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A M Wobus

Publications and source records attributed to A M Wobus.

At least 37 records · Page 2Linked to original sources
PubMed1999

Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development.

beta-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37 degreesC. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal ICaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaL by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+ channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gs protein accounted for the ICaL insensitivity to beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

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.

Adaptation, Physiological↗
PubMed1999

Establishment of pluripotent cell lines from vertebrate species--present status and future prospects.

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro.

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the beta1 integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack beta1 integrin (beta1-/-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta1 integrin function during myogenesis and neurogenesis we studied differentiation of beta1-/- ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta1-/- cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (beta1+/-) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta1 integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from beta1-/- ES cells appeared earlier than wildtype and heterozygous (beta1+/-) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in beta1-/- embryoid bodies. However, neuronal outgrowth was retarded in the absence of beta1 integrin. No functional difference between wildtype and beta1-/- cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of beta1 integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in beta1-/- cells, we analyzed in wildtype and beta1-/- cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and beta1-/- cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta1-/- cells may account for the accelerated neuronal differentiation in beta1-/- ES cells.

Animals↗
PubMed1998

Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.

Agrin↗
PubMed1998

Spontaneous calcium oscillations in embryonic stem cell-derived primitive endodermal cells.

In vitro differentiation of mouse embryonic stem cells within three-dimensional cell aggregates called embryoid bodies parallels the development of postimplantation embryos at the egg cylinder stage, where visceral and parietal endoderm diverge from the primitive endoderm. We have investigated spontaneous [Ca2+]i oscillations by means of confocal laser-scanning microscopy in primitive endodermal cell layers of embryoid bodies during their differentiation to parietal and visceral endoderm. The frequency of [Ca2+]i oscillations increased from day 4 to day 19 of development, whereas their duration decreased from day 3 to days 16-17. Oscillations depended on both extracellular Ca2+ and Ca2+ release from intracellular stores as they were abolished in Ca(2+)-free solution and in the prescence of Ni2+ and thapsigargin. Signal transduction operated via the phospholipase C (PLC)-mediated inositol 1,4,5-triphosphate (InsP3) pathway with a negative feedback loop via protein kinase C (PKC) as U73,122, a blocker of PLC; bisindolylmaleimide 1, staurosporine, and H-7, blockers of PKC; and 10 mM caffeine totally inhibited [Ca2+]i spiking. Thimerosal, which hypersensitizes the InsP3 receptor, as well as vasopressin and bradykinin, which act via the InsP3 pathway, increased the frequency of [Ca2+]i spikes. In the prescence of brefeldin A (50 microM) or monensin (20 microM), which both inhibit endo/exocytotic vesicle pathways, an immediate transient increase in spiking activity was followed by a decline within 1 to 2 h. In the presence of brefeldin A or thapsigargin or in the absence of extracellular Ca2+, endocytotic vesicles were absent, suggesting that oscillating [Ca2+]i transients are involved in the exo/endocytotic vesicle shuttle.

Animals↗
PubMed1997

Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes.

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-, atrium- and ventricle-like type, which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes, we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected, which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA, both, in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression, a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes. It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MHC and MLC-2v mRNA in embryoid bodies in early, but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA, whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected.

Alitretinoin↗
PubMed1997

From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model.

Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.

Animals↗
PubMed1997

Development of G protein-mediated Ca2+ channel regulation in mouse embryonic stem cell-derived neurons.

Besides other mechanisms, the influx of Ca2+ into embryonic neurons controls growth and differentiation processes. To study the expression and regulation of voltage-gated Ca2+ channels during early neurogenesis, we measured whole-cell Ca2+ currents (I(Ca)) in neurons developing from pluripotent embryonic stem cells. Various receptor agonists, including somatostatin and baclofen, reversibly inhibited I(Ca) in embryonic stem cell-derived neurons. The effects of somatostatin and baclofen were abolished by pretreatment of cells with pertussis toxin and mimicked by intracellular infusion of guanosine 5'-O-(3-thiotriphosphate), suggesting the involvement of pertussis toxin-sensitive G proteins in I(Ca) inhibition. Investigations at different stages of neuronal differentiation showed that somatostatin efficiently suppressed L- and N-type Ca2+ channels in immature as well as mature neurons. In contrast, inhibition of L- and N-type channels by baclofen was rarely observed at the early stage. In terminally differentiated neurons, responses to baclofen were as prominent as those to somatostatin but were confined to N-type Ca2+ channels. The stage-dependent sensitivity of voltage-gated Ca2+ channels to somatostatin and baclofen was not due to differential expression of G alpha(o) isoforms, as revealed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. These findings demonstrate that specific neurotransmitters such as somatostatin regulate voltage-gated Ca2+ channels via G proteins during the early stages of neurogenesis, thus providing a mechanism for the epigenetic control of neuronal differentiation.

Animals↗
PubMed1996

Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and beta 1 integrin-deficient cells.

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture, differentiation of embryonic or simple epithelial cells occurred in both ES cell populations, as detected by expression of keratins 8, 18, and 19. From 21 days, expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast, keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface, consistent with the association of these a subunits with beta 1. Furthermore, alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro, they did form keratinocyte cysts expressing alpha 6 beta 4, keratins 1 and 14, and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore, beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy, extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures, but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Primordial germ cell-derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated stem cells with respect to differentiation capacity and cell cycle distribution.

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for their in vitro differentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G1-phase and shorter S- and G2/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cells in vitro resemble that of totipotent ES cells.

Animals↗
PubMed1996

Differentiation and integrity of cardiac muscle cells are impaired in the absence of beta 1 integrin.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting peacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of beta 1 integrin. Fourth, beta 1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with beta 1-null cells contain cell debris. The number of beta 1-null cells decrease from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via beta 1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

Animals↗
PubMed1995

M-twist expression inhibits mouse embryonic stem cell-derived myogenic differentiation in vitro.

The mouse M-twist gene codes for a basic helix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modified SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, the development of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cells appeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotinic acetylcholine receptor and the cell adhesion molecule M-cadherin were found to appear with a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitory effect on skeletal muscle cell development depending on the level of exogenous M-twist expression.

Animals↗