Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.

Recent studies using stable isotope labeling with amino acids in culture (SILAC) in quantitative proteomics have made mention of the problematic conversion of isotopecoded arginine to proline in cells. The resulting converted proline peptide divides the heavy peptide ion signal causing inaccuracy when compared with the light peptide ion signal. This is of particular concern as it can effect up to half of all peptides in a proteomic experiment. Strategies to both compensate for and limit the inadvertent conversion have been demonstrated, but none have been shown to prevent it. Additionally, these methods combined with SILAC labeling in general have proven problematic in their large scale application to sensitive cell types including embryonic stem cells (ESCs) from the mouse and human. Here, we show that by providing as little as 200 mg/liter L-proline in SILAC media, the conversion of arginine to proline can be rendered completely undetectable. At the same time, there was no compromise in labeling with isotope-coded arginine, indicating there is no observable back conversion from the proline supplement. As a result, when supplemented with proline, correct interpretation of "light" and "heavy" peptide ratios could be achieved even in the worst cases of conversion. By extending these principles to ESC culture protocols and reagents we were able to routinely SILAC label both mouse and human ESCs in the absence of feeder cells and without compromising the pluripotent phenotype. This study provides the simplest protocol to prevent proline artifacts in SILAC labeling experiments with arginine. Moreover, it presents a robust, feeder cell-free, protocol for performing SILAC experiments on ESCs from both the mouse and the human.

The practicum constitutes an integral part of many professional courses in higher education; and is manifest in several different forms depending on the discipline: field experience, cooperative education, sandwich programs, internships, clerkships, clinical practicum, and the like. This paper provides an overview of different ways in which the practicum has been conceptualised, implemented and evaluated in higher education. It focuses attention on the purpose and value of the practicum; the relationship between the practicum and the learning outcomes of a course as a whole; and the structure and placement of the practicum within a course. Findings indicate that whilst the practicum is widely accepted as a valuable and successful component of professional education, it has a number of shortcomings; and the lack of good quality research into the practicum makes it difficult to draw unequivocal conclusions. A number of questions are posed to guide further research into the role of supervision during practicum placements; the kinds of learning goals and outcomes that are best achieved through the practicum; and the impact on student learning of the length and structure of the practicum.

The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometrybased method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion -mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Human embryonic stem cells (hESCs)1 are non-transformed cell lines that can proliferate indefinitely in culture, although maintaining the potential to form all primary human cell types (pluripotency) (1, 2). These cells, which originate from the inner cell mass of pre-implantation blastocysts, represent a unique source of human cells for cell replacement therapies and for creating model human systems for understanding disease and development (3). Like other mammalian ESCs, hESCs were originally derived and propagated on replication-deficient mouse embryonic fibroblast (MEF) feeder cells in serum (2, 4), with varying efficiencies (5). At the heart of this variability is a lack of understanding of the regulatory pathways and growth factors that govern hESC self-renewal and pluripotency (6). This ambiguity restricts the application of hESCs in both research and therapeutic applications.We hypothesize that under optimal hESC culture conditions, there exist autocrine and paracrine growth factors, produced both by the feeder cells and the hESCs themselves, that establish the complex microenvironment required to retain hESC potential in culture. Previous genomic-based studies suggested the presence of such networks of hESC transcriptional regulation (7); however, these networks were not correlated to the extracellular microenvironment that ultimately controls hESC fate. Moreover, prior attempts to identify proteins within the hESC microenvironment using MSbased approaches produced few potential candidate regulators and provided little new insight or tangible improvements upon hESC line derivation and culture (6, 8 -11).From the ‡Don Rix Protein Identification Facility,

An innovative medical curriculum at the University of New South Wales (UNSW) has been developed through a highly collaborative process aimed at building faculty ownership and ongoing sustainability. The result is a novel capability-based program that features early clinical experience and small-group teaching, which offers students considerable flexibility and achieves a high degree of alignment between graduate outcomes, learning activities and assessments. Graduate capabilities that focus student learning on generic outcomes are described (critical evaluation, reflection, communication and teamwork) along with traditional outcomes in biomedical science, social aspects, clinical performance and ethics. Each two-year phase promotes a distinctive learning process to support and develop autonomous learning across six years. The approaches emphasize important adult education themes: student autonomy; learning from experience; collaborative learning; and adult teacher-learner relationships. Teaching in each phase draws on stages of the human life cycle to provide an explicit organization for the vertical integration of knowledge and skills. A learning environment that values the social nature of learning is fostered through the program's design and assessment system, which supports interdisciplinary integration and rewards students who exhibit self-direction. Assessment incorporates criterion referencing, interdisciplinary examinations, a balance between continuous and barrier assessments, peer feedback and performance assessments of clinical competence. A portfolio examination in each phase, in which students submit evidence of reflection and achievement for each capability, ensures overall alignment.

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The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis.In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MSbased proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrixassociated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach. Molecular & Cellular Proteomics

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