Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS), but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs) are shown to effectively direct in vitro differentiation of neural stem cells (NSCs) predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs) treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~. 75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~. 150. ns) micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons) and O4 (for oligodendrocytes), while the expression of GFAP (for astrocytes) remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO) production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives. © 2013.

The importance of science fiction and other STEM-related mass media in young people’s decisions to enrol in university STEM courses

This paper presents Australian results from the Interests and Recruitment in Science (IRIS) study with respect to the influence of STEM-related mass media, including science fiction, on students’ decisions to enrol in university STEM courses. The study found that across the full cohort (N=2999), students tended to attribute far greater influence to science-related documentaries/channels such as Life on Earth and the Discovery Channel, etc. than to science-fiction movies or STEM-related TV dramas. Males were more inclined than females to consider science fiction/fantasy books and films and popular science books/magazines as having been important in their decisions. Students taking physics/astronomy tended to rate the importance of science fiction/fantasy books and films higher than students in other courses. The implications of these results for our understanding of influences on STEM enrolments are discussed.

Wood density : a tool to find complementary species for the design of mixed species plantations

The successful establishment and growth of mixed-species forest plantations requires that complementary or facilitatory species be identified. This can be difficult in many tropical areas because the growth characteristics of endemic species are often unknown, particularly when grown at potentially higher densities in plantations than in natural forests. Here, we investigate whether wood density is a useful and readily accessible trait for choosing complementary species for mixed species plantations. Wood density represents the carbon investment per unit volume of stem with a trade-off generally found between fast (low wood density) and slow (high wood density) growing species. To do this, we use data collected from 18 highly diverse mixed species plantations (4–23 mostly native species) aged from 6 to 11 years at the time of data collection located on Leyte Island, Philippines. We found significant negative correlations between wood densities and the height of the most abundant species, as well as with measures of overall stand growth and tree diameter size distribution. Not only do species with denser woods have slower growth rates, but also mixed-species plantations with higher average wood density and higher stem density were also less productive, at least in these young plantations. Similarly, stands with a high diversity in wood densities were less productive. There is growing interest in making greater use of native multi-species mixtures in smallholder and community planting programs in the tropics, and our results show databases of wood density values may help improve their design. In the early development stages of plantations, canopy closure and rapid height growth are usually key silvicultural targets, and wood density values can predict the rapid height development of species. If plantations are being grown for the livelihood of small landholders then the best target is to choose some species with different wood densities. This allows an early harvest of low-wood density species for early income, and will also reduce competition for slower growing trees with higher wood densities for later income generation.

The attack of the clones: Patent law and stem cell research

This article considers the integral role played by patent law in respect of stem cell research. It highlights concerns about commercialization, access to essential medicines and bioethics. The article maintains that there is a fundamental ambiguity in the Patents Act 1990 (Cth) as to whether stem cell research is patentable subject matter. There is a need to revise the legislation in light of the establishment of the National Stem Cell Centre and the passing of the Research Involving Embryos Act 2002 (Cth). The article raises concerns about the strong patent protection secured by the Wisconsin Alumni Research Foundation and Geron Corporation in respect of stem cell research in the United States. It contends that a number of legal reforms could safeguard access to stem cell lines, and resulting drugs and therapies. Finally, this article explores how ethical concerns are addressed within the framework of the European Biotechnology Directive. It examines the decision of the European Patent Office in relation to the so-called Edinburgh patent, and the inquiry of the European Group on Ethics in Science and New Technologies into The Ethical Aspects of Patenting Involving Human Stem Cells.

Stochastic dynamics of interacting haematopoietic stem cell niche lineages

Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.

Lung cancer stem cells: The root of resistance

In the absence of specific treatable mutations, platinum-based chemotherapy remains the gold standard of treatment for lung cancer patients. However, 5-year survival rates remain poor due to the development of resistance and eventual relapse. Resistance to conventional cytotoxic therapies presents a significant clinical challenge in the treatment of this disease. The cancer stem cell (CSC) hypothesis suggests that tumors are arranged in a hierarchical structure, with the presence of a small subset of stem-like cells that are responsible for tumor initiation and growth. This CSC population has a number of key properties such as the ability to asymmetrically divide, differentiate and self-renew, in addition to having increased intrinsic resistance to therapy. While cytotoxic chemotherapy kills the bulk of tumor cells, CSCs are spared and have the ability to recapitulate the heterogenic tumor mass. The identification of lung CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted therapies that may ultimately improve clinical outcomes in lung cancer patients. This review will focus on lung CSC markers, their role in resistance and their relevance as targets for future therapies.

Characterization of expiration air jets and droplet size distributions immediately at the mouth opening

Size distributions of expiratory droplets expelled during coughing and speaking and the velocities of the expiration air jets of healthy volunteers were measured. Droplet size was measured using the Interferometric Mie imaging (IMI) technique while the Particle Image Velocimetry (PIV) technique was used for measuring air velocity. These techniques allowed measurements in close proximity to the mouth and avoided air sampling losses. The average expiration air velocity was 11.7 m/s for coughing and 3.9 m/s for speaking. Under the experimental setting, evaporation and condensation effects had negligible impact on the measured droplet size. The geometric mean diameter of droplets from coughing was 13.5m and it was 16.0m for speaking (counting 1 to 100). The estimated total number of droplets expelled ranged from 947 – 2085 per cough and 112 – 6720 for speaking. The estimated droplet concentrations for coughing ranged from 2.4 - 5.2cm-3 per cough and 0.004 – 0.223 cm-3 for speaking.

Stem cell technologies : regulation, patents and problems

Human embryonic stem cell research promises to deliver in the future a whole range of therapeutic treatments, but currently governments in different jurisdictions must try to regulate this burgeoning area. Part of the problem has been, and continues to be, polarised community opinion on the use of human embryonic stem cells for research. This article compares the approaches of the Australian, United Kingdom and United States governments in regulating human embryonic stem cell research. To date, these governments have approached the issue through implementing legislation or policy to control research. Similarly, the three jurisdictions have viewed the patentability of human embryonic stem cell technologies in their own ways with different policies being adopted by the three patent offices. This article examines these different approaches and discusses the inevitable concerns that have been raised due to the lack of a universal approach in relation to the regulation of research; the patenting of stem cell technologies; and the effects patents granted are having on further human embryonic stem cell research.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Biomechanical force displacement analysis of strength of fixation of tracker holding devices to bone in computer aided joint replacement

Computer aided joint replacement surgery has become very popular during recent years and is being done in increasing numbers all over the world. The accuracy of the system depends to a major extent, on accurate registration and immobility of the tracker attachment devices to the bone. This study was designed to asses the forces needed to displace the tracker attachment devices in the bone simulators. Bone simulators were used to maintain the uniformity of the bone structure during the study. The fixation devices tested were 3mm diameter self drilling, self tapping threaded pin, 4mm diameter self tapping cortical threaded pin, 5mm diameter self tapping cancellous threaded pin and a triplanar fixation device ‘ortholock’ used with three 3mm pins. All the devices were tested for pull out, translational and rotational forces in unicortical and bicortical fixation modes. Also tested was the normal bang strength and forces generated by leaning on the devices. The forces required to produce translation increased with the increasing diameter of the pins. These were 105N, 185N, and 225N for the unicortical fixations and 130N, 200N, 225N for the bicortical fixations for 3mm, 4mm and 5mm diameter pins respectively. The forces required to pull out the pins were 1475N, 1650N, 2050N for the unicortical, 1020N, 3044N and 3042N for the bicortical fixated 3mm, 4mm and 5mm diameter pins. The ortholock translational and pull out strength was tested to 900N and 920N respectively and still it did not fail. Rotatory forces required to displace the tracker on pins was to the magnitude of 30N before failure. The ortholock device had rotational forces applied up to 135N and still did not fail. The manual leaning forces and the sudden bang forces generated were of the magnitude of 210N and 150N respectively. The strength of the fixation pins increases with increasing diameter from three to five mm for the translational forces. There is no significant difference in pull out forces of four mm and five mm diameter pins though it is more that the three mm diameter pins. This is because of the failure of material at that stage rather than the fixation device. The rotatory forces required to displace the tracker are very small and much less that that can be produced by the surgeon or assistants in single pins. Although the ortholock device was tested to 135N in rotation without failing, one has to be very careful not to put any forces during the operation on the tracker devices to ensure the accuracy of the procedure.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Selective growth of carbon nanotubes on bare silicon : MWNT sinthesys on patterned substrates without predeposition of metal catalyst

Carbon nanotubes (CNTs) are expected to become the ideal constituent of many technologes, in particular for future generation electronics. This considerable interest is due to their unique electrical and mechanical properties. They show indeed super-high current-carrying capacity, ballistic electron transport and good field-emission properties. Then, these superior features make CNTs the most promising building blocks for electronic devices, as organic solar cells and organic light emitting devices (OLED). By using Focused Ion Beam (FIB) patterning it is possible to a obtain a high control on position, relative distances and diameter of CNTs. The present work shows how to grow three-dimensional architecture made of vertical-aligned CNTs directly on silicon. Thanks to the higher activity of a pre-patterned surface the synthesis process results very quick, cheap and simple. Such large area growths of CNTs could be used in preliminary test for application as electrodes for organic solar cells.

Characterisation and culturing of adipose-derived precursor cells

This book focuses on practical applications for using adult and embryonic stem cells in the pharmaceutical development process. It emphasizes new technologies to help overcome the bottlenecks in developing stem cells as therapeutic agents. A key reference for professionals working in stem cell science, it presents the general principles and methodologies in stem cell research and covers topics such as derivitization and characterization of stem cells, stem cell culture and maintenance, stem cell engineering, applications of high-throughput screening, and stem cell genetic modification with their use for drug delivery.

Melt electrospinning of polycaprolactone and its blends with poly(ethylene glycol)

Melt electrospinning is one aspect of electrospinning with relatively little published literature, although the technique avoids solvent accumulation and/or toxicity which is favoured in certain applications. In the study reported, we melt-electrospun blends of poly(ε-caprolactone) (PCL) and an amphiphilic diblock copolymer consisting of poly(ethylene glycol) and PCL segments (PEG-block-PCL). A custom-made electrospinning apparatus was built and various combinations of instrument parameters such as voltage and polymer feeding rate were investigated. Pure PEG-block-PCL copolymer melt electrospinning did not result in consistent and uniform fibres due to the low molecular weight, while blends of PCL and PEG-block-PCL, for some parameter combinations and certain weight ratios of the two components, were able to produce continuous fibres significantly thinner (average diameter of ca 2 µm) compared to pure PCL. The PCL fibres obtained had average diameters ranging from 6 to 33 µm and meshes were uniform for the lowest voltage employed while mesh uniformity decreased when the voltage was increased. This approach shows that PCL and blends of PEG-block-PCL and PCL can be readily processed by melt electrospinning to obtain fibrous meshes with varied average diameters and morphologies that are of interest for tissue engineering purposes. Copyright © 2010 Society of Chemical Industry