Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells

BACKGROUND: Bone marrow stromal cells and radial glia are two stem cell types with neural phenotypic plasticity. Bone marrow mesenchymal stem cells can differentiate into osteocytes, chondrocytes and adipocytes, but can also differentiate into non-mesenchymal cell, i.e. neural cells in appropriate in vivo and in vitro experimental conditions. Likewise, radial glial cells are the progenitors of many neurons in the developing cortex, but can also generate astrocytes. Both cell types express nestin, an intermediate filament protein which is the hallmark of neural precursors. RESULTS: In this study, we demonstrate that thrombin, a multifunctional serine protease, stimulates the growth of radial glial cells (RG) and mesenchymal stem cells (MSCs) in a dose-dependent manner. In RG, the mitogenic effect of thrombin is correlated with increased expression of nestin but in MSCs, this mitogenic effect is associated with nestin down-regulation. Both cell types express the PAR-1 type receptor for Thrombin and the effect of Thrombin on both cell types can be mimicked by its analogue TRAP-6 activating specifically this receptor subtype or by serum which contains various amount of thrombin. Moreover, we also demonstrate that serum deprivation-induced expression of nestin in MSCs is inhibited by high cell density (> 50,000 cells/cm2). CONCLUSION: This work shows that thrombin stimulates the growth of both RG and MSCs and that nestin expression by MSCs and RG is regulated in opposite manner by thrombin in vitro. Thrombin effect is thus associated in both cell types with a proliferating, undifferentiated state but in RG this involves the induction of nestin expression, a marker of immaturity for neural progenitors. In MSCs however, nestin expression, as it corresponds to a progression from the mesenchymal "undifferentiated", proliferating phenotype toward acquisition of a neural fate, is inhibited by the mitogenic signal

Skeletal-specific expression of Fgd1 during bone formation and skeletal defects in faciogenital dysplasia (FGDY; Aarskog syndrome)

FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42; FGD1 mutations result in Faciogenital Dysplasia (FGDY, Aarskog syndrome), an X-linked developmental disorder that adversely affects the formation of multiple skeletal structures. To further define the role of FGD1 in skeletal development, we examined its expression in developing mouse embryos and correlated this pattern with FGDY skeletal defects. In this study, we show that Fgd 1, the mouse FGD1 ortholog, is initially expressed during the onset of ossification during embryogenesis. Fgd 1 is expressed in regions of active bone formation in the trabeculae and diaphyseal cortices of developing long bones. The onset of Fgd 1 expression correlates with the expression of bone sialo-protein, a protein specifically expressed in osteoblasts at the onset of matrix mineralization; an analysis of serial sections shows that Fgd 1 is expressed in tissues containing calcified and mineralized extracellular matrix. Fgd1 protein is specifically expressed in cultured osteoblast and osteoblast-like cells including MC3T3-E1 cells and human osteosarcoma cells but not in other mesodermal cells; immunohistochemical studies confirm the presence of Fgd1 protein in mouse calvarial cells. Postnatally, Fgd 1 is expressed more broadly in skeletal tissue with expression in the perichondrium, resting chondrocytes, and joint capsule fibroblasts. The data indicate that Fgd 1 is expressed in a variety of regions of incipient and active endochondral and intramembranous ossification including the craniofacial bones, vertebrae, ribs, long bones and phalanges. The observed pattern of Fgd 1 expression correlates with FGDY skeletal manifestations and provides an embryologic basis for the prevalence of observed skeletal defects. The observation that the induction of Fgd 1 expression coincides with the initiation of ossification strongly suggests that FGD1 signaling plays a role in ossification and bone formation; it also suggests that FGD1 signaling does not play a role in the earlier phases of skeletogenesis. With the observation that FGD1 mutations result in the skeletal dysplasia FGDY, accumulated data indicate that FGD1 signaling plays a critical role in ossification and skeletal development. © 2000 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35167/1/1015_ftp.pd

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells-0

<p><b>Copyright information:</b></p><p>Taken from "Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells"</p><p>http://www.biomedcentral.com/1471-2202/8/104</p><p>BMC Neuroscience 2007;8():104-104.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2231362.</p><p></p>ession. Then, the cells were renewed in DEM supplemented with different concentrations of thrombin (0 (A, rat MSCs), 90, 180, 450 (B, rat MSCs) and 900 pM), in DEM + 10% FBS or MEM + 10% horse serum (L) or in DEM + 900 pM of thrombin with 10M of FUdR (K) for a further 3 days period and then fixed and labelled with anti-nestin antibody (green). Nuclei were counterstained with TOPRO-3 (red) and then counted. In these conditions, there is a significant decrease of nestin expression both in rat MSCs (C) with 180 pM (*, p < 0,05), 450 and 900 pM of thrombin (**, p < 0,01) and in mouse MSCs (F) from 180 pM of thrombin (**). (L) There is however no difference when HS in MEM (18% of cell expressing nestin) is used instead of FBS in DEM (10% of cell expressing nestin). These results were confirmed by Western blotting (D), where nestin expression in rat MSCs cultivated in DEM, DEM + 1 nM of thrombin and DEM + 10% FBS was compared after 3 days of induction of nestin expression. The actin immunostaining controls the amount of protein loaded on the gel in each condition and allows to calculate the ratio between the intensity of the nestin and actin signals and to normalize data to the level of nestin expression in rat MSCs treated with DEM only (E). Rat MSCs were cultured for 3 days in serum-free medium to induce nestin expression. Then, those cells were renewed in DEM supplemented with different concentration of TRAP6, for a further 3 days period and then fixed and labelled with nestin antibody. Nuclei counterstained with TOPRO-3 and nestin-positive MSCs were then counted. (G) In these conditions, there is a significant decrease of nestin expression from 1 μM TRAP6 (*, p < 0,05; **, p < 0,01 from 30 μM, n = 3, ANOVA1). (H) To measure the effect of thrombin on the apoptosis of rat MSCs, cells were cultivated for 3 days in serum-free medium to induce nestin expression. Then, those cells were renewed in DEM with various concentrations of thrombin. After a new 3 days period, cells were fixed and submitted to a TUNEL labelling (green). Cell nuclei were counterstained with DAPI. For the positive control of the TUNEL assay, rat MSCs previously treated with DNAse were used (I). (J) To measure the effect of thrombin on cell density, cells were treated with the indicated concentrations of thrombin, then fixed, labelled with DAPI and counted in 10 microscopic fields at 10× magnification. Scale bar in A, B, H, I = 40 μm

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells-2

<p><b>Copyright information:</b></p><p>Taken from "Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells"</p><p>http://www.biomedcentral.com/1471-2202/8/104</p><p>BMC Neuroscience 2007;8():104-104.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2231362.</p><p></p>medium (C, D, F). Then, the cells were fixed and RG were labelled with anti-nestin (green), anti-PAR1 (red) and anti-GFAP (blue) antibody (panel A-D) while rat MSCs were stained with anti-nestin (green) and anti-PAR1 (red) antibody (panel E, F). Scale bar (in A = 10 μm) applies to all images. Expression of thrombin receptor PAR1mRNA in mouse RG (G) and rat MSCs and astrocytes (H) was analyzed by RT-PCR using optimized primers for each species

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells-3

<p><b>Copyright information:</b></p><p>Taken from "Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells"</p><p>http://www.biomedcentral.com/1471-2202/8/104</p><p>BMC Neuroscience 2007;8():104-104.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2231362.</p><p></p>ese cells were then fixed, labelled with anti-nestin antibody (green), counterstained with TOPRO-3 (red) and positive cells were counted in triplicate cultures. A and B show typical results of anti-nestin immunostaining in culture seeded at initial densities of 20 and 50·10cells/cm. From 50000 rMSC/cm, nestin expression decreases rapidly (C). In this experiment, results are expressed as percentages of nestin-positive cells in each culture conditions (n = 3, ANOVA1). These results were confirmed by Western blotting analysis. Rat MSCs were plated during 3 days in serum-free medium at different densities (20 (D), 50 (E) or 100 (F)·10cells/cm) and representative images were taken with a phase contrast microscope. Protein extracts were separated and revealed with anti-nestin and anti-actin antibodies. A decrease of nestin expression is apparent from an initial density of 50·10cells/cm(G). Actin immunostaining controls the protein amounts loaded on the gel in each condition (H). Rat MSCs were cultivated at a density of 10,000 rat MSCs/cmtogether with different densities of rat MSCs in a physically separated millicell co-culture device. No significant difference was observed when the proportion of rat MSCs immunolabelled for nestin was compared in each condition (I) (n = 3, ANOVA1). Serum added in a millicell device as a positive control strongly inhibits nestin expression by rMSCs in the same setup. Scale bar A, B, D, E, F = 40 μm

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells-1

<p><b>Copyright information:</b></p><p>Taken from "Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells"</p><p>http://www.biomedcentral.com/1471-2202/8/104</p><p>BMC Neuroscience 2007;8():104-104.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2231362.</p><p></p>their nestin expression (B), or in serum-free medium plus 900 pM Thrombin (C) or 100 μM TRAP6 (D). Then, the cells were fixed and labelled with anti-nestin (green) and anti-GFAP (red) antibody. Nuclei were counterstained with TOPRO-3 (blue). In the presence of thrombin or TRAP6, the expression of nestin is maintained together with that of GFAP which is co-expressed in a varying proportion of the cells. Additional cell cultures that were treated in parallel with varying concentrations of thrombin (90 to 900 pM, E) or TRAP6 (0.3 to 100 μM, F) were collected and total protein extracts were prepared. Nestin concentrations in those extracts was then measured by Western blots as described in Material and Methods. Scale bar (in A = 40 μm) applies to all images