Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage.

Present state and future perspectives of using pluripotent stem cells in toxicology research.

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro-cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative.

The Janus face of pluripotent stem cells--connection between pluripotency and tumourigenicity.

Pluripotent stem cells have gained special attraction because of their almost unlimited proliferation and differentiation capacity in vitro. These properties substantiate the potential of pluripotent stem cells in basic research and regenerative medicine. Here three types of in vitro cultured pluripotent stem cells (embryonic carcinoma, embryonic stem and induced pluripotent stem cells) are compared in their historical context with respect to their different origin and properties.

Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

Background: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage.

Human embryonic stem cell lines and their use in international research.

Research in human pluripotent stem cells, including human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), is one of the most dynamic research fields. Despite the high public attention, especially for hESC research, there is only scattered information on the number of hESC lines and the degree, dynamics, and diversification of their use on a global level. In this study we present data on the current number of publicly disclosed hESC lines, on the extent and impact of experimental work involving hESCs, and on the use of specific hESC lines in international research.

Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry.

We have previously shown that mouse embryonic stem (ES) cells differentiate into insulin-positive cells via multi-lineage progenitors. Here, we used Affymetrix chips and quantitative RT-PCR analysis to determine transcriptional profiles of undifferentiated wildtype (wt) and Pax4 expressing (Pax4+) ES cells and differentiated cells of committed progenitor and advanced stages. From undifferentiated to the committed stage, 237 (wt) and 263 (Pax4+) transcripts were 5- or more-fold up-regulated, whereas from the committed to the advanced stage, 28 (wt) and 5 (Pax4+) transcripts, respectively, were two- or more-fold up-regulated.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin.

Background: Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells.

Methods: Differentiating mouse ES cells were treated with suramin (500 µM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools.

Induced human pluripotent stem cells: promises and open questions.

Adult cells have been reprogrammed into induced pluripotent stem (iPS) cells by introducing pluripotency-associated transcription factors. Here, we discuss recent advances and challenges of in vitro reprogramming and future prospects of iPS cells for their use in diagnosis and cell therapy. The generation of patient-specific iPS cells for clinical application requires alternative strategies, because genome-integrating viral vectors may cause insertional mutagenesis.

Mouse ES cells over-expressing the transcription factor NeuroD1 show increased differentiation towards endocrine lineages and insulin-expressing cells.

Embryonic stem (ES) cells which constitutively express the Pdx-1, Ngn-3, NeuroD1, Nkx2.2, and Nkx6.1 transcription factors were engineered by means of lentiviral vectors, following a multi-step infection procedure to successively generate ES cell lines expressing one, two, and three factors, respectively.

Differentiation analysis of pluripotent mouse embryonic stem (ES) cells in vitro.

Pluripotent embryonic stem (ES) cells are characterized by their almost unlimited potential to self-renew and to differentiate into virtually any cell type of the organism. Here we describe basic protocols for the in vitro differentiation of mouse ES cells into cells of the cardiac, neuronal, pancreatic, and hepatic lineage. The protocols include (1) the formation of embryoid bodies (EBs) followed by (2) the spontaneous differentiation of EBs into progenitor cells of the ecto-, endo-, and mesodermal germ layer and (3) the directed differentiation of early progenitors into the respective lineages.

Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors-the influence of differentiation factors and culture conditions.

The differentiation of murine and human embryonic stem (ES) cells into pancreatic cell types has been shown by several methods including spontaneous differentiation, formation of multi-lineage progenitors, lineage selection or transgene expression. However, these strategies led to a mixture of cells of all three primary germ layers and only a low percentage of definitive endoderm cells giving rise to pancreas, liver, lung and intestine. To reproducibly generate functional insulin-producing cells, ES cells have to be differentiated via definitive endoderm and pancreatic endocrine progenitors recapitulating the in vivo development.

The transcription factor repertoire of Flt3+ hematopoietic stem cells.

Hematopoietic stem cells maintain the development of all mature blood cells throughout life due to their sustained self-renewal capacity and multilineage differentiation potential. During development into specific cell lineages, the options of stem cells and multipotent progenitor cells become increasingly restricted concomitant with a successive decline in self-renewal potential. Here we describe an Flt3+CD11b+ multipotent progenitor that can be amplified in vitro with a specific combination of cytokines to yield homogeneous populations in high cell numbers.

B-MYB is essential for normal cell cycle progression and chromosomal stability of embryonic stem cells.

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1.

Methodology/principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M.

Cardiomyogenic stem and progenitor cell plasticity and the dissection of cardiopoiesis.

Cell-based therapies hold promise of repairing an injured heart, and the description of stem and progenitor cells with cardiomyogenic potential is critical to its realization. At the vanguard of these efforts are analyses of embryonic stem cells, which clearly have the capacity to generate large numbers of cardiomyocytes in vitro. Through the use of this model system, a number of signaling mechanisms have been worked out that describes at least partially the process of cardiopoiesis.

Linkage of pluripotent stem cell-associated transcripts to regulatory gene networks.

Knowledge of the transcriptional circuitry responsible for pluripotentiality and self-renewal in embryonic stem cells is tantamount to understanding early mammalian development and a prerequisite to determining their therapeutic potential. Various techniques have employed genomics to identify transcripts that were abundant in stem cells, in an attempt to define the molecular basis of 'stemness'. In this study, we have extended traditional genomic analyses to identify cis-elements that might be implicated in the control of embryonic stem cell-restricted gene promoters.

Pluripotency associated genes are reactivated by chromatin-modifying agents in neurosphere cells.

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin-modifying agents trichostatin A (TSA) and 5-Aza-2'-deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long-term, multilineage, and transplantable.

Pluripotency of embryonic stem cells.

Embryonic stem (ES) cells derived from pre-implantation embryos have the potential to differentiate into any cell type derived from the three germ layers of ectoderm (epidermal tissues and nerves), mesoderm (muscle, bone, blood), and endoderm (liver, pancreas, gastrointestinal tract, lungs), including fetal and adult cells. Alone, these cells do not develop into a viable fetus or adult animal because they do not retain the potential to contribute to extraembryonic tissue, and in vitro, they lack spatial and temporal signaling cues essential to normal in vivo development. The basis of pluripotentiality resides in conserved regulatory networks composed of numerous transcription factors and multiple signaling cascades.

Dioxin affects glucose transport via the arylhydrocarbon receptor signal cascade in pluripotent embryonic carcinoma cells.

Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells.

A chip-based platform for the in vitro generation of tissues in three-dimensional organization.

We describe a multi-purpose platform for the three-dimensional cultivation of tissues. The device is composed of polymer chips featuring a microstructured area of 1-2 cm(2). The chip is constructed either as a grid of micro-containers measuring 120-300 x 300 x 300 microm (h x l x w), or as an array of round recesses (300 microm diameter, 300 microm deep).

Differentiation of mouse embryonic stem cells to insulin-producing cells.

Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d.

WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD133+ cells in vitro.

Cord blood-derived CD133+ cells have a degree of non-hematopoietic potential and express transcripts of pluripotency markers including Oct-4, Sox-2, Rex-1, and leukemia inhibitory factor (LIF) receptor, as well as markers of progenitor cells, such as HoxB4, brachyury, and nestin. Having shown by transcriptome analysis that the mouse embryonic fibroblast (MEF) cells routinely used to maintain pluripotent embryonic stem cells express transcripts of the WNT/BMP families of signaling factors, we have assessed the effects on proliferation and differentiation of CD133+ cells of medium conditioned (CM) by MEF, by NIH3T3, and by NIH3T3 cells stably expressing WNT1, WNT3a, WNT4, WNT5a, and WNT11. Cultivation of CD133+ cells in MEF-CM led to a significant increase in cell number after 7 days of culture, while WNT-1, WNT3a-, and WNT11-CM increased the cell number significantly by 14 days of culture.