Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers

A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro. The beta-adrenoceptor agonists (-)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forskolin and isobutylmethylxanthine (IBMX), the alpha 1-adrenoceptor agonist (-)phenylephrine, and the heart glycoside digitoxin induced a positive, the muscarinic cholinoceptor agonist carbachol and L-type Ca2+ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response. In early differentiated cardiomyocytes beta 1-, alpha 1-, but not beta 2-adrenoceptors, cholinoceptors, as well as L-type Ca2+ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes beta 2-adrenoceptors and digitoxin responses were also functionally expressed. The contractions of spontaneously beating cardiomyocytes were concomitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinus-node cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L-type Ca2+ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms. The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro

delta-CA2+/Calmodulin-dependent protein kinase II Expression pattern in adult mouse heart and cardiogeneic differentiation of embryonic stem cells

{delta}-isoforms of the Ca2+/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 δ10, 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

Etablierung eines in vitro-Modells neuronaler Zellen. Teilprojekt 1: Zell-und molekularbiologische Charakterisierung definierter neuronaler Zellpopulationen aus embryonalen Stamm (ES)-Zellen Abschlussbericht

SIGLEAvailable from TIB Hannover: F01B1569 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro

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

We have previously shown that mouse embryonic stem (ES) cells differentiate into insulin-positive cells via multi-lineage progenitors. Here, we used Affymetrix chips and quantitative RT-PCR analysis to determine transcriptional profiles of undifferentiated wildtype (wt) and Pax4 expressing (Pax4+) ES cells and differentiated cells of committed progenitor and advanced stages. From undifferentiated to the committed stage, 237 (wt) and 263 (Pax4+) transcripts were 5- or more-fold up-regulated, whereas from the committed to the advanced stage, 28 (wt) and 5 (Pax4+) transcripts, respectively, were two- or more-fold up-regulated. Transcripts were classified into main subclasses including transcriptional regulation, signalling/growth factors, adhesion/extracellular matrix, membrane/transport, metabolism and organogenesis. Remarkably, endoderm-specific Sox17 and early pancreas-specific Isl1 transcripts were up-regulated at an earlier stage of multi-lineage progenitors, whereas highly up-regulated probe sets and transcripts of genes involved in endoderm, pancreatic, hepatic, angiogenic and neural differentiation were detected at the committed progenitor stage. Pax4+ cells showed specific differences in transcript up-regulation and a lower amount of up-regulated neural-specific transcripts in comparison to wt cells, but no enhanced gene expression complexity. Immunocytochemical analysis of selected proteins involved in endoderm and pancreatic differentiation, such as chromogranin B, transthyretin, Foxa1 and neuronatin revealed co-expression with insulin- or C-peptide-positive cells. The comparison of transcript profiles of ES cells differentiating in vitro with those of the embryonic and adult pancreas in vivo suggested that in vitro differentiated cells resemble an embryonal stage of development, supporting the view that ES-derived pancreatic cells are unable to complete pancreatic differentiation in vitro