Biology subjects

Sutcliffe, M.

Publications and source records attributed to Sutcliffe, M..

8 recordsLinked to original sources

bioRxiv2025

Isolation and characterization of synaptic structures from human neural organoids

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However, methodologies for isolating functional synaptic structures from these models remain limited. Here, we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air-liquid interface cerebral organoids (ALI-COs) at distinct developmental stages (day 90 and 150). Notably, the method avoids density gradients, requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g. GAP43) and classical synaptosomal proteins (e.g. PCLO, BSN, SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane-enclosed GCPs with fibrous content and mitochondria in day 90 isolates, and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl-induced depolarization, which triggered phosphorylation changes in growth cone proteins (GAP43, MARCKS, MARCKSL1), cytoskeletal regulators (DCLK1, SHTN1, MARK4, MAP1B) and protein kinases (CAMK2G, PRKCE) in day 90 GCPs, as well as classical synaptic vesicle cycle proteins (SYN1, DNM1, RPH3A) at day 150. Overall, this study establishes a centrifugation-based protocol for isolating growth cones and immature synapses from human organoids, capturing key stages of synaptic development and enabling scalable, patient-compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders.

neuroscience↗

bioRxiv2023

Multi-omic analysis of guided and unguided forebrain organoids reveal differences in cellular composition and metabolic profiles

Neural organoids are invaluable model systems for studying neurodevelopment and neurological diseases. For this purpose, reproducible differentiation protocols are needed that minimize inter-organoid variability whilst generating neural organoids that physiologically resemble the brain area of interest. Currently, two main approaches are used: guided, where the differentiation towards neuroectoderm and subsequently specific CNS regions is driven by applying extrinsic signalling molecules, and unguided, where the intrinsic capability of pluripotent stem cells to generate neuroectoderm without external signalling is promoted. Despite the importance for the field, the resulting differences between these models have not been directly investigated. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated using a guided and unguided approach focusing on proteomic, lipidomic and metabolomic differences. Furthermore, we characterised differences in phosphorylation and sialylation states of proteins, two key post-translational modifications (PTMs) in neurodevelopment, and performed single cell transcriptomics (scRNAseq). The multi-omic analysis revealed considerable differences in neuronal-, synaptic and glial content, indicating that guided forebrain organoids contain a larger proportion of neurons, including GABAergic interneurons, and synapses whereas unguided organoids contain significantly more GFAP+ cells and choroid plexus. Furthermore, substantial differences in mitochondrial- and metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid {beta}-oxidation in unguided forebrain organoids and a higher reliance on glycolysis in guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences arising when generating guided and unguided forebrain organoids and provide an important resource for the organoid field studying neurodevelopment and -disease.

neuroscience↗

bioRxiv2020

The supramolecular landscape of growing human axons

During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.

cell biology↗

bioRxiv2020

An early cell shape transition drives evolutionary expansion of the human forebrain

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that the differentiation of neuroepithelial cells to neurogenic radial glia is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition. Temporally resolved RNA-seq from human and gorilla organoids reveals differences in gene expression patterns associated with cell morphogenesis, and in particular highlights ZEB2, a known regulator of epithelial-mesenchymal transition and cell shape. We show, through loss- and gain-of-function experiments, that ZEB2 promotes the progression of neuroepithelial differentiation, and its ectopic overexpression in human is sufficient to trigger a premature transition. Thus, by mimicking the nonhuman ape expression in human organoids, we are able to force the acquisition of nonhuman ape architecture, establishing for the first time, an instructive role of neuroepithelial cell shape in human brain expansion.

evolutionary biology↗

bioRxiv2020

Pan-cancer Drivers are Recurrent Transcriptional Regulatory Heterogeneities in Early-stage Luminal Breast Cancer

The heterogeneous composition of solid tumors is known to impact disease progression and response to therapy. Malignant cells coexist in different regulatory states that can be accessed transcriptomically by single-cell RNA sequencing, but these methods have many caveats related to sensitivity, noise, and sample handling. We revised a statistical fluctuation analysis called stochastic profiling to combine with 10-cell RNA sequencing, which was designed for laser-capture microdissection (LCM) and extended here for immuno-LCM. When applied to a cohort of late-onset, early-stage luminal breast cancers, the integrated approach identified thousands of candidate regulatory heterogeneities. Intersecting the candidates from different tumors yielded a relatively stable set of 710 recurrent heterogeneously expressed genes (RHEGs) that were significantly variable in >50% of patients. RHEGs were not strongly confounded by dissociation artifacts, cell cycle oscillations, or driving mutations for breast cancer. Rather, we detected RHEG enrichments for epithelial-to-mesenchymal transition genes and, unexpectedly, the latest pan-cancer assembly of driver genes across cancer types other than breast. Heterogeneous transcriptional regulation conceivably provides a faster, reversible mechanism for malignant cells to evaluate the effects of potential oncogenes or tumor suppressors on cancer hallmarks. Statement of significanceProfiling intratumor heterogeneity of luminal breast carcinoma cells identifies a recurrent set of genes suggesting sporadic activation of pathways known to drive other types of cancer.

cancer biology↗

bioRxiv2020

Fragmentation of Small-cell Lung Cancer Regulatory States in Heterotypic Microenvironments

Small-cell lung cancers derive from pulmonary neuroendocrine cells, which have stemlike properties to reprogram into other cell types upon lung injury. It is difficult to uncouple transcriptional plasticity of these transformed cells from genetic changes that evolve in primary tumors or secondary metastases. Profiling of single cells also problematic if the required sample dissociation activates injury-like signaling and reprogramming. Here, we defined cell-state heterogeneities in situ through laser capture microdissection-based 10-cell transcriptomics coupled with stochastic-profiling fluctuation analysis. Using labeled cells from a small-cell lung cancer mouse model initiated by neuroendocrine deletion of Rb1-Trp53, we profiled variations in transcript abundance to identify cell-to-cell differences in regulatory state in vitro and in vivo. Fluctuating transcripts in spheroid culture were partly shared among Rb1-Trp53-null models, and heterogeneities increased considerably when cells were delivered intravenously to colonize the liver. Colonization of immunocompromised animals drove fractional appearance of alveolar type II-like markers and poised cells for paracrine stimulation from immune cells and hepatocytes. Immunocompetency further exaggerated the fragmentation of tumor states in the liver, yielding mixed stromal signatures evident in bulk sequencing from autochthonous tumors and metastases. Dozens of transcript heterogeneities recur irrespective of biological context; their mapped orthologs brought together observations of murine and human small-cell lung cancer. Candidate heterogeneities recurrent in the liver also stratified primary human tumors into discrete groups not readily explained by molecular subtype but with prognostic relevance. We conclude that heterotypic interactions in the liver and lung are an accelerant for intratumor heterogeneity in small-cell lung cancer. Statement of significanceThe single-cell regulatory heterogeneity of small-cell lung cancer becomes increasingly elaborate in the liver, a common metastatic site for the disease.

cancer biology↗

bioRxiv2020

Single-cell Bottlenecks and Dead-ends During Glioma Premalignancy

Cancer evolves from premalignant clones that accumulate mutations and adopt unusual cell states to achieve transformation. Previously, we pinpointed the oligodendrocyte precursor cell (OPC) as a cell-of-origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection and determined gene-expression changes by bulk RNA sequencing and a fluctuation analysis, called stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, while bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected very few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, we identified mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) along with DNA-damage markers suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss. At 150 days, Rin1 was microdeleted in some gliomas and downregulated in all others. Replication stress may pose a considerable bottleneck that must be resolved for gliomas to initiate. Statement of significanceIn situ stochastic profiling of heterogeneous cell states in a mouse model of glioma uncovers regulatory confusion in a glioma cell-of-origin and defines a state of replication stress that precedes tumor initiation.

cancer biology↗

bioRxiv2018

Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output

Three-dimensional neural organoids are emerging tools with the potential for improving our understanding of human brain development and neurological disorders. Recent advances in this field have demonstrated their capacity to model neurogenesis1,2, neuronal migration and positioning3,4, and even response to sensory input5. However, it remains to be seen whether these tissues can model axon guidance dynamics and the formation of complex connectivity with functional neuronal output. Here, we have established a longterm air-liquid interface culture paradigm that leads to improved neuronal survival and allows for imaging of axon guidance. Over time, these cultures spontaneously form thick axon tracts capable of projecting over long distances. Axon bundles display various morphological behaviors including intracortical projection within and across the organoid, growth cone turning, decussation, and projection away from the organoid. Single-cell RNA-sequencing reveals the full repertoire of cortical neuronal identities, and retrograde labelling demonstrates these tract morphologies match the appropriate molecular identities, namely callosal and corticofugal neuron types. We show that these neurons are functionally mature, generate active networks within the organoid, and that extracortical projecting tracts innervate and activate mouse spinal cord-muscle explants. Muscle contractions can be evoked by stimulation of the organoid, while axotomy of the innervating tracts abolishes the muscle contraction response, demonstrating dependence on connection with the organoid. Overall, these results reveal a remarkable selforganization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and response to injury.

neuroscience↗