[Human embryonic stem cells within the context of international research activity]. / Humane embryonale Stammzellen im Kontext internationaler Forschungsaktivitäten.

Research involving pluripotent human embryonic stem cells (hESCs) is a rapidly growing field of science. Since hESCs originate from early human embryos, alternative methods for producing pluripotent cells have been developed. This article introduces some of those strategies and, in addition, covers international efforts to establish consistent international standards for cultivation, characterization and preservation of hESCs. Furthermore, global trends to form networks in the field of stem cell research as well as endeavors to harmonize ethical standards for hESC research are presented. Finally, potential applications of hESCs in the field of pharmacology/toxicology are discussed as well as recent results of animal studies using hESCs.

Nestin expression--a property of multi-lineage progenitor cells?

Tissue-specific progenitor cells are characterized by proliferation and differentiation, but, in contrast to embryonic stem (ES) cells, have limited capacities for self-renewal and no tumourigenic potential. These latter traits make progenitor cells an ideal source for regenerative cell therapies. In this review, we describe what is currently known about nestin, an intermediate filament first identified in neuroepithelial stem cells. During embryogenesis, nestin is expressed in migrating and proliferating cells, whereas in adult tissues, nestin is mainly restricted to areas of regeneration. We show that nestin is abundant in ES-derived progenitor cells that have the potential to develop into neuroectodermal, endodermal and mesodermal lineages. Although it remains unclear what factors regulate in vitro and in vivo expression of nestin, we conclude that nestin represents a characteristic marker of multi-lineage progenitor cells and suggest that its presence in cells may indicate multi-potentiality and regenerative potential.

Membrane segregation and downregulation of raft markers during sarcolemmal differentiation in skeletal muscle cells.

Muscle contraction implies flexibility in combination with force resistance and requires a high degree of sarcolemmal organization. Smooth muscle cells differentiate largely from mesenchymal precursor cells and gradually assume a highly periodic sarcolemmal organization. Skeletal muscle undergoes an even more striking differentiation programme, leading to cell fusion and alignment into myofibrils. The lipid bilayer of each cell type is further segregated into raft and non-raft microdomains of distinct lipid composition. Considering the extent of developmental rearrangement in skeletal muscle, we investigated sarcolemmal microdomain organization in skeletal and smooth muscle cells. The rafts in both muscle types are characterized by marker proteins belonging to the annexin family which localize to the inner membrane leaflet, as well as glycosyl-phosphatidyl-inositol (GPI)-anchored enzymes attached to the outer leaflet. We demonstrate that the profound structural rearrangements that occur during skeletal muscle maturation coincide with a striking decrease in membrane lipid segregation, downregulation of annexins 2 and 6, and a significant decrease in raft-associated 5'-nucleotidase activity. The relative paucity of lipid rafts in mature skeletal in contrast to smooth muscle suggests that the organization of sarcolemmal microdomains contributes to the muscle-specific differences in stimulatory responses and contractile properties.

Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects.

Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.

Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells.

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.

Potential of embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.

Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.

Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.

Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro.

The effects of lithium chloride (LiCl) on differentiation of mouse embryonic stem (ES) cells were investigated in order to evaluate the ES cell test (EST) used in a European Union validation study for screening of embryotoxic agents in vitro. We show that LiCl inhibited concentration-dependently the differentiation of ES cells into cardiac and myogenic cells. Whereas the inhibition of cardiac differentiation by high concentrations of LiCl was obvious at day 5 + 5, decreased skeletal muscle cell differentiation was observed only at day 5 + 8. Semi-quantitative RT-PCR analyses revealed significantly lower levels of mRNA encoding cardiac-specific alpha-myosin heavy chain and skeletal muscle-specific myoD. By morphological investigation, an influence of lithium on neuronal differentiation was not evident. However, mRNA levels of genes encoding synaptophysin and the 160 kDa neurofilament protein were increased by high LiCl concentrations, whereas mRNA levels of mash-1 and Engrailed-1 were decreased, suggesting a specific influence of lithium on neuronal differentiation. Furthermore, LiCl treatment resulted in a slight, but non-significant increase of beta-catenin levels in ES cell-derived embryoid bodies. Our results demonstrate that the ES cell test, EST may be suitable to detect inhibitory effects of test compounds especially on cardiac differentiation, whereas effects on neuronal cells would not be detected. Therefore, we propose that morphological analyses of cardiac differentiation alone are insufficient to detect embryotoxic effects. The assay of other cell lineages at different developmental stages, and expression analyses of tissue-specific genes should also be employed.

Expression and cellular distribution of alpha(v)integrins in beta(1)integrin-deficient embryonic stem cell-derived cardiac cells.

beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.

Overexpression of insulin-like growth factor-II in mouse embryonic stem cells promotes myogenic differentiation.

Embryonic stem (ES) cells derived from androgenetic or parthenogenetic mouse embryos are important tools for studying the roles of imprinted genes in early development. Androgenetic ES cells have been shown to preferentially differentiate into the myogenic lineage both in vitro and after formation of teratocarcinomas in vivo. To clarify if the maternally imprinted Igf2 gene which is expected to be overexpressed in androgenetic ES cells is sufficient to induce myogenic differentiation, R1 ES cells were transfected with human IGF-II expression vectors. Stable ES cell clones exhibiting human IGF-II mRNA and protein expression were studied vs ES cell clones without IGF-II overexpression in a standard in vitro differentiation system involving culture in "hanging drops" and observation of differentiation of the recovered embryoid bodies (EBs). EBs derived from IGF-II overexpressing ES cells showed stimulated myogenic differentiation evident by the appearance of myoblasts already 3 days after plating and by higher levels of skeletal muscle-specific transcripts (myf5, myoD, myogenin) at earlier stages. Our study demonstrates for the first time that overexpression of IGF-II enhances and accelerates myogenic differentiation of ES cells, which has implications for ES cell-derived tissue engineering.

delta-Ca(2+)/calmodulin-dependent protein kinase II expression pattern in adult mouse heart and cardiogenic differentiation of embryonic stem cells.

delta-isoforms of the Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) are considered to substantially influence cardiac functions. However, no data exist on the expression of these isoforms in the mouse heart. We analyzed the transcript pattern of non-neuronally expressed delta-isoforms in heart and skeletal muscle of adult mice by RT-PCR. For members of the delta-CaMKII subclass with both variable domains (subclass II), weak transcriptional expression of isoforms delta(2) and delta(3) was found in the heart. In skeletal muscle no delta(3)-specific transcript was detectable. In cardiac tissue, stronger signals result from amplifications of delta(9) and from members of the subclass I lacking the second variable domain. Western blotting was performed using a subclass II-specific antibody. In murine cardiac and skeletal muscle tissue a delta-CaMKII protein pattern was obtained similar to that described for rat. To gain insight into the expression of delta-CaMKII during the earliest steps of cardiogenic differentiation, we analyzed the transcript pattern of murine embryonic stem cell-derived cardiomyocytes in various differentiation stages. Reproducible RT-PCR signals could be obtained for delta(6) and delta(10), both belonging to the delta-CaMKII subclass I. Transcripts for delta(6) were ubiquitously expressed, whereas transcripts for delta(10) were detectable in increasing amounts after 7-10 days of the onset of cardiogenic differentiation. Our results point to a differentiation-dependent expression of the two delta-CaMKII subclasses, and also to differences in the expression of individual members of subclass I during the early stages of cardiogenic differentiation.

Design, optimization, realization, and analysis of an in vitro system for the exposure of embryonic stem cells at 1.71 GHz.

The aim of this study was to develop an exposure system which enables in vitro experiments to be conducted under variously modulated radiofrequency exposures. Based on the evaluation of different possible systems, it was decided to realize a system based on rectangular waveguides. The system was optimized for the following parameters: (1) homogeneity of the cell exposure, (2) simultaneous exposure of several Petri dishes, (3) efficiency, (4) strict environmental control, (5) quick and easy access to the Petri dishes, (6) cost, and (7) simple operation by non-engineering personnel. The implemented control software enables investigation of a wide spectrum of amplitude modulation schemes between 0.1 Hz and 1 kHz, including the modulation schemes of current and future digital mobile communication systems as well as other exposure protocols. The system described has been initially utilized for a study on the differentiation and cell functions of embryonic stem cells. Detailed numerical and experimental dosimetry and environmental tests have demonstrated that it meets all target objectives. The entire system including the sham exposure system fits into a single incubator. It enables the carrying out of various experiments designed to test biological responses to RF exposures at 1.2-1.7 GHz by using various modulation schemes and long term exposure protocols as well as simultaneous data logging.

Establishment of SSEA-1- and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth.

Here, we demonstrate long-term cultivation of alkaline phosphatase-positive rat embryonic stem-like (RES) cell lines. RES cells were characterized by their typical growth in highly compacted cell clusters, which were found to be sensitive against enzymatic dissociation. RES cells expressed stage-specific embryonic antigen-1 (SSEA-1) and transcription factor Oct-4, but Oct-4 mRNA was detected at lower levels compared to mouse ES cells. Once established to tissue culture, RES cells were able to grow in the absence of feeder cells under clonal conditions. Cytokines of the interleukin-6 family known to maintain the undifferentiated state of mouse ES cells were comparatively analyzed for their capacity to maintain the undifferentiated growth of two cell lines, RES-1 and RES-15, in a clonal assay. Rat ciliary neurotrophic factor (rCNTF), human oncostatin M (hOSM), and interleukin-6 and soluble interleukin-6 receptor (IL-6/sIL-6R) were found to support clonal growth of RES cells, but the cytokines did not reach the efficiency of the colony forming ability of leukemia inhibitory factor (LIF). When RES-1 and RES-15 cells were cultivated without feeder cells, SSEA-1 expression was maintained after clonal growth in the presence of LIF and LIF + rCNTF, respectively. Oct-4 mRNA was significantly detected in RES-15 cells when cultivated in the absence of feeder cells in media substituted by LIF and/or IL-6/sIL-6R, as well as without cytokines. In summary, rat embryonic stem-like cell lines could be established from rat blastocysts and were able to proliferate as undifferentiated alkaline phosphatase-positive cells. Embryonal stem cell properties, such as SSEA-1 and Oct-4 expression, were maintained by members of the IL-6 family of cytokines, but most significantly by LIF.

Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4.

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.

Selection of ventricular-like cardiomyocytes from ES cells in vitro.

Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.

Induction of cellular differentiation by retinoic acid in vitro.

Cellular differentiation by the vitamin A derivative retinoic acid (RA) has been studied with undifferentiated pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells in vitro. Both cellular systems are suitable to study differentiation of various cell types, because they recapitulate early stages of mouse embryogenesis. In vivo, RA was identified as a morphogenic and teratogenic compound and furthermore as a signalling molecule influencing gene expression in a complex manner via a family of RA receptors. Here, we summarize in vitro studies with ES and EC cells in comparison to in vivo studies that have contributed to our understanding how RA influences differentiation and regulates gene expression. We demonstrate that modulation of ES cell differentiation in vitro by RA depends on the concentration and developmental stage of application which is comparable to its stage-dependent influence on embryonic development in vivo.