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The interaction of beta1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the beta1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major beta1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and beta1 integrin -/- neurones - as determined by MAP-2- and HNK-1-immunoreactive processes - was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in beta1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as beta1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.

Endostatin was described as an anti-angiogenic factor. Therefore endostatin looked to be a new way in anti-angiogenic treatment of cancer. Unfortunately, up to now no objective response were seen in clinical trials using endostatin. We compared two different endothelial cell types. Human umbilical vein endothelial cells (HUVEC) and endothelial cells derived from differentiated embryonic stem cells (eESC) were tested in view of endostatin induced proliferation, apoptosis, migration and endostatin binding. Both endothelial cell types had shown an opposite response to endostatin for all observed parameters in dependency of the used concentration. The quantity of HUVEC cells was slightly reduced to 84 +/- 8% by treating with 50 ng/ml endostatin whereas the eESC's showed a significant increase up to 142 +/- 12% under same conditions (p = 0.01). The observation that endostatin is able to evoke non-uniform response for proliferation, cell mount and migration of endothelial cells, with different endostatin binding characteristic, leads to the assumption that endostatin effect is strongly dependent from endothelial cell type. Furthermore the cell biological response at lower concentration on angiogenic eESC gives evidence for an angiogenic modulatory rather than a predicted anti-angiogenic role of endostatin.

Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity "gutless" adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.

There are remarkable changes of calcium binding proteins and voltage dependent Ca(2+) 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 Ca(2+) 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 Ca(2+) homeostasis, and clarifies contradictory reports on the development of Ca(2+) channel expression using primary cultures of neurones already committed to certain brain compartments.

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.

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis., (Copyright 1999 S. Karger AG, Basel)

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and beta1 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, beta1 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 beta1 integrin for both morphological and chemical differentiation.

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.

Bone marrow derived stromal cells are of mesenchymal origin and precursor cells for skeletal tissue components such as chondroblasts and osteoblasts. Furthermore, under experimental conditions, a differentiation potency into myogenic and neuronal cells could be demonstrated. Because of their multipotency these cells represent a population of non-haematogen stem cells that can be regarded as an alternative to human embryonic stem cells for future autologous cell replacement therapies. For a closer look at the differentiation capacity of these cells, rat and human bone marrow stromal cells were isolated from the femur bone and kept in the cell culture applying different cultivation protocols. In a cultivation medium with a serum content of 20%, the majority of these cells express a variety of neuronal markers such as ß-III Tubulin and NeuN as well as the astrocyte marker GFAP, while a minority of about 20% express the marker for neural precursor cells nestin. Cultivation in a chemically defined serum free medium results in the differentiation of a markedly higher percentage of nestin positive neural precursor-like cells. Using bFGF in combination with B27 these cells can be forced to form three dimensionally organized spheres. In order to elucidate a possible therapeutical potency of the bone marrow derived cells the synthesis of neurotrophic factors such as BDNF and NGF were analysed using the ELISA technique. Furthermore, they can be infected using a third generation adenoviral vector with high efficiency and show migratory activity in vitro. After injection of bone marrow derived mesenchymal stem cells into the lateral ventricle of adult rats they adhere to the ependymocytes and pass the ependymal barrier in order to settle in the subventricular space. [ABSTRACT FROM AUTHOR]