Co-producing Randomized Controlled Trials: How Do We Work Together?

In the light of the declaration “Nothing about us without us” (Charlton, 2000), interest in co-production, and coproduced research is expanding. Good work has been done establishing principles for co-production (Hickey et al., 2018) and for good quality involvement (Involve, 2013; 4Pi, 2015) and describing how this works in practice in mental health research (Gillard et al., 2012a,b, 2013). In the published literature, co-production has worked well in qualitative research projects in which there is often methodological flexibility. However, to change treatment guidelines in the UK, e.g., the National Institute for Health and Care Excellence guidelines, and influence service commissioning, high quality quantitative research is also needed. This type of research is characterized by formal methodological rules, which pose challenges for the scope of co-production. In this paper we describe the significant challenges and solutions we adopted to design and deliver a coproduced randomized controlled trial of mental health peer support. Given the methodological rigidity of a randomized controlled trial, establishing clearly which methodological and practical decisions and processes can be coproduced, by whom, and how, has been vital to our ongoing co-production as the project has progressed and the team has expanded. Creating and maintaining space for the supported dialogue, reflection, and culture that co-production requires has been vital. This paper aims to make our learning accessible to a wide audience of people developing co-production of knowledge in this field

Autonomous object harvesting using synchronized optoelectronic microrobots

Optoelectronic tweezer-driven microrobots (OETdMs) are a versatile micromanipulation technology based on the application of light induced dielectrophoresis to move small dielectric structures (microrobots) across a photoconductive substrate. The microrobots in turn can be used to exert forces on secondary objects and carry out a wide range of micromanipulation operations, including collecting, transporting and depositing microscopic cargos. In contrast to alternative (direct) micromanipulation techniques, OETdMs are relatively gentle, making them particularly well suited to interacting with sensitive objects such as biological cells. However, at present such systems are used exclusively under manual control by a human operator. This limits the capacity for simultaneous control of multiple microrobots, reducing both experimental throughput and the possibility of cooperative multi-robot operations. In this article, we describe an approach to automated targeting and path planning to enable open-loop control of multiple microrobots. We demonstrate the performance of the method in practice, using microrobots to simultaneously collect, transport and deposit silica microspheres. Using computational simulations based on real microscopic image data, we investigate the capacity of microrobots to collect target cells from within a dissociated tissue culture. Our results indicate the feasibility of using OETdMs to autonomously carry out micromanipulation tasks within complex, unstructured environments

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

Characterization of primary neurospheres generated from mouse ventral rostral hindbrain

Serotonergic (5-HT) neurons of the reticular formation play a key role in the modulation of behavior, and their dysfunction is associated with severe neurological and psychiatric disorders, such as depression and schizophrenia. However, the molecular mechanisms underlying the differentiation of the progenitor cells and the specification of the 5-HT phenotype are not fully understood. A primary neurosphere cell-culture system from mouse ventral rostral hindbrain at embryonic day 12 was therefore established. The generated primary neurospheres comprised progenitor cells and fully differentiated neurons. Bromodeoxyuridine incorporation experiments in combination with immunocytochemistry for neural markers revealed the proliferation capacity of the neural multipotent hindbrain progenitors within neurospheres and their ability to differentiate toward the neuronal lineage and serotonergic phenotype. Gene expression analysis by reverse transcription with the polymerase chain reaction showed that the neurospheres were regionally specified, as reflected by the expression of the transcription factors Gata2 and Pet1. Treatment of dissociated primary neurospheres with exogenous Shh significantly increased the number of 5-HT-immunopositive cells compared with controls, whereas neutralization of endogenous Shh significantly decreased the number of 5-HT neurons. Thus, the primary neurosphere culture system presented here allows the expansion of hindbrain progenitor cells and the experimental control of their differentiation toward the serotonergic phenotype. This culture system is therefore a useful model for in vitro studies dealing with the development of 5-HT neurons

Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation

Stem cells persist throughout life by self-renewing in numerous tissues including the central(1) and peripheral(2) nervous systems. This raises the issue of whether there is a conserved mechanism to effect self-renewing divisions. Deficiency in the polycomb family transcriptional repressor Bmi-1 leads to progressive postnatal growth retardation and neurological defects(3). Here we show that Bmi-1 is required for the self-renewal of stem cells in the peripheral and central nervous systems but not for their survival or differentiation. The reduced self-renewal of Bmi-1-deficient neural stem cells leads to their postnatal depletion. In the absence of Bmi-1, the cyclin-dependent kinase inhibitor gene p16(Ink4a) is upregulated in neural stem cells, reducing the rate of proliferation. p16(Ink4a) deficiency partially reverses the self-renewal defect in Bmi-1(-/-) neural stem cells. This conserved requirement for Bmi-1 to promote self-renewal and to repress p16(Ink4a) expression suggests that a common mechanism regulates the self-renewal and postnatal persistence of diverse types of stem cell. Restricted neural progenitors from the gut and forebrain proliferate normally in the absence of Bmi-1. Thus, Bmi-1 dependence distinguishes stem cell self-renewal from restricted progenitor proliferation in these tissues.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62726/1/nature02060.pd

Developing and testing a principle-based fidelity index for peer support in mental health services

Purpose Evidence suggests that the distinctive relational qualities of peer support—compared to clinical-patient relationships—can be eroded in regulated healthcare environments. Measurement of fidelity in trials of peer support is lacking. This paper reports the development and testing of a fidelity index for one-to-one peer support in mental health services, designed to assess fidelity to principles that characterise the distinctiveness of peer support. Methods A draft index was developed using expert panels of service user researchers and people doing peer support, informed by an evidence-based, peer support principles framework. Two rounds of testing took place in 24 mental health services providing peer support in a range of settings. Fidelity was assessed through interviews with peer workers, their supervisors and people receiving peer support. Responses were tested for spread and internal consistency, independently double rated for inter-rater reliability, with feedback from interviewees and service user researchers used to refine the index. Results A fidelity index for one-to-one peer support in mental health services was produced with good psychometric properties. Fidelity is assessed in four principle-based domains; building trusting relationships based on shared lived experience; reciprocity and mutuality; leadership, choice and control; building strengths and making connections to community. Conclusions The index offers potential to improve the evidence base for peer support in mental health services, enabling future trials to assess fidelity of interventions to peer support principles, and service providers a means of ensuring that peer support retains its distinctive qualities as it is introduced into mental health services

Asymmetric and symmetric stem-cell divisions in development and cancer

Much has been made of the idea that asymmetric cell division is a defining characteristic of stem cells that enables them to simultaneously perpetuate themselves (self-renew) and generate differentiated progeny. Yet many stem cells can divide symmetrically, particularly when they are expanding in number during development or after injury. Thus, asymmetric division is not necessary for stem-cell identity but rather is a tool that stem cells can use to maintain appropriate numbers of progeny. The facultative use of symmetric or asymmetric divisions by stem cells may be a key adaptation that is crucial for adult regenerative capacity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62868/1/nature04956.pd

CD133+ adult human retinal cells remain undifferentiated in Leukaemia Inhibitory Factor (LIF)

<p>Abstract</p> <p>Background</p> <p>CD133 is a cell surface marker of haematopoietic stem and progenitor cells. Leukaemia inhibitory factor (LIF), sustains proliferation and not differentiation of embryonic stem cells. We used CD133 to purify adult human retinal cells and aimed to determine what effect LIF had on these cultures and whether they still had the ability to generate neurospheres.</p> <p>Methods</p> <p>Retinal cell suspensions were derived from adult human post-mortem tissue with ethical approval. With magnetic automated cell sorting (MACS) CD133⁺retinal cells were enriched from post mortem adult human retina. CD133⁺retinal cell phenotype was analysed by flow cytometry and cultured cells were observed for proliferative capacity, neuropshere generation and differentiation with or without LIF supplementation.</p> <p>Results</p> <p>We demonstrated purification (to 95%) of CD133⁺cells from adult human postmortem retina. Proliferating cells were identified through BrdU incorporation and expression of the proliferation markers Ki67 and Cyclin D1. CD133⁺retinal cells differentiated whilst forming neurospheres containing appropriate lineage markers including glia, neurons and photoreceptors. LIF maintained CD133⁺retinal cells in a proliferative and relatively undifferentiated state (Ki67, Cyclin D1 expression) without significant neurosphere generation. Differentiation whilst forming neurospheres was re-established on LIF withdrawal.</p> <p>Conclusion</p> <p>These data support the evidence that CD133 expression characterises a population of cells within the resident adult human retina which have progenitor cell properties and that their turnover and differentiation is influenced by LIF. This may explain differences in retinal responses observed following disease or injury.</p

The Fetal Hypothalamus Has the Potential to Generate Cells with a Gonadotropin Releasing Hormone (GnRH) Phenotype

Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation