Biology subjects

Hauser, S.

Publications and source records attributed to Hauser, S..

11 recordsLinked to original sources

bioRxiv2026

A synthetic xeno-free extracellular matrix surrogate supports robust human induced pluripotent stem cell maintenance and multilineage differentiation

Animal-derived surface coatings present the gold standard in stem cell culture. However, they come with major drawbacks such as lot-to-lot inconsistency and an ethically problematic production. Approaches to develop synthetic coating alternatives have largely failed to recapitulate biological functions essential to PSC-self renewal and differentiation. In this study, we have investigated the suitability of a fully synthetic, chemically defined extracellular matrix-mimicking coating named BIOINSTRUCT for hiPSC culture. BIOINSTRUCT was employed during maintenance culture of IMR90-4 cells, as well as directed differentiation of HIHCNi008-A, ACTN2-GFP, and UKKI-011-A cells. BIOINSTRUCT preserved hiPSC characteristics over the experimental period of 10 passages similar to Matrigel. Spontaneous differentiation resulted in the formation of ectodermal, mesodermal and endodermal derivatives, confirming the retention of pluripotency. Directed differentiation on BIOINSTRUCT enabled the generation of neuronal, cardiac and pancreatic endocrine cells. Altogether, our data underscore BIOINSTRUCT as a promising alternative to animal-derived extracellular matrix coatings.

cell biology↗

bioRxiv2026

Deep axonal proteomics of human iPSC-derived neurons by microfluidic separation and DIA-MS

One of the defining features of neurons is their compartmentalisation into soma, dendrites and axon. Axons are highly specialised for the transmission of signals over long distances but also exhibit unique vulnerabilities in the context of neurodegeneration, implying a specialised axonal proteome. However, in-depth proteomic characterisation of axons has been limited by the difficulty of isolating pure axonal material in sufficient quantities for conventional mass spectrometry analysis. Here, we combine microfluidic-based axon-soma separation with data-independent acquisition mass spectrometry (DIA-MS) on an Orbitrap Astral mass spectrometer for deep profiling of compartment-resolved proteomes of human induced pluripotent stem cell (iPSC)-derived cortical neurons. We quantify over 9,000 proteins in the somatodendritic compartment and [~]6,000 proteins in the axonal compartment, to our knowledge, representing the deepest human axonal proteome reported to date. Differential abundance analysis identifies 1,250 axon-enriched proteins with strong enrichment of axon-related pathways including vesicle-mediated transport, synaptic vesicle dynamics, and cytoskeletal organisation. Extending this workflow to iPSC-derived lower motor neurons, we reveal a core set of 417 axon-enriched proteins shared between cortical and lower motor neurons, alongside subtype-specific axonal signatures whose functional differences become evident only through compartment-restricted analysis. We show broad coverage of genes for the neurodegenerative diseases hereditary spastic paraplegia and amyotrophic lateral sclerosis, establishing a quantitative reference for interpreting how disease-linked mutations may differentially affect axonal versus somatodendritic proteostasis. This workflow and resource are readily applicable to other neuronal subtypes and disease models, paving the way for studying axonal proteome dynamics in health and disease. HighlightsO_LIDeepest human axonal proteome with [~]6,000 proteins from iPSC-derived neurons C_LIO_LIMicrofluidic separation paired with DIA mass spectrometry C_LIO_LIAxon-specific signatures masked in whole-cell analyses are revealed C_LIO_LICore set of 417 axon-enriched proteins shared across neuron subtypes C_LIO_LIFramework for studying axon-selective vulnerability in neurodegeneration C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/739061v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@191493dorg.highwire.dtl.DTLVardef@15f459forg.highwire.dtl.DTLVardef@1d3e61forg.highwire.dtl.DTLVardef@a9262a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Comprehensive investigation of AAV tropism across human iPSC-derived neuronal subtypes

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

neuroscience↗

bioRxiv2024

Long-Read RNA-sequencing reveals transcript-specific regulation in human-derived cortical neurons

Long-read RNA sequencing has transformed transcriptome analysis by enabling comprehensive mapping of full-length transcripts, providing an unprecedented resolution of transcript diversity, alternative splicing, and transcript-specific regulation. In this study, we employed nanopore long-read RNA sequencing to profile the transcriptomes of human fibroblasts, induced pluripotent stem cells, and stem cell-derived cortical neurons, identifying extensive transcript diversity with 15,072 transcripts in stem cell-derived cortical neurons, 13,048 in fibroblasts, and 12,759 in induced pluripotent stem cells. Our analyses uncovered 35,519 differential transcript expression events and 5,135 differential transcript usage events, underscoring the complexity of transcriptomic regulation across these cell types. Importantly, by integrating differential transcript expression and usage analyses, we gained deeper insights into transcript dynamics that are not captured by gene-level expression analysis alone. Notably, differential transcript usage analysis highlighted transcript-specific changes in disease-relevant genes such as APP, KIF2A, and BSCL2, associated with Alzheimers disease, neuronal migration disorders, and degenerative axonopathies, respectively. This added resolution emphasizes the significance of transcript- level variations that often remain hidden in traditional differential gene expression analyses. Overall, our work provides a framework for understanding transcript diversity in both pluripotent and specialized cell types, which can be used to investigate transcriptomic changes in disease states. Additionally, this study underscores the utility of differential transcript usage analysis in advancing our understanding of neurodevelopmental and neurodegenerative diseases, paving the way for identifying transcript-specific therapeutic targets.

neuroscience↗

bioRxiv2024

Most L1CAM is not associated with extracellular vesicles in human biofluids and iPSC-derived neurons

Transmembrane L1 cell adhesion molecule (L1CAM) is widely used as a marker to enrich for neuron-derived extracellular vesicles (EVs), especially in plasma. However, this approach lacks sufficient robust validation. This study aimed to assess whether human biofluids are indeed enriched for EVs, particularly neuron-derived EVs, by L1CAM immunoaffinity, utilizing multiple sources (plasma, CSF, conditioned media from iPSC-derived neurons [iNCM]) and different methods (mass spectrometry [MS], nanoparticle tracking analysis [NTA]). Following a systematic multi-step validation approach, we confirmed isolation of generic EV populations using size-exclusion chromatography (SEC) and polymer-aided precipitation (PPT) - two most commonly applied EV isolation methods - from all sources. Neurofilament light (NfL) was detected in both CSF and blood-derived EVs, indicating their neuronal origin. However, L1CAM immunoprecipitation did not yield enrichment of L1CAM in EV fractions. Instead, it was predominantly found in its free-floating form. Additionally, MS-based proteomic analysis of CSF-derived EVs also did not show L1CAM enrichment. Our study validates EV isolation from diverse biofluid sources by several isolation approaches and confirms that some EV subpopulations in human biofluids are of neuronal origin. Thorough testing across multiple sources by different orthogonal methods, however, does not support L1CAM as a marker to reliably enrich for a specific subpopulation of EVs, particularly of neuronal origin.

neuroscience↗

bioRxiv2024

Unraveling Axonal Transcriptional Landscapes: Insights from iPSC-Derived Cortical Neurons and Implications for Motor Neuron Degeneration

Neuronal function and pathology are deeply influenced by the distinct molecular profiles of the axon and soma. Traditional studies have often overlooked these differences due to the technical challenges of compartment specific analysis. In this study, we employ a robust RNA-sequencing (RNA-seq) approach, using microfluidic devices, to generate high-quality axonal transcriptomes from iPSC-derived cortical neurons (CNs). We achieve high specificity of axonal fractions, ensuring sample purity without contamination. Comparative analysis revealed a unique and specific transcriptional landscape in axonal compartments, characterized by diverse transcript types, including protein-coding mRNAs, RNAs encoding ribosomal proteins (RPs), mitochondrial-encoded RNAs, and long non-coding RNAs (lncRNAs). Previous works have reported the existence of transcription factors (TFs) in the axon. Here, we detect a set of TFs specific to the axon and indicative of their active participation in transcriptional regulation. To investigate transcripts and pathways essential for central motor neuron (MN) degeneration and maintenance we analyzed KIF1C-knockout (KO) CNs, modeling hereditary spastic paraplegia (HSP), a disorder associated with prominent length-dependent degeneration of central MN axons. We found that several key factors crucial for survival and health were absent in KIF1C-KO axons, highlighting a possible role of these also in other neurodegenerative diseases. Taken together, this study underscores the utility of microfluidic devices in studying compartment-specific transcriptomics in human neuronal models and reveals complex molecular dynamics of axonal biology. The impact of KIF1C on the axonal transcriptome not only deepens our understanding of MN diseases but also presents a promising avenue for exploration of compartment specific disease mechanisms.

neuroscience↗

bioRxiv2022

Human organoid model of PCH2a recapitulates brain region-specific pathology

Pontocerebellar hypoplasia type 2 a (PCH2a) is a rare, autosomal recessive pediatric disorder with limited treatment options. Its anatomical hallmark is the hypoplasia of the cerebellum and pons accompanied by progressive microcephaly. PCH2a results from a homozygous founder variant in TSEN54, which encodes a tRNA splicing endonuclease (TSEN) complex subunit. Despite the ubiquitous expression of the TSEN complex, the tissue-specific pathological mechanism of PCH2a remains unknown due to a lack of model system. In this study, we developed human models of PCH2a using brain region-specific organoids. We therefore obtained skin biopsies from three affected males with genetically confirmed PCH2a and derived induced pluripotent stem cells (iPSCs). Proliferation and cell death rates were not altered in PCH2a iPSCs. We subsequently differentiated cerebellar and neocortical organoids from control and PCH2a iPSCs. Mirroring clinical neuroimaging findings, PCH2a cerebellar organoids were reduced in size compared to controls starting early in differentiation. We observed milder growth deficits in neocortical PCH2a organoids. While PCH2a cerebellar organoids did not upregulate apoptosis, their stem cell zones showed altered proliferation kinetics, with increased proliferation at day 30 and reduced proliferation at day 50 compared to controls. In summary, we have generated a human model of PCH2a, which provides the foundation for deciphering brain region-specific disease mechanisms.

neuroscience↗

bioRxiv2022

Psychometric and subcortical neurometric measures of temporal discrimination in rhesus macaques

Temporal envelope fluctuations are abundant in nature and are critical for perception of complex sounds. While psychophysical sinusoidal amplitude modulation (SAM) processing studies have characterized the perception of SAM, and neurophysiological studies report a subcortical transformation from temporal to rate-based code, no studies have characterized this transformation in unanesthetized animals or in nonhuman primates. To address this, we recorded single-unit responses and compared derived neurometric measures in the cochlear nucleus (CN) and inferior colliculus (IC) to psychometric measures of modulation frequency (MF) discrimination in macaques. IC and CN neurons often exhibited tuned responses to SAM in their rate and spike-timing. Neurometric thresholds spanned a large range (2-200 Hz {Delta} MF). The lowest 40% of IC thresholds were less than or equal to psychometric thresholds, regardless of which code was used, while CN thresholds were greater than psychometric thresholds. Discrimination at 10-20 Hz could be explained by indiscriminately pooling 30 units in either structure, while discrimination at higher MFs was best explained by more selective pooling. This suggests that pooled brainstem activity was sufficient for AM discrimination. Psychometric and neurometric thresholds decreased as a function of stimulus duration, but IC and CN thresholds were greater and more variable than behavior at durations less than 500 ms. This slower subcortical temporal integration compared to behavior was consistent with a drift diffusion model which reproduced individual differences in performance and can constrain future neurophysiological studies of temporal integration. These measures provide an account of AM perception at the neurophysiological, computational, and behavioral levels. Significance statementListening in everyday environments tasks the brain with extracting information from sound envelopes. This process involves both sensory encoding and decision-making. Different neural codes for envelope representation have been well characterized in the auditory midbrain and cortex, but studies of the brainstem have usually been conducted in anesthetized rodents or cats. Moreover, these candidate neural codes have been studied in isolation from the decision-making process. In this study, we found that population activity in the primate subcortical auditory system contains sufficient information for discriminating sound envelope and applied a biologically plausible model of decision-making to sound envelope discrimination performance from rhesus macaques, a species with great phylogenetic and perceptual similarity to humans.

neuroscience↗

bioRxiv2021

Human KIR+CD8+ T cells target pathogenic T cells in Celiac disease and are active in autoimmune diseases and COVID-19

Previous reports show that Ly49+CD8+ T cells can suppress autoimmunity in mouse models of autoimmune diseases. Here we find a markedly increased frequency of CD8+ T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various autoimmune diseases. Moreover, KIR+CD8+ T cells can efficiently eliminate pathogenic gliadin-specific CD4+ T cells from Celiac disease (CeD) patients leukocytes in vitro. Furthermore, we observe elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Expanded KIR+CD8+ T cells from these different diseases display shared phenotypes and similar T cell receptor sequences. These results characterize a regulatory CD8+ T cell subset in humans, broadly active in both autoimmune and infectious diseases, which we hypothesize functions to control self-reactive or otherwise pathogenic T cells. One-Sentence SummaryHere we identified KIR+CD8+ T cells as a regulatory CD8+ T cell subset in humans that suppresses self-reactive or otherwise pathogenic CD4+ T cells.

immunology↗

bioRxiv2021

The crucial role of genome-wide genetic variation in conservation

The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on the conservation of genome-wide genetic variation, and that this approach should be replaced with another that focuses instead on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that the conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations towards extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide variation on long-term population viability will only worsen the biodiversity crisis.

evolutionary biology↗

bioRxiv2020

Clinical metagenomics bioinformatics pipeline for the identification of hospital-acquired pneumonia pathogens antibiotic resistance genes from bronchoalveolar lavage samples

BackgroundShortening the time-to-result for pathogen detection and identification and antibiotic susceptibility testing for patients with Hospital-Acquired and Ventilator-Associated pneumonia (HAP-VAP) is of great interest. For this purpose, clinical metagenomics is a promising non-hypothesis driven alternative to traditional culture-based solutions: when mature, it would allow direct sequencing all microbial genomes present in a BronchoAlveolar Lavage (BAL) sample with the purpose of simultaneously identifying pathogens and Antibiotic Resistance Genes (ARG). In this study, we describe a new bioinformatics method to detect pathogens and their ARG with good accuracy, both in mono- and polymicrobial samples. MethodsThe standard approach (hereafter called TBo), that consists in taxonomic binning of metagenomic reads followed by an assembly step, suffers from lack of sensitivity for ARG detection. Thus, we propose a new bioinformatics approach (called TBwDM) with both models and databases optimized for HAP-VAP, that performs reads mapping against ARG reference database in parallel to taxonomic binning, and joint reads assembly. ResultsIn in-silico simulated monomicrobial samples, the recall for ARG detection increased from 51% with TBo to 97.3% with TBwDM; in simulated polymicrobial infections, it increased from 41.8% to 82%. In real sequenced BAL samples (mono and polymicrobial), detected pathogens were also confirmed by traditional culture approaches. Moreover, both recall and precision for ARG detection were higher with TBwDM than with TBo (35 points difference for recall, and 7 points difference for precision). ConclusionsWe present a new bioinformatics pipeline to identify pathogens and ARG in BAL samples from patients with HAP-VAP, with higher sensitivity for ARG recovery than standard approaches and the ability to link ARG to their host pathogens.

bioinformatics↗