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.

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.

Correction to “The Importance of Convexity in Learning With Squared Loss” [Sep 98 1974-1980]

The paper "the importance of convexity in learning with squared loss" gave a lower bound on the sample complexity of learning with quadratic loss using a nonconvex function class. The proof contains an error. We show that the lower bound is true under a stronger condition that holds for many cases of interest.

Optimal strategies and minimax lower bounds for online convex games

A number of learning problems can be cast as an Online Convex Game: on each round, a learner makes a prediction x from a convex set, the environment plays a loss function f, and the learner’s long-term goal is to minimize regret. Algorithms have been proposed by Zinkevich, when f is assumed to be convex, and Hazan et al., when f is assumed to be strongly convex, that have provably low regret. We consider these two settings and analyze such games from a minimax perspective, proving minimax strategies and lower bounds in each case. These results prove that the existing algorithms are essentially optimal.

Learning the Kernel Matrix with Semi-Definite 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 definite 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 semi-definite programming (SDP) techniques. When applied to a kernel matrix associated with both training and test data this gives a powerful transductive algorithm -- using the labelled part of the data one can learn an embedding also for the unlabelled 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 to learn the 2-norm soft margin parameter in support vector machines, solving another important open problem. Finally, the novel approach presented in the paper is supported by positive empirical results.

Scoping work integrated learning purposes, practices and issues

A range of terms is used in Australian higher education institutions to describe learning approaches and teaching models that provide students with opportunities to engage in learning connected to the world of work. The umbrella term currently being used widely is Work Integrated Learning (WIL). The common aim of approaches captured under the term WIL is to integrate discipline specific knowledge learnt in university setting with that learnt in the practice of work through purposefully designed curriculum. In endeavours to extend WIL opportunities for students, universities are currently exploring authentic learning experiences, both within and outside of university settings. Some universities describe these approaches as ‘real world learning’ or ‘professional learning’. Others refer to ‘social engagement’ with the community and focus on building social capital and citizenship through curriculum design that enables students to engage with the professions through a range of learning experiences. This chapter discusses the context for, the scope, purposes, characteristics and effectiveness of WIL across Australian universities as derived from a national scoping study. This study, undertaken in response to a high level of interest in WIL, involved data collection from academic and professional staff, and students at nearly all Australian universities. Participants in the study consistently reported the benefits, especially in relation to the student learning experience. Responses highlight the importance of strong partnerships between stakeholders to facilitate effective learning outcomes and a range of issues that shape the quality of approaches and models being adopted, in promoting professional learning.

Work integrated learning for life : encouraging agentic engagement

In a rapidly changing world where new work patterns impact on our health, relationships and social fabric, it is critical that we reconsider the role universities could or should play in helping students prepare for the complexities of the 21st century. Efforts to respond to economic imperatives such as the skills shortage have seen a rush to embed work integrated and career development learning in the curriculum as well as a strengthening of the discourse that the university’s role is primarily to produce industry ready or ‘oven ready and self basting’ graduates (Atkins, 1999). This narrow focus on ‘giving industry what industry wants’ (Patrick, Peach & Pocknee, 2009) ignores the importance of helping students develop the types of skills and dispositions they will need. To enable students to thrive not just survive socially and economically in a radically unknowable world, where knowledge becomes obsolete, we need to be ready to develop new futures (Barnett, 2004). This paper considers the concept of ‘work’, the role it plays in our lives, and our aspirations to build sustainable, socially connected communities. We revisit the assumptions underlying the employability argument (Atkins, 1999) in the light of changing notions of work (Hagel, Seely Brown & Davison, 2010), and the need for higher education to contribute to a better and more sustainable society (Pocock, 2003). Specifically we present initiatives developed from work integrated learning (WIL) programs in the United Kingdom and Australia, where WIL programs are framed within the broader context of real world and life-wide curriculum (Jackson, 2010), and where transferable skills and elements of work-related learning programs prepare students for less certain job futures. Such approaches encourage students to take an agentic role (Billett & Pavlova, 2005) in selecting their work possibilities to develop resilience and capabilities to deal with new and challenging situations, assisting students to become who they want to be not just what they want to be. The theoretical and operational implications and challenges of shaping real world and life-wide curriculum will be investigated in more depth in the next phase of this research.

The WIL (Work Integrated Learning) report : a national scoping study [Final Report]

This report provides an account of the first large-scale scoping study of work integrated learning (WIL) in contemporary Australian higher education. The explicit aim of the project was to identify issues and map a broad and growing picture of WIL across Australia and to identify ways of improving the student learning experience in relation to WIL. The project was undertaken in response to high levels of interest in WIL, which is seen by universities both as a valid pedagogy and as a means to respond to demands by employers for work-ready graduates, and demands by students for employable knowledge and skills. Over a period of eight months of rapid data collection, 35 universities and almost 600 participants contributed to the project. Participants consistently reported the positive benefits of WIL and provided evidence of commitment and innovative practice in relation to enhancing student learning experiences. Participants provided evidence of strong partnerships between stakeholders and highlighted the importance of these relationships in facilitating effective learning outcomes for students. They also identified a range of issues and challenges that face the sector in growing WIL opportunities; these issues and challenges will shape the quality of WIL experiences. While the majority of comments focused on issues involved in ensuring quality placements, it was recognised that placements are just one way to ensure the integration of work with learning. Also, the WIL experience is highly contextualised and impacted by the expectations of students, employers, the professions, the university and government policy.

Malnutrition and clinical outcomes in elderly patients from a Singapore acute hospital

Older adults, especially those acutely ill, are vulnerable to developing malnutrition due to a range of risk factors. The high prevalence and extensive consequences of malnutrition in hospitalised older adults have been reported extensively. However, there are few well-designed longitudinal studies that report the independent relationship between malnutrition and clinical outcomes after adjustment for a wide range of covariates. Acutely ill older adults are exceptionally prone to nutritional decline during hospitalisation, but few reports have studied this change and impact on clinical outcomes. In the rapidly ageing Singapore population, all this evidence is lacking, and the characteristics associated with the risk of malnutrition are also not well-documented. Despite the evidence on malnutrition prevalence, it is often under-recognised and under-treated. It is therefore crucial that validated nutrition screening and assessment tools are used for early identification of malnutrition. Although many nutrition screening and assessment tools are available, there is no universally accepted method for defining malnutrition risk and nutritional status. Most existing tools have been validated amongst Caucasians using various approaches, but they are rarely reported in the Asian elderly and none has been validated in Singapore. Due to the multiethnicity, cultural, and language differences in Singapore older adults, the results from non-Asian validation studies may not be applicable. Therefore it is important to identify validated population and setting specific nutrition screening and assessment methods to accurately detect and diagnose malnutrition in Singapore. The aims of this study are therefore to: i) characterise hospitalised elderly in a Singapore acute hospital; ii) describe the extent and impact of admission malnutrition; iii) identify and evaluate suitable methods for nutritional screening and assessment; and iv) examine changes in nutritional status during admission and their impact on clinical outcomes. A total of 281 participants, with a mean (+SD) age of 81.3 (+7.6) years, were recruited from three geriatric wards in Tan Tock Seng Hospital over a period of eight months. They were predominantly Chinese (83%) and community-dwellers (97%). They were screened within 72 hours of admission by a single dietetic technician using four nutrition screening tools [Tan Tock Seng Hospital Nutrition Screening Tool (TTSH NST), Nutritional Risk Screening 2002 (NRS 2002), Mini Nutritional Assessment-Short Form (MNA-SF), and Short Nutritional Assessment Questionnaire (SNAQ©)] that were administered in no particular order. The total scores were not computed during the screening process so that the dietetic technician was blinded to the results of all the tools. Nutritional status was assessed by a single dietitian, who was blinded to the screening results, using four malnutrition assessment methods [Subjective Global Assessment (SGA), Mini Nutritional Assessment (MNA), body mass index (BMI), and corrected arm muscle area (CAMA)]. The SGA rating was completed prior to computation of the total MNA score to minimise bias. Participants were reassessed for weight, arm anthropometry (mid-arm circumference, triceps skinfold thickness), and SGA rating at discharge from the ward. The nutritional assessment tools and indices were validated against clinical outcomes (length of stay (LOS) >11days, discharge to higher level care, 3-month readmission, 6-month mortality, and 6-month Modified Barthel Index) using multivariate logistic regression. The covariates included age, gender, race, dementia (defined using DSM IV criteria), depression (defined using a single question “Do you often feel sad or depressed?”), severity of illness (defined using a modified version of the Severity of Illness Index), comorbidities (defined using Charlson Comorbidity Index, number of prescribed drugs and admission functional status (measured using Modified Barthel Index; MBI). The nutrition screening tools were validated against the SGA, which was found to be the most appropriate nutritional assessment tool from this study (refer section 5.6) Prevalence of malnutrition on admission was 35% (defined by SGA), and it was significantly associated with characteristics such as swallowing impairment (malnourished vs well-nourished: 20% vs 5%), poor appetite (77% vs 24%), dementia (44% vs 28%), depression (34% vs 22%), and poor functional status (MBI 48.3+29.8 vs 65.1+25.4). The SGA had the highest completion rate (100%) and was predictive of the highest number of clinical outcomes: LOS >11days (OR 2.11, 95% CI [1.17- 3.83]), 3-month readmission (OR 1.90, 95% CI [1.05-3.42]) and 6-month mortality (OR 3.04, 95% CI [1.28-7.18]), independent of a comprehensive range of covariates including functional status, disease severity and cognitive function. SGA is therefore the most appropriate nutritional assessment tool for defining malnutrition. The TTSH NST was identified as the most suitable nutritional screening tool with the best diagnostic performance against the SGA (AUC 0.865, sensitivity 84%, specificity 79%). Overall, 44% of participants experienced weight loss during hospitalisation, and 27% had weight loss >1% per week over median LOS 9 days (range 2-50). Wellnourished (45%) and malnourished (43%) participants were equally prone to experiencing decline in nutritional status (defined by weight loss >1% per week). Those with reduced nutritional status were more likely to be discharged to higher level care (adjusted OR 2.46, 95% CI [1.27-4.70]). This study is the first to characterise malnourished hospitalised older adults in Singapore. It is also one of the very few studies to (a) evaluate the association of admission malnutrition with clinical outcomes in a multivariate model; (b) determine the change in their nutritional status during admission; and (c) evaluate the validity of nutritional screening and assessment tools amongst hospitalised older adults in an Asian population. Results clearly highlight that admission malnutrition and deterioration in nutritional status are prevalent and are associated with adverse clinical outcomes in hospitalised older adults. With older adults being vulnerable to risks and consequences of malnutrition, it is important that they are systematically screened so timely and appropriate intervention can be provided. The findings highlighted in this thesis provide an evidence base for, and confirm the validity of the current nutrition screening and assessment tools used among hospitalised older adults in Singapore. As the older adults may have developed malnutrition prior to hospital admission, or experienced clinically significant weight loss of >1% per week of hospitalisation, screening of the elderly should be initiated in the community and continuous nutritional monitoring should extend beyond hospitalisation.

Domains of rural social work practice

Even though there is substantial agreement about the nature of rural contexts, practice principles, and factors influencing practice we still do not have a framework for organising this knowledge in a way that can directly inform the practitioner in their day-to-day work. In this paper, we introduce the concepts 'practice domains', 'domain location', and 'domain alignment' that, taken together, provide such a framework. We suggest that each practitioner works within a number of practice domains. A domain is a discourse about practice comprising narratives about how a social worker should practise and which factors they should take most account of in their practice decision making. Each practitioner, and each practice process, can be located somewhere within each domain (domain location) and also situated amongst domains according to their relative alignment with each of them (domain alignment). In this paper, we present this framework and show how it is useful for practitioners in understanding practice, identifying factors influencing it, and making practice decisions in immediate, concrete situations.