Percentage Of BrdU-positive Cells Research Articles

Comparative characterization and osteogenic / adipogenic differentiation of mesenchymal stem cells derived from male rat hair follicles and bone marrow

Clinical applications of cell therapy and tissue regeneration under different conditions need a multiplicity of adult stem cell sources. Up to date, little is available on the comparative isolation, characterization, proliferation, rapid amplification, and osteogenic/adipogenic differentiation of rat mesenchymal stem cells (MSCs) isolated from living bulge cells of the hair follicle (HF) and bone marrow (BM) from the same animal. This work hopes to use HF-MSCs as an additional adult stem cell source for research and application. After reaching 80% confluence, the cell counting, viability %, and yields of HF-MSCs and BM-MSCs were nearly similar. The viability % was 91.41 ± 2.98 and 93.11 ± 3.06 while the cells yield of initial seeding was 33.15 ± 2.76 and 34.22 ± 3.99 and of second passage was 28.76 ± 1.01 and 29.56 ± 3.11 for HF-MSCs and BM-MSCs respectively. Clusters of differentiation (CDs) analysis revealed that HF-MSCs were positively expressed CD34, CD73 and CD200 and negatively expressed CD45. BM-MSCs were positively expressed CD73 and CD200 and negatively expressed of CD34 and CD45. The proliferation of HF-MSCs and BM-MSCs was determined by means of incorporation of Brd-U, population doubling time (PDT) assays and the quantity of formazan release. The percentage of Brd-U positive cells and PDT were relatively similar in both types of cells. The proliferation, as expressed by the quantity of formazan assay in confluent cells, revealed that the quantity of release by BM-MSCs was slightly higher than HF-MSCs. Adipogenic differentiated BM-MSCs showed moderate accumulation of oil red-O stained lipid droplets when compared to that of HF-MSCs which exhibited high stain. The total lipid concentration was significantly higher in adipogenic differentiated HF-MSCs than BM-MSCs (P < 0.05). It was found that activity of bone alkaline phosphatase and calcium concentration were significantly higher (P < 0.01 and P < 0.05 respectively) in osteogenic differentiated BM-MSCs than that of HF-MSCs. The present findings demonstrate that the HF-MSCs are very similar in most tested characteristics to BM-MSCs with the exception of differentiation. Additionally; no issues have been reported during the collection of HF-MSCs. Therefore, the HF may represent a suitable and accessible source for adult stem cells and can be considered an ideal cell source for adipogenesis research.

Methylprednisolone inhibits the proliferation of endogenous neural stem cells in nonhuman primates with spinal cord injury

OBJECTIVE The aim of this work was to investigate the effects of methylprednisolone on the proliferation of endogenous neural stem cells (ENSCs) in nonhuman primates with spinal cord injury (SCI). METHODS A total of 14 healthy cynomolgus monkeys ( Macaca fascicularis) (4-5 years of age) were randomly divided into 3 groups: the control group (n = 6), SCI group (n = 6), and methylprednisolone therapy group (n = 2). Only laminectomy was performed in the control animals at T-10. SCI was induced in monkeys using Allen's weight-drop method (50 mm × 50 g) to injure the posterior portion of the spinal cord at T-10. In the methylprednisolone therapy group, monkeys were intravenously infused with methylprednisolone (30 mg/kg) immediately after SCI. All animals were intravenously infused with 5-bromo-2-deoxyuridine (BrdU) (50 mg/kg/day) for 3 days prior to study end point. The small intestine was dissected for immunohistochemical examination. After 3, 7, and 14 days, the spinal cord segments of the control and SCI groups were dissected to prepare frozen and paraffin sections. The proliferation of ENSCs was evaluated using BrdU and nestin immunofluorescence staining. RESULTS Histological examination showed that a larger number of mucosa epithelial cells in the small intestine of all groups were BrdU positive. Nestin-positive ependymal cells are increased around the central canal after SCI. After 3, 7, and 14 days of SCI, BrdU-positive ependymal cells in the SCI group were significantly increased compared with the control group, and the percentage of BrdU-positive cells in the left/right ventral horns and dorsal horn was significantly higher than that of the control group. Seven days after SCI, the percentages of both BrdU-positive ependymal cells around the central canal and BrdU- and nestin-double positive cells in the left/right ventral horns and dorsal horn were significantly lower in the methylprednisolone therapy group than in the SCI group. CONCLUSIONS While ENSCs proliferate significantly after SCI in nonhuman primates, methylprednisolone can inhibit the proliferation of ependymal cells after SCI.

Experimental study on DEHP affect the neurodevelopment through interfering with placental thyroid hormones transport

Objective: The present study was represented by di-(2-ethylhexyl) phthalate (DEHP), to explore the role of thyroid hormones (THs) disruption in the connection of placenta and neurodevelopmental toxicity. Methods: During fetal mice neural tube closed (pregnancy 9.5 days, E9.5d) to begin synthesis of THs (E15.5 d), all pregnant mice were administered with different concentration of DEHP (0、10、50、200 mg/kg) by gavage once a day(10 mice per group). All pregnant mice were conducted with BrdU administration in E14d by subcutaneous injection. Seven pregnant mice from each group were scarified after anesthesia in E15.5 d, serum and amniotic fluid were collected to determinate the levels of THs(T(3), T(4), FT(3) and FT(4)) by the automatic biochemical analyzer, detecting fetal mice placental protein expression of monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptide 1C1 (OATP1C1) and deiodinaseⅡ&Ⅲ (DIO(2), DIO(3)) by Western blot. Each group of the remaining three pregnant mices were killed after anesthesia in E18d, take the male fetal brain, BrdU immunohistochemistry was used to detect the proliferation and migration of fetal brain cortical neurons. Results: There was no abnormalities in diet, water intake, body weight and general activity of pregnant mice in each treatment group, and there were no difference in the general physiolo. Results There was no abnormalities in diet, water intake, body weight and general activity of pregnant mice in each treatment group, and there were no difference in the general physiological development status of body weight, brain weight, brain body ratio between the mice of each group. There was no statistically significant differences in serum T(3), T(4), FT(3), FT(4) and amniotic fluid FT(4) in pregnant mice of each group (P>0.05), Compared with the control group, the FT(3) levels in the amniotic fluid of the DEHP 50 and 200 mg/kg groups were significantly decreased(P<0.05). Compared with the control group, the placental MCT8 and DIO(2) protein levels of male fetal mice in the DEHP 50 and 200 mg/kg group decreased, and the level of OATP1C1 protein in 200 mg/kg group decreased(P<0.05), and there was no statistically significant difference in DIO(3) protein levels among all groups (P>0.05). Compared with the control group, the number of BrdU positive cells in the cerebral cortex of male mice in DEHP 200 mg/kg group decreased, 56.5% was distributed in VZ-SVZ layer, and the percentage of BrdU positive cells in the IZ layer of 50 mg/kg group increased (P<0.05). Conclusion: DEHP 50, 200 mg/kg may affect the proliferation and migration of neural cells in the developing brain, which may be related to its interference with thyroid hormone by placental transport.

Wnt/β-catenin signaling is involved in the Icariin induced proliferation of bone marrow mesenchymal stem cells

ObjectiveTo investigate the effect of icariin on proliferation of bone marrow mesenchymal stem cells (BMSCs) in Sprague-Dawley (SD) rats. MethodsBMSCs were obtained from SD rat bone marrow with differential time adherent method. Its characteristic was identified through differentiation cell surface antigens and the multi-lineage (osteo/adipo/chondo) differentiation potential. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and 5-Bromo-2-Deoxyuridine (BrdU) incorporation were applied to detect the effect of icariin on BMSCs proliferation. Flow cytometry was used to detect proliferation index of BMSCs. The mRNA level and the distribution of β-catenin were evaluated by Real-time Polymerase Chain Reaction (PCR) and Immunofluorescent staining respectively. Western blot was used to detect protein expression levels of β-catenin, glycogen synthase kinase-3 beta (GSK-3β), phospho-glycogen synthase kinase-3 beta (pGSK-3β) and cyclinD1. ResultsIcariin promoted BMSCs proliferation at the concentration of 0.05-2.0 mg/L. The percentage of BrdU positive cells of BMSCs was increased from 40.98% to 70.42%, and the proliferation index value was increased from 8.9% to 17.5% with the treatment of 0.05 mg/L icariin, which significance values were both less than 0.05. Compared with the control group, total and nuclear β-catenin proteins, as well as β-catenin mRNA expression, were all increased with icariin treatment. Meanwhile, the phosphorylation level of GSK-3β and cyclinD1 protein expressions were also increased in BMSCs with icariin treatment. ConclusionThe findings of the present study demonstrated that low dosage of icariin could promote BMSCs proliferation. The activation of Wnt/β-catenin pathways was involved in this process.

Evaluation of the osteogenic potential of Hancornia speciosa latex in rat calvaria and its phytochemical profile

Ethnopharmacological relevanceHancornia speciosa Gomes, commonly known as Mangabeira, is a Brazilian native fruit tree belonging to the Apocynaceae family. In folk medicine, the latex obtained from Mangabeira's trunk has been used as an adjunct therapy for bone fractures. Few pharmacological studies on the Hancornia speciosa latex have been developed and despite its popular use for bone healing there is no data about its biological effect on bone. Aim of the studyThe present study aimed to investigate the osteogenic potential of Hancornia speciosa latex in rat calvaria, as well as its phytochemical profile. Materials and methodsA neutral gel composition containing 5% latex was topical applied to a critical size bone defect and over intact calvaria of rats. Areas of newly formed bone on the borders of the defect and of calvaria periosteum were quantified, as well as the percentage of BrdU-positive cells and total cells in the periosteum at different periods of time after latex application. The cytotoxicity of the latex aqueous phase was evaluated in rat calvarial cells in vitro by MTT assay and its phytochemical profile was investigated by ESI-MS/MS. ResultsThe area of newly formed bone on the borders of the calvaria defect was larger in rats that received latex at 15 and 30 days of healing. After 3 days of latex application over the intact calvaria, the periosteum area was increased and newly formed bone was observed after 5 and 11 days. There was also an increase in periosteum cell proliferation and population followed latex application on calvaria (p<0.05). The latex aqueous phase limited rat calvarial cell viability in vitro in concentrations larger than 0.6mg/mL. Chlorogenic acid and naringenin-7-O-glucoside were identified in the latex aqueous phase, along with catechin and procyanidin compounds. ConclusionThere was a stimulus for periosteum cell proliferation and bone formation when Hancornia speciosa latex was topically applied on rat calvaria. In addition, chlorogenic acid and naringenin-7-O-glucoside present in Hancornia speciosa latex may contribute to its effects on bone formation.

Loss of NR4A1 Accelerates the Development of Aggressive Lymphomas in Myc Induced Cancerogenesis

Introduction and Aim:We recently described a reduced NR4A1 and NR4A3 expression chronic lymphocytic leukemia, follicular lymphoma, and diffuse large B-cell lymphoma compared to normal controls. Our survival analysis of aggressive lymphomas revealed that low NR4A1 expression was associated with poor cancer specific survival. Over-expression of NR4A1 in lymphoma cell lines led to a significantly higher proportion of lymphoma cells undergoing apoptosis and abrogated tumor growth in xenografts1. The aim of this study is to define the role of NR4A1 as a tumor suppressor in the development of lymphoid malignancies in vivo.Methods:To identify, whether the loss of NR4A1 has an impact in Myc driven lymphomagenesis we crossed EµMyc mice with NR4A1-/- mice and performed phenotypical analysis including measurement of tumor development, survival and immunophenotypic determination of the newly developed lymphomas by FACS analysis. To further investigate the impact of NR4A1 loss on the oncogenic potential of EµMyc lymphoma cells we isolated viable tumor cells (B220+ and 7AAD-) and cultured them for 72h with or without lipopolysaccharide (LPS) and determined the number of viable cells and their viability (B220 and 7AAD-staining by flow cytometry analysis) after 24h, 48h and 72h. Finally, expression levels of NR4A1, NR4A3 and Myc with or without NR4A1 loss were evaluated by using RT-qPCR.Results:EµMyc mice with NR4A1 loss (EµMyc NR4A1-/-, n=46) developed visible tumors significantly faster compared to EµMyc mice with NR4A1 (EµMyc NR4A1+/+, n=75) (median = 44 days for EµMyc NR4A1 -/- vs. 107 days for EµMyc NR4A1+/+; p<0.001). Additionally, EµMyc NR4A1-/- mice showed a significantly shorter life span (median survival = 77 days) compared to EµMyc NR4A1 +/+ mice (median survival = 156 days; p<0.001). By comparing the immunophenotype of the newly developed lymphoma between the two groups (EµMyc NR4A1+/+, n=17 and EµMyc NR4A1 /- , n=19), no significant difference was observed. Interestingly, EµMyc NR4A1-/- mice showed an increased frequency of strong CD93 expression (10 of 18, respectively, vs. 2 of 17 EµMyc NR4A1 +/+ mice, p=0.004). Since most of the EµMyc NR4A1-/- lymphoma were IgM negative (7 of 10) it might be speculated that NR4A1 loss leads to a more immature phenotype of the lymphoma. The number of viable B220+ lymphoma cells isolated from EµMyc NR4A1-/- mice was higher compared to B220+ lymphoma cells isolated from EµMyc NR4A1+/+ mice after 72h in culture with or without LPS (p=0.056; p=0,052). This was accompagnied by a higher in vitro proliferation rate as demonstrated by a higher percentage of BrdU positive cells of the EµMyc NR4A1-/- mice compared to B220+ EµMyc NR4A1+/+ cells with and without LPS stimulation (p= 0,064, p=0,038). Interestingly, we detected a 12 fold higher NR4A3 mRNA expression (p=0,038) in EµMyc NR4A1-/- tumors compared to EµMyc NR4A1+/+.Conclusion: Our data demonstrate that NR4A1 possesses tumor suppressive properties and that loss of NR4A1 accelerates Myc driven lymphomagenesis. Furthermore, this study indicates that deletion of NR4A1 confers a more aggressive behavior and increases the oncogenic potential of EµMyc driven lymphoma cells.1. Deutsch AJ, Rinner B, Wenzl K, et al. NR4A1-mediated apoptosis suppresses lymphomagenesis and is associated with a favorable cancer specific survival in patients with aggressive B-cell lymphomas. Blood. 2014. DisclosuresNo relevant conflicts of interest to declare.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Abstract 16708: Cathelicidin Related Antimicrobial Peptide Treated Bone Marrow Derived Mononuclear Cells Improves Cardiac Function in a Mouse Model of Acute Myocardial Infarction: Potential Therapeutic Targets in Ischemic Heart Disease

Introduction: Limited stem cell retention following intracoronary administration for ischemic heart disease has reduced the clinical efficacy of this novel therapy. Cathelicidins have been shown to prime BMMNC migration towards low gradients of SDF-1 suggesting a potential role in BMMNC retention. We sought to assess the safety and efficacy of BMMNC pre-treatment with CRAMP for treatment of acute ischemic heart disease. METHODS: BMMNCs isolated from GFP mice were incubated with recombinant CRAMP (2.5 μg/ml) or placebo for 1 hour followed by chemotaxis studies towards low levels of SDF-1 (2 ng/ml) using a Boyden chamber in vitro. During the in vivo studies, mice were randomized into 3 groups: AMI followed by injection of phosphate buffered saline (PBS), BMMNCs alone, or BMMNCs incubated with CRAMP. Scar size, survival and retention of injected BMNNCs were examined by immunohistochemistry at 5 weeks. Left ventricular function was measured by echocardiography at baseline, 48 hours, and 5 weeks after MI. Changes in infarct size between 5 days and 5 weeks after AMI was assessed by cardiac MRI utilizing delayed gadolinium enhancement. RESULTS: Treatment of BMNNCs with CRAMP enhanced their migration towards low, yet physiological, levels of SDF-1 (Fig 1A). In vivo, a greater proportion of cell survival and retention was observed in the BMNNC+CRAMP group than in the BMNNC-alone group (Fig 1B) and this was associated with higher percentage of BrdU positive cells (Fig 1C). Moreover, BMNNC+CRAMP administration led to significantly better survival, improvement of cardiac function (Fig 1D-H) and reduction in infarct size compared with other control groups (Fig 1I). CONCLUSIONS: Cathelicidins enhance BMMNC retention after intramyocardial administration for acute ischemic heart disease resulting in enhanced recovery. Therapies employing this strategy may represent an effective method for improving cardiac recovery and survival rate after AMI in human studies.

Extracellular signal-regulated kinases 1/2 and Akt contribute to triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

Triclosan is a broad spectrum anti-bacterial agent widely used in many personal care products, household items, medical devices, and clinical settings. Human exposure to triclosan is mainly through oral and dermal routes. In previous studies, we found that sub-chronic dermal exposure of B6C3F1 mice to triclosan induced epidermal hyperplasia and focal necrosis; however, the mechanisms for these responses remain elusive. In this study, using mouse epidermis-derived JB6 Cl 41-5a cells, we found that triclosan stimulated cell growth in a concentration- and time-dependent manner. Enhanced cell proliferation was demonstrated by a substantial increase in the percentage of BrdU-positive cells, an elevation in the protein levels of cyclin D1 and cyclin A, and a reduction in the protein level of p27(Kip1). Western blotting analysis revealed that triclosan induced the activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38, and Akt. Pre-treatment of the cells with PD184352, an inhibitor of the upstream kinase MEK1/2, or with wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked triclosan-mediated phosphorylation of ERK1/2 and Akt, respectively, and substantially suppressed triclosan-stimulated cell proliferation, whereas the JNK inhibitor SP600125 or the p38 inhibitor SB203580 had little to no effect on triclosan-stimulated cell proliferation. The phosphorylation activation of ERK1/2 and Akt was further confirmed on the skin of mice dermally administered triclosan. These data suggest that the activation of ERK1/2 and Akt is involved in triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

AB0224 Oaz regulate B-cell functions through the inhibition of CCL2 expression in mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) had been indicated to exert suppressive effects on B-cell proliferation and differentiation through released humoral factor(s) yet to be defined, which is impaired in patients with...

260 INITIAL CHARACTERIZATION OF THE CELLULAR BASIS FOR BLADDER REGENERATION IN A RODENT MODEL

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2012260 INITIAL CHARACTERIZATION OF THE CELLULAR BASIS FOR BLADDER REGENERATION IN A RODENT MODEL Charles Peyton, David Burmeister, Karl-Erik Andersson, and George Christ Charles PeytonCharles Peyton Winston Salem, NC More articles by this author , David BurmeisterDavid Burmeister Winston Salem, NC More articles by this author , Karl-Erik AnderssonKarl-Erik Andersson Winston Salem, NC More articles by this author , and George ChristGeorge Christ Winston Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.317AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The significant regenerative capacity of the bladder has facilitated clinical application of tissue engineering technologies for bladder augmentation/reconstruction. Recent studies in a rodent model documented de novo functional and structural bladder regeneration following removal of 70-80% of the bladder (subtotal cystectomy/STC). The aim of this study was to characterize the early cellular response that mediates bladder regeneration following STC in this physiologically relevant mammalian organ system. METHODS Trigone-sparing STC was performed on 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7 days post-STC, bladders were harvested 2 hours after IP injection of 50 mg/kg Bromodeoxyridine (BrdU, a common index of DNA-replication). Fluorescent BrdU labeling was used to quantify BrdU-positive cells within the urothelium, lamina propria and muscularis propria. Immunohistochemistry was performed to evaluate the sonic hedgehog (Shh) pathway and its related transcription factors Gli-1 and bone morphogenic factor-4 (BMP-4). Normal rat bladders served as controls. RESULTS Sparse BrdU labeling was observed in control bladders (Fig. 1A). The percentage of BrdU-positive cells was significantly higher post-STC compared to controls at all time points (Fig. 1C). Interestingly, a time-dependent shift in the location of BrdU-positive cells was observed such that BrdU-positivity was significantly elevated in the urothelium at 1-day post-STC and lower in the muscularis at 3 and 5-days, with similar expression across all layers by 7-days (Fig. 1D). Increased positive staining for Shh, Gli-1 and BMP-4 was noted post-STC compared to normal bladder. CONCLUSIONS STC induced a robust and global proliferative response with quantifiable spatiotemporal characteristics. Up-regulation of key targets in the Shh pathway was also observed. Presumably, increased cellular proliferation and expression of key targets in the Shh developmental signaling pathway provide, at least in part, the cellular basis for functional bladder recovery after STC. Describing the origin, migration and identity of these cells will improve our mechanistic understanding of the intrinsic regenerative capacity of the bladder and could potentially lead to more efficacious therapies. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e105-e106 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Charles Peyton Winston Salem, NC More articles by this author David Burmeister Winston Salem, NC More articles by this author Karl-Erik Andersson Winston Salem, NC More articles by this author George Christ Winston Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Osteoporosis increases chondrocyte proliferation without a change in apoptosis during fracture healing in an ovariectomized rat model

Osteoporotic fractures commonly occur in the elderly. Current studies regarding cell proliferation and apoptosis during osteoporotic fracture healing are limited. In this study, we established an osteoporotic fracture healing model. Bone loss and callus formation were monitored with DXA, cell proliferation was examined using immunohistochemistry with BrdU monoclonal antibody and apoptotic cells were detected using the TUNEL method. Both cell proliferation and apoptosis occurred during the entire period of the study. BrdU immunostaining showed a decreasing tendency in the process of fracture healing. On days 20 and 30 post-fracture, the percentage of BrdU-positive cells in ovariectomized rats was significantly higher compared to sham-operated rats. TUNEL-positive chondrocytes reached a peak on day 20 post-fracture. There was no significant difference between the two groups. Our results indicate that osteoporosis markedly delays the fracture healing process, mostly due to increased chondrocyte proliferation without a change in chondrocyte apoptosis.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

The survival promoting peptide Y-P30 promotes cellular migration

Y-P30, the 30 amino acid N-terminal peptide of the dermcidin gene, has been found to promote neuronal survival and differentiation. Its early presence in development and import to the fetal brain led to the hypothesis that Y-P30 has an influence on proliferation, differentiation and migration. Neurospheres derived from neural stem cells isolated from E13 mouse cortex and striatal ganglionic eminences were treated with Y-P30, however, the proportion of progenitors, neurons and astrocytes generated in differentiation assays was not altered. A short Y-P30 treatment of undifferentiated striatal and cortical neurospheres failed to alter the proportion of BrdU-positive cells. A longer treatment reduced the percentage of BrdU-positive cells and GABA-immunoreactive neurons only in striatal spheres. The presence of Y-P30 enhanced migration of T24 human bladder carcinoma cells in a wound-healing assay in vitro. Further, Y-P30 enhanced migration of T24 cells, rat primary cortical astrocytes and PC12 cells in chemotactic Boyden chamber assays. Together, these findings suggest that a major function of Y-P30 is to promote migration of neural and non-neural cell types.

Loss of the chromatin regulator MRG15 limits neural stem/progenitor cell proliferation via increased expression of the p21 Cdk inhibitor

Chromatin regulation is crucial for many biological processes such as transcriptional regulation, DNA replication, and DNA damage repair. We have found that it is also important for neural stem/progenitor cell (NSC) function and neurogenesis. Here, we demonstrate that expression of the cyclin-dependent kinase inhibitor p21 is specifically up-regulated in Mrg15 deficient NSCs. Knockdown of p21 expression by p21 shRNA results in restoration of cell proliferation. This indicates that p21 is directly involved in the growth defects observed in Mrg15 deficient NSCs. Activated p53 accumulates in Mrg15 deficient NSCs and this most likely accounts for the up-regulation of p21 expression in the cells. We observed decreased p53 and p21 levels and a concomitant increase in the percentage of BrdU positive cells in Mrg15 null cultures following expression of p53 shRNA. DNA damage foci, as indicated by immunostaining for γH2AX and 53BP1, are detectable in a sub-population of Mrg15 deficient NSC cultures under normal growing conditions and the majority of p21-positive cells are also positive for 53BP1 foci. Furthermore, Mrg15 deficient NSCs exhibit severe defects in DNA damage response following ionizing radiation. Our observations highlight the importance of chromatin regulation and DNA damage response in NSC function and maintenance.

Expression of hedgehog signaling pathway in squamous cell carcinoma.

Objective To investigate the expression of Ptch-1 and Gli-1, hedgehog pathway-related genes in squamous cell carcinoma (SCC), and the effect of cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the proliferation of a SCC cell line Tca. Methods Skin samples were resected from 42 patients with SCC and 10 normal human controls. Immunohistochemistry and in situ hybridization were employed to study the expression and distribution of Ptch-1 and Gli-1 in these specimens. Tca cells were incubated with cyclopamine (1, 2, 5, 10 μmol/mL) for 48 hours, or cyclopamine (5 μmol/mL) for 1-8 days. The same concentrations of lycopersicin served as the control treatment. Then, MTT assay was performed to detect the proliferation of Tca cells. A fraction of Tca cells were cultured in the presence of 5 μmol/mL cyclopamine for 72 hours followed by BrdU assay for the evaluation of cell growth and proliferation. Results A significant increment was shown in the expression of both Patch-1 and Gli-1 by immunohistochemistry (χ2= 5.656, 6.732, P<0.05, 0.01, respectively) and in situ hybridization (χ2=6.787, 9.600, respectively, both P<0.01) in SCC tissue compared with the control specimens. And both of them were predominantly distributed in the cytoplasm of SCC cells. As MTT assay revealed, cyclopamine notably inhibited the proliferation of Tea cells, and the effect increased with the concentration and action time of cyclopamine. Further more, the percentage of BrdU-positive cells was 26% in cyclopamine-treated Tca cells, significantly higher than that in the blank control cells (77%) and lycopersicin-treated cellls (72%). Conclusions Hedgehog signaling pathway is activated in the lesions of SCC, and inhibition of the pathway may facilitate the treatment of SCC. Key words: Carcinoma, squamous cell; Signal transducting; Hedgehogs

Protection of the Intestinal Epithelium from Conditioning with R-spondin1 Ameliorates Graft-Versus-Host Disease

Damage to the gastrointestinal (GI) tract by pretransplant conditioning regimen plays a critical role in amplifying graft-versus-host disease (GVHD). Thus protection of the GI tract from conditioning may represent a novel approach to prevent GVHD. R-Spondin1 (R-Spo1) is a novel class of soluble activator for Wnt/□-catenin signaling, and has potent and specific proliferative effects on the intestinal crypt cells; injection of R-Spo1 protects mice from chemotherapy-induced intestinal mucositis. We therefore hypothesized that administration of R-Spo1 could modulate GVHD by reducing the GI tract damage and improve outcome of allogeneic bone marrow transplantation (BMT). Lethally irradiated B6D2F1 (H-2b/d) mice were injected with 5 × 106 BM and 2 × 106 T cells from MHC-mismatched B6 (H-2b) donors on day 0. Mice were intravenously injected with 200 μg of R-Spo1 or diluent from days −3 to −1 and +1 to +3 after BMT. In vivo labeling assay of mitotic cells with BrdU demonstrated that the proliferative index, as determined by the percentages of BrdU-positive cells among crypt epithelial cells, was significantly greater in the small intestine of R-Spo1 treated mice than controls 4 days after BMT (57% ± 3% vs 48% ± 1%, P<0.05). Analysis of the mesenteric lymph nodes and spleens on day +7 demonstrated significantly reduced expansion of donor T cells in R-Spo1 treated recipients in association with reduced serum levels of IFN-□ and TNF-□ on day +7 when compared to controls (Table). GVHD was severe in allogeneic controls, with 12.5% survival by day +40, whereas 62.5% of R-Spo1-treated animals survived this period (Table). Histopathologic examination of the small and large bowel and liver showed significantly reduced GVHD pathology in R-Spo1 treated animals than in controls (Table). A flowcytometric analysis of the spleen and thymus after BMT showed that administration of R-Spo1 did not impair donor cell engraftment and T and B cell immune reconstitution. We next evaluated the impact of R-Spo1 on graft-versus-leukemia (GVL) effects. BMT was performed similarly as above with the addition of 5 × 104 host-type P815 leukemia cells (H-2d). All recipients of T cell-depleted BM died from leukemia by day +20 after BMT, while no leukemia death was observed in R-Spo1 treated allogeneic animals. Overall, R-Spo1 treatment improved outcome of allogeneic BMT by reducing GVHD, while maintaining immune reconstitution and GVL effects. Thus, administration of R-Spo1 reduces the GI tract damage and suppresses donor T cell activation and systemic GVHD, supporting a hypothesis that the GI tract plays a major role in the amplification of systemic GVHD. Brief treatment with R-Spo1 may serve as an effective adjunct to clinical regimens of GVHD prophylaxis.Pathology ScoresGroupSurvivals on day+40 (%)Small bowelLarge bowelLiverINF □ (ng/ml)TNF □ (pg/ml)TCD diluent1002.4± 0.93.5± 1.00.3± 0.3NDND+T diluent12.58.3± 2.77.3± 1.92.0± 0.86.0 ± 2.4103.7 ± 9.9+T R-Spo162.5*3.4±1.9**3.9± 0.3**0.8± 0.7**2.3 ± 1.5**55.4 ± 6.6**TCD: T cell-depleted BMT, +T: T cell-repleted BMT, ND: not detectedData are expressed as mean ± SD. *P<0.01 vs control, **P<0.05 vs control

Donepezil, an acetylcholinesterase inhibitor, enhances adult hippocampal neurogenesis

Donepezil hydrochloride is a potent and selective acetylcholinesterase inhibitor and has been treated for Alzheimer's disease, in which the cholinergic dysfunction is observed. Recently, the degeneration of medial septal cholinergic nuclei in adult rat suppressed the neurogenesis in hippocampal dentate gyrus (DG) was reported. Then, we determined whether donepezil which activated the brain cholinergic system could modulate hippocampal neurogenesis in normal rats. After the injection of 5′-bromo-2′-deoxyuridine (BrdU) to label dividing cells, we orally treated with donepezil (0.5 or 2 mg/kg) once a day for 4 weeks. In the other group, we performed 4-week subcutaneous infusion of scopolamine (0.75 or 3 mg/day), a muscarinic acetylcholine receptor blocker. The doses of donepezil and scopolamine we used in this study were reported to activate and inhibit cholinergic activity in rats, respectively. One day after the completion of drug treatment, the animals were sacrificed, and immunohistochemical analysis was performed. Donepezil increased, but scopolamine decreased, the number of BrdU-positive cells in the DG as compared with the vehicle-treated control. Neither drug had any effects on the percentage of BrdU-positive cells that were also positive for a neuronal marker NeuN, nor the number of proliferating cell nuclear antigen-positive cells in the DG. These results indicate that donepezil enhances and scopolamine suppresses the survival of newborn neurons in the DG without affecting the proliferation of neural progenitor cell and the neuronal differentiation. We also found that chronic treatment of donepezil enhanced, and scopolamine suppressed phosphorylation of cAMP response element binding protein (CREB), which was involved in cell survival, in the DG. These results suggest that donepezil activates the central cholinergic transmission and enhances the survival of newborn neurons in the DG via CREB signaling.

Comparison of the labeling efficiency of BrdU, DiI and FISH labeling techniques in bone marrow stromal cells

Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1±8.6%, 97.6±3.4% and 90.5±3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9±18.0% and 18.4±6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1±4.0% and 94.5±3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0±3.0% and 89.1±9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study.

TRB2, a Mouse Tribbles Ortholog, Suppresses Adipocyte Differentiation by Inhibiting AKT and C/EBPβ

Adipocyte differentiation is regulated by a complex array of extracellular signals, intracellular mediators and transcription factors. Here we describe suppression of adipocyte differentiation by TRBs, mammalian orthologs of Drosophila Tribbles. Whereas all the three TRBs were expressed in 3T3-L1 preadipocytes, TRB2 and TRB3, but not TRB1, were immediately down-regulated by differentiation stimuli. Forced expression of TRB2 and TRB3 inhibited adipocyte differentiation at an early stage. Akt activation is a key event in adipogenesis and was severely inhibited by TRB3 in 3T3-L1 cells. However, the inhibition by TRB2 was mild compared with severe inhibition by TRB3, though TRB2 suppressed adipogenesis as strongly as TRB3. Interestingly, TRB2 but not TRB3 reduced the level of C/EBPbeta, a transcription factor required for an early stage of adipogenesis, through a proteasome-dependent mechanism. Furthermore, knockdown of endogenous TRB2 by siRNA allowed 3T3-L1 cells to differentiate without full differentiation stimuli. These results suggest that inhibition of Akt activation in combination with degradation of C/EBPbeta is the basis for the strong inhibitory effect of TRB2 on adipogenesis.