Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

The role of academic community in higher learning : Alternatives to a drive-thru education

The purpose of our paper is to illustrate the fundamental importance of developing academic community among first year students. We argue that a sense of academic community is of fundamental importance in combating the effects of the neo-liberal economic discourse on higher education, and that the values of higher education are incongruent with those of economic rationalism. The discursive commodification of the student, and of education itself, works against the formation of community, both within the university environment and in the wider society. We argue that, at present, the dominant discourse shaping the social practice of higher education is that of neo-liberal economics. Community values stand in opposition to the dominant discourse, and are integral to the long-term survival of a socially critical and socially responsive society. We conclude that the importance of establishing a sense of academic community during the first year of university is justified by its ultimate value to society.

Book Review: "Rethinking Learning in Early Childhood Education" written by Nicola Yelland, Libby Lee, Maureen O’Rourke & Cathie Harrison

There is a growing body of work that responds to the impact of the rapid uptake of information and communication technology (ICT) on education (Buckingham, 2003; Cheung, 2003; Cuban, 2003; Leung, 2003; Prensky, 2005; Green & Hannon, 2007; Brooks-Gunn & Donahue, 2008; Lyman et al, 2008). Mostly, this work has been positioned in the context of upper-primary or secondary classrooms. More recently, there has been a growing call for research about the impact of ICT on the early years or in early childhood contexts. This text initiates a response to that call. The authors concur that today’s children are a generation who create, learn, work, play and communicate very differently from their parents and teachers (Buckingham, 2003), and that classroom activity needs to reflect this difference.

Ezine and iRadio as knowledge creation metaphors for scaffolding learning in physical and virtual learning spaces

eZine and iRadio represent metaphors for multimedia communication on the Internet. Participating students experience a simulated Internet publishing environment in both their classroom and virtual learning environment. This chapter presents an autoethnographic account highlighting the voices of the learning designer and the teacher and provides evidence of the planning and implementation of two tertiary music elective courses over three iterations of each course. A blended learning environment was incorporated within each elective music course and a collaborative approach to development between lecturers, tutors, learning and technological designers using an iterative research design. The research suggests that learning design which provides real world examples and resources integrating authentic task design into their unit can provide meaningful and engaging experiences for students. The dialogue between learning designers and teachers and iterative review of the learning process and student outcomes, we believe, has engaged students meaningfully to achieve transferable learning outcomes.

Reframing the development of corporate learning

Due to increasing recognition by industry that partnerships with universities can lead to more effective knowledge and skills acquisition and deployment, corporate learning programmes are currently experiencing a resurgence of interest. Rethinking of corporations’ approaches to what has traditionally been classed as ‘training’ has resulted in a new focus on learning and the adoption of philosophies that underlie the academic paradigm. This paper reports on two studies of collaboration between major international engineering corporations and an Australian university, the aim of which was to up-skill the workforce in response to changing markets. The paper highlights the differences between the models of learning adopted in such collaboration and those in more conventional, university-based environments. The learning programmes combine the ADDIE (analysis, design, develop, implement and evaluate) development and workplace learning models. Adaptations that have added value for industry partners and recommendations as to how these can be evolved to cope with change are discussed. The learning is contextualised by industry- based subject matter experts working in close collaboration with university experts and learning designers to develop programmes that are reflective of current and future needs in the organisation. Results derived from user feedback indicate that the learning programmes are effectively aligned with the needs of the industry partners whilst simultaneously upholding academic ideals. In other words, it is possible to combine academic and more traditional approaches to develop corporate learning programmes that satisfy requirements in the workplace. Emerging from the study, a new conceptual framework for the development of corporate learning is presented.

Three Interventions on culture-led regeneration in Gwanju, Korea

These three interventions, given over a three day period in 2010, concern the proposed multifunctional exhibition hall in Gwanju, Korea. The three interventions cover some theoretical and historical issues, but also consider the practical aspects of such a project.

Cities, culture and “Transitional Economies”: Developing cultural industries in St Petersburg

In this chapter I look at some issues around the transfer of cultural industry policy between two very different national contexts, the UK and Russia. Specifically it draws on a partnership project between Manchester and St. Petersburg financed by the European Union as part of a program to promote economic development through knowledge transfer between Europe and the countries of the former Soviet Union. This specific project attempted to place the cultural industries squarely within the dimension of economic development, and drew on the expertise of Manchester’s Creative Industries Development Service and other partners to effect this policy transfer

Yorkshire Cities and Culture : A Review of Current Thinking

This report was commissioned by the Yorkshire Cultural Observatory and the Yorkshire & Humber Key Cities group. It is not a strategy document but an attempt to give an overview of the current thinking within academia and policy-making about the cultural agenda for regions and regional cities in the UK. In particular it looks at the challenges for Yorkshire cities in the context of the current and potential regional cultural offer. The report is a snapshot of current academic and policy thinking, but it also draws on a series of interviews conducted with policymakers in the five key cities as well as regional agencies. These interviews were limited in number and are not meant to be a comprehensive consultation exercise. Rather they acted to focus some of the issues raised by the literature and policy review and to develop suggestions around priority areas for the region.

Building on the past for the future : assessment for learning as a field of exchange

Standardised testing does not recognise the creativity and skills of marginalised youth. This paper presents the development of an innovative approach to assessment designed for the re-engagement of at risk youth who have left formal schooling and are now in an alternative education institution. An electronic portfolio system (EPS) has been developed to capture, record and build on the broad range of students’ cultural and social capital. The assessment as a field of exchange model draws on categories from sociological fields of capital and reconceptualises an eportfolio and social networking hybrid system as a sociocultural zone of learning and development. The EPS, and assessment for learning more generally, are conceptualised as social fields for the exchange of capital (Bourdieu 1977, 1990). The research is underpinned by a sociocultural theoretical perspective that focuses on how students and teachers at the Flexible Learning Centre (FLC) develop and learn, within the zone of proximal development (Vygotsky, 1978). The EPS is seen to be highly effective in the engagement and social interaction between students, teachers and institutions. It is argued throughout this paper that the EPS provides a structurally identifiable space, an arena of social activity, or a field of exchange. The students, teachers and the FLC within this field are producing cultural capital exchanges. The term efield (exchange field) has been coined to refer to this constructed abstract space. Initial results from the trial show a general tendency towards engagement with the EPS and potential for the attainment of socially valued cultural capital in the form of school credentials.

Neural network learning:theoretical foundations

This important work describes recent theoretical advances in the study of artificial neural networks. It explores probabilistic models of supervised learning problems, and addresses the key statistical and computational questions. Chapters survey research on pattern classification with binary-output networks, including a discussion of the relevance of the Vapnik Chervonenkis dimension, and of estimates of the dimension for several neural network models. In addition, Anthony and Bartlett develop a model of classification by real-output networks, and demonstrate the usefulness of classification with a "large margin." The authors explain the role of scale-sensitive versions of the Vapnik Chervonenkis dimension in large margin classification, and in real prediction. Key chapters also discuss the computational complexity of neural network learning, describing a variety of hardness results, and outlining two efficient, constructive learning algorithms. The book is self-contained and accessible to researchers and graduate students in computer science, engineering, and mathematics

Learning the kernel matrix with semidefinite programming

Kernel-based learning algorithms work by embedding the data into a Euclidean space, and then searching for linear relations among the embedded data points. The embedding is performed implicitly, by specifying the inner products between each pair of points in the embedding space. This information is contained in the so-called kernel matrix, a symmetric and positive semidefinite matrix that encodes the relative positions of all points. Specifying this matrix amounts to specifying the geometry of the embedding space and inducing a notion of similarity in the input space - classical model selection problems in machine learning. In this paper we show how the kernel matrix can be learned from data via semidefinite programming (SDP) techniques. When applied to a kernel matrix associated with both training and test data this gives a powerful transductive algorithm -using the labeled part of the data one can learn an embedding also for the unlabeled part. The similarity between test points is inferred from training points and their labels. Importantly, these learning problems are convex, so we obtain a method for learning both the model class and the function without local minima. Furthermore, this approach leads directly to a convex method for learning the 2-norm soft margin parameter in support vector machines, solving an important open problem.

Learning and teaching from the edge to centre stage : Critical factors in embedding sustainable university-wide engagement in external awards and grants funding initiatives.

Teaching awards, grants and fellowships are strategies used to recognise outstanding contributions to learning and teaching, encourage innovation, and to shift learning and teaching from the edge to centre stage. Examples range from school, faculty and institutional award and grant schemes to national schemes such as those offered by the Australian Learning and Teaching Council (ALTC), the Carnegie Foundation for the Advancement of Teaching in the United States, and the Fund for the Development of Teaching and Learning in higher education in the United Kingdom. The Queensland University of Technology (QUT) has experienced outstanding success in all areas of the ALTC funding since the inception of the Carrick Institute for Learning and Teaching in 2004. This paper reports on a study of the critical factors that have enabled sustainable and resilient institutional engagement with ALTC programs. As a lens for examining the QUT environment and practices, the study draws upon the five conditions of the framework for effective dissemination of innovation developed by Southwell, Gannaway, Orrell, Chalmers and Abraham (2005, 2010): 1. Effective, multi-level leadership and management 2. Climate of readiness for change 3. Availability of resources 4. Comprehensive systems in institutions and funding bodies 5. Funding design The discussion on the critical factors and practical and strategic lessons learnt for successful university-wide engagement offer insights for university leaders and staff who are responsible for learning and teaching award, grant and associated internal and external funding schemes.

Multitask learning with expert advice

We consider the problem of prediction with expert advice in the setting where a forecaster is presented with several online prediction tasks. Instead of competing against the best expert separately on each task, we assume the tasks are related, and thus we expect that a few experts will perform well on the entire set of tasks. That is, our forecaster would like, on each task, to compete against the best expert chosen from a small set of experts. While we describe the “ideal” algorithm and its performance bound, we show that the computation required for this algorithm is as hard as computation of a matrix permanent. We present an efficient algorithm based on mixing priors, and prove a bound that is nearly as good for the sequential task presentation case. We also consider a harder case where the task may change arbitrarily from round to round, and we develop an efficient approximate randomized algorithm based on Markov chain Monte Carlo techniques.

A learning-based approach to reactive security

Despite the conventional wisdom that proactive security is superior to reactive security, we show that reactive security can be competitive with proactive security as long as the reactive defender learns from past attacks instead of myopically overreacting to the last attack. Our game-theoretic model follows common practice in the security literature by making worst-case assumptions about the attacker: we grant the attacker complete knowledge of the defender’s strategy and do not require the attacker to act rationally. In this model, we bound the competitive ratio between a reactive defense algorithm (which is inspired by online learning theory) and the best fixed proactive defense. Additionally, we show that, unlike proactive defenses, this reactive strategy is robust to a lack of information about the attacker’s incentives and knowledge.