Franz-Josef Klinz

Basic Fibroblast Growth Factor Controls Migration in Human Mesenchymal Stem Cells

Little is known about the migration of mesenchymal stem cells (MSCs). Some therapeutic approaches had demonstrated that MSCs were able to regenerate injured tissues when applied from different sites of application. This implies that MSCs are not only able to migrate but also that the direction of migration is controlled. Factors that are involved in the control of the migration of MSCs are widely unknown. The migratory ability of isolated MSCs was tested in different conditions. The migratory capability was examined using Boyden chamber assay in the presence or absence of basic fibroblast growth factor (bFGF), erythropoietin, interleukin‐6, stromal cell‐derived factor‐β, and vascular endothelial growth factor. bFGF in particular was able to increase the migratory activity of MSCs through activation of the Akt/protein kinase B (PKB) pathway. The results were supported by analyzing the orientation of the cytoskeleton. In the presence of a bFGF gradient, the actin filaments developed a parallelized pattern that was strongly related to the gradient. Surprisingly, the influence of bFGF was not only an attraction but also routing of MSCs. The bFGF gradient experiment showed that low concentrations of bFGF lead to an attraction of the cells, whereas higher concentrations resulted in repulsion. This ambivalent effect of bFGF provides the possibility to a purposeful routing of MSCs.

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.

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.

Selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis

Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation‐dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor‐cell‐derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS‐II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.

Cytokinesis failure and successful multipolar mitoses drive aneuploidy in glioblastoma cells

Glioblastoma (GB) is the most frequent human brain tumor and is associated with a poor prognosis. Multipolar mitosis and spindles have occasionally been observed in cultured glioblastoma cells and in glioblastoma tissues, but their mode of origin and relevance have remained unclear. In the present study, we investigated a novel GB cell line (SGB4) exhibiting mitotic aberrations and established a functional link between cytokinesis failure, centrosome amplification, multipolar mitosis and aneuploidy in glioblastoma. Long-term live cell imaging showed that >3% of mitotic SGB4 cells underwent multipolar mitosis (tripolar>tetrapolar>pentapolar). A significant amount of daugther cells generated by multipolar mitosis were viable and completed several rounds of mitosis. Pedigree analysis of mitotic events revealed that in many cases a bipolar mitosis with failed cytokinesis occurred prior to a multipolar mitosis. Additionally, we observed that SGB4 cells were also able to undergo a bipolar mitosis after failed cytokinesis. Colchicine-induced mitotic arrest and metaphase spreads demonstrated that SGB4 cells had a modal chromosome number of 58 ranging from 23 to 170. Approximately 82% of SGB4 cells were hyperdiploid (47-57 chromosomes) or hypotriploid (58-68 chromosomes). In conclusion, SGB4 cells passed through multipolar cell divisions and generated viable progeny by reductive mitoses. Our results identified cytokinesis failure occurring before and after multipolar or bipolar mitoses as important mechanisms to generate chromosomal heterogeneity in glioblastoma cells.

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.

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Transcription factor CREB is phosphorylated in human molar odontoblasts and cementoblasts in vivo.

A wide variety of stimuli can trigger activation of the transcription factor CREB (cAMP-responsive element binding protein), pointing toward a central role for CREB in the integration of various signaling inputs. No data are available on the expression and phosphorylation of CREB in mammalian teeth. Using immunohistochemical analysis of free-floating sections, we show here that CREB was strongly expressed and phosphorylated at Ser-133 within the nucleus of a subpopulation of adult human molar odontoblasts. Many dental pulp stromal cells and periodontal ligament fibroblasts expressed CREB and showed phosphorylation of CREB at Ser-133. In addition, cementoblasts displayed nuclear expression and phosphorylation of CREB at Ser-133. The epithelial rests of Malassez revealed strong nuclear expression of CREB, but phosphorylation at Ser-133 was variable. Our results provide the first evidence that the constitutively phosphorylated transcription factor CREB is involved in the biomineralization process of adult human molar odontoblasts and cementoblasts.

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca2+-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca2+-binding proteins in the ERM has not so far been characterized. Among the three Ca2+-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca2+ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca2+ is regulated only by calretinin. The expression of Ca2+-binding proteins is restricted in a developmental manner in the ERM.

The Constitutive Activation of Extracellular Signal-Regulated Kinase 1 and 2 in Periodontal Ligament Nerve Fibers

BACKGROUND The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization. METHODS Decalcified free-floating sections of rat molars with PDL were incubated using total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p-ERK1/2. To test whether p-ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p-ERK1/2 with calcitonin gene-related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point-counting method. RESULTS In cervical, midroot, and apical zones of the PDL, t-ERK1/2- and p-ERK1/2-labeled nerve fibers were found in close association with blood vessels. The p-ERK1/2-labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p-ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin. CONCLUSIONS The perivascular distribution of t-ERK1/2 and p-ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p-ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.