Biology subjects

Dalby, M. J.

Publications and source records attributed to Dalby, M. J..

15 recordsLinked to original sources

bioRxiv2026

iPSC-Derived Chondroprogenitors as a Promising Cell Source for Cartilage Engineering: A Comparison with MSCs in Unmodified and Peptide-Functionalized Alginate Hydrogels

Articular cartilage degeneration is a hallmark of degenerative joint diseases, yet its limited regenerative capacity poses significant challenges for tissue engineering. While mesenchymal stem cells (MSCs) are the most commonly employed cell type in cartilage tissue engineering, they exhibit donor variability, restricted expansion capacity, and tendencies toward fibrocartilage formation and hypertrophic differentiation. Human induced pluripotent stem cell-derived chondroprogenitors (iCPs) represent a promising alternative, offering scalable production of developmentally relevant cells with intrinsic chondrogenic commitment. However, their performance within three-dimensional biomaterial scaffolds remains largely unexplored. Here, we compared chondrogenic differentiation of iCPs and MSCs within alginate hydrogels of varying stiffness (0.37 - 4.55 kPa) exhibiting physiologically relevant stress relaxation properties. Intermediate stiffness (2% alginate, ~2.17 kPa) optimally supported chondrogenesis for both cell types. While MSCs differentiated as single cells, iCPs spontaneously self-organized into cartilaginous aggregates without requiring a separate pellet pre-culture step, showing significantly higher hyaline indices and reduced COL10 expression, despite initial low viability in hydrogels. To further enhance chondrogenesis, we functionalized 2% alginate gels with RGD and HAVDI peptides mimicking integrin- and cadherin-mediated signaling. HAVDI/RGD functionalization significantly enhanced hyaline cartilage marker expression in both cell types, with iCPs exhibiting superior matrix composition characterized by elevated aggrecan and SOX9 expression and reduced COL10 and MMP13 compared to MSCs. These findings establish iCPs as a promising cell source for cartilage tissue engineering and disease modeling, particularly within biomaterials integrating mechanical and bioactive cues that recapitulate the native cartilage microenvironment.

cell biology↗

bioRxiv2026

Culture enhanced MSCs to boost haematopoietic engraftment in a model of the post-leukaemic bone marrow niche.

Relapse remains a leading cause of treatment failure in acute myeloid leukaemia (AML), making haematopoietic stem cell transplantation (HSCT) the only curative option for many patients. Yet HSCT efficacy is often limited by impaired engraftment, driven by AML-induced remodelling of the bone marrow stem cell niche. Mesenchymal stromal cells (MSCs) are key mediators of niche formation and could, in principle, restore a supportive microenvironment when introduced alongside HSC therapy; but this strategy remains largely untested. A key obstacle to MSC-based therapy is that MSCs acquire a senescent, pro-inflammatory phenotype during standard in vitro expansion. We addressed this by engineering a polymer-laminin presentation system that suppresses senescence and preserves a proliferative, regenerative MSC phenotype during expansion. Then, to investigate potential cell therapy use, we developed a bioengineered in vitro model as a new approach methodology (NAM) for studying disease-driven niche modification. The system consists of MSC spheroids embedded in a synthetic hydrogel within a transwell platform, allowing controlled co-culture of healthy or AML-derived haematopoietic cells, therapeutic MSCs, and chemotherapeutic agents. Using this platform, we modelled an AML-like niche and showed that MSCs expanded via the polymer-laminin system, when introduced alongside HSCs, significantly improved HSCT engraftment relative to both standard-expanded MSCs and HSCT performed without MSC support. These results establish MSC phenotype maintenance as a critical determinant of therapeutic efficacy, and position this NAM as a platform for pre-clinical screening of niche-targeted therapies in AML.

bioengineering↗

bioRxiv2025

Decoding the microbiome and resistome of advanced chronic liver disease through long-read metagenomics

IntroductionPatients with advanced chronic liver disease (ACLD) and underlying cirrhosis frequently require repeated courses of antimicrobial therapy, with both the frequency and spectrum of antimicrobial exposure increasing alongside disease progression. In this population, impaired immune function and increased intestinal barrier dysfunction contribute to a heightened susceptibility of multidrug-resistant bacterial infections. ObjectiveTo comprehensively characterise the gastrointestinal microbiome and antimicrobial resistance gene (ARG) landscape across the clinical spectrum of ACLD, and to identify microbial and resistome signatures associated with disease severity. DesignWe employed long-read metagenomic sequencing (Oxford Nanopore Technologies) to profile the gastrointestinal microbiome and resistome across distinct ACLD stages: acute-on-chronic liver failure (ACLF), decompensated cirrhosis (DC), and stable cirrhosis, compared to healthy controls. ResultsACLF patients had pronounced levels of Enterococcus faecium, with six samples out of 28 showing over 95% relative abundance, suggesting its potential as a bacterial biomarker for advanced cirrhosis. We reconstructed 28 high-quality MAGs of E. faecium from cirrhosis patients, 17 of which originated from ACLF cases. This dominance of E. faecium correlated with substantially reduced microbial diversity and a marked depletion of key commensal taxa, including Blautia, Akkermansia, Faecalibacterium, and Bifidobacterium. Resistome analysis revealed significant enrichment of clinically relevant ARGs in DC and ACLF, including those conferring resistance to aminoglycosides, beta-lactams, and glycopeptides, correlating with prior antimicrobial exposure. ConclusionLong-read metagenomics enables high-resolution characterisation of microbial and resistome dynamics across ACLD severities. By capturing taxonomic shifts, functional potential, and ARG enrichment, this approach provides valuable insights into microbiome trajectories linked with disease severity, informing mechanistic research and potential clinical interventions. Impact statementChronic liver disease is a major global health burden, responsible for approximately 2 million deaths each year. Advanced chronic liver disease profoundly disrupts the gut microbiome, often exacerbated by repeated antibiotic exposure, promoting the persistence of antimicrobial-resistant organisms. Leveraging Oxford Nanopore Technologies long-read metagenomic sequencing, this study delivers high-resolution insights into the gut microbiome and resistome across progressive stages of cirrhosis. We reveal Enterococcus faecium dominance as a defining feature of cirrhosis, accompanied by severe loss of commensal diversity and enrichment of clinically significant resistance genes. These findings underscore the clinical utility of culture-independent metagenomic profiling for detecting pathogenic taxa and resistance determinants within the gut ecosystem of high-risk patients. Our work highlights the translational potential of long-read metagenomics as an accessible tool for pathogen surveillance, antimicrobial stewardship, and precision infection management in advanced liver disease.

microbiology↗

bioRxiv2025

Bioengineered bone marrows that can support stem cells and remodel to mimic human disease metabolism and chemotherapeutic effects

Blood cancer drug discovery is reliant on poorly predictive and costly animal models. Therefore, bioengineered, human cell containing, models are attractive to Pharma. Designing effective bone marrow (BM) models is complicated as they need to both regulate haematopoietic stem cell (HSC) phenotype and be able to undergo remodelling to mimic the blood cancer microenvironment. Here, we develop synthetic hybrid niches using poly(ethylacrylate) to organise laminin on hard, bone mimicking, surfaces and interface with soft, marrow mimicking, polyethylene glycol-fibronectin hydrogels. Optimisation of mesenchymal stromal cell (MSC) mechanobiology within the model offers both support for HSCs and remodelling in response to acute myeloid leukaemia derived cells. The remodelling has many parallels to in vivo and patient data including increased dependency on nestin+ve MSCs, enhanced cytoprotection and increased taurine metabolism. We also use the model to demonstrate, as has been seen in vivo, that targeting taurine enhances effects of chemotherapy.

bioengineering↗

bioRxiv2024

Developing and Investigating a Nanovibration Intervention for the Prevention/Reversal of Bone Loss Following Spinal Cord Injury

Osteoporosis disrupts the fine-tuned balance between bone formation and resorption leading to reductions in bone quantity and quality, ultimately leading to increased fracture risk. Prevention and treatment of osteoporotic fractures is essential, for reductions in mortality, morbidity and the economic burden, particularly considering the ageing global population. Extreme bone loss that mimics time-accelerated osteoporosis develops in the paralysed limbs following complete spinal cord injury (SCI). In vitro nanoscale vibration (1 kHz, 30- or 90 nm amplitude) has been shown to drive differentiation of mesenchymal stem cells towards osteoblast-like phenotypes, enhancing osteogenesis, and inhibiting osteoclastogenesis, simultaneously. Here we develop and characterise a wearable device designed to deliver continuous nano-amplitude vibration to the hindlimb long bones of rats with complete SCI. We investigate whether a clinically feasible dose of nanovibration (4-hours/day, 5-days/week for 6 weeks) is effective at reversing the established SCI-induced osteoporosis. Laser interferometry and finite element analysis confirmed transmission of nanovibration into the bone, and micro-computed tomography and serum bone formation and resorption markers assessed effectiveness. The intervention did not reverse SCI-induced osteoporosis. However, serum analysis indicated an elevated concentration of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in rats receiving 40 nm amplitude nanovibration, suggesting increased synthesis of type 1 collagen, the major organic component of bone. Therefore, enhanced doses of nanovibrational stimulus may yet prove beneficial in attenuating/reversing osteoporosis, particularly in less severe forms of osteoporosis.

bioengineering↗

bioRxiv2023

Gut microbiota and faecal cytokine profiles of rural Cambodian infants and links to early life nutrition and diarrhoeal episodes.

The gut microbiota of infants in low-middle income countries like Cambodia remain underrepresented in microbiome research. This study aimed to explore the faecal gut microbiota composition and faecal cytokine profiles in a cohort of infants living in a rural province of Cambodia and explore the impact of sample storage conditions and infant environment on microbiota composition. Faecal samples collected at three time points from 32 infants (96 samples in total) after 7 months of age were analysed using 16S rRNA amplicon sequencing to determine the composition of the microbiota. Bacterial strains were isolated and subjected to whole genome sequencing and genomic analysis and concentrations of faecal cytokines were also measured. Initially, we compared the effects of two sample collection methods due to the challenges of faecal sample collection and storage in a rural location. Storage of faecal samples in a DNA preservation solution retained a greater abundance of Bacteroides. Analyses of microbiota composition of samples stored in DNA preservation solution indicated that Bifidobacterium was the most abundant genus with Bifidobacterium longum the most abundant species, particularly in breastfed infants. Most infants had detectable pathogenic taxa indicating frequent pathogen exposure, with Shigella and Klebsiella more abundant in infants with recent diarrheal illness. We did not detect antibiotic-associated perturbations in the gut microbiota, and no associations were found between the gut microbiota and infant growth. Genomic analysis of isolated strains indicated the presence of gene clusters encoding the ability to digest human milk oligosaccharides in B. longum and Bifidobacterium breve isolates. The presence of antibiotic-resistant genes was also identified in potentially pathogenic species, as well as in beneficial genera including Bifidobacterium. Faecal cytokine analysis showed higher concentrations of Interlukin-1alpha and vascular endothelial growth factor in breastfed infants, which may influence the infant gut mucosal immune system. This study provides insights into an underrepresented population of rural Cambodian infants, emphasising the impact of pathogen exposure and breastfeeding on gut microbiota composition and faecal immune profiles.

microbiology↗

bioRxiv2023

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome. Impact StatementDetermining the functional capabilities of microbial communities within different human microbiomes is important to understand their impacts on health. Extraction of sufficient DNA is challenging, especially from low biomass samples, such as skin swabs suitable for shotgun metagenomics, which is needed for taxonomic resolution and functional information. Here we describe an optimised DNA extraction method that produces enough DNA from skin swabs, suitable for shotgun metagenomics, and demonstrate it can be used to effectively characterise the skin microbiota. This method will allow future studies to identify taxonomic and functional changes in the skin microbiota which is needed to develop interventions to improve and maintain skin health. Data SummaryAll sequence data and codes can be accessed at: NCBI Bio Project ID: PRJNA937622 DOI: https://github.com/quadram-institute-bioscience/coronahit_guppy DOI: https://github.com/ilianaserghiou/Serghiou-et-al.-2023-Codes

genomics↗

bioRxiv2022

Extracellular vesicle cargo metabolome changes in response to the mesenchymal stromal cell microenvironment and influences cell quiescence and activation in a human breast cancer cell model

Breast cancer is the leading cause of cancer mortality in women worldwide and commonly metastasizes to the bone marrow, drastically reducing patient prognosis and survival. In the bone marrow niche, metastatic cells can enter into a dormant state, thereby evading immune surveillance and treatment, and can be reactivated to enter a proliferative state due to poorly understood cues. Mesenchymal stromal cells (MSCs) maintain cells in this niche partly by secreting extracellular matrix and paracrine factors and by responding to regenerative cues. MSCs also produce extracellular vesicles (EVs) that carry a range of cargoes, some of which are implicated in cell signalling. Here, we investigate if the changing metabolic state of MSCs alters the cargoes they package into EVs, and how these changing cargoes act on dormant breast cancer cells (BCCs) using an in vitro BCC spheroid model and a scratch assay to create a regenerative demand on MSCs. Our findings show that EVs produced by standard MSCs contain glycolytic metabolites that maintain BCC dormancy. When MSCs are placed under a regenerative demand and increase their respiration to fuel differentiation, these metabolites disappear from the EV cargo and their absence encourages rapid growth in the BCC spheroids. This work implicates EVs in cancer cell dormancy in the bone marrow niche and indicates that pressures on the niche, such as regeneration, can be a driver of BCC activation.

cell biology↗

bioRxiv2022

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via allogeneic stem cell transplantation (alloSCT), to engraft and repopulate the blood system. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs (ST-HSC) and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs or pericytes). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1 expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1 expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

bioengineering↗

bioRxiv2021

Maternal microbiota Bifidobacterium promotes placental vascularization, nutrient transport and fetal growth in mice

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

physiology↗

bioRxiv2020

The use of nanovibration to discover specific and potent bioactive metabolites that stimulate osteogenic differentiation in mesenchymal stem cells

Bioactive metabolites have wide-ranging biological activities and are a potential source of future research and therapeutic tools. Here, we use nanovibrational stimulation to induce the osteogenic differentiation of MSCs, in the absence of off-target differentiation. We show that this differentiation method, which does not rely on the addition of exogenous growth factors to the culture media, provides an artefact-free approach to identifying bioactive metabolites that specifically and potently induce osteogenesis. We first identify a highly specific metabolite as the endogenous steroid, cholesterol sulphate. Next, a screen of other small molecules with a similar steroid scaffold identified fludrocortisone acetate as being both specific and having highly potent osteogenic-inducing activity. These findings demonstrate that physical priciples can be used to identify bioactive metabolites and then metabolite potency can be optimised by examining structure-function relationship.

bioengineering↗

bioRxiv2019

Bifidobacterium breve UCC2003 exopolysaccharide modulates the early life microbiota by acting as a dietary substrate

Members of the genus Bifidobacterium represent an important bacterial group for promoting health during early life. Previous studies have indicated that bifidobacterial exopolysaccharides (EPS) are involved in host interactions, with purified EPS also suggested to modulate microbe-microbe interactions by acting as a nutrient substrate. To further explore the role of EPS as a potential dietary component, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. Bifidobacterium breve UCC2003 was utilised as a representative early life bifidobacterial strain, and a corresponding isogenic EPS-deletion mutant (B. breve UCC2003 EPS-). Initial transcriptomics analysis of the EPS mutant vs. parent B. breve UCC2003 strain highlighted differential expression in a discrete number of genes, including the eps biosynthetic cluster, though overall growth dynamics between the two strains were unaffected. Model colon vessels were inoculated with B. breve strains and microbiome dynamics were monitored using metataxonomic (via 16S rRNA sequencing) and metabolomic (via 1H NMR) approaches. Baseline early life microbiota profiles were similar between vessels, with persistence of B. breve (EPS+ and EPS-) observed between 0-36h. Within the EPS-positive vessel there was a significant shift in microbiome and metabolite profiles until the end of the study (405h); we observed increases of Escherichia and Tyzzerella, and short-chain fatty acids including acetate, propionate and formate, including further correlations between taxa and metabolites which were not observed in the EPS-negative vessel. These data indicate that the B. breve UCC2003 EPS is potentially being metabolised by members of the infant microbial community, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow for development of EPS-specific strategies to beneficially alter the early life microbiota to promote infant health.

microbiology↗

bioRxiv2019

The early life microbiota protects neonatal mice from pathological small intestinal epithelial cell shedding

The gut microbiota plays a crucial role in regulating and maintaining the epithelial barrier, particularly during early life. Notably, patients with chronic intestinal inflammation have a dysregulated process of renewal and replenishment of the intestinal epithelial cell (IEC) barrier, which is linked to disturbances in the gut microbiota. To date, there are no studies focussed on understanding the impact of inflammatory cell shedding events during the early life developmental window, and which host and microbial factors mediate these responses. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period (day 14, 21, 29 and week 8). Surprisingly neonatal mice (day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of LPS, with day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses, although we did observe that neonatal mice had elevated anti-inflammatory (IL-10) responses. Notably, when we profiled microbiota and metabolites from these mice, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Faecal microbiota transplant (FMT) and antibiotic studies confirmed the importance of early life gut microbiota in cell shedding responses. In these studies, neonates treated with antibiotics restored LPS-induced small intestinal cell shedding, whereas adult FMT alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlight areas for further investigation to probe underlying mechanisms to drive therapeutic development within the context of chronic inflammatory intestinal diseases.

microbiology↗

bioRxiv2019

Engineered full-length Fibronectin-based hydrogels sequester and present growth factors to promote regenerative responses in vitro and in vivo

Extracellular matrix (ECM)-derived matrices such as Matrigel are used to culture numerous cell types in vitro and can recapitulate certain ECM functions that support cell growth and differentiation. However, ECM-derived matrices suffer lot-to-lot variability, undefined composition and lack of controlled physical properties. There is a need to develop rationally designed synthetic matrices that can also recapitulate ECM roles. Synthetic matrices have certain limitations as they use synthetic peptides or fragments whereas the ECM consists of full proteins. Here, we report the development of degradable, PEG-based hydrogels of controlled stiffness that incorporate full-length fibronectin (FN) to enable solid-phase presentation of growth factors in a physiological manner. We demonstrate, in vitro and in vivo, the effect of incorporating vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2), in these hydrogels to enhance angiogenesis and bone regeneration, respectively. We show that the solid-state presentation of growth factors enables very low growth factor doses to achieve regenerative effects.

bioengineering↗

bioRxiv2019

Design, construction and characterisation of a novel nanovibrational bioreactor and cultureware for osteogenesis

In regenerative medicine, techniques which control stem cell lineage commitment are a rapidly expanding field of interest. Recently, nanoscale mechanical stimulation of mesenchymal stem cells (MSCs) has been shown to activate mechanotransduction pathways stimulating osteogenesis in 2D and 3D culture. This has the potential to revolutionise bone graft procedures by creating cellular graft material from autologous or allogeneic sources of MSCs without using chemical induction. With the increased interest in mechanical stimulation of cells and huge potential for clinical use, it is apparent that researchers and clinicians require a scalable bioreactor system that provides consistently reproducible results with a simple turnkey approach. A novel bioreactor system is presented that consists of: a bioreactor vibration plate, calibrated and optimised for nanometre vibrations at 1 kHz, a power supply unit, which supplies a 1 kHz sine wave signal necessary to generate approximately 30 nm of vibration amplitude, and custom 6-well cultureware with toroidal shaped magnets incorporated in the base of each well for conformal attachment to the bioreactors magnetic vibration plate. The cultureware and vibration plate were designed using finite element analysis to determine the modal and harmonic responses, and validated by interferometric measurement. This helps ensure that the vibration plate and cultureware, and thus collagen and MSCs, all move as a rigid body, avoiding large deformations close to the resonant frequency of the vibration plate and vibration damping beyond the resonance. Assessment of osteogenic protein expression was performed to confirm differentiation of MSCs after initial biological experiments with the system, as well as atomic force microscopy of the 3D gel constructs during vibrational stimulation to verify that strain hardening of the gel did not occur. This shows that cell differentiation was the result of the nanovibrational stimulation provided by the bioreactor alone, and that other cell differentiating factors, such as stiffening of the collagen gel, did not contribute.

cell biology↗