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.     

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.     

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote™ as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote™, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote™ is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.     

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Multicolor karyotype analyses of mouse embryonic stem cells

Summary The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointment due to inadequate ES cell sources. Both authors contributed equally to this work.     

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formarion and their subsequent differentiation in a single three dimensional environment.     

Transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryonic stem cells and embryoid bodies

Ceramides (Cers) are important in embryogenesis, but no comprehensive analysis of gene expression for Cer metabolism nor the Cer amounts and subspecies has been conducted with an often used model: mouse embryonic stem cells (mESCs) versus embroid bodies (EBs). Measuring the mRNA levels by quantitative RT-PCR and the amounts of the respective metabolites by LC-ESI/MS/MS, notable differences between R1 mESCs and EBs were: EBs have higher mRNAs for CerS1 and CerS3, which synthesize C18- and C≥24-carbons dihydroceramides (DH)Cer, respectively; EBs have higher CerS2 (for C24:0- and C24:1-); and EBs have lower CerS5 + CerS6 (for C16-). In agreement with these findings, EBs have (DH)Cer with higher proportions of C18-, C24- and C26- and less C16-fatty acids, and longer (DH)Cer are also seen in monohexosylCers and sphingomyelins. EBs had higher mRNAs for fatty acyl-CoA elongases that produce C18-, C24-, and C26-fatty acyl-CoAs (Elovl3 and Elovl6), and higher amounts of these cosubstrates for CerS. Thus, these studies have found generally good agreement between genomic and metabolomic data in defining that conversion of mESCs to EBs is accompanied by a large number of changes in gene expression and subspecies distributions for both sphingolipids and fatty acyl-CoAs.     

In vitro expression of natriuretic peptides in cardiomyocytes differentiated from monkey embryonic stem cells

Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis.     

DNA羟甲基化在小鼠胚胎干细胞中的研究进展

简要总结DNA羟甲基化在小鼠胚胎干细胞(mouse embryonic stem cells,mESC)中的最新研究进展.DNA甲基化(DNAmethylation)影响染色质的结构与功能,在发育与疾病发生过程中具有重要作用.2009年Tahiliani等发现TET1可以催化甲基化胞嘧啶(5-methylcytosine,5mC)氧化为羟甲基化胞嘧啶(5-hydroxymethylcytosine,5hmC).DNA羟甲基化(DNAhydroxymethylation)被认为是调节DNA甲基化的一种重要方式,成为了表观遗传学的研究热点之一.     

Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder-cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by (1) ability to grow in an undifferentiated state on feeder layers, (2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and (3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well-characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease. © 1993 Wiley-Liss, Inc.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1–7 days of differentiation), mid-stage EBs groups (9–15 days of differentiation), maturing EBs groups (17–45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.     

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage.     

DNA repair in murine embryonic stem cells and differentiated cells

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.     

Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies

Human embryonic stem cells (hESCs) have the potential to provide alternative sources for pancreatic islet grafts. In the present study we have investigated the influence of Activin A and Activin B on the expression of the pancreas marker gene Pdx1 in hESCs differentiated as embryoid bodies (EBs). We report here that Activin B in a dose depend manner markedly up-regulates Pdx1 expression as compared to Activin A and untreated cultures. Pdx1(+) cells co-express FOXA2 but lacks, however, co-expression with nkx6.1, a marker combination that in the present study is shown precisely to identify embryonic and fetal pancreas anlage in humans. Pdx1(+) cells are found in cell clusters also expressing Serpina1 and FABP1, suggesting activation of intestinal/liver developmental programs. Moreover, Activin B up-regulates Sonic Hedgehog (Shh) and its target Gli1, which during normal development is suppressed in the pancreatic anlage. In conclusion, Activin B is a potent inducer of Pdx1 as well as Shh in differentiating hESCs. The data suggest that additional suppression of Shh signaling may be required to allow for proper specification of pancreatic cell lineages in hESCs.