p120-Catenin Down-Regulation and Epidermal Growth Factor Receptor Overexpression Results in a Transformed Epithelium That Mimics Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis due to its highly invasive and metastatic potential. The molecular pathogenesis underlying the invasive mechanism of ESCC is not well known because of the lack of existing models to study this disease. p120-Catenin (p120ctn) and the epidermal growth factor receptor (EGFR) have each been implicated in several cancers, including ESCC. p120ctn is down-regulated in 60% of ESCC tumors, whereas EGFR is the most commonly overexpressed oncogene in ESCC. For these reasons, we investigated the cooperation between p120ctn and EGFR and its effect on ESCC invasion. We show that p120ctn down-regulation is commonly associated with EGFR overexpression. By using a three-dimensional culture system, we demonstrate that the inverse relationship between p120ctn and EGFR has biological implications. Specifically, p120ctn down-regulation coupled with EGFR overexpression in human esophageal keratinocytes (EPC1-PE) was required to promote invasion. Morphological comparison of EPC1-PE cells grown in three-dimensional culture and human ESCC revealed identical features, including significantly increased cellularity, nuclear grade, and proliferation. Molecular characteristics were measured by keratin expression patterns, which were nearly identical between EPC1-PE cells in three-dimensional culture and ESCC samples. Altogether, our analyses have demonstrated that p120ctn down-regulation and EGFR overexpression are able to mimic human ESCC in a relevant three-dimensional culture model

Investigating the Vascularization of Tissue-Engineered Bone Constructs Using Dental Pulp Cells and 45S5 Bioglass(®) Scaffolds.

Identification of a suitable cell source combined with an appropriate 3D scaffold is an essential prerequisite for successful engineering of skeletal tissues. Both osteogenesis and angiogenesis are key processes for bone regeneration. This study investigated the vascularization potential of a novel combination of human dental pulp stromal cells (HDPSCs) with 45S5 Bioglass(®) scaffolds for tissue-engineered mineral constructs in vivo and in vitro. 45S5 Bioglass scaffolds were produced by the foam replication technique with the standard composition of 45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt% CaO, and 6 wt% P2O5. HDPSCs were cultured in monolayers and on porous 45S5 Bioglass scaffolds under angiogenic and osteogenic conditions for 2-4 weeks. HDPSCs expressed endothelial gene markers (CD34, CD31/PECAM1, and VEGFR2) under both conditions in the monolayer. A combination of HDPSCs with 45S5 Bioglass enhanced the expression of these gene markers. Positive immunostaining for CD31/PECAM1 and VEGFR2 and negative staining for CD34 supported the gene expression data, while histology revealed evidence of endothelial cell-like morphology within the constructs. More organized tubular structures, resembling microvessels, were seen in the constructs after 8 weeks of implantation in vivo. In conclusion, this study suggests that the combination of HDPSCs with 45S5 Bioglass scaffolds offers a promising strategy for regenerating vascularized bone grafts

Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines

Raman spectroscopy could offer non-invasive, rapid and an objective nature to cancer diagnostics. However, much work in this field has focused on resolving differences between cancerous and non-cancerous tissues, and lacks the reproducibility and interpretation to be put into clinical practice. Much work is needed on basic cellular differences between malignancy and normal. This would allow the establishment of a clinically relevant cellular based model to translate to tissue classification. Raman spectroscopy provides a very detailed biochemical analysis of the target material and to 'unlock' this potential requires sophisticated mathematical modelling such as neural networks as an adjunct to data interpretation. Commercially obtained cancerous and non-cancerous cells, cultured in the laboratory were used in Raman spectral measurements. Data trends were visualised through PCA and then subjected to neural network analysis based on self-organising maps; consisting of m maps, where m is the number of classes to be recognised. Each map approximates the statistical distribution of a given class. The neural network analysis provided a 95% accuracy for identification of the cancerous cell line and 92% accuracy for normal cell line. In this preliminay study we have demonstrated th ability to distinguish between "normal" and cancerous commercial cell lines. This encourages future work to establish the reasons underpinning these spectral differences and to move forward to more complex systems involving tissues. We have also shown that the use of sophisticated mathematical modelling allows a high degree of discrimination of 'raw' spectral data

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

Aseptic Raman spectroscopy can detect changes associated with the culture of human dental pulp stromal cells in osteoinductive culture.

There is an unmet need for the non-invasive characterisation of stem cells to facilitate the translation of cell-based therapies. Raman spectroscopy has proven utility in stem cell characterisation but as yet no method has been reported capable of taking repeated Raman measurements of living cells aseptically over time. The aim of this study was to determine if Raman spectroscopy could be used to monitor changes in a well characterised cell population (human dental pulp stromal cells (DPSCs)) by taking repeated Raman measurements from the same cell populations in osteoinductive culture over time and under aseptic conditions. DPSCs were isolated from extracted premolar teeth from 3 consenting donors. Following in vitro expansion, DPSCs were maintained for 28 days in osteo-inductive medium. Raman spectra were acquired from the cells at days 0, 3, 7, 10, 14 and 28. Principal component analysis (PCA) was carried out to assess if there was any temporal spectral variation. At day 28, osteoinduction was confirmed using alizarin red staining and qRT-PCR for alkaline phosphatase and osteocalcin. Alizarin red staining was positive in all samples at day 28 and significant increases in alkaline phosphatase (p < 0.001) and osteocalcin (p < 0.05) gene expression were also observed compared with day 0. PCA of the Raman data demonstrated trends in PC1 from days 0-10, influenced by protein associated features and PC2 from days 10-28, influenced by DNA/RNA associated features. We conclude that spectroscopy can be used to monitor changes in Raman signature with time associated with the osteoinduction of DPSCs using repeated measurements via an aseptic methodology

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway, also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth