Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δß splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

Microenvironmental regulation of telomerase isoforms in human embryonic stem cells.

Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δß hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.

Does a summative portfolio foster the development of capabilities such as reflective practice and understanding ethics? An evaluation from two medical schools.

Portfolios need to be evaluated to determine whether they encourage students to develop in capabilities such as reflective practice and ethical judgment. The aims of this study were (i) to determine whether preparing a portfolio helps promote students' development in a range of capabilities including understanding ethical and legal principles, reflective practice and effective communication, and (ii) to determine to what extent the format of the portfolio affected the outcome by comparing the experiences of students at two different medical schools. A questionnaire was designed to evaluate undergraduate medical students' experiences of completing a portfolio at two medical schools. A total of 526 (45% response rate) students answered the on-line questionnaire. Students from both medical schools gave the highest ranking for the portfolio as a trigger for reflective practice. 63% of students agreed their portfolio helped them develop reflective practice skills (p < 0.001), whereas only 22% disagreed. 48% of students agreed portfolios helped them understand ethical and legal principles whereas 29% disagreed (p < 0.001). In contrast, only 34% of students thought the portfolio helped them to develop effective communication. Students perceive portfolio preparation as an effective learning tool for the development of capabilities such as understanding ethical and legal principles and reflective practice, whereas other capabilities such as effective communication require complementary techniques and other modes of assessment.

Proteomic analysis of extracellular matrices used in stem cell culture.

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel™. From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.

Proteomics of human embryonic stem cells.

Human embryonic stem cells (hESCs) offer exciting potential in regenerative medicine for the treatment of a host of diseases including cancer, Alzheimer's and Parkinson's disease. They also provide insight into human development and disease and can be used as models for drug discovery and toxicity analyses. The key properties of hESCs that make them so promising for medical use are that they have the ability to self-renew indefinitely in culture and they are pluripotent, which means that they can differentiate into any of more than 200 human cell types. Since proteins are the effectors of cellular processes, it is important to investigate hESC expression at the protein level as well as at the transcript level. In addition, post-translational modifications, such as phosphorylation, may influence the activity of pivotal proteins in hESCs, and this information can only be determined by studying the proteome. In this review, we summarize the results obtained from several proteomics analyses of hESCs that have been reported in the last few years.

Matrigel: a complex protein mixture required for optimal growth of cell culture.

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.

An innovative outcomes-based medical education program built on adult learning principles.

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.