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.

Medical science and the law

Advances in medical science have presented both law and bioethics with some of the most fascinating questions of our time. As science continues to forge ahead into new frontiers, in fields such as reproductive technology, human genetics, cloning technologies, and stem cell research, questions have arisen over the role for law in regulating this new terrain. The speed with which medical science has advanced, and continues to advance, can make it difficult to formulate appropriate regulatory responses. The rapid pace of scientific change and the increasing complexity of the science can present hurdles and barriers to the engagement of the public with science and the legal and ethical issues raised by it.

Inventing life: Intellectual property and the new biology

In 2009, the National Research Council of the National Academies released a report on A New Biology for the 21st Century. The council preferred the term ‘New Biology’ to capture the convergence and integration of the various disciplines of biology. The National Research Council stressed: ‘The essence of the New Biology, as defined by the committee, is integration—re-integration of the many sub-disciplines of biology, and the integration into biology of physicists, chemists, computer scientists, engineers, and mathematicians to create a research community with the capacity to tackle a broad range of scientific and societal problems.’ They define the ‘New Biology’ as ‘integrating life science research with physical science, engineering, computational science, and mathematics’. The National Research Council reflected: 'Biology is at a point of inflection. Years of research have generated detailed information about the components of the complex systems that characterize life––genes, cells, organisms, ecosystems––and this knowledge has begun to fuse into greater understanding of how all those components work together as systems. Powerful tools are allowing biologists to probe complex systems in ever greater detail, from molecular events in individual cells to global biogeochemical cycles. Integration within biology and increasingly fruitful collaboration with physical, earth, and computational scientists, mathematicians, and engineers are making it possible to predict and control the activities of biological systems in ever greater detail.' The National Research Council contended that the New Biology could address a number of pressing challenges. First, it stressed that the New Biology could ‘generate food plants to adapt and grow sustainably in changing environments’. Second, the New Biology could ‘understand and sustain ecosystem function and biodiversity in the face of rapid change’. Third, the New Biology could ‘expand sustainable alternatives to fossil fuels’. Moreover, it was hoped that the New Biology could lead to a better understanding of individual health: ‘The New Biology can accelerate fundamental understanding of the systems that underlie health and the development of the tools and technologies that will in turn lead to more efficient approaches to developing therapeutics and enabling individualized, predictive medicine.’ Biological research has certainly been changing direction in response to changing societal problems. Over the last decade, increasing awareness of the impacts of climate change and dwindling supplies of fossil fuels can be seen to have generated investment in fields such as biofuels, climate-ready crops and storage of agricultural genetic resources. In considering biotechnology’s role in the twenty-first century, biological future-predictor Carlson’s firm Biodesic states: ‘The problems the world faces today – ecosystem responses to global warming, geriatric care in the developed world or infectious diseases in the developing world, the efficient production of more goods using less energy and fewer raw materials – all depend on understanding and then applying biology as a technology.’ This collection considers the roles of intellectual property law in regulating emerging technologies in the biological sciences. Stephen Hilgartner comments that patent law plays a significant part in social negotiations about the shape of emerging technological systems or artefacts: 'Emerging technology – especially in such hotbeds of change as the life sciences, information technology, biomedicine, and nanotechnology – became a site of contention where competing groups pursued incompatible normative visions. Indeed, as people recognized that questions about the shape of technological systems were nothing less than questions about the future shape of societies, science and technology achieved central significance in contemporary democracies. In this context, states face ongoing difficulties trying to mediate these tensions and establish mechanisms for addressing problems of representation and participation in the sociopolitical process that shapes emerging technology.' The introduction to the collection will provide a thumbnail, comparative overview of recent developments in intellectual property and biotechnology – as a foundation to the collection. Section I of this introduction considers recent developments in United States patent law, policy and practice with respect to biotechnology – in particular, highlighting the Myriad Genetics dispute and the decision of the Supreme Court of the United States in Bilski v. Kappos. Section II considers the cross-currents in Canadian jurisprudence in intellectual property and biotechnology. Section III surveys developments in the European Union – and the interpretation of the European Biotechnology Directive. Section IV focuses upon Australia and New Zealand, and considers the policy responses to the controversy of Genetic Technologies Limited’s patents in respect of non-coding DNA and genomic mapping. Section V outlines the parts of the collection and the contents of the chapters.

The last taboo: Patenting human beings

This article considers the efforts of the Australian Law Reform Commission to clarify the meaning of section 18(2) of the Australian Patents Act 1990 (Cth): ’Human beings and the biological processes for their generation are not patentable inventions.' It provides a critique of the proposals of the Commission with respect to patent law and stem cell research. The Commission has recommended that IP Australia should develop examination guidelines to explain how the criteria for patentability apply to inventions involving stem cell technologies. It has advised the Australian Government that the practice code of the United Kingdom Patent Office (UKPO) would be a good model for such guidelines, with its distinction between totipotent and pluripotent stem cells. Arguably, though, there is a need to codify this proposal in a legislative directive, and not merely in examination guidelines. The Commission has been reluctant to take account of the ethical considerations with respect to patent law and stem cell research. There could be greater scope for such considerations, by the use of expert advisory boards, opposition proceedings and the requirement of informed consent. The Commission has put forward a number of general and specific recommendations to enhance access to patented stem cell technologies. It recommends the development of a research exemption, and the modernisation of compulsory licensing and crown use provisions. It also explores the establishment of a stem cell bank and the promulgation of guidelines by funding agencies. Such proposals to promote greater public access to stem cell research are to be welcomed.

Intellectual property and emerging technologies: The new biology

This unique and comprehensive collection investigates the challenges posed to intellectual property by recent paradigm shifts in biology. It explores the legal ramifications of emerging technologies, such as genomics, synthetic biology, stem cell research, nanotechnology, and biodiscovery. Extensive contributions examine recent controversial court decisions in patent law – such as Bilski v. Kappos, and the litigation over Myriad’s patents in respect of BRCA1 and BRCA2 – while other papers explore sui generis fields, such as access to genetic resources, plant breeders' rights, and traditional knowledge. The collection considers the potential and the risks of the new biology for global challenges – such as access to health-care, the protection of the environment and biodiversity, climate change, and food security. It also considers Big Science projects – such as biobanks, the 1000 Genomes Project, and the Doomsday Vault. The inter-disciplinary research brings together the work of scholars from Australia, Canada, Europe, the UK and the US and involves not only legal analysis of case law and policy developments, but also historical, comparative, sociological, and ethical methodologies. Intellectual Property and Emerging Technologies will appeal to policy-makers, legal practitioners, business managers, inventors, scientists and researchers.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Generation and characterisation of Cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature

Introduction: Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods: An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results: Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion: Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. © 2013 Barr et al.

Research needed on cultural capital, STEM endeavours and instructional innovations : a call for "collaborations between nations"

New Australian curriculum documents and government initiatives advocate the inclusion of Asian perspectives, which is highly relevant to the STEM fields. For Australia and other countries, STEM education is an opportunity to develop competencies towards employment in high-demand areas, yet the world’s knowledge of STEM is changing rapidly, requiring continuous analysis to meet market demands. This paper presents the need for “collaborations between nations” through research to advance each country’s STEM agenda towards further globalisation of education with the sharing of knowledge. Research is needed on views of what constitutes cultural capital for STEM, which also involves understanding past and current STEM endeavours occurring within various countries. Most importantly for STEM education is uncovering instructional innovations aligned with countries’ cultures and STEM endeavours. Research questions are provided in this paper to stimulate ideas for investigating in these fields. Economically, and as demonstrated recently by Greece and Spain, countries throughout the world can no longer operate independently for advancing standards of living. The world needs to recognise interdependence not only in trade and resources but also through the knowledge base that exists within countries. Learning together globally means transitioning from independence to interdependence in STEM education that will help each country meet global demands.

Mesenchymal stem cells and nano-structured surfaces

Mesenchymal stem cells (MSCs) represent multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). Their multi-potency provides a great promise as a cell source for tissue engineering and cell-based therapy for many diseases, particularly bone diseases and bone formation. To be able to direct and modulate the differentiation of MSCs into the desired cell types in situ in the tissue, nanotechnology is introduced and used to facilitate or promote cell growth and differentiation. These nano-materials can provide a fine structure and tuneable surface in nanoscales to help the cell adhesion and promote the cell growth and differentiation of MSCs. This could be a dominant direction in future for stem cells based therapy or tissue engineering for various diseases. Therefore, the isolation, manipulation, and differentiation of MSCs are very important steps to make meaningful use of MSCs for disease treatments. In this chapter, we have described a method of isolating MSC from human bone marrow, and how to culture and differentiate them in vitro. We have also provided research methods on how to use MSCs in an in vitro model and how to observe MSC biological response on the surface of nano-scaled materials.

Toward biomimetic materials in bone regeneration : functional behavior of mesenchymal stem cells on a broad spectrum of extracellular matrix components

Bone defect treatments can be augmented by mesenchymal stem cell (MSC) based therapies. MSC interaction with the extracellular matrix (ECM) of the surrounding tissue regulates their functional behavior. Understanding of these specific regulatory mechanisms is essential for the therapeutic stimulation of MSC in vivo. However, these interactions are presently only partially understood. This study examined in parallel, for the first time, the effects on the functional behavior of MSCs of 13 ECM components from bone, cartilage and hematoma compared to a control protein, and hence draws conclusions for rational biomaterial design. ECM components specifically modulated MSC adhesion, migration, proliferation, and osteogenic differentiation, for example, fibronectin facilitated migration, adhesion, and proliferation, but not osteogenic differentiation, whereas fibrinogen enhanced adhesion and proliferation, but not migration. Subsequently, the integrin expression pattern of MSCs was determined and related to the cell behavior on specific ECM components. Finally, on this basis, peptide sequences are reported for the potential stimulation of MSC functions. Based on the results of this study, ECM component coatings could be designed to specifically guide cell functions.

Progress in epithelial-mesenchymal transition research

The field of research of epithelial-mesenchymal transitions, EMT, and its reverse, mesenchymal-epithelial transitions, MET, has expanded very rapidly indeed from its beginnings, heralded by Professor Betty Hay in the 1970s and 1980s. This expansion has involved the realisation that the EMT was not just an interesting phenomenon of early developmental morphogenetic cell behaviour, but bore remarkable resemblance to clinically crucial pathological events in cancer invasion. Not surprisingly, this discipline soon became numerically dominant in the EMT publication field. Simultaneously, the EMT concept has been extended to normal physiological wound healing. Exploration revealed that these resemblances were more than skin deep: the same sets of growth factors, receptors, transcription factors, epigenetic marks and signalling pathways turned up repeatedly in EMTs and METs in a variety of contexts, both pathological and normal. This molecular genetic research in turn uncovered similarities of the EMT signature to that of fibrosis, a set of diseases which is of enormous clinical importance, rivalling that of cancer. Most recently, and more surprisingly, the EMT signature has shown considerable similarity to that found in stem cell and cancer stem cell biology.

Targeted disruption of the JAK2/STAT3 pathway in combination with systemic administration of paclitaxel inhibits the priming of ovarian cancer stem cells leading to a reduced tumor burden

Chemotherapy resistance associated with recurrent disease is the major cause of poor survival of ovarian cancer patients. We have recently demonstrated activation of the JAK2/STAT3 pathway and the enhancement of a cancer stem cell (CSC)-like phenotype in ovarian cancer cells treated in vitro with chemotherapeutic agents. To elucidate further these mechanisms in vivo,we used a two-tiered paclitaxel treatment approach in nude mice inoculated with ovarian cancer cells. In the first approach, we demonstrate that a single intraperitoneal administration of paclitaxel in mice 7 days after subcutaneous transplantation of the HEY ovarian cancer cell line resulted in a significant increase in the expression of CA125, Oct4, and CD117 in mice xenografts compared to control mice xenografts which did not receive paclitaxel. In the second approach, mice were administered once weekly with paclitaxel and/or a daily dose of the JAK2-specific inhibitor, CYT387, over 4weeks. Mice receiving paclitaxel only demonstrated a significant decrease in tumor volume compared to control mice. At the molecular level, mouse tumors remaining after paclitaxel administration showed a significant increase in the expression of Oct4 and CD117 coinciding with a significant activation of the JAK2/STAT3 pathway compared to control tumors. The addition of CYT387 with paclitaxel resulted in the suppression of JAK2/STAT3 activation and abrogation of Oct4 and CD117 expression in mouse xenografts. This coincided with significantly smaller tumors in mice administered CYT387 in addition to paclitaxel, compared to the control group and the group of mice receiving paclitaxel only. These data suggest that the systemic administration of paclitaxel enhances Oct4- and CD117-associated CSC-like marker expression in surviving cancer cells in vivo, which can be suppressed by the addition of the JAK2-specific inhibitor CYT387, leading to a significantly smaller tumor burden. These novel findings have the potential for the development of CSC-targeted therapy to improve the treatment outcomes of ovarian cancer patients.

Mesenchymal stem cells as mediators of the neuronal cell niche

This study examined the role of heparan sulfate proteoglycans (HSPGs) in neural lineage differentiation of human mesenchymal stem cells (hMSCs). Several HSPGs were identified as potential new targets controlling neural fate specification and may be applied to the development of improved models to examine and repair brain damage. hMSCs were characterised throughout extended in vitro expansion for neural lineage potential (neurons, astrocytes, oligodendrocytes) and differentiated using terminal differentiation and intermediate sphere formation. Brain damage and neurological disorders caused by injury or disease affect a large number of people often resulting in lifelong disabilities. Multipotent mesenchymal stem cells have a large capacity for self-renewal and provide an excellent model to examine the regulation and contribution of both stem cells and their surrounding microenvironment to the repair of neural tissue damage.

Cancer stem cells in oesophageal squamous cell carcinoma: Identification, prognostic and treatment perspectives

Cancer stem cells (CSCs) are a vital subpopulation of cells to target for the treatment of cancers. In oesophageal squamous cell carcinoma (ESCC), there are several markers such as CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, Side population (SP), CD271 and CD90 that have been proposed to identify the cancer stem cells in individual cancer masses. It has also been demonstrated that stem cell markers like ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133 and podoplanin are associated with patient's prognosis, pathological stages, cancer recurrence and therapy resistance. Finding new cancer stem cell targets or designing drugs to manipulate the known molecular targets in CSCs could be useful for improvements in clinical outcomes of the disease. To conclude, data suggest that CSCs in oesophageal squamous cell carcinoma are related to resistance to therapy and poor prognosis of patients with ESCC. Therefore, innovative insights into CSC biology and CSC-targeted therapies will help to achieve more effective management of patients with oesophageal squamous cell carcinoma.

Concise review: Humanized models of tumor immunology in the 21st century: Convergence of cancer research and tissue engineering

Despite positive testing in animal studies, more than 80% of novel drug candidates fail to proof their efficacy when tested in humans. This is primarily due to the use of preclinical models that are not able to recapitulate the physiological or pathological processes in humans. Hence, one of the key challenges in the field of translational medicine is to “make the model organism mouse more human.” To get answers to questions that would be prognostic of outcomes in human medicine, the mouse's genome can be altered in order to create a more permissive host that allows the engraftment of human cell systems. It has been shown in the past that these strategies can improve our understanding of tumor immunology. However, the translational benefits of these platforms have still to be proven. In the 21st century, several research groups and consortia around the world take up the challenge to improve our understanding of how to humanize the animal's genetic code, its cells and, based on tissue engineering principles, its extracellular microenvironment, its tissues, or entire organs with the ultimate goal to foster the translation of new therapeutic strategies from bench to bedside. This article provides an overview of the state of the art of humanized models of tumor immunology and highlights future developments in the field such as the application of tissue engineering and regenerative medicine strategies to further enhance humanized murine model systems.