Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation

<p>Abstract</p> <p>Background</p> <p>Although the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4⁺T cell recognition of alveolar antigen. This model was utilized to assess the profile of inflammatory mediators expressed by alveolar epithelial target cells triggered by antigen-specific recognition in CD4⁺T cell-mediated lung inflammation.</p> <p>Methods</p> <p>We established a method that allows the flow cytometric negative selection and isolation of primary AEC II of high viability and purity. Genome wide transcriptional profiling was performed on mRNA isolated from AEC II isolated from healthy mice and from mice with acute and chronic CD4⁺T cell-mediated pulmonary inflammation.</p> <p>Results</p> <p>T cell-mediated inflammation was associated with expression of a broad array of cytokine and chemokine genes by AEC II cell, indicating a potential contribution of epithelial-derived chemoattractants to the inflammatory cell parenchymal infiltration. Morphologically, there was an increase in the size of activated epithelial cells, and on the molecular level, comparative transcriptome analyses of AEC II from inflamed versus normal lungs provide a detailed characterization of the specific inflammatory genes expressed in AEC II induced in the context of CD4⁺T cell-mediated pneumonitis.</p> <p>Conclusion</p> <p>An important contribution of AEC II gene expression to the orchestration and regulation of interstitial pneumonitis is suggested by the panoply of inflammatory genes expressed by this cell population, and this may provide insight into the molecular pathogenesis of pulmonary inflammatory states. CD4⁺T cell recognition of antigen presented by AEC II cells appears to be a potent trigger for activation of the alveolar cell inflammatory transcriptosome.</p

Human Dental Pulp Stem Cells Hook into Biocoral Scaffold Forming an Engineered Biocomplex

The aim of this study was to evaluate the behavior of human Dental Pulp Stem Cells (DPSCs), as well as human osteoblasts, when challenged on a Biocoral scaffold, which is a porous natural hydroxyapatite. For this purpose, human DPSCs were seeded onto a three-dimensional (3D) Biocoral scaffold or on flask surface (control). Either normal or rotative (3D) cultures were performed. Scanning electron microscopic analyses, at 8, 24 and 48 h of culture showed that cells did not adhere on the external surface, but moved into the cavities inside the Biocoral structure. After 7, 15 and 30 days of culture, morphological and molecular analyses suggested that the Biocoral scaffold leads DPSCs to hook into the cavities where these cells quickly start to secrete the extra cellular matrix (ECM) and differentiate into osteoblasts. Control human osteoblasts also moved into the internal cavities where they secreted the ECM. Histological sections revealed a diffuse bone formation inside the Biocoral samples seeded with DPSCs or human osteoblasts, where the original scaffold and the new secreted biomaterial were completely integrated and cells were found within the remaining cavities. In addition, RT-PCR analyses showed a significant increase of osteoblast-related gene expression and, above all, of those genes highly expressed in mineralized tissues, including osteocalcin, OPN and BSP. Furthermore, the effects on the interaction between osteogenesis and angiogenesis were observed and substantiated by ELISA assays. Taken together, our results provide clear evidence that DPSCs differentiated into osteoblasts, forming a biocomplex made of Biocoral, ECM and differentiated cells

Development of a Surface Plasmon Resonance Biosensor for Real-Time Detection of Osteogenic Differentiation in Live Mesenchymal Stem Cells

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis

Age-related changes in rat bone-marrow mesenchymal stem cell plasticity

<p>Abstract</p> <p>Background</p> <p>The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients.</p> <p>Results</p> <p>We demonstrated that the expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs.</p> <p>Conclusion</p> <p>The results suggest age related differences in the differentiation capacity of BM-MSCs. These changes may affect the efficacy of BM-MSCs for use in stem cell therapy.</p

Characterization of a rat osteotomy model with impaired healing

<p>Abstract</p> <p>Background</p> <p>Delayed union or nonunion are frequent and feared complications in fracture treatment. Animal models of impaired bone healing are rare. Moreover, specific descriptions are limited although understanding of the biological course of pathogenesis of fracture nonunion is essential for therapeutic approaches.</p> <p>Methods</p> <p>A rat tibial osteotomy model with subsequent intramedullary stabilization was performed. The healing progress of the osteotomy model was compared to a previously described closed fracture model. Histological analyses, biomechanical testing and radiological screening were undertaken during the observation period of 84 days (d) to verify the status of the healing process. In this context, particular attention was paid to a comparison of bone slices by histological and immunohistological (IHC) methods at early points in time, <it>i.e</it>. at 5 and 10 d post bone defect.</p> <p>Results</p> <p>In contrast to the closed fracture technique osteotomy led to delayed union or nonunion until 84 d post intervention. The dimensions of whole reactive callus and the amounts of vessels in defined regions of the callus differed significantly between osteotomized and fractured animals at 10 d post surgery. A lower fraction of newly formed bone and cartilaginous tissue was obvious during this period in osteotomized animals and more inflammatory cells were observed in the callus. Newly formed bone tissue accumulated slowly on the anterior tibial side with both techniques. New formation of reparative cartilage was obviously inhibited on the anterior side, the surgical approach side, in osteotomized animals only.</p> <p>Conclusion</p> <p>Tibial osteotomy with intramedullary stabilisation in rats leads to pronounced delayed union and nonunion until 84 d post intervention. The early onset of this delay can already be detected histologically within 10 d post surgery. Moreover, the osteotomy technique is associated with cellular and vascular signs of persistent inflammation within the first 10 d after bone defect and may be a contributory factor to impaired healing. The model would be excellent to test agents to promote fracture healing.</p

Next-generation sequencing

Next-generation sequencing (also known as massively parallel sequencing) technologies are revolutionising our ability to characterise cancers at the genomic, transcriptomic and epigenetic levels. Cataloguing all mutations, copy number aberrations and somatic rearrangements in an entire cancer genome at base pair resolution can now be performed in a matter of weeks. Furthermore, massively parallel sequencing can be used as a means for unbiased transcriptomic analysis of mRNAs, small RNAs and noncoding RNAs, genome-wide methylation assays and high-throughput chromatin immunoprecipitation assays. Here, I discuss the potential impact of this technology on breast cancer research and the challenges that come with this technological breakthrough

The effect of mechanical loading on osteogenesis of human dental pulp stromal cells in a novel in vitro model

Tooth loss often results in alveolar bone resorption because of lack of mechanical stimulation. Thus, the mechanism of mechanical loading on stem cell osteogenesis is crucial for alveolar bone regeneration. We have investigated the effect of mechanical loading on osteogenesis in human dental pulp stromal cells (hDPSCs) in a novel in vitro model. Briefly, 1 × 107 hDPSCs were seeded into 1 ml 3 % agarose gel in a 48-well-plate. A loading tube was then placed in the middle of the gel to mimic tooth-chewing movement (1 Hz, 3 × 30 min per day, n = 3). A non-loading group was used as a control. At various time points, the distribution of live/dead cells within the gel was confirmed by fluorescence markers and confocal microscopy. The correlation and interaction between the factors (e.g. force, time, depth and distance) were statistically analysed. The samples were processed for histology and immunohistochemistry. After 1-3 weeks of culture in the in-house-designed in vitro bioreactor, fluorescence imaging confirmed that additional mechanical loading increased the viable cell numbers over time as compared with the control. Cells of various phenotypes formed different patterns away from the reaction tube. The cells in the middle part of the gel showed enhanced alkaline phosphatase staining at week 1 but reduced staining at weeks 2 and 3. Additional loading enhanced Sirius Red and type I collagen staining compared with the control. We have thus successfully developed a novel in-house-designed in vitro bioreactor mimicking the biting force to enhance hDPSC osteogenesis in an agarose scaffold and to promote bone formation and/or prevent bone resorption