Mechanistic and metabolic insights into bioengineering the bone marrow niche in vitro

Stem cell balance of proliferation, differentiation and self-renewal, is regulated by the microenvironment in which they reside, termed the stem cell niche (Schofield, 1978). Niche microenvironments provide physical and functional regulatory cues that control fundamental cell intrinsic and extrinsic mechanisms. In adults, the process of haematopoiesis is sustained by a population of haematopoietic stem cells (HSCs) that are found primarily in the bone marrow (BM) (Jagannathan-Bogdan and Zon, 2013). The BM niche also houses populations of mesenchymal stromal and perivascular cells (MSPCs), that are themselves regulated by the niche, and are fundamental cellular constituents in HSC regulation (Pinho and Frenette, 2019). Both HSCs and MSPCs hold enormous clinical potential. HSCs have the ability to reconstitute the entire blood and immune system (Jagannathan-Bogdan and Zon, 2013), whereas MSPCs contain immunosuppression capacity and have the ability to regenerate damaged and diseased tissue (Caplan, 1991; Uccelli et al., 2008). However, there are still important hurdles that must be overcome before the potential of these cells are fully realised. The regenerative capacity of these stem cells is quickly lost upon ex vivo culture, meaning achieving clinically relevant numbers of cells is challenging (Dalby et al., 2018; Zon, 2008). Although BM MSPCs (such as nestin+ MSPCs) contain HSC support activity, their ability to maintain HSCs ex vivo is only modest due to loss of expression of these niche factors in culture (Kunisaki et al., 2013; Nakahara et al., 2019). The absence of sustained self-renewal or maintenance of the stem cell phenotype could be related to the lack of integration of biophysical and biochemical cues required for stem cell regulation, provided by the native BM niche microenvironment in vivo. This has led to a focus on biomaterial and engineering strategies that aim to recapitulate BM niche properties in vitro (Müller et al., 2014). It is envisaged that bioengineered artificial niches will offer protocols for ex vivo expansion and maintenance of HSCs without the need for high risk protocols (e.g. genetic manipulations (Nakahara et al., 2019)), but also platforms on which to study the fundamental mechanisms that control self-renewal in both HSCs and MSPCs in the niche. In this thesis, biomaterial strategies were employed to mimic aspects of the BM niche microenvironment. First retention of HSC support activity in a population of MSPCs was investigated and the metabolic mechanisms that may support this phenotype were probed. The ability of the system to support HSC maintenance in vitro was then assessed. Poly(ethyl acrylate) (PEA) is a polymer that causes spontaneous unfolding of the extracellular matrix protein (ECM) fibronectin (FN). These physiological-like networks expose key binding sites on the FN molecule, which can be harnessed for cell adhesion and growth factor tethering (Cheng et al., 2018; Llopis-hernández et al., 2016). Noting that low-stiffness matrices support nestin expression in MSPCs (Engler et al., 2006), a key niche marker (Kunisaki et al., 2013; Pinho et al., 2013), low-stiffness collagen hydrogels were introduced into the system. We were able to use this system to promote a population of nestin+ MSPCs that express key HSC support factors and were able to maintain a population of HSCs in vitro. The nestin+ MSPCs utilise hypoxic-like metabolic mechanisms in response to low-stiffness, that may be important in retaining this BM niche-like phenotype in long term in vitro culture

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Current approaches for modulation of the nanoscale interface in the regulation of cell behavior

Regulation of cell behavior in response to nanoscale features has been the focus of much research in recent years and the successful generation of nanoscale features capable of mimicking the natural nanoscale interface has been of great interest in the field of biomaterials research. In this review, we discuss relevant nanofabrication techniques and how they are combined with bioengineering applications to mimic the natural extracellular matrix (ECM) and create valuable nanoscale interfaces

The Irish Study of Sexual Health and relationships Sub-Report 2: Sexual Health Challenges and Related Service Provision

The ISSHR was commissioned by the Department of Health and Children and the Crisis Pregnancy Agency in response to a recommendation by the National AIDS Strategy Committee. It is the largest nationally representative study of sexual knowledge, attitudes and behaviour ever undertaken in Ireland. International evidence indicates that aspects of sexual health, such as contraception, crisis pregnancy and sexually transmitted infections, should be examined jointly. To this end, the Crisis Pregnancy Agency and the Department of Health and Children instigated the ISSHR. The ISSHR findings have been outlined in a suite of reports – the Main Report, the Summary Report and three sub-reports; the latter provide detailed information in defined areas of interest. This, the second sub-report, focuses on the area of sexual risk-taking and the use of relevant services. The Crisis Pregnancy Agency (CPA) and the sexual-health sector in general need robust evidence in order to develop sexual-health policies, to plan strategies and to inform the effective promotion of sexual-health messages. The ISSHR findings will be invaluable not only to the work of the CPA in preventing crisis pregnancy, but also to that of other organisations concerned with promoting sexual health , providing sexual-health services, preventing sexually transmitted infections, and providing sex education for young people

The plot thickens: the emerging role of matrix viscosity in cell mechanotransduction

Cell mechanotransduction is an area of intense research focus. Until now, very limited tools have existed to study how cells respond to changes in the extracellular matrix beyond, for example, mechanical deformation studies and twisting cytometry. However, emerging are a range of elastic, viscoelastic and even purely viscous materials that deform and dissipate on cellular length and timescales. This article reviews developments in these materials, typically translating from 2D model surfaces to 3D microenvironments and explores how cells interact with them. Specifically, it focuses on emerging concepts such as the molecular clutch model, how different extracellular matrix proteins engage the clutch under viscoelastic‐stress relaxation conditions, and how mechanotransduction can drive transcriptional control through regulators such as YAP/TAZ

Researching belonging with people with learning disabilities:Self-building active community lives in the context of personalisation

We wanted to understand more about how people with learning disabilities are building active community lives to help belonging. We spoke to 39 people from 29 different support organisations, 7 local authority representatives and 43 people with learning disabilities. They said belonging was about having the time to connect with other people in “everyday” places, being part of a supportive network and having the right choice and information. Belonging is like a cake. It needs the right ingredients. These ingredients include the right combination of people, places and times. Because of cuts to funding, many people with learning disabilities lack the right support, choice and information to access their communities. This is not belonging. ​. Abstract: Background This journal article draws on findings from a research project that examined how people with learning disabilities and their allies were seeking to build a sense of belonging. We wanted to focus on the concept of “belonging” in the context of personalisation and reduced government social care funding. Specifically, we sought to understand how people with learning disabilities and their supporters were coming together to “self-build” networks of support including friendship clubs and self-advocacy groups to enable a greater sense of belonging in their local communities. Methods Qualitative interviews were conducted with seven local authority representatives across four case study areas in the UK, as well as 39 staff across 29 organisations providing a range of day and evening support and activities. We also talked to 43 people with learning disabilities across the four areas about their experiences. Findings Our findings demonstrate how belonging involves a complex configuration of actors, places, times, relationships and institutional roles (much like the ingredients in a cake). The ways in which belonging intersects with agency and choice was also identified as an important and novel finding of our study. Conclusion While belonging is often presented to people as a desirable and realisable outcome of social inclusion policies, cuts in funding and a lack of appropriate support frustrate people's desires to meaningfully belong with other people in their local community. This demonstrates the importance of supporting social environments that meet people's needs for social connectedness and belonging

The influence of nanotopography on cell behaviour through interactions with the extracellular matrix - A review

[EN] Nanotopography presents an effective physical approach for biomaterial cell manipulation mediated through material-extracellular matrix interactions. The extracellular matrix that exists in the cellular microenvironment is crucial for guiding cell behaviours, such as determination of integrin ligation and interaction with growth factors. These interactions with the extracellular matrix regulate downstream mechanotransductive pathways, such as rearrangements in the cytoskeleton and activation of signal cascades. Protein adsorption onto nanotopography strongly influences the conformation and distribution density of extracellular matrix and, therefore, subsequent cell responses. In this review, we first discuss the interactive mechanisms of protein physical adsorption on nanotopography. Secondly, we summarise advances in creating nanotopographical features to instruct desired cell behaviours. Lastly, we focus on the cellular mechanotransductive pathways initiated by nanotopography. This review provides an overview of the current state-of-the-art designs of nanotopography aiming to provide better biomedical materials for the future.We acknowledge support from the Leverhulme Trust through gran t RPG-2019-252 and the Engineering and Physical Sciences Research Council (EPSRC) grant EP/P001114/1.Luo, J.; Walker, M.; Xiao, Y.; Donnelly, H.; Dalby, MJ.; Salmerón Sánchez, M. (2022). The influence of nanotopography on cell behaviour through interactions with the extracellular matrix - A review. Bioactive materials. 15:145-159. https://doi.org/10.1016/j.bioactmat.2021.11.0241451591

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L(482)F) or cleavage and polyadenylation specificity factor 3 (N(232)H), in the Trypanosoma cruzi proteasome β5 subunit (G(208)S), or in Leishmania donovani CRK12 (G(572)D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G(492) in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G(492) edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy

The role of community-based Hubs in reef restoration: Collaborative monitoring at Moore Reef

The Cairns-Port Douglas region is home to multiple coral rehabilitation and stewardship projects supported by scientists, Traditional Owners, and a range of local stakeholders. The Cairns-Port Douglas Reef Hub has been a platform for collaboration across Traditional Owners, tourism operators, not-for-profits and scientists from the Reef Restoration and Adaptation Program (AIMS and CSIRO) to design and deliver a project at Moore Reef that assesses how new techniques for assisted coral recovery can be applied in rubble habitats. The collaborative project evaluates the viability of newly engineered coral seeding devices developed by AIMS, for deploying coral recruits that were spawned in the National Sea Simulator in December 2022 to sites at Moore Reef close to tourist pontoons. This project provides important data to inform future scaling up of restoration activities and provides a model for active involvement of a range of partners. Through this work, the project builds understanding around key ingredients for best-practice, place-based engagement opportunities for Reef communities and the general public