Christian Andressen

Nitric oxide synthase expression and role during cardiomyogenesis

OBJECTIVE The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role. DESIGN The qualitative and quantitative expression of NOS isoforms during different stages of cardiac development was evaluated using immunocytochemistry and dot blots, respectively. The functional relevance of NOS expression during cardiomyogenesis was investigated using the in vitro ES cell-differentiation model and selective pharmacological agents. RESULTS On day 7.5 of embryonic development (E7.5) none of the NOS isoforms were expressed in the embryo, whereas the inducible (iNOS), as well as the endothelial (eNOS) isoforms were detected in the extraembryonic parts. In contrast, starting from E9.5 rat and murine embryos displayed prominent iNOS and eNOS expression. This was correlated with high expression of soluble guanylylcyclase (sGC) as well as high cyclic GMP (cGMP) content. During further development after E14.5 both, iNOS as well as eNOS, started to be downregulated and shortly prior to birth reduced staining for eNOS was found, whereas iNOS was hardly detectable. We further investigated whether NO plays a role for cardiomyogenesis, using in vitro ES cell-derived cardiomyocytes differentiating within embryoid bodies (EBs). The NOS expression pattern in these cells paralleled the one detected in vivo. We demonstrate that continuous incubation of EBs with the NOS inhibitors L-NMMA (2-10 mM) or L-NA (2-10 mM) for 4 to 9 days after plating resulted in a pronounced differentiation arrest of cardiomyocytes, whereas this effect could be reversed by coapplication of the NO-donor spermine-NONOate (10 microM). CONCLUSIONS Both, iNOS and eNOS isoforms are prominently expressed during early stages of cardiomyogenesis. Around E14.5 NOS expression starts to decline. Moreover, the NO-generation is required for cardiomyogenesis since NOS inhibitors prevent the maturation of terminally differentiated cardiomyocytes using the ES cell system.

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the β‐adrenoceptor‐mediated increase in myocardial contractility and heart rate by decreasing the L‐type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP‐dependent phosphorylation of voltage‐dependent L‐type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 ±3% (n=34) in early stage cardiomyocytes lacking functional β ‐adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L‐NMA, L‐NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP‐mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized β‐adrenoceptor‐stimulated ICa by 43 ±4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO‐producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.—Ji, G. J., Fleischmann, B. K., Bloch, W., Feelisch, M., Andressen, C., Addicks, K., Hescheler, J. Regulation of the L‐type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase‐mediated inhibition. FASEB J. 13, 313–324 (1999)

Cardiac specific expression of the green fluorescent protein during early murine embryonic development

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac α‐actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac α‐actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Nestin‐Specific Green Fluorescent Protein Expression in Embryonic Stem Cell‐Derived Neural Precursor Cells Used for Transplantation

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long‐lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell‐derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence‐activated cell sorting of EGFP‐labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.

NO synthase-II is transiently expressed in embryonic mouse olfactory receptor neurons

Among three NO synthase (NOS) isoforms only the inducible NOS-II was localized in developing olfactory receptor neurons of the mouse. First NOS-II immunoreactive receptor cells including their processes were detected by embryonic day 11 when the olfactory pit starts to invaginate. Cellular staining lasted until embryonic day 16, and was reduced during the next few days. At embryonic day 20 no reactivity was found in the olfactory epithelium, whereas centripetal nerve fibers remained positive. This transient expression of NOS-II implies a role for the differentiation of early olfactory receptor neurons and synaptic plasticity.

Temporospatial Relationships between Macroglia and Microglia during in vitro Differentiation of Murine Stem Cells

Embryonic stem (ES) cells of the permanent line BLC6 derived from a 129/Sv Gat mouse blastocyst were differentiated as spheroid aggregates (embryoid bodies, EBs) in the presence of retionic acid. After 2 days in suspension, EBs were plated on gelatine-coated glass coverslips and cultivated for 5, 9, and 16 days post plating (DPP) in normal medium. In this study we investigated whether the well-known retinoic acid-induced differentiation of ES cells into neurons (identified by immunostaining for neuron-specific enolase and synaptophysin) was accompanied by cells expressing astroglial (GFAP), oligodendroglial (O4), and microglial (5C6, galectin-3) markers. Whereas differentiation of neurons was closely related to their centrifugal migration towards the periphery of the EBs, the maturation of neuroglia followed a strict time-dependent manner. At 5 DPP, only neurons but no cells expressing glia-specific markers, were observed. At 9 DPP, GFAP-positive and O4-positive macroglial cells appeared. At 16 DPP, microglial cells (5C6-positive and galectin-3-positive) occurred. The established dynamic of relationships between neuronal and nonneuronal cells shows that the model of EBs is similar to the sequence differentiation of the nervous tissue. Thus, enabling in vivo observation of neurons, astrocytes, oligodendrocytes, and microglia, the model of EBs provides a basis for further investigations on the relationships between neurons and neuroglia under various experimental conditions.

Embryonic stem-cell derived neurones express a maturation dependent pattern of voltage-gated calcium channels and calcium-binding proteins

There are remarkable changes of calcium binding proteins and voltage dependent Ca2+ channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi‐ and multipolar neurones, expressing all Ca2+ channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N‐ and L‐type channel contribution in favour of P/Q‐ and R‐type participation in bi‐ and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca2+ homeostasis, and clarifies contradictory reports on the development of Ca2+ channel expression using primary cultures of neurones already committed to certain brain compartments.

β1 integrin deficiency impairs migration and differentiation of mouse embryonic stem cell derived neurons

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and β1 integrin-deficient (G201) embryonic stem cell derived neurons. In D3 preparations complex networks of functionally coupled neurons with bi- and multipolar morphologies develop. In contrast, neuronal differentiation is retarded in G201 derived neurons, recognised by limited migration and restricted morphological differentiation. Furthermore, β1 integrin deficiency causes a delay in expression of major neurotransmitters like GABA and glutamate as well as of synaptophysin. These findings indicate a prominent role of β1 integrin for both morphological and chemical differentiation.

Demarcation of prosencephalic regions by CD15-positive radial glia

A subpopulation of radial glial cells has been identified in the mouse prosencephalon during the second half of embryonic development. This subpopulation, specified by the putative cell adhesion molecule CD15 (Lex, FAL), is arranged in a segmented pattern within the telencephalon and diencephalon. Glial processes, spanning the prosencephalic wall, first appear at E10.5 and remain clearly visible until E19, when staining of discrete nuclei begins to appear. Registration of the correspondence between ventricular and pial surfaces, however, is still possible due to the persistence of individual CD15‐positive fibres. These can be traced even when the initial simple linear (radial) orientation between ventricular and pial surfaces becomes complicated and distorted. After birth, CD15 immunoreactivity is distributed in a mosaic pattern in the forebrain. Because radial glial cells provide a scaffolding system for postmitotic neurones, the pattern of CD15‐positive fibres in the embryonic prosencephalon may also demarcate future discrete regions of the postnatal brain.