Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure.

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.

[Axonal damage and its significance for the concept of neurodegeneration in multiple sclerosis]. / Die Bedeutung axonaler Pathologie für das Konzept der Neurodegeneration bei der Multiplen Sklerose.

In spite of tremendous scientific effort, the mechanisms underlying multiple sclerosis (MS) still remain to be elucidated. The prevalent pathogenetic concept adheres to the assumption of a strict hierarchical sequence of the triad inflammation, demyelination and axonal damage. However, recent studies have provided evidence that axonal pathology can occur independently of inflammation and demyelination. The present article critically re-evaluates the traditional paradigm of MS pathology. Potential cellular, humoral and metabolic mechanisms of axonal pathology are delineated and the development of isolated axonal damage is assessed. A better understanding of the pathological processes underlying MS is likely to result in an improvement of current therapeutic strategies. These should not only target the inflammatory, but also the neurodegenerative component of the disease.

Irreversible inflammation is associated with decreased levels of the alpha1-, beta1-, and alpha2-subunits of sGC in human odontoblasts.

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αß heterodimer, and two heterodimer isoforms (α(1)ß(1), α(2)ß(1)) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against ß-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α(1)-, ß(1)-, and α(2)-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α(1)-, ß(1)-, and α(2)-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α(1)-, ß(1)-, and α(2)-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.

[The significance of a B cell-dependent immunopathology in multiple sclerosis]. / Die Relevanz einer B-Zell-abhängigen Immunpathologie für die Multiple Sklerose.

In spite of keen clinical and neuroscientific interest, the aetiology and immunopathology of multiple sclerosis (MS) remain to be elucidated. The present work seeks to give insight into the important, but thus far underestimated contribution of B cells to the disease. Emphasis will be placed on the role of B cells as producers of autoantibodies and as antigen presenting cells. In addition, the development of ectopic B cell follicles in the CNS and their potential correlation with the course of the disease and MS severity will be discussed. Finally, regulatory functions of a B cell-dependent immunopathology should be mentioned. A better understanding of the complex pathomechanisms of MS will allow for therapeutic options that are causative. Potential targets of a B cell-oriented therapy will be delineated in the following review. We hereby aim at triggering a critical re-evaluation of traditional paradigms assigned to MS, appreciating the importance of B cells in the disease.

In vivo MRI stem cell tracking requires balancing of detection limit and cell viability.

Cell-based therapy using adult mesenchymal stem cells (MSCs) has already been the subject of clinical trials, but for further development and optimization the distribution and integration of the engrafted cells into host tissues have to be monitored. Today, for this purpose magnetic resonance imaging (MRI) is the most suitable technique, and micron-sized iron oxide particles (MPIOs) used for labeling are favorable due to their low detection limit. However, constitutional data concerning labeling efficiency, cell viability, and function are lacking. We demonstrate that cell viability and migratory potential of bone marrow mesenchymal stromal cells (BMSCs) are negatively correlated with incorporated MPIOs, presumably due to interference with the actin cytoskeleton. Nevertheless, labeling of BMSCs with low amounts of MPIOs results in maintained cellular function and sufficient contrast for in vivo observation of single cells by MRI in a rat glioma model. Conclusively, though careful titration is indicated, MPIOs are a promising tool for in vivo cell tracking and evaluation of cell-based therapies.

Time-dependent activation of ERK1/2 in nerve terminals of the dentin-pulp complex following bradykinin treatment.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors, and the inflammatory mediator bradykinin (BK) led to a reduced threshold in the nociceptor terminals, activating intracellular signaling by phosphorylating receptors and ion channels. The effects of BK on the non-transcriptional modulation of the ERK1/2 in the peripheral nociceptor terminals, including in nerve endings of the dentin-pulp complex, are unknown. The time-dependent effects of BK (10(-7) M) on the ERK1/2 phosphorylation in nerve terminals of the dentin-pulp complex were investigated by quantitative and double immunolabeling with organ bath experiments. In nerve terminals, total and p-ERK1/2 were detected. In comparison with the controls, the numbers of p-ERK1/2-positive nerve endings increased after 1 and 3 min and decreased after 10 min of BK treatment. Analysis of the data indicates that BK induces phosphorylation-mediated local activation of ERK1/2 in nerve terminals modulating nociception in the dentin-pulp complex.

Neuronal characteristics of amniotic fluid derived cells after adenoviral transformation.

Efficient transformation of primary human amniocytes by E1 gene functions of human adenovirus serotype 5 (Ad5) yield in stable cell lines, which exhibit morphological features of epithelial like cells. A thorough investigation using immunocytochemistry confirmed the expression of epithelial cell markers. The analysis also revealed the expression of neuronal and glial marker proteins, such as nestin, vimentin, A2B5 and GFAP. Using RT-PCR, transcripts of the neurotrophic factors nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), and neurotrophin 3 (NT-3) could be detected. Neurotrophic factors could also be detected in the cell culture supernatants of transformed amniocytes. In line with previous experimental data on a human Ad5 E1-transformed embryonal kidney cell line (HEK-293), the results suggest a co-expression of epithelial and neuronal marker proteins in E1-transformed human amniotic fluid derived cells and thus a preferential transformation into neuronal-like cells.

Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse.

BACKGROUND: Retinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse. METHODS: For the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho(-/-)) animals used as controls. The second group consisted of rho(-/-) mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections. RESULTS: Histological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected. CONCLUSIONS: Our data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Bradykinin mediates phosphorylation of eNOS in odontoblasts.

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10(-7) M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10(-6) M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex.

By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.

Transient expression of NOS-II during development of the murine enteric nervous system.

In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca(2+) -dependent and Ca(2+) -independent NOS isoforms.

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases.

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

Morphology and density of initial lymphatics in human myocardium determined by immunohistochemistry.

BACKGROUND: Myocardial edema is a common finding in congestive heart failure, transplant rejection, and cardiomyopathy. Although pathological alterations in the lymphatic morphology are part of the underlying disease, knowledge on these changes is limited. However, lymphatic morphology may be investigated by immunohistochemical staining for fms-like tyrosine 4 kinase (flt-4), which is specific for lymphatic endothelium in adult tissue. METHODS: We used myocardial tissue of ventricular out flow tract taken from five human semilunar valves harvested as allografts but unsuitable for implantation for analysis, performing immunohistochemical staining for flt-4 with a commercially available antibody. Lymphatic morphometry was completed according Gundersen method. RESULTS: Immunohistochemical staining for flt-4 resulted in successful labeling of lymph capillaries in adult human myocardium. Lymph capillary density was calculated as 50.7 +/- 12.5 per mm2 and average diameter was 3.7 +/- 0.7 microm. Conclusions. Lymph capillary morphology in human myocardium may be successfully determined by immunohistochemical staining for flt-4. Tissue samples as small as myocardial biopsies may be used for analysis. Using this method, morphological changes in myocardial lymphatics may be investigated in various cardiovascular diseases.

Neural precursor cells as carriers for a gene therapeutical approach in tumor therapy.

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.

MAP kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB expression and activity in the human corpus cavernosum.

Nitric oxide (NO) is an important mediator in the cavernosal smooth muscle relaxation that causes erections. The purpose of this study was to examine the existence, distribution and phosphorylation stage of two recently discovered key enzymes for NO regulation in human cavernosal tissue, the MAP Kinase 1/2 (Erk 1/2) and the serine/threonine specific protein kinase Akt/PKB. The expression of the enzymes was examined in corpus cavernosum specimens taken from both potent men and from patients with long-term impotence. There was a distinct difference in the activation stage of the MAP Kinase 1/2 (Erk 1/2) between endothelium and smooth muscle cells in potent patients. This finding gives evidence for a cell-type-specific regulation of the eNOS-dependent NO release. Furthermore, we found a higher basal level of active MAP Kinase 1/2 (Erk 1/2) in impotent patients. This finding gives the first evidence for an inhibitory influence of MAP Kinase 1/2 (Erk 1/2) on cavernosal eNOS activity.

NOS-II is involved in early differentiation of murine cortical, retinal and ES cell-derived neurons-an immunocytochemical and functional approach.

Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.

Intracerebral transplantation and successful integration of astrocytes following genetic modification with a high-capacity adenoviral vector.

To investigate the ability of genetically modified astrocytes to integrate into adult rat brain, two spontaneously immortalized cell lines and the allogenic nontumorigenic glioma cell line F98 were transduced with a high-capacity adenoviral vector (HC-Adv) expressing the EGFP gene from the hCMV promoter. In organotypic slice cultures the transduced astrocytes were shown to integrate into the brain tissue. Following transplantation of the transduced astrocytes into the striatum of adult rats, the transplanted cells survived at least for 6 weeks, continuously expressed the EGFP transgene, in close neighborhood with cells of the recipient tissue executing their differentiation capacity along the glial lineage. Thus, HC-Adv transduced astrocytes are promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases.

Nitric oxide synthase expression and function in embryonic and adult cardiomyocytes.

Nitric oxide (NO) is an important signalling molecule that plays a relevant role in different cell systems, among them the adult heart. The effects of NO are primarily mediated through modulation of Ca(2+) homeostasis, myofibrillar contractility, and metabolic regulation in cardiomyocytes. Recent evidence also suggests an important role of NO for cardiomyogenesis by modulating proliferation and differentiation and regulating cardiac function. In the embryonic, but also the healthy and diseased, adult mammalian heart, the inducible (iNOS) and the endothelial (eNOS) nitric oxide synthases (NOS) are detected. However, the expression pattern of NO and its function differ during development. Furthermore, under pathophysiological conditions NOS expression can also change and cause impairment of cardiac performance and cytotoxic effects. The present review focuses on the role and function of NO during cardiomyogenesis, the mechanisms responsible for eNOS availability, and the paracrine effects of NO generated by cardiomyocytes.

Functional interaction of caveolin-1 and eNOS in myocardial capillary endothelium revealed by immunoelectron microscopy.

Immunogold labeling on samples of isolated perfused rat hearts embedded by an innovative low-temperature LR White procedure provided detailed insight into the interaction of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level. Separately, the localization of caveolin-1 and eNOS at caveolae under steady state conditions was visualized. A double-labeling experiment supported their close co-localization. Short-term bradykinin stimulation caused a detectable dissociation of eNOS from caveolin and its redistribution to different cell compartments, whereas caveolin itself remained stationary at caveolae. Morphometric analysis revealed that more than 80% of detectable eNOS was co-localized with caveolin-1 at caveolae under control conditions. After brief stimulation for 2 min with 10(-7) M bradykinin, only 26% of the eNOS signals were associated with caveolin-1 and randomly distributed over the endothelial cells. After stimulation, eNOS was found at the plasmalemmal and intracellular membranes, freely in the cytoplasm, and at outer mitochondrial membranes.