The transplantation of insulin-producing cells is a promising option for the treatment of patients suffering from diabetes. Because the amount of transplantable surrogate islets is limited, pluripotent stem cells characterized by self-renewal and differentiation capacity offer unique alternatives for regenerative medicine. Over the last years, embryonic stem (ES) cells isolated from early embryos of mouse and human origin have been used for in vitro differentiation into the endoderm and pancreatic lineages. The application of various differentiation strategies resulted in the formation of insulin-producing cells, but only partially, ES-derived insulin-secreting clusters were able to normalize the blood glucose level of diabetic mice. Recently, induced pluripotent stem (iPS) cells have been established by reprogramming of adult cells via the transfer of pluripotency-associated genes. These iPS cells were also capable to differentiate into insulin-positive cells. Here, we present a short overview about the strategies established for the generation of insulin-producing cells from murine and human pluripotent ES and iPS cells. The data demonstrate the achievements that have been obtained with respect to the production of islet-like cells, but also underline that future efforts are needed to improve their functional status and to translate laboratory data into clinical application.

Human embryonic stem cells and, more generally, human pluripotent stem cells have attracted much interest for nearly 15 years. The German Stem Cell Act that came into effect on 1 July 2002 strictly regulates research in human embryonic stem cells. This article introduces currently available human pluripotent stem cell types, gives an overview on potential options to use these cells beyond research and summarizes present research activities involving human pluripotent stem cells in Germany. In particular, German research projects with the aim of establishing novel cell models for the detection of human-specific developmental toxicity in vitro are discussed and efforts to develop human germ cells from human pluripotent stem cells are presented.

Research on human pluripotent stem cells is rapidly progressing. In the present chapter the current state of research on human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) is analyzed with respect to the number of publicly disclosed cell lines and to the extent and impact of published scientific work involving these cells. Our data reveal that activities in both research fields have markedly increased over the past years and that there are no indications for a diminished role of hESCs in international research as a consequence of the availableness of hiPSCs. Moreover, recent data presented here confirm the global dominance of only a few hESC lines for research, and preferences for distinct hESCs in different countries. Possible causes for these phenomena are discussed.

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. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.

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. RESULTS:Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. CONCLUSION:We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.

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 article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed.

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. It became evident that tumourigenicity is an inherent property of pluripotent cells based on p53 down-regulation, expression of tumour-related genes and high telomerase activity that allow unlimited proliferation. In addition, culture-adapted genetic and epigenetic changes may induce tumourigenicity of pluripotent cells. The use of stem cells in regenerative medicine, however, requires non-malignant cell types and strategies that circumvent stages of malignancy.Reprogramming strategies of adult somatic cells that avoid the tumourigenic state of pluripotency may offer alternatives for future biomedical application.

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. The results are based on the evaluation of nearly 1,000 research papers published by the end of 2008, which describe experimental work on hESCs, and of a comprehensive database of published hESC lines. The average impact of hESC research papers is high at 7.422, with a predominance of research output by the United States. Of at least 1,071 original hESC lines derived up to November 2009 at 87 institutions in 24 countries, only a fraction is thoroughly characterized. Our data show the global predominance of a few hESC lines in research, but also reveal remarkable country-specific differences. Comparison of hESC and hiPSC application did not show a diminished role for hESC research, but rather revealed that, up to this time, both fields continue to expand, exist independently, and partially overlap.

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. Moreover, when suitable iPS cell lines will be available, efficient and selective differentiation protocols are needed to generate transplantable grafts. Finally, we point to the requirement of a regulatory framework necessary for the commercial use of iPS 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. Each ES cell line was allowed to differentiate into nestin+/Isl-1+ endocrine precursors, then into more mature pancreatic cells, and subsequently analysed for expression of Glc, Ins, and Sst, markers of alpha, beta and delta cells, respectively. Each ES cell line generated displayed a unique pattern of gene expression. The ES cell line expressing NeuroD1 displayed vastly elevated levels of Glc, Ins-1, Ins-2 and Sst, and showed an increase in Pdx-1, Pax-4, Nkx6.1, Isl-1, Glut-2 and GK transcript levels. Furthermore, immunofluorescence analysis revealed that differentiation of NeuroD1-expressing ES cells in nestin+/Isl-1+ multilineage progenitors, followed by the formation of C-peptide+/insulin+ clusters, was accelerated. Together, these results indicate that stable expression of NeuroD1 in ES cells facilitates differentiation into endocrine and insulin-producing cells.

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. Activin A, a member of the transforming growth factor beta superfamily, has been shown to induce definitive endoderm cells dependent on concentration, culture conditions and time of application. Moreover, serum components or contamination by feeder cells as well as differentiation and proliferation factors are critical for successful generation of activin A-induced ES cells into endoderm and pancreatic cells. The review presents an overview on those factors that influence activin A activity on endoderm and endocrine progenitor cells and determines the role of signaling factors in the differentiation process into the pancreatic lineage.

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. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.