Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types.

Pluripotent embryonic stem cells (ESC, ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique, alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC, as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinusnodal, atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state, action potentials of very long duration (0.3-1 s) were found, which may represent developmentally controlled transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.

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.

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.

Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.

Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.

Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells.

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 microM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na+ suppressed the adrenaline-induced inward current, while reducing extracellular Cl- had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na+, K+, Cs+ >> Ca2+. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.

Embryonic Stem Cell-Derived Cardiac Differentiation: Modulation of Differentiation and "Loss-of-Function" Analysis In Vitro.

Mouse embryonic stem (ES) cells, when cultivated as embryo-like aggregates, are able to differentiate in vitro into derivatives of all primary germ layers. These include functionally active cardiomyocytes representing ventricle-like, atrium-like, and pacemaker-like cells. During differentiation, a developmentally controlled expression pattern of cardiac-specific genes, proteins, action potentials, ion channels, and receptors is found. This developmental pattern can be modulated in vitro by differentiation factors such as retinoic acid or by "gain-of-function" and "loss-of-function" approaches. The latter strategy was successfully used for the characterization of cardiac phenotypes of ß(1) integrin-deficient ES cells after differentiation into the cardiogenic lineage.

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.

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.

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.

Cation channels in oocytes and early states of development: a novel type of nonselective cation channel activated by adrenaline in a clonal mesoderm-like cell line (MES-1).

The expression of receptors and ion channels alters during growth, maturation, and after fertilization of oocytes reflecting functional changes. Besides voltage-dependent ion channels, oocyte membranes possess an IP3-activated cation channel mediating a prolonged Ca2+ influx. The Ca2+ is thought to be involved in maturation and fertilization. Alternatively, mono- and divalent cations can enter oocytes via stretch-activated channels. The oocyte channel population is further modified during subsequent embryogenesis, suggesting that ionic channels obviously become expressed at specific states of embryological differentiation and in tissue-specific manner. The resulting differences in functional ion channel populations of adult cells underlie the large diversity of cells and their function. Conversely, differentiation and cell proliferation themselves depend on ion transport. Ca2+ ions have been shown to play a pivotal role in these processes. Nonselective cation channels represent one possible pathway for Ca2+ entry into the cell and, therefore, might be involved in the regulation of embryological development. Undifferentiated embryonal carcinoma cells (P19), visceral endoderm-like cells (END-2), epithelioid ectoderm-like cells (EPI-7), mesoderm-like cells (MES-1), and parietal yolk sac cells (PYS-2) have been used as a model to study the expression of ionic channels during early development. In MES-1 cells a nonselective cation current was activated by adrenaline. Interestingly, the intracellular pathway for activation of these channels involved the cascade of activation of the cAMP-dependent protein kinase (PKA) resulting in protein phosphorylation. This mechanism is well known for Ca2+ channel stimulation in cardiac and skeletal muscle both originating from the mesoderm.

Lower mutation frequencies are induced by ENU in undifferentiated embryonic cells than in differentiated cells of the mouse in vitro.

The pluripotent embryonic carcinoma cells of line P19 established from undifferentiated cells of the early mouse embryo and their differentiated progeny, the epithelioid ectoderm-like EPI-7 cells, were investigated for the induction of mutations at the HPRT locus by the alkylating agent N-ethyl-N-nitrosourea (ENU). We showed that the cytotoxic effects of ENU after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells. The IC50 values of ENU in the two cell lines amounted to 0.6 mg/ml and 0.09 mg/ml for P19 and EPI-7 cells, respectively. The induction of 6-thioguanine-resistant mutants by ENU (1.0 mg/ml) determined after an expression time of 8 days for both cell lines resulted in similar mutation frequencies. Using expression times of 8 days for P19 and 11.75 days for EPI-7 cells, taking into account the longer generation time of differentiated EPI-7 cells (13.7 +/- 3.6 h) in comparison to undifferentiated P19 cells (9.3 +/- 0.9 h), ENU induced significantly higher mutant frequencies in EPI-7 cells (4865 mutants/10(6) cells) than in P19 cells (282 mutants/10(6) cells). Our results and data from the literature on UV irradiation-induced repair support the idea that the induction of lower mutation frequencies in embryonic cells may correlate with different proliferation capacities, cell cycle parameters and/or different mechanisms of DNA repair in embryonic stem cells and differentiated cells, respectively.

The effect of the mode of administration of nitrogen mustard and cytosine arabinoside on the production of chromosomal aberrations in mouse bone marrow and ascites tumour cells.

The cytogenetic effects of intraperitoneally (i.p.) and subcutaneously (s.c.) administered nitrogen mustard (HN2) and cytosine arabinoside (ara-C) on bone-marrow and ascites tumour cells of mice were studied. Ehrlich ascites tumour-bearing mice were treated with the mutagens, and cytological preparations were made from ascites tumour and bone-marrow cells of the same animal. The following parameters were investigated: frequencies of mitotic and chromosomal aberrations, time of aberration maxima and aberration spectra. HN2 (0.68 mg/kg b.w.), when given i.p., induced in ascites tumour cells a strong inhibition of mitotic frequency and very high aberration rates, whereas in bone marrow no aberrant chromosomes were observed. On the other hand, after s.c. administration, the same dose induced more aberrant metaphases in bone marrow than in tumour cells. Ara-C (315 mg/kg b.w.) resulted, after s.c. administration, in higher aberration frequencies both in ascites and bone-marrow cells compared with i.p. treatment. All ascites tumour cells showed higher aberration requencies than bone-marrow cells. In bone marrow the aberration maximum occurred as soon as 6 h after treatment. Furthermore, clear differences with respect ot the types of aberration found in the two systems were evident. The differences caused by the different modes of administration in two different types of cell are discussed in terms of metabolic inactivation and differences of the two tissues with respect to karyotype, cell cycle time and repair capacity.

Low mutagenic effects of mitomycin C in undifferentiated embryonic P19 cells are correlated with efficient cell cycle control.

Pluripotent undifferentiated embryonic carcinoma cells of line P19 and their differentiated progeny, epithelioid ectoderm-like EPI-7 cells, showed different responses to mitomycin C (MMC) with respect to induction of micronuclei, mutations at the HPRT-locus and cell cycle control. Cytotoxic effects of MMC after a 5-h treatment were lower in undifferentiated P19 cells than in differentiated EPI-7 cells with IC50 values of 1.3 and 0.25 microM for P19 and EPI-7 cells, respectively. MMC did not induce 6-thioguanine-resistant mutants in P19 cells but significantly increased the mutation frequency in EPI-7 cells with concentrations of 0.25, 0.5 and 1.0 microM MMC. Micronuclei determined by flow-cytometry were induced by MMC in both cell lines at equitoxic concentrations of 4.5 (P19) and 0.75 (EPI-7) microM, reducing the viability in both cell lines to 10%. Whereas the induction of micronuclei in P19 cells was maximal 28 h after treatment and declined thereafter, micronucleus induction peaked 48 h post treatment in EPI-7 cells and remained significantly increased even 67 h after the treatment. Flow-cytometric determination of the distribution of MMC-treated P19 and EPI-7 within the cell cycle revealed a distinct G2/M-block in P19 cells, whereas EPI-7 cells showed normal progression through S-phase and a negligible G2/M-block. Therefore, we conclude that the lower effectivity of MMC to induce gene mutations and micronuclei in P19 cells seemed to be correlated with a more efficient cell cycle control in undifferentiated compared to differentiated EPI-7 cells.

Primordial germ cell-derived embryonic germ cells of the mouse-in vitro model for cytotoxicity studies with chemical mutagens.

Different screening methods to detect the toxic effects of xenobiotics using cells from vertebrates and invertebrates in cytotoxicity and viability assays have been developed, but up to now appropriate in vitro methods with mammalian germ cells have not been available. In the present study the primordial germ (PG) cell-derived permanent embryonic germ (EG) cell line EG-1 was used as in vitro model in toxicity studies with chemical mutagens. EG-1 cells and embryonic stem cells of line D3 were comparatively investigated for their cell survival in response to N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and mitomycin C (MMC) and the results compared with those obtained for undifferentiated embryonic carcinoma cells of line P19 and differentiated epithelioid EPI-7 cells. As a prerequisite for in vitro toxicity and viability studies the cultivation conditions for EG-1 and D3 cells in the absence of a feeder layer were improved by a conditioned medium, increasing the plating efficiency from 0.08% to 17.5% and from 21.1% to 25.1% for EG-1 and D3 cells, respectively. The resulting mean generation time (MGT) of 16.9 hr for EG-1 cells was identical to the generation time of PG cells in vivo, and was not significantly different from the MGT of D3 (15.6 hr) and EPI-7 (13.7 hr) cells, but significantly longer than the MGT of P19 cells (9.3 hr). Calculations of the concentrations resulting in vitro in a 50% decrease in cell survival demonstrated that EG-1 cells were more sensitive to the toxic effects of ENU, MNNG and MMC than D3 and P19 cells and, with the exception of MNNG, also more sensitive than EPI-7 cells. It is proposed that EG cells are used as a model system to screen for toxic effects of teratogenic and embryotoxic chemical agents in vitro.

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.

In vitro cellular models for cardiac development and pharmacotoxicology.

Permanent cultures of cardiac cells described so far have limited value for studying cell biology and pharmacology of the developing heart because of the loss of proliferative capacity and cardiac-specific properties of cardiomyocytes during long-term cultivation. Pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells cultivated as permanent lines offer a new approach for studying cardiogenic differentiation in vitro. We describe cardiogenesis in vitro by differentiating EC and ES cells by way of embryo-like aggregates (embryoid bodies) into spontaneously beating cardiomyocytes. During cardiomyocyte differentiation three distinct developmental stages were defined by expression of specific action potentials and ionic currents measured by the whole-cell patch-clamp technique. Whereas early differentiated cardiomyocytes are characterized by action potentials and ionic currents typical for early pacemaker cells, terminally differentiated cardiomyocytes show action potentials and ionic currents inherent to ventricular-, atrial- or sinus nodal-like cells. These functional characteristics are in accordance with the expression of alpha- and beta-cardiac myosin heavy chain at early differentiation stages and the additional expression of ventricular-specific MLC-2V and atrial-specific ANF genes at terminal stages demonstrated by reverse transcription polymerase chain reaction (RT-PCR) analysis. Pharmacological studies performed by measuring chronotropic responses and by analysing the Ca(2+) channel activity correspond to data obtained with cardiac cells from living organisms. For testing the influence of exogenous compounds on cardiac differentiation the teratogenic compound retinoic acid (RA) was applied during distinct stages of embryoid body development. A temporally controlled influence of RA on cardiac differentiation and expression of cardiac-specific genes was found. We conclude that ES cell-derived cardiomyocytes provide an excellent cellular model to study early cardiac development and to perform pharmacological and embryotoxicological investigations.

Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels.

P19 embryonal carcinoma cells were differentiated via embryolike aggregates (embryoid bodies) into spontaneously beating myocytes. During the whole process of differentiation the functional expression of cardiac-specific receptors and ionic channels was characterized by measuring the chronotropic reactivity, action potentials, and ionic currents in response to various cardioactive drugs. Positive chronotropic effects obtained at different maximal effective concentrations of adrenoceptor-mediated agonists indicated differential adrenoceptor expression during the in vitro development of cardiomyocyte-like cells. No cardiac-specific response was obtained with the muscarinic cholinoceptor agonist carbachol. Single beating cells were enzymatically isolated and investigated by the patch-clamp technique. Pacemaker action potentials similar to those of embryonal cardiomyocytes exhibited amplitudes ranging from 50 to 85 mV. The action potentials were synchronous to the mechanical contractions and, comparable to the chronotropic effects, were modulated by BayK 8644, isradipine, and adrenaline. The functional expression of L-type Ca2+ channels was demonstrated by the Ca2+ channel blockers isradipine, nisoldipine, gallopamil, and diltiazem causing negative chronotropic responses, as well as by the Ca2+ channel activator BayK 8644 causing positive chronotropic responses. These effects gradually increased with time of differentiation. The expression of L-type Ca2+ channels and of nicotinic acetylcholine receptors was confirmed in voltage-clamp experiments. The study demonstrates that P19 embryonal carcinoma cells can be induced to differentiate into cardiomyocyte-like cells comparable to embryonal and neonatal heart cells lacking the muscarinic cholinoceptor response only.