Human preadipocyte proliferation and differentiation

Obesity represents a major health, social and economic burden to many developing and Westernized communities, with the prevalence increasing at a rate exceeding almost all other medical conditions. Despite major recent advances in our understanding of adipose tissue metabolism and dynamics, we still have limited insight into the regulation of adipose tissue mass in humans. Any significant increase in adipose tissue mass requires proliferation and differentiation of precursor cells (preadipocytes) present in the stromo-vascular compartment of adipose tissue. These processes are very complex and an increasing number of growth factors and hormones have been shown to modulate the expression of genes involved in preadipocyte proliferation and differentiation. A number of transcription factors, including the C/EBP family and PP ARy, have been identified as integral to adipose tissue development and preadipocyte differentiation. Together PP ARy and C/EBPa regulate important events in the activation and maintenance of the terminally differentiated phenotype. The ability of PP ARy to increase transcription through its DNA recognition site is dependent on the binding of ligands. This suggests that an endogenous PP ARy ligand may be an important regulator of adipogenesis. Adipose tissue functions as both the major site of energy storage in the body and as an endocrine organ synthesizing and secreting a number of important molecules involved in regulation of energy balance. For optimum functioning therefore, adipose tissue requires extensive vascularization and previous studies have shown that growth of adipose tissue is preceded by development of a microvascular network. This suggests that paracrine interactions between constituent cells in adipose tissue may be involved in both new capillary formation and fat cell growth. To address this hypothesis the work in this project was aimed at (a) further development of a method for inducing preadipocyte differentiation in subcultured human cells; (b) establishing a method for simultaneous isolation and separate culture of both preadipocytes and microvascular endothelial cells from the same adipose tissue biopsies; (c) to determine, using conditioned medium and co-culture techniques, if endothelial cell-derived factors influence the proliferation and/or differentiation of human preadipocytes; and (d) commence characterization of factors that may be responsible for any observed paracrine effects on aspects of human adipogenesis. Major findings of these studies were as follows: (A) Inclusion of either linoleic acid (a long-chain fatty acid reported to be a naturally occurring ligand for PP ARy) or Rosiglitazone (a member of the thiazolidinedione class of insulin-sensitizing drugs and a synthetic PPARy ligand) in differentiation medium had markedly different effects on preadipocyte differentiation. These studies showed that human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation, and that thiazolidinediones and fatty acids may exert their adipogenic and lipogenic effects via different biochemical pathways. It was concluded that Rosiglitazone is a more potent inducer of human preadipocyte differentiation than linoleic acid. (B) A method for isolation and culture of both endothelial cells and preadipocytes from the same adipose tissue biopsy was developed. Adipose-derived microvascular endothelial cells were found to produce factor/s, which enhance both proliferation and differentiation of human preadipocytes. (C) The adipogenic effects of microvascular endothelial cells can be mimicked by exposure of preadipocytes to members of the Fibroblast Growth Factor family, specifically ~-ECGF and FGF-1. (D) Co-culture of human preadipocytes with endothelial cells or exposure of preadipocytes to either ~-ECGF or FGF-1 were found to 'prime' human preadipocytes, during their proliferative phase of growth, for thiazolidinedione-induced differentiation. (E) FGF -1 was not found to be acting as a ligand for PP ARy in this system. Findings from this project represent a significant step forward in our understanding of factors involved in growth of human adipose tissue and may lead to the development of therapeutic strategies aimed at modifying the process. Such strategies would have potential clinical utility in the treatment of obesity and obesity related disorders such as Type II Diabetes.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

The influence of fibrin based hydrogels on the chondrogenic differentiation of human bone marrow stromal cells

Mesenchymal Stem Cells (MSC) are frequently incorporated into osteochondral implants and cell seeding is often facilitated with hydrogels which exert a profound influence on the chondrogenic differentiation of MSC. An attempt was made to elucidate this effect by comparing the chondrogenic differentiation of Bone Marrow Stromal Cells (BMSC) in fibrin and fibrin alginate composites. A biphasic osteochondral model which simulated the native in vivo environment was employed in the study. In the first stage of the experiment, BMSC was encapsulated in fibrin, Fibrin Alginate 0.3% (FA0.3) and 0.6% (FA0.6). Chondrogenic differentiation within these cell-hydrogel pellets was compared against that of standard cell pellets under inductive conditions and the matrices which supported chondrogenesis were used in the cartilage phase of biphasic constructs. Neo-cartilage growth was monitored in these cocultures. It was observed that hydrogel encapsulation influenced mesenchymal condensation which preceded chondrogenic differentiation. Early cell agglomeration was observed in fibrin as compared to fibrin alginate composites. These fibrin encapsulated cells differentiated into chondrocytes which secreted aggrecan and collagen II. When the alginate content rose from 0.3 to 0.6%, chondrogenic differentiation declined with a reduction in the expression of collagen II and aggrecan. Fibrin and FA0.3 were tested in the cartilage phase of the biphasic osteochondral constructs and the former supported superior cartilage growth with higher cellularity, total Glycosaminoglycan (GAG) and collagen II levels. The FA0.3 cartilage phase was found to be fragmented and partially calcified. The use of fibrin for cartilage repair was advocated as it facilitated BMSC chondrogenesis and cartilaginous growth in an osteochondral environment.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 2 3 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time realtime polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast- like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.

Autoethnography in vocational psychology : wearing your class on your sleeve

This paper addresses reflective practice in research and practice and takes the issue of consciousness of social class in vocational psychology as a working example. It is argued that the discipline’s appreciation of social class can be advanced through application of the qualitative research method autoethnography. Excerpts from an autoethnographic study are used to explore the method’s potential. This reflexive research method is presented as a potential vehicle to improve vocational psychologists’ own class consciousness, and to concomitantly enhance their capacity to grasp social class within their own spheres of research and practice. It is recommended that autoethnography be used for research, training, and professional development for vocational psychologists.

Embracing diversity : empowering preservice teachers for teaching gifted and talented students

This study investigated preservice teachers’ perceptions for teaching and sustaining gifted and talented students while developing, modifying and implementing activities to cater for the diverse learner. Participants were surveyed at the end of a gifted and talented education program on their perceptions to differentiate the curriculum for meeting the needs of the student (n=22). SPSS data analysis with the five-part Likert scale indicated these preservice teachers agreed or strongly agreed they had developed skills in curriculum planning (91%) with well-designed activities (96%), and lesson preparation skills (96%). They also claimed they were enthusiastic for teaching (91%) and understanding of school practices and policies (96%). However, 46% agreed they had knowledge of syllabus documents with 50% claiming an ability to provide written feedback on student’s learning. Furthermore, nearly two-thirds suggested they had educational language from the syllabus and effective student management strategies. Preservice teachers require more direction on how to cater for diversity and begin creating sustainable societies by building knowledge from direct GAT experiences. Designing diagnostic surveys associated with university coursework can be used to determine further development for specific preservice teacher development in GAT education. Preservice teachers need to create opportunities for students to realise their potential by involving cognitive challenges through a differentiated curriculum. Differentiation requires modification of four primary areas of curriculum development (Maker, 1975) content (what we teach), process (how we teach), product (what we expect the students to do or show) and learning environment (where we teach/our class culture). Ashman and Elkins (2009) and Glasson (2008) emphasise the need for preservice teachers, teachers and other professionals to be able to identify what gifted and talented (GAT) students know and how they learn in relation to effective teaching. Glasson (2008) recommends that educators keep up to date with practices in pedagogy, support, monitoring and profiling of GAT students to create an environment conducive to achieving. Oral feedback is one method to communicate to learners about their progress but has advantages and disadvantages for some students. Oral feedback provides immediate information to the student on progress and performance (Ashman & Elkins, 2009). However, preservice teachers must have clear understandings of key concepts to assist the GAT student. Implementing teaching strategies to engage innovate and extend students is valuable to the preservice teacher in focusing on GAT student learning in the classroom (Killen, 2007). Practical teaching strategies (Harris & Hemming, 2008; Tomlinson et al., 1994) facilitate diverse ways for assisting GAT students to achieve learning outcomes. Such strategies include activities to enhance creativity, co-operative learning and problem-solving activities (Chessman, 2005; NSW Department of Education and Training, 2004; Taylor & Milton, 2006) for GAT students to develop a sense of identity, belonging and self esteem towards becoming an autonomous learner. Preservice teachers need to understand that GAT students learn in a different way and therefore should be assessed differently. Assessment can be through diverse options to demonstrate the student’s competence, demonstrate their understanding of the material in a way that highlights their natural abilities (Glasson, 2008; Mack, 2008). Preservice teachers often are unprepared to assess students understanding but this may be overcome with teacher education training promoting effective communication and collaboration in the classroom, including the provision of a variety of assessment strategies to improve teaching and learning (Callahan et al., 2003; Tomlinson et al., 1994). It is also critical that preservice teachers have enthusiasm for teaching to demonstrate inclusion, involvement and the excitement to communicate to GAT students in the learning process (Baum, 2002). Evaluating and reflecting on teaching practices must be part of a preservice teacher’s repertoire for GAT education. Evaluating teaching practices can assist to further enhance student learning (Mayer, 2008). Evaluation gauges the success or otherwise of specific activities and teaching in general (Mayer, 2008), and ensures that preservice teachers and teachers are well prepared and maintain their commitment to their students and the community. Long and Harris (1999) advocate that reflective practices assist teachers in creating improvements in educational practices. Reflective practices help preservice teachers and teachers to improve their ability to pursue improved learning outcomes and professional growth (Long & Harris, 1999). Context This study is set at a small regional campus of a large university in Queensland. As a way to address departmental policies and the need to prepare preservice teachers for engaging a diverse range of learners (see Queensland College of Teachers, Professional Standards for Teachers, 2006), preservice teachers at this campus completed four elective units within their Bachelor of Education (primary) degree. The electives include: 1. Middle years students and schools 2. Teaching strategies for engaging learners 3. Teaching students with learning difficulties, and 4. Middle-years curriculum, pedagogy and assessment. In the university-based component of this unit, preservice teachers engaged in learning about middle years students and schools, and gained knowledge of government policies pertaining to GAT students. Further explored within in this unit was the importance of: collaboration between teachers, parents/carers and school personnel in supporting middle years GAT students; incorporating challenging learning experiences that promoted higher order thinking and problem solving skills; real world learning experiences for students and; the alignment and design of curriculum, pedagogy and assessment that is relevant to the students development, interests and needs. The participants were third-year Bachelor of Education (primary) preservice teachers who were completing an elective unit as part of the middle years of schooling learning with a focus on GAT students. They were assigned one student from a local school. In the six subsequent ninety minute weekly lessons, the preservice teachers were responsible for designing learning activities that would engage and extend the GAT students. Furthermore, preservice teachers made decisions about suitable pedagogical approaches and designed the assessment task to align with the curriculum and the developmental needs of their middle years GAT student. This research aims to describe preservice teachers’ perceptions of their education for teaching gifted and talented students.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.