Conceptualising and Teaching Biomedical Uncertainty to Medical Students: an Exploratory Qualitative Study.

Introduction: Certainty/uncertainty in medicine is a topic of popular debate. This study aims to understand how biomedical uncertainty is conceptualised by academic medical educators and how it is taught in a medical school in the UK. Methods: This is an exploratory qualitative study grounded in ethnographic principles. This study is based on 10 observations of teaching sessions and seven semi-structured qualitative interviews with medical educators from various biomedical disciplines in a UK medical school. The data set was analysed via a thematic analysis. Results: Four main themes were identified after analysis: (1) ubiquity of biomedical uncertainty, (2) constraints to teaching biomedical uncertainty, (3) the 'medic filter' and (4) fluid distinction: core versus additional knowledge. While medical educators had differing understandings of how biomedical uncertainty is articulated in their disciplines, its presence was ubiquitous. This ubiquity did not translate into teaching due to time constraints and assessment strategies. The 'medic filter' emerged as a strategy that educators employed to decide what to include in their teaching. They made distinctions between core and additional knowledge which were defined in varied ways across disciplines. Additional knowledge often encapsulated biomedical uncertainty. Discussion: Even though the perspective that knowledge is socially constructed is not novel in medical education, it is neither universally valued nor universally applied. Moving beyond situativity theories and into broader debates in social sciences provides new opportunities to discuss the nature of scientific knowledge in medical education. We invite a move away from situated learning to situated knowledge.

Effects of Surface Chemistry Interaction on Primary Neural Stem Cell Neurosphere Responses.

The characteristics of a material's surface are extremely important when considering their interactions with biological species. Despite surface chemistry playing a critical role in mediating the responses of cells, there remains no single rule which dictates absolute performance; this is particularly challenging when considering the response of differing cell types to a range of materials. Here, we highlight the functional behavior of neural stem cells presented as neurospheres, with respect to a range of alkane-based self-assembled monolayers presenting different functional groups: OH, CO2H, NH2, phenyl, CH3, SH, and laminin. The influence of chemical cues was examined in terms of neurosphere spreading on each of these defined surfaces (cell adhesion and migration capacity) and neuronal versus glial marker expression. Measurements were made over a time series of 3, 5, and 7 days, showing a dynamic nature to the initial responses observed after seeding. While OH surfaces presented an excellent platform for glial migration, larger proportions of cells expressing neuronal ß3-tubulin were found on SH- and laminin-coated surfaces. Axonal elongation was found to be initially similar on all surfaces with neurite lengths having a wider spread predominantly on NH2- and laminin-presenting surfaces. A generalized trend could not be found to correlate cellular responses with surface wettability, lipophilicity (log P), or charge/ionizability (pK a). These results highlight the potential for chemical cues to direct primary neural stem cell responses in contact with the defined materials. New biomaterials which control specific cell culture characteristics in vitro will streamline the up-scale manufacture of cellular therapies, with the enrichment of the required populations resulting from a defined material interaction.

Nicotinamide restricts neural precursor proliferation to enhance catecholaminergic neuronal subtype differentiation from mouse embryonic stem cells.

Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant's risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson's disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide's action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development.

The Influence of Nicotinamide on Health and Disease in the Central Nervous System.

Nicotinamide, the amide form of vitamin B3 (niacin), has long been associated with neuronal development, survival, and function in the central nervous system (CNS), being implicated in both neuronal death and neuroprotection. Here, we summarise a body of research investigating the role of nicotinamide in neuronal health within the CNS, with a focus on studies that have shown a neuroprotective effect. Nicotinamide appears to play a role in protecting neurons from traumatic injury, ischaemia, and stroke, as well as being implicated in 3 key neurodegenerative conditions: Alzheimer's, Parkinson's, and Huntington's diseases. A key factor is the bioavailability of nicotinamide, with low concentrations leading to neurological deficits and dementia and high levels potentially causing neurotoxicity. Finally, nicotinamide's potential mechanisms of action are discussed, including the general maintenance of cellular energy levels and the more specific inhibition of molecules such as the nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin 1 (SIRT1).

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

INTRODUCTION: Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington's disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. METHODS: Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. RESULTS: Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated ßIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons. CONCLUSIONS: Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.

The Role of Vitamin D3 in the Development and Neuroprotection of Midbrain Dopamine Neurons.

Vitamin D has long been synonymous with bone health. More recently, new health benefits are continually being associated with vitamin D, including a burgeoning field on neuroprotective properties. This has generated a huge explosion of interest in recent years in the potential for vitamin D to be used not only as a therapeutic in neurodegenerative disease, including Parkinson's disease, but also as biomarkers and for risk association. With an emphasis on Parkinson's disease, this chapter will discuss recent evidence supporting the assertion that vitamin D can be a useful therapeutic agent used as an intervention therapy to be combined with existing treatments; and the case for further development of novel treatments utilizing the potential of vitamin D. In addition, we present novel, previously unpublished evidence showing that in a unilateral model of Parkinson's disease, vitamin D can not only reduce the extent of denervation, but that this is also reflected in functional benefit to the animals. The potential of vitamin D is slowly being realized; in the future, it will be widely associated with far more than just bone health and may even contribute to an elusive treatment of neurodegenerative illness.

Nicotinamide promotes neuronal differentiation of mouse embryonic stem cells in vitro.

Factors controlling proliferation and differentiation are crucial in advancement of neural cell-based experimental neurodegenerative therapies. In this regard, nicotinamide has been shown to determine the fate of neural cells, enhance neuralization, and influence DNA repair and apoptosis. This study investigated whether the biologically active vitamin B3 metabolite, nicotinamide, could direct the differentiation of mouse embryonic stem cells, cultured as monolayers, into neurons at either early or late stages of development. Interestingly, we observed a dose-responsive increase in the percentage of neurons when nicotinamide was added at early stages to the cells undergoing differentiation (days 0-7). Nicotinamide (10 mM) had a significant effect on neuronal differentiation, increasing the ßIII-tubulin-positive neuronal population and concomitantly decreasing the total number of cells in culture, measured by quantification of 4',6-diamidino-2-phenylindole (DAPI)-positive cells. Nicotinamide added between days 7 and 14 had no effect on neuronal induction. High levels of nicotinamide (20 mM) induced cytotoxicity and cell death. Current work is focusing on elucidating the mechanism(s) mediating neural specification by nicotinamide--that is, induction of cell-cycle exit and/or selective apoptosis in non-neural populations. Preliminary data suggest a reduction in the proportion of proliferating cells in nicotinamide-treated cultures--that is, nicotinamide enhances cell-cycle exit, thereby promoting neuronal differentiation. Future work will focus on evaluating the effect of nicotinamide on the differentiation of midbrain dopamine neurons, towards a therapy for Parkinson's disease.

Calcitriol imparts neuroprotection in vitro to midbrain dopaminergic neurons by upregulating GDNF expression.

During development a tightly controlled signaling cascade dictates the differentiation, maturation and survival of developing neurons. Understanding this signaling mechanism is important for developing therapies for neurodegenerative illnesses. In previous work we have sought to understand the complex signaling pathways responsible for the development of midbrain dopamine neurons using a proteomic approach. One protein we have identified as being expressed in developing midbrain tissue is the vitamin D receptor. Therefore we investigated the effect of the biologically active vitamin D3 metabolite, calcitriol, on primary fetal ventral mesencephalic cultures of dopamine neurons. We observed a dose responsive increase in numbers of rat primary dopamine neurons when calcitriol was added to culture media. Western blot data showed that calcitriol upregulated the expression of glial derived neurotrophic factor (GDNF). Blocking GDNF signaling could prevent calcitriol's ability to increase numbers of dopamine neurons. An apoptosis assay and cell birth dating experiment revealed that calcitriol increases the number of dopamine neurons through neuroprotection and not increased differentiation. This could have implications for future neuroprotective PD therapies.

Stem cell therapy for ischaemic stroke: translation from preclinical studies to clinical treatment.

No pharmacological intervention has been shown convincingly to improve neurological outcome in stroke patients after the brain tissue is infarcted. While conventional therapeutic strategies focus on preventing brain damage, stem cell treatment has the potential to repair the injured brain tissue. Stem cells not only produce a source of trophic molecules to minimize brain damage caused by ischaemia/reperfusion and promote recovery, but also potentially turn to new cells to replace those lost in ischaemic core. Although preclinical studies have shown promise, stem cell therapy for stroke treatment in human is still at an early stage and it is difficult to draw conclusions from current clinical trials about the efficacy of the different treatments used in humans. This article reviews the potential of various types of stem cells, from embryonic to adult to induced pluripotent stem cells, in stroke therapy, highlights new evidence from the ongoing clinical trials and discusses some of the problems associated with translating stem cell technology to a clinical therapy for stroke.

Remote and local control of stimuli responsive materials for therapeutic applications.

Materials offering the ability to change their characteristics in response to presented stimuli have demonstrated application in the biomedical arena, allowing control over drug delivery, protein adsorption and cell attachment to materials. Many of these smart systems are reversible, giving rise to finer control over material properties and biological interaction, useful for various therapeutic treatment strategies. Many smart materials intended for biological interaction are based around pH or thermo-responsive materials, although the use of magnetic materials, particularly in neural regeneration, has increased over the past decade. This review draws together a background of literature describing the design principles and mechanisms of smart materials. Discussion centres on recent literature regarding pH-, thermo-, magnetic and dual responsive materials, and their current applications for the treatment of neural tissue.

Transplanting intact donor tissue enhances dopamine cell survival and the predictability of motor improvements in a rat model of Parkinson's disease.

Primary cell transplantation is currently the gold standard for cell replacement in Parkinson's disease. However, the number of donors needed to treat a single patient is high, and the functional outcome is sometimes variable. The present work explores the possibility of enhancing the viability and/or functionality of small amounts of ventral mesencephalic (VM) donor tissue by reducing its perturbation during preparation and implantation. Briefly, unilaterally lesioned rats received either: (1) an intact piece of half an embryonic day 13 (E13) rat VM; (2) dissociated cells from half an E13 rat VM; or (3) no transplant. D-amphetamine- induced rotations revealed that animals receiving pieces of VM tissue or dissociated cells showed significant improvement in ipsilateral rotation 4 weeks post transplantation. By 6 weeks post transplantation, animals receiving pieces of VM tissue showed a trend for further improvement, while those receiving dissociated cells remained at their 4 week scores. Postmortem cell counts showed that the number of dopaminergic neurons in dissociated cell transplants was significantly lower than that surviving in transplants of intact tissue. When assessing the correlation between the number of dopamine cells in each transplant, and the improvement in rotation bias in experimental animals, it was shown that transplants of whole pieces of VM tissue offered greater predictability of graft function based on their dopamine cell content. Such results suggest that maintaining the integrity of VM tissue during implantation improves dopamine cell content, and that the dopamine cell content of whole tissue grafts offers a more predictable outcome of graft function in an animal model of Parkinson's disease.

GABAergic neurons from mouse embryonic stem cells possess functional properties of striatal neurons in vitro, and develop into striatal neurons in vivo in a mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disease where GABAergic medium spiny neurons (MSNs) in the striatum degenerate. Embryonic stem cell-derived neural transplantation may provide an appropriate therapy for HD. Here we aimed to develop a suitable protocol to obtain a high percentage of functional GABAergic neurons from mouse embryonic stem cells (mESCs), and then tested their differentiation potential in vivo. The monolayer method was compared with the embryoid body and five stage method for its efficiency in generating GABAergic neurons from mESCs. All three methods yielded a similar percentage of GABAergic neurons from mESCs. Monolayer method-derived GABAergic neurons expressed the MSN marker dopamine- and cyclic AMP-regulated phosphoprotein (DARPP32). The pluripotent stem cell population could be eliminated in vitro by treating cells with puromycin and retinoic acid. Using patch-clamp recordings, the functional properties of GABAergic neurons derived from mESCs were compared to GABAergic neurons derived from primary lateral ganglionic eminence. Both types of neurons showed active membrane properties (voltage-gated Na(+) and K(+) currents, Na(+)-dependent action potentials, and spontaneous postsynaptic currents) and possessed functional glutamatergic receptors and transporters. mESC-derived neural progenitors were transplanted into a mouse model of HD. Grafted cells differentiated to mature neurons expressing glutamate decarboxylase, dopamine type 1 receptors, and DARPP32. Also, neural precursors and dividing populations were found in the grafts. In summary, mESCs are able to differentiate efficiently into functional GABAergic neurons using defined in vitro conditions, and these survive and differentiate following grafting to a mouse model of HD.