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To study the effects of inducement on the expression of mouse embryonic stem cells SF1-G imprinted genes, Kcnq1 and Cd-kn1c during the course of differentiation into islet-like cells in vitro. Mouse embryonic fibroblasts (MEFs) were isolated from pregnant mice embryos and fibroblast feeder cells were prepared by treating 3-5th generations MEFs with Mitomycin C. Moreover, mouse embryonic stem cells were induced to differentiate into islet-like cells directly. RT-PCR and Immunofluorescence staining were used to test the expression of islet cell-specific markers. Cells were collected at various stages throughout the differentiation process and the imprinted genes Kcnq1 and Cdkn1c were tested by reverse transcription-polymerase chain reaction fragment length polymorphism (RT-PCR/RFLP). In the present study, we found that cells appear islet cell-specific gene expression. Furthermore, immunofluorescence shows us that the islet cell-specific hormone protein can be measured at stage, which confirms that the embryonic stem cells can be successfully induced into islet-like cells in vitro. RT-PCR/RFLP analysis showsthat imprinted genes Kcnq1 and Cdkn1c are biallelic expression in the differentiated cells, suggestive of loss of imprinting (LOI), while these genes demonstrate maternal monoallelic expression in the undifferentiated cells' continued subculture; this marks the maintenance of imprinting (MOI). Our data indicate that mouse embryonic stem cells are induced into islet-like cells in vitro. The gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells during the induction in vitro. [ABSTRACT FROM AUTHOR]

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38ß, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38ß protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38ß is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells. [ABSTRACT FROM AUTHOR]

Numerous studies have determined that physical cues, especially the nanotopography of materials, play key roles in directing stem cell differentiation. However, most research on nanoarrays for stem cell fate regulation is based on nonbiodegradable materials, such as silicon wafers, TiO2, and poly(methyl methacrylate), which are rarely used as tissue engineering biomaterials. In this study, we prepared biodegradable polylactic acid (PLA) nanopillar arrays with different diameters but the same center-to-center distance using a series of anodic aluminum oxide nanowell arrays as templates. Human adipose-derived stem cells (hADSCs) were selected to investigate the effect of the diameter of PLA nanopillar arrays on stem cell differentiation. By culturing hADSCs without the assistance of any growth factors or osteogenic-induced media, the differentiation tendencies of hADSCs on the nanopillar arrays were assessed at the gene and protein levels. The assessment results suggested that the osteogenic differentiation of hADSCs can be driven by nanopillar arrays, especially by nanopillar arrays with a diameter of 200 nm. Moreover, an in vivo animal model of the samples demonstrated that PLA film with the 200 nm pillar array exhibits an improved ectopic osteogenic ability compared with the planar PLA film after 4 weeks of ectopic implantation. This study has provided a new variable to investigate in the interaction between stem cells and nanoarray structures, which will guide the bone regeneration clinical research field. This work paves the way for the utility of degradable biopolymer nanoarrays with specific geometrical and mechanical signals in biomedical applications, such as patches and strips for spine fusion, bone crack repair, and restoration of tooth enamel. [ABSTRACT FROM AUTHOR]

The molecular mechanisms coordinating cell cycle exit with cell differentiation and organogenesis are a crucial, yet poorly understood, aspect of normal development. The mammalian cyclin-dependent kinase inhibitor p27Kip1 is required for the correct timing of cell cycle exit in developing tissues, and thus plays a crucial role in this process. Although studies of p27Kip1 regulation have revealed important posttranscriptional mechanisms regulating p27Kip1 abundance, little is known about how developmental patterns of p27Kip1 expression, and thus cell cycle exit, are achieved. Here, we show that during inner ear development transcriptional regulation of p27Kip1 is the primary determinant of a wave of cell cycle exit that dictates the number of postmitotic progenitors destined to give rise to the hair cells and supporting cells of the organ of Corti. Interestingly, transcriptional induction from the p27Kip1 gene occurs normally in p27Kip1-null mice, indicating that developmental regulation of p27Kip1 transcription is independent of the timing of cell cycle exit. In addition, cell-type-specific patterns of p27Kip1 transcriptional regulation are observed in the mature organ of Corti and retina, suggesting that this mechanism is important in differential regulation of the postmitotic state. This report establishes a link between the spatial and temporal pattern of p27Kip1 transcription and the control of cell number during sensory organ morphogenesis. [ABSTRACT FROM AUTHOR]

Background: Literature data indicate that brain-derived neurotrophic factor (BDNF), cyclic-AMP response element-binding protein (CREB) and phospho-CREB (pCREB) may have a place in depression. BDNF belongs to the neurotrophin family that plays an important role in proliferation, survival and differentiation of different cell populations in the mammalian nervous system. The herbal mixture used in the present study consists of Euphoria longana, Houttuynia cordata and Dioscorea japonica. The purpose of the present study was to determine the neuroprotective effect of herbal mixture. We also tested the hypothesis that administration of herbs reverses memory deficits and promotes the protein expression of BDNF in the mouse brain. Methods: Mice were randomized into four different treatment groups (n = 10/group). Normal and stress groups received regular lab chow without stress and under stress conditions, respectively, for 3 weeks. The animals in the stress group were immobilized for 4 hours a day for 2 weeks. Different doses of herbal mixture (206 and 618 mg/kg) were administered for 3 weeks to those mice under stress conditions. Mice were analyzed by behavioral tests and immunoblotting examination in the hippocampus and cortex. An additional in vitro investigation was performed to examine whether herbs induce neurotoxicity in a human neuroblastoma cell line, SH-SY5Y cells. Results: No significant toxicity of herbs on human neuroblastoma cells was observed. These herbs demonstrated an inductive effect on the expression of BDNF, pCREB and pAkt. For spatial working memory test, herbal mixture fed mice exhibited an increased level of spontaneous alternation (p < 0.01) compared to those in stress conditions. Moreover, herbal mixture produced highly significant (p < 0.01) reduction in the immobility time in the tail suspension test. Mice in the herbal mixture groups demonstrated lower serum corticosterone concentration than mice in the stress group (p < 0.05). Effects of the oral administration of herbal mixture on protein levels of BDNF in the hippocampi and cortices were significant. Conclusions: Our study showed that herbal mixture administration has antidepressant effects in mice. It is proposed that adverse events such as stress and depression can modulate the expression of molecular players of cellular plasticity in the brain. [ABSTRACT FROM AUTHOR]

Background Parkinson’s disease (PD) is caused by the gradual loss of dopamine-producing cells in the brain. This study evaluated the potential neuroprotective role of puerarin (PR) on dopamine (DA)-producing cells in vitro and in vivo . Method In vitro , the effects of PR on proliferation and differentiation and DA releases of mesenchymal stem cells (MSCs) were assayed by CCK-8, flow cytometry, real-time PCR and ELISA respectively. Then the differentiated cells were labeled with enhanced green fluorescent protein (EGFP) and administrated into PD animal models induced by 6-OHDA. The proliferation and differentiation of labeled cells were identified by fluorescence microscopy and immunostaining. Results In vitro , after being treated with different concentrations of PR for 1 week, the TUJ1, TH and DAT protein and mRNA expression and DA releases increased significantly. In vivo , after transplantation of PR-treated DA-producing cells, the symptoms of PD improved significantly from the second week after transplantation; more transplanted cells survived and migrated to wider region along injection line; more transplanted cells proliferated and differentiated into TH + cells; more DA was detected in the striatum during 6 weeks’ observation. Conclusion The results suggest that PR promote DA neuron survival, proliferation and differentiation. [ABSTRACT FROM AUTHOR]

T regulatory (Treg) cells play crucial roles in the regulation of cellular immunity. The development of Treg cells depends on signals from TCRs and IL-2Rs and is influenced by a variety of transcription factors. The basic helix-loop-helix proteins are known to influence TCR signaling thresholds. Whether this property impacts Treg differentiation is not understood. In this study, we interrogated the role of basic helix-loop-helix proteins in the production of Treg cells using the CD4 promoter-driven Id1 transgene. We found that Treg cells continued to accumulate as Id1 transgenic mice aged, resulting in a significant increase in Treg cell counts in the thymus as well as in the periphery compared with wild-type controls. Data from mixed bone marrow assays suggest that Id1 acts intrinsically on developing Treg cells. We made a connection between Id1 expression and CD28 costimulatory signaling because Id1 transgene expression facilitated the formation of Treg precursors in CD28-/- mice and the in vitro differentiation of Treg cells on thymic dendritic cells despite the blockade of costimulation by anti-CD80/CD86. Id1 expression also allowed in vitro Treg differentiation without anti-CD28 costimulation, which was at least in part due to enhanced production of IL-2. Notably, with full strength of costimulatory signals, however, Id1 expression caused modest but significant suppression of Treg induction. Finally, we demonstrate that Id1 transgenic mice were less susceptible to the induction of experimental autoimmune encephalomyelitis, thus illustrating the impact of Id1-mediated augmentation of Treg cell levels on cellular immunity. [ABSTRACT FROM AUTHOR]

Proteinuria is a common feature for almost all glomerular diseases and reflects the severity of the glomerular lesion. The presence of a large amount of proteins in tubular fluid, however, may also contribute to the development of RIF (renal interstitial fibrosis). Endocytosis of albumin in proximal tubular cells triggers PKC (protein kinase C)-dependent generation of reactive oxygen species and secretion of chemokines. As a family including 12 isozymes, which PKC isozymes participate in RIF is still unclear. EMT (epithelial-mesenchymal transdifferentiation) of RTECs (renal tubular epithelial cells) plays a crucial role in the progress of RIF induced by proteinuria. In the present study, we investigated the role of classical PKC isozymes in the proteinuria-induced EMT of RTECs. Employing immunochemical staining, we found that PKC-α, -βI and -βII were expressed in glomerulus and in RTECs in both normal and diseased renal tissues, while PKC-γ was only expressed in podocytes in the glomerulus. Treatment of HK-2 cells with extracted urinary proteins resulted in EMT, as evidenced by morphological changes, decreased E-cadherin expression, increased α-SMA (α-smooth muscle actin) expression, as well as production of type I collagen and fibronectin. Western blot analysis of PKC isozymes in the cytosolic compared with membrane fraction revealed translocation of PKC-α and -βI, but not PKC-βII, in HK-2 cells undergoing EMT. Pretreatment with selective PKC-α inhibitor G-6976 or PKC-β inhibitor significantly attenuated EMT induced by urinary proteins. In summary, the present study suggested that PKC-α and -bI play critical roles in the EMT of RTECs in response to urinary proteins. [ABSTRACT FROM AUTHOR]