Biology subjects

Gentleman, E.

Publications and source records attributed to Gentleman, E..

5 recordsLinked to original sources

bioRxiv2025

ALS mutations do not alter perineuronal net formation in human stem cell-derived motor neurons

Perineuronal nets (PNNs) are extracellular matrix structures that stabilise synaptic inputs and regulate neuronal plasticity. Although PNN dysregulation is observed in several neurological disorders, their relevance to amyotrophic lateral sclerosis (ALS) remains unclear. In particular, the extent to which PNN alterations in ALS are motor neuron (MN)-intrinsic is unknown. We investigated whether human pluripotent stem cell-derived MNs form PNN-like structures in vitro, and whether ALS-associated mutations alter this process. We show that MNs generate PNN-like structures containing hyaluronan, tenascin-R, and aggrecan and that their formation and gene expression were not altered by ALS mutations. To explore whether PNN dysregulation reflects contributions from other cell types or selective MN vulnerability, we conducted meta-analyses of transcriptomic datasets from pluripotent stem cell-derived astrocytes carrying ALS-associated mutations, as well as datasets comparing MN populations with differential susceptibility to ALS. These analyses revealed no consistent differences in PNN-related gene expression in human stem cell-derived MN. In contrast, transcriptomic analyses of human post-mortem ALS tissues revealed significant dysregulation of PNN-related genes, including core PNN components and linker proteins. These findings suggest that PNN changes in ALS are not MN-intrinsic, but may result from interactions with other cell types in the central nervous system such as glia.

neuroscience↗

bioRxiv2023

Label-free 3-D molecular imaging of living tissues using Raman Spectral Projection Tomography

The ability to image tissues in three-dimensions (3-D) with label-free molecular contrast at mesoscale would be a valuable capability in biology and biomedicine. Here, we introduce Raman spectral projection tomography (RSPT) for volumetric molecular imaging with sub-millimeter spatial resolution. We have developed a RSPT imaging instrument capable of providing 3-D molecular contrast in transparent and semi-transparent samples. A computational pipeline for multivariate reconstruction was established to extract label-free spatial molecular information from Raman projection data. We demonstrate imaging and visualization of phantoms of various complex shapes with label-free molecular contrast. Finally, we apply RSPT as a novel tool for imaging of molecular gradients and extracellular matrix heterogeneities in fixed and live tissue-engineered constructs and explanted native tissues. RSPT imaging opens new possibilities for label-free molecular monitoring of tissues.

bioengineering↗

bioRxiv2023

Morphogen-driven human iPSCs differentiation in 3D in vitro models of gastrulation is precluded by physical confinement.

In early human development, gastrulation is tightly associated with lineage specification. The interplay between mechanical forces and biochemical signals during these processes is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model of gastrulation that uses spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures, and observe the emergence of organised tri-germ layers. In these unconfined cultures, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels and hiPSCs-derived models of early development to define the drivers causing changes in shape and emergence of germ layers.

cell biology↗

bioRxiv2020

ILC1-derived TGFβ1 drives intestinal remodelling

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we developed gut organoid co-cultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate for the first time that murine and human ILC1 secrete TGF{beta}1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

immunology↗

bioRxiv2017

Collective cell behaviour in mechanosensing of substrate thickness

Extracellular matrix stiffness has a profound effect on the behaviour of many cell types. Adherent cells apply contractile forces to the material on which they adhere, and sense the resistance of the material to deformation - its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 {micro}m) thicknesses. Here, we hypothesised that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic ECM-functionalised polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker labelled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 m. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater halfmaximal response at 54 m. Depth-sensing was dependent on ROCK-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.

biophysics↗