建立绿色荧光蛋白标记的小鼠胚胎干细胞系及向心肌样细胞的分化

带有GFP基因的ESD3细胞系是一个良好的可以用于研究体内和体外细胞分化和组织产生的模型。用磷酸钙共沉淀法将质粒pEGFP-N2导入小鼠胚胎干细胞D3细胞系中 ,在荧光显微镜下以 488nm激发光检查阳性克隆 ,并进行初步扩增。经G4 18筛选后 ,机械挑取EGFP强阳性表达的克隆 ,并在丝裂霉素C处理的小鼠胚胎成纤维细胞的饲养层上 ,在无选择性压力的条件下 ,进一步扩大培养 ,获得纯化的转染细胞系。20代以后 ,转染细胞仍然表达绿色荧光蛋白。PCR检测表明 8代和 18代转染细胞均携带有GFP标志基因。对稳定表达EGFP的干细胞系进行碱性磷酸酶染色、拟胚体和畸胎瘤形成的检测 ,证明这些细胞具有干细胞的特征。经拟胚体 ,可进一步分化成具有搏动能力的心肌细胞 ,分化百分率为 30 %～ 4 0 % ,较未转染细胞 60 %～ 70 %的分化率低 ,造成低分化率机制还不清楚。这些细胞在激光共聚焦显微镜下呈绿色荧光 ,免疫组化染色显示具心肌细胞特异的cTnT分子标志。该EGFP标记的干细胞系带有可进行原位、实时检测的绿色荧光 ,可应用于细胞移植和体内分化的研究.     

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Establishment of customized mouse stem cell lines by sequential nuclear transfer

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from nuclear transfer （NT） embryos, may play a major role in the new era of regenerative medicine. In this study we established forty nuclear transfer-ESC （NTESC） lines that were derived from NT embryos of different donor cell types or passages. We found that NT-ESCs were capable of forming embryoid bodies. In addition, NT-ESCs expressed pluripotency stem cell markers in vitro and could differentiate into embryonic tissues in vivo. NT embryos from early passage RI donor cells were able to form full term developed pups, whereas those from late passage RI ES donor cells lost the potential for reprogramming that is essential for live birth. We subsequently established sequential NT-RI-ESC lines that were developed from NT blastocyst of late passage R 1 ESC donors. However, these NT-R I-ESC lines, when used as nuclear transfer donors at their early passages, failed to result in live pups. This indicates that the therapeutic cloning process using sequential NT-ESCs may not rescue the developmental deficiencies that resided in previous donor generations.     

CARDIAC MUSCLE CELL DIFFERENTIATION IN VITRO FROM A CHARACTERISTIC CELL LINE ISOLATED FROM MOUSE TERATOCARCINOMA

A characteristic in vitro cell line, SEBIII, was isolated from the mouse teratocarcinoma OTT6050. The SEBIII line, which maintains the structure of the embryoid body, grows rapidly in suspension culture. The chromosome number is near diploid. SEBIII cells, which were transferred to the peritoneal cavity of 129/Sv strain mice and left for one week, differentiated into several tissues in the subsequent in vitro culture. Of the tissues differentiated, the most dominant was the spontaneously pulsating muscle cells. Electron microscopic observations of these muscle cells revealed the presence of myofilaments with Z bands and intercalated disks. The nature of the factor(s) which induce the differentiation of SEBIII EB into cardiac muscle cells is discussed.     

The pro-inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells

Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram-negative bacteria. LPS treatment dose-dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α-actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose-dependent increased protein expression of toll-like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL-1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK-242 which abolished the LPS-induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro-inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.     

Embryonic stem cells as a novel cell source of cell-based biosensors

To investigate the use of embryonic stem cells as biosensor elements, mouse embryoid bodies were cultured on the surface of the light-addressable potentiometric sensor and induce to in vitro differentiate into cardiomyocytes and neurons. Extracellular potentials of the cells were recorded by sensor, to detect stem cells potential applications in drugs screening. The experimental results show that known cardiac stimulants (isoproterenol) and relaxants (carbamylcholine) have characteristic effects on the cardiomyocytes in terms of the changes of beat frequency, amplitude and duration. Thus, the embryonic stem cells potentially represent a renewable cell source for the cell-based biosensors.     

Wnt11 facilitates embryonic stem cell differentiation to Nkx2.5-positive cardiomyocytes

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.     

定向诱导小鼠ES细胞向心肌细胞的分化

为了提高体外诱导ES细胞向心肌细胞分化的效率 ,对以往的诱导方法加以改进 ,采用直接悬浮培养和 0 8%DMSO诱导 ,建立了简便、高效的定向诱导ES细胞向心肌细胞分化的体系 .诱导第 9d起可见自发性、有节律跳动的类胚体出现 ,第 14d达到高峰 ,约有 70 %的拟胚体产生跳动 .用RT PCR的方法在跳动的拟胚体中检测到心肌细胞特异性标志物的表达 ,采用免疫荧光染色的方法在蛋白水平检测到心肌特异的α辅肌动蛋白 (α actinin)的表达 ,并可见清晰肌小节 ,表明在改进的体外诱导条件下ES细胞可分化为成熟的心肌细胞 .     

Pluripotent differentiation in vitro of murine Es-D3 embryonic stem cells

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.     

猪羊水干细胞特异向跳动心肌细胞诱导分化

为了筛选并建立一种由猪羊水干细胞向心肌细胞分化的有效方法,以猪羊水干细胞为研究对象,以5-氮胞苷 (5-aza) 和维生素C (Vc) 为诱导剂,对猪羊水干细胞形成的类胚体 (EBs) 进行诱导分化。应用免疫荧光、RT-PCR、透射电镜技术检测跳动细胞团中心肌特异性标记的表达情况。结果显示,在猪羊水干细胞形成的类胚体中加入心肌细胞诱导剂,10 d后即见到节律性跳动的细胞团,t检验发现0.1 mmol/L Vc加5 μmol/L 5-aza联合诱导组的诱导效率最高,达33％。免疫荧光结果显示跳动心肌细胞团表达细胞骨架蛋白α-actin和肌钙蛋白Tnni3。RT-PCR检测跳动心肌细胞团,发现心肌细胞特异性标记分子TbX5、Gata4、α-MHC、Tnni3均呈阳性表达。借助透射电镜观察诱导后的跳动样细胞团,能清晰可见其中的肌丝、糖原粒、糖原池等结构。说明5-氮胞苷和维生素C可以促进猪羊水干细胞向心肌细胞的诱导分化。     

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.     

Modulation of sarcomere organization during embryonic stem cell-derived cardiomyocyte differentiation

Myofibrillogenesis - sarcomeres - mouse embryonic stem cells - cardiomyocytes - beta1 integrin Mouse embryonic stem (ES) cells, when cultivated as embryoid bodies, differentiate in vitro into cardiomyocytes of ventricle-, atrium- and pacemaker-like cell types characterized by developmentally controlled expression of cardiac-specific genes, structural proteins and ion channels. Using this model system, we show here, (I) that during cardiac myofibrillogenesis sarcomeric proteins are organized in a developmentally regulated manner following the order: titin (Z-disk), alpha-actinin, myomesin, titin (M-band), myosin heavy chain, alpha-actin, cardiac troponin T and M-protein, recapitulating the sarcomeric organization in the chicken embryonal heart in vivo. Our data support the view that the formation of I-Z-I complexes is developmentally delayed with respect to A-band assembly. We show (2) that the process of cardiogenic differentiation in vitro is influenced by medium components: Using a culture medium supplemented with glucose, amino acids, vitamins and selenium ions, we were able to increase the efficiency of cardiac differentiation of wild-type, as well as of beta1 integrin-deficient (beta1-/-) ES cells, and to improve the degree of organization of sarcomeric structures in wild-type and in beta1-/- cardiac cells. The data demonstrate the plasticity of cardiogenesis during the differentiation of wild-type and of genetically modified ES cells.     

Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.