 X C3H/He)F1-Wv/Wx or to congenic +/+ mice, neutrophilia developed in mice of both genotypes. However, there was a significant difference between the degree of neutrophilia that developed in them; there was a 107-fold increase in the +/+ mice, but only a 28-fold increase in the Wv/Wx mice four weeks after tumor transplantation. This result is consistent with the concept that doubly heterozygous W mice have multipotential stem cells with diminished ability to respond to stimulation. The unperturbed condition may not provide a sufficient stimulus to demonstrate the defect in neutrophil production in doubly heterozygous W mutant mice.     

c-kit is required for cardiomyocyte terminal differentiation

c-kit, the transmembrane tyrosine kinase receptor for stem cell factor, is required for melanocyte and mast cell development, hematopoiesis, and differentiation of spermatogonial stem cells. We show here that in the heart, c-kit is expressed not only by cardiac stem cells but also by cardiomyocytes, commencing immediately after birth and terminating a few days later, coincident with the onset of cardiomyocyte terminal differentiation. To examine the function of c-kit in cardiomyocyte terminal differentiation, we used compound heterozygous mice carrying the W (null) and W(v) (dominant negative) mutations of c-kit. In vivo, adult W/W(v) cardiomyocytes are phenotypically indistinguishable from their wild-type counterparts. After acute pressure overload adult W/W(v) cardiomyocytes reenter the cell cycle and proliferate, leading to left ventricular growth; furthermore in transgenic mice with cardiomyocyte-restricted overexpression of the dominant negative W(v) mutant, pressure overload causes cardiomyocytes to reenter the cell cycle. In contrast, in wild-type mice left ventricular growth after pressure overload results mainly from cardiomyocyte hypertrophy. Importantly, W/W(v) mice with pressure overload-induced cardiomyocyte hyperplasia had improved left ventricular function and survival. In W/W(v) mice, c-kit dysfunction also resulted in an approximately 14-fold decrease (P<0.01) in the number of c-kit(+)/GATA4(+) cardiac progenitors. These findings identify novel functions for c-kit: promotion of cardiac stem cell differentiation and regulation of cardiomyocyte terminal differentiation.     

Knock-in mouse model of dilated cardiomyopathy caused by troponin mutation

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca(2+) sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca(2+) transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca(2+) transient was augmented to compensate for decreased myofilament Ca(2+) sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca(2+) sensitivity could not be fully compensated by only increasing the intracellular Ca(2+) transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca(2+) sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca(2+) desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca(2+) sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.     

Analysis of Na+/Ca2+ exchanger knockout mice

The Na(+)/Ca(2+) exchanger (NCX) on the plasma membrane is thought to be the main calcium extrusion system from the cytosol to the extracellular space in many mammalian excitable cells including cardiac myocytes. However, the precise roles of NCX are still unclear because of lack of its specific inhibitors. We generated NCX1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous mutant died at 9.5 days post coitum. Embryonic hearts did not beat and cardiac myocytes showed apoptosis. These results suggest that NCX1 is required for heart beats and survival of cardiac myocytes in embryos. Heterozygous mutant mice were viable and indistinguishable from wild type mice. mRNA and protein levels in the heart of heterozygous mutant were half as much as wild type mice. In response to pressure overload, mutant mice showed better systolic and diastolic relaxation functions than wild type mice. Intracellular Ca(2+) measurement revealed an increase in calcium content of cytoplasm and sarcoplasmic reticulum (SR) and RNA analysis revealed preserved SR Ca(2+) ATPase expression in the ventricle of mutant mice. These results suggest that NCX plays an important role in cardiac performance in these pathological situations.     

Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo

OBJECTIVE: Hematopoietic stem cells are important clinically, both as targets of disease and as reagents for cellular therapy. Studies in hematopoietic stem cell biology have been hampered by difficulties in purifying and manipulating these cells. To facilitate these studies, we sought to develop a system for targeting genes of interest to the hematopoietic stem cell compartment in transgenic mice. MATERIALS AND METHODS: We used Sca-1, a glycosyl phosphatidylinositol-anchored protein expressed on the surface of all hematopoietic stem cells in commonly used inbred mouse strains. We created a mutant Sca-1 allele in which the enhanced green fluorescent protein (EGFP) cDNA is integrated into the Sca-1 locus by homologous recombination in embryonic stem cells. RESULTS: EGFP protein is detectable in all hematopoietic tissues of mice heterozygous for the mutant Sca-1 allele. Growth and development of these mice are normal. No adverse effects of long-term, high-level EGFP expression were noted. Sca-1 positive cells coexpress EGFP in all tissues and lineages examined, as predicted by the targeting strategy. Sca-1 and EGFP expression are coordinately up-regulated in splenocytes from mutant mice. The Lin(-)EGFP(+) bone marrow population contains all progenitor activity in Sca-1(+)(/EGFP) mice. The Lin(-)EGFP(+) bone marrow cells are equivalent to Lin(-)Sca-1(+) cells in long-term repopulation and serial transplantation assays. CONCLUSION: The hematopoietic stem cell compartment appears to be targeted in Sca-1(+)(/EGFP) mutant mice. This system should be useful for studying the normal biology of hematopoietic stem cells and for targeting other genes to this cellular compartment.     

Comparison of two murine models of familial hypertrophic cardiomyopathy

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.     

A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

The sarcoplasmic reticulum Ca(2+)-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca(2+)-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were identified. By middle age, heterozygous individuals developed left ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias, with overt heart failure in some cases. Transgenic mice overexpressing the mutant PLN-R14Del recapitulated human cardiomyopathy exhibiting similar histopathologic abnormalities and premature death. Coexpression of the normal and mutant-PLN in HEK-293 cells resulted in sarcoplasmic reticulum Ca(2+)-ATPase superinhibition. The dominant effect of the PLN-R14Del mutation could not be fully removed, even upon phosphorylation by protein kinase A. Thus, by chronic suppression of sarcoplasmic reticulum Ca(2+)-ATPase activity, the nonreversible superinhibitory function of mutant PLN-R14Del may lead to inherited dilated cardiomyopathy and premature death in both humans and mice.     

Cardiac-specific haploinsufficiency of beta-catenin attenuates cardiac hypertrophy but enhances fetal gene expression in response to aortic constriction

In addition to its role in cell adhesion, beta-catenin is an important signaling molecule in the Wnt/Wingless signaling pathway. Recent studies have indicated that beta-catenin is stabilized by hypertrophic stimuli and may regulate cardiac hypertrophic responses. To explore the role and requirement of beta-catenin in cardiac development and hypertrophy, we deleted the beta-catenin gene specifically in cardiac myocytes by crossing loxP-floxed beta-catenin mice with transgenic mice expressing a Cre recombinase under the control of the alpha-myosin heavy chain promoter. No homozygous beta-catenin-deleted mice were born alive and died before embryonic day 14.5, indicating significant and irreplaceable roles of beta-catenin in embryonic heart development. Heterozygous beta-catenin-deleted mice, however, demonstrated no structural and functional abnormality. The response of heterozygous beta-catenin-deleted mice to transverse aortic constriction, however, was significantly attenuated with decreased heart weight and heart weight/body weight ratio compared to controls with intact beta-catenin genes. Hemodynamic analysis revealed that there was no difference in cardiac function between wild-type and heterozygous beta-catenin-deleted mice. On the other hand, the expression of fetal genes, beta-myosin heavy chain, atrial and brain natriuretic peptides was significantly higher in heterozygous beta-catenin-deleted mice when compared to wild-type beta-catenin mice. These results suggest that the cytoplasmic level of beta-catenin modulates hypertrophic response and fetal gene reprogramming after pressure overload.     

Endonuclease G is required for early embryogenesis and normal apoptosis in mice

Endonuclease G (EndoG) is a nuclear-encoded mitochondrial protein reported to be important for both nuclear DNA fragmentation during apoptosis and mitochondrial DNA replication. To evaluate the in vivo function of EndoG, we have investigated the effects of EndoG deficiency in cells and mice. We found that EndoG homozygous mutant embryos die between embryonic days 2.5 and 3.5. Mitochondrial DNA copy numbers in ovulated oocytes from EndoG heterozygous mutant and wild-type mice are similar, suggesting that EndoG is involved in a cellular function unrelated to mitochondrial DNA replication. Interestingly, we found that cells from EndoG heterozygous mutant mice exhibit increased resistance to both tumor necrosis factor alpha- and staurosporine-induced cell death. Moreover, spontaneous cell death of spermatogonia in EndoG heterozygous mutant mice is significantly reduced compared with wild-type mice. DNA fragmentation is also reduced in EndoG+/- thymocytes and splenocytes compared with wild-type cells, as well as in EndoG+/- thymus in vivo compared with that of the wild-type mice, on activation of apoptosis. These findings indicate that EndoG is essential during early embryogenesis and plays a critical role in normal apoptosis and nuclear DNA fragmentation.     

Desmoglein 2 mutant mice develop cardiac fibrosis and dilation

Desmosomes are cell–cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.     

Strategies to promote donor cell survival: combining preconditioning approach with stem cell transplantation

Stem cell transplantation has emerged as a potential modality in cardiovascular therapeutics due to their inherent characteristics of self-renewal, unlimited capacity for proliferation and ability to cross lineage restrictions and adopt different phenotypes. Constrained by extensive death in the unfriendly milieu of ischemic myocardium, the results of heart cell therapy in experimental animal models as well as clinical studies have been less than optimal. Several factors which play a role in early cell death after engraftment in the ischemic myocardium include: absence of survival factors in the transplanted heart, disruption of cell-cell interaction coupled with loss of survival signals from matrix attachments, insufficient vascular supply and elaboration of inflammatory cytokines resulting from ischemia and/or cell death. This article reviews various signaling pathways involved in triggering highly complex forms of cell death and provides critical appreciation of different novel anti-death strategies developed from the knowledge gained from using an ischemic preconditioning approach. The use of pharmacological preconditioning for up-regulation of pro-survival proteins and cardiogenic markers in the transplanted stem cells will be discussed.     

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.     

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.     

Calcineurin Abeta gene targeting predisposes the myocardium to acute ischemia-induced apoptosis and dysfunction

Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Abeta gene-targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Abeta-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Abeta-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.