Biology subjects

Meyer, D.

Publications and source records attributed to Meyer, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Evaluating Aggregated Gene Level eQTL Scores

Genetic feature engineering, used in methods such as transcriptome-wide association study, supports gene-trait association testing by aggregating single variants into gene-level features predictive of expression. To evaluate how different model architectures, LD filtering thresholds, and variant prioritization methods affect expression prediction quality, we trained over 3 million models and evaluated their performance in independent cohorts. Using the best performing models to impute expression and immunotherapy response as an example trait, we found a significant association with the reactive oxygen species pathway (p=0.032). Our model training workflow will support genetic feature engineering towards improved complex trait modeling.

bioinformatics↗

bioRxiv2026

Is APOE ε2 always a protective allele? Deviations in Hardy-Weinberg equilibrium in admixed Brazilian elderly individuals

The APOE gene is a critical determinant of human healthspan and longevity, with the rare{varepsilon} 2 allele traditionally viewed as a universal protective factor against Alzheimer s disease (AD) and a driver of exceptional lifespan. However, this protective paradigm is predominantly derived from European-centric cohorts, leaving the evolutionary and clinical impacts of{varepsilon} 2 across diverse, highly admixed populations largely unknown due to a lack of local ancestry (LA) resolution. To investigate how local genomic backgrounds modulate APOE survival dynamics we analyzed two Brazilian sample collection of older adults from Sao Paulo city: the Biobank for Aging Studies (BAS, n = 716), a post-mortem autopsy study of naturally deceased individuals; and the Health, Well-being and Aging Study (SABE, n = 952), a census-based elderly sample collection. We evaluated deviations from Hardy-Weinberg equilibrium (HWE) using robust permutation-based models to capture ongoing selective and mortality pressures at the APOE locus. While global APOE frequencies adhered to HWE, integrating LA unveiled striking, mirrored ancestral deviations. Our findings reveal that APOE {varepsilon}2 homozygotes with African ancestry significantly contribute to deviations from HWE in the BAS, with an excess of {varepsilon}2AFR/{varepsilon}2AFR homozygotes observed (p = 0.0196). These distinct HWE deviations demonstrate that an African LA background acts as a genetic buffer, attenuating the phenotypic extreme effects of APOE alleles. Furthermore, we observed an excess of the{varepsilon} 4 European haplotypes in the BAS, which is consistent with a mortality pressure allelic effect in the European LA context. Conversely, the{varepsilon} 4AFR/{varepsilon}4AFR combination was overrepresented in the SABE. While this buffering mechanism mitigates{varepsilon} 4 toxicity, it simultaneously dampens the exceptional longevity advantage typically conferred by the{varepsilon} 2 allele, leading to its neutral accumulation in the post-mortem cohort. Our study challenges the "one-size-fits-all" assumption of APOE biomarkers, demonstrating that{varepsilon} 2 protective mechanisms are context-dependent and modulated by local genomic backgrounds in admixed populations.

genetics↗

bioRxiv2026

Gut-derived metabolic reprogramming drives immune aging and tissue degeneration

Aging is characterized by changes in gut microbiome, metabolic imbalance and chronic inflammation, yet how these processes integrate to drive tissue degeneration remains poorly defined. Using age-related macular degeneration (AMD) as a model of tissue aging, we identify a diet-induced metabolic-immune axis that promotes systemic and retinal degeneration. In mice, a high-fat, cholesterol-enriched (HFC) diet induced perturbations in the gut structural integrity and microbiome repertoire, as well as systemic metabolic aging signatures, prominently marked by reduced circulating histidine. Plasma histidine levels were similarly decreased in AMD patients and inversely correlated with body mass index (BMI) in control donors. These diet-induced gut microbiome changes and subsequent metabolic alterations promoted peripheral innate immune reprogramming, with expansion of inflammatory neutrophils and monocytes that infiltrated the outer retina in a mouse model. Mechanistically, the gut-derived IGF1R/AKT2 signaling acts as a central regulator of global epigenetic remodeling and systemic immune aging under high-fat conditions in C. elegans. In a mouse model with an age-dependent dry AMD-like pathology, distinct retinal pigment epithelium (RPE) subpopulations exhibited downregulation of the histidine transporter SLC7A5, linking metabolic stress to activation of MIF/CD74-dependent inflammatory signaling between RPE and infiltrating immune cells. Histidine supplementation or AKT2 phospho-state modulation attenuated systemic immune activation and rescued retinal degeneration. These findings identify histidine-axis dysregulation as a mechanistic bridge between diet-induced microbiome changes, metabolic stress, immune aging, and retinal degeneration.

cell biology↗

bioRxiv2026

Enhancing volumetric microscopy with blind computational correction of spatially variant aberrations using neural field representation

Studying biological processes across multi-millimeter scales requires imaging systems that combine high spatial resolution with a large field of view (FOV). However, optical aberrations degrade image quality, particularly in large-FOV systems where distortions gradually worsen toward the periphery. Existing methods for correcting field-dependent aberrations are limited and often impractical. Hardware-based solutions such as adaptive optics demand additional components and sample manipulation, increasing system complexity and experimental burden. Computational approaches, while promising, typically rely on exhaustive point spread function (PSF) calibration or large training datasets, restricting their applicability. We introduce a self-supervised algorithm that simultaneously estimates spatially varying aberrations and reconstructs 3D sample structure from a single blurred image--without PSF calibration or training data. Our method was implemented on optically cleared mouse hippocampal neurons and cortical vasculature imaged with oblique plane microscopy (OPM), as well as in vivo mouse retinal vasculature captured with adaptive optics scanning laser ophthalmoscopy (AOSLO), where PSF calibration is infeasible. The algorithm predicts and corrects aberrations across multi-millimeter FOVs, enhances contrast in low signal-to-noise regions, and reveals critical structural details in neuronal and vascular systems that are obscured in raw images. This approach enables high-resolution, large-scale imaging without hardware modifications, expanding the accessibility of advanced microscopy techniques.

bioinformatics↗

bioRxiv2025

Versatile high-speed volumetric imaging from microscopic to macroscopic scale by self-adaptive oblique plane microscopy

There is an increasing need for large-scale high-speed volumetric recording in complex multi-cellular model systems to define dynamic processes. Oblique plane microscopy (OPM) provides a solution that features oblique illumination, rapid optical scanning, and remote focusing to achieve real-time 4D microscopy. OPM implements light sheet imaging via a single primary objective lens, making the entire space below the objective accessible for large specimens, such as living mouse brain. Yet it is challenging to adopt OPM beyond a microscopic scale (i.e. size < 1mm), limiting its broad applications. Here we present a self-adaptive OPM that leverages Abbes sine condition to unlock its flexibility across a range of field-of-views (FOVs) (up to 8 mm2) and resolutions (down to 2.2 {micro}m3). This versatility enables brain-wide single neuron volumetric calcium imaging in behaving larval zebrafish (1x0.4 mm2 FOV at 5 Hz) and capillary blood cell tracking in living mouse brain (>3x3 mm2 FOV) with a sweeping 0.32 mm wide volume section at 100 Hz. In optically cleared mouse brain, the flexibility allows a screen-and-zoom capability by sequentially imaging the whole brain at low-and-high magnifications to locate and resolve subcellular structures such as dendritic tress and spines. By offering a switchable imaging resolution, volume, and speed, the self-adaptive OPM achieves a versatile platform for studying a wide range of multi-cellular model system, whether in vivo or fixed and optically cleared.

bioengineering↗

bioRxiv2025

A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

The retinal pigment epithelium (RPE) sustains lifelong proteostasis under chronic stress, yet how post-mitotic cells activate autophagy when canonical kinase pathways suppress it remains unresolved. Here we demonstrate that AKT1, conventionally regarded as an autophagy suppressor, drives autophagy through a non-canonical transcriptional mechanism triggered by isoform imbalance. In RPE and age-related macular degeneration (AMD) models, AKT2 hyperactivation destabilizes mTORC2 and engages S6K-mediated IRS-1 inhibition, creating a feedforward autophagic arrest. Compensatory AKT1 activation via mTORC2 phosphorylates telomerase reverse transcriptase (TERT) at Serine 824, driving nuclear translocation that is independent of telomere maintenance. In the nucleus, TERT assembles with FOXO3 and MYC into a tripartite transcriptional complex that occupies the EIF2AK3 promoter, enabling PERK transcriptional activation. This activity converts the unfolded protein response from pro-apoptotic to cytoprotective: PERK-ATF4 signaling drives biogenesis of core autophagy machinery while simultaneously inducing selective ERphagy through the receptors TEX264 and CCPG1, which prevents pathological PERK clustering and preserves tubular ER architecture in diseased RPE. Using C. elegans phosphorylation-deficient mutants, mouse models and AMD patient induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE), we establish that AKT1-mediated TERT phosphorylation is an evolutionarily conserved prerequisite for FOXO/DAF-16 nuclear function and lysosomal homeostasis in post-mitotic cells. Pharmacologic targeting of AKT2 with a first-in-class dual-pocket allosteric inhibitor selectively enhances AKT1 compensation, restoring macroautophagic and ERphagy flux across disease models. These findings reveal a kinase-to-transcription axis that reprograms organelle quality control and identify the AKT1-TERT-PERK-ATF4 pathway as a therapeutic target in proteostasis-driven disease.

cell biology↗

bioRxiv2025

Characterizing Co-Circulating Respiratory Virus Genomic Diversity in Switzerland with Hybrid-Capture Sequencing and Phylogenetic Reconstructions: Insights into the 2023/24 Season

Respiratory viruses circulate yearly with strain-specific patterns. Although SARS-CoV-2 and Influenza A/B genomic surveillance is well-developed, most respiratory viruses are unevenly monitored, lacking geographical diversity to capture wider population dynamics. Consequently, insights into respiratory virus evolution are limited. We investigated the genetic diversity of these viruses within one country. During the 2023/24 season, we conducted whole-genome sequencing of 1129 clinical samples using a hybrid-capture protocol. These samples were pre-tested by real-time PCR panels throughout Switzerland. Leveraging publicly-available full-length genomes, we constructed background datasets representative of geographical diversity and built phylogenies for all viruses with more than 40 new high-quality genomes from this study. We detected 981 viruses and recovered 461 high-quality genomes, including 18 co-infections, from 437 PCR-positive and 6 PCR-negative samples. All viruses detected by PCR were also detected by sequencing in 56% of samples. The four most prevalent viruses were Influenza A/H1N1, SARS-CoV-2, RSV-A, and HPIV-3, and their seasonal spread was consistent with wastewater monitoring and influenza-like illness reports. Swiss viral genomes were representative of the global genomic diversity, with evidence for multiple introductions into Switzerland, and we identified putative Swiss clusters. In this proof-of-concept study, we focus on 3 viruses (Influenza A/H1N1, RSV-A/B, and HPIV-3), and we demonstrate the streamlined implementation of a broad respiratory virus genomic surveillance workflow with an off-the-shelf protocol and publicly-available software. In addition, we highlight additional evolutionary insights that can only be derived from genomic surveillance. Going forward, this dataset will be a useful resource for future investigations into respiratory viral genomic diversity.

genetics↗

bioRxiv2025

Human genetic variation shapes the response of neurons to interferons

Inflammation is increasingly recognized as important to neuropathology, including more classic neuroimmune disease as well as neurodegenerative and neuropsychiatric disorders. Interferons (IFN) are important mediators of central nervous system inflammation. Individuals appear to vary in susceptibility to neuroinflammatory pathology, suggesting that identifying human genetic modifiers of the neuronal IFN response might provide insight into disease pathophysiology. To identify potential modifiers, we stimulated neuronal "cellular villages" of iPSC-derived neurons from over one hundred donors with IFN-alpha (IFNa) or IFN-gamma (IFNg). We then correlated allele states of common variable SNPs to gene expression to identify hundreds of expression quantitative trait loci (eQTLs), many of which emerged specifically upon IFN treatment. We characterized the distinct but overlapping neuronal transcriptional responses to IFNa and IFNg, and identified specific response QTLs. Functional annotation of STAT1 binding to the genome in response to IFN stimulus identified STAT1 binding sites as enriched for response-regulating human genetic variation and also enabled identification of loci with IFN-dependent allele-specific binding of STAT1. These results demonstrate how human genetic variation can influence IFN-dependent mechanisms in neurons in disease-relevant ways.

molecular biology↗

bioRxiv2025

Suite3D: Volumetric cell detection for two-photon microscopy

In two-photon imaging of neuronal activity it is increasingly common to acquire 3-dimensional volumes. However, these volumes are typically processed plane by plane, leading to uncorrected axial movement, duplicated cells across planes, reduced signal-to-noise ratio per cell, and missed cells. To overcome these limitations, we introduce Suite3D, a volumetric cell detection pipeline. Suite3D corrects for rigid and non-rigid 3D brain motion, both lateral and axial. It detects neurons using 3D correlation, improving detectability. Finally, it performs 3D segmentation, identifying cells across imaging planes. We validated Suite3D with data from conventional multi-plane microscopes and advanced volumetric microscopes, at multiple resolutions and in multiple brain regions, and with ground truth anatomical labelling. Suite3D successfully detected cells appearing on multiple imaging planes, improving cell detectability and signal quality, avoiding duplications, and running faster than a prior volumetric pipeline. Suite3D offers a powerful solution for analyzing volumetric two-photon data.

neuroscience↗

bioRxiv2025

diffONT: predicting methylation-specific PCR biomarkers based on nanopore sequencing data for clinical application

DNA methylation is known to act as biomarker applicable for clinical diagnostics, especially in cancer detection. Methylation-specific PCR (MSP) is a widely used approach to screen patient samples fast and efficiently for differential methylation. During MSP, methylated regions are selectively amplified with specific primers. With nanopore sequencing, knowledge about DNA methylation is generated during direct DNA sequencing, without any need for pretreatment of the DNA. Multiple methods, mainly developed for whole-genome bisulfite sequencing (WGBS) data, exist to predict differentially methylated regions (DMRs) in the genome. However, the predicted DMRs are often very large, and not sufficiently discriminating to generate meaningful results in MSP creating a gap between theoretical cancer marker research and practical application, as no tool currently provides methylation difference predictions tailored for PCR-based diagnostics. Here we present diffONT, which predicts differentially methylated primer regions, directly suitable for MSP primer design and thus allowing a direct translation into practical approaches. diffONT takes into account (i) the specific length of primer and amplicon regions, (ii) the fact that one condition should be unmethylated, and (iii) a minimal required amount of differentially methylated cytosines within the primer regions. Based on two nanopore sequencing data sets we compared the results of diffONT to metilene, DSS and pycoMeth. We show that the regions predicted by diffONT are more specific towards hypermethylated regions and more usable for MSP. diffONT accelerates the design of methylation-specific diagnostic assays, bridging the gap between theoretical research and clinical application.

bioinformatics↗

bioRxiv2024

Rad51 determines pathway usage in post-replication repair

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro. A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 [A] resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

molecular biology↗

bioRxiv2024

Toll-like receptor 3 orchestrates a conserved mechanism of heart regeneration

The humans heart responds to tissue damage with persistent fibrotic scarring. Unlike humans, zebrafish can repair cardiac injury and re-grow heart tissue throughout life. Recently, Toll-like receptor 3 (Tlr3) was identified as an important mediator of cardiac regeneration in neonatal mice. However, no functional analysis of tlr3 knock-out mutant zebrafish in respect to cardiac regeneration has yet been performed. We hypothesize that TLR3 signalling plays a central, conserved role in driving cardiac regeneration upon injury. Therefore, we focused on tlr3 mediated cardiac regeneration in zebrafish, ultimately discovering an evolutionary conserved mechanism of heart repair. Using histological, behavioural, and RNA-Sequencing analysis, we uncovered a conserved mechanism of tlr3 mediated cardiac repair after myocardial injury. Upon myocardial cryoinjury subjection, survival is decreased in tlr3-/- fish as compared to wildtype controls. Tlr3-/- zebrafish fail to recruit immune cells to the injured ventricle, resulting in impaired DNA repair and transcriptional reprogramming of cardiomyocytes. Mechanistically, we uncover an evolutionary conserved mechanism of tlr3 activation in fibroblasts promoting monocyte migration towards an injured ventricular area. Our data reveal tlr3 as a novel therapeutic target to promote cardiac regeneration. Every experiment including human participants has been approved by the ethics committee of the Medical University of Innsbruck (Ref. Nr.: 1262/2023). All experiments including the use of laboratory animals have been approved by the federal ministry of education, science, and research of Austria (Ref. Nr.: 2020-0.345.504).

molecular biology↗

bioRxiv2024

Concerted neuron-astrocyte gene expression declines in aging and schizophrenia

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a striking relationship between peoples cortical neurons and cortical astrocytes. We used single-nucleus RNA-seq to analyze the prefrontal cortex of 191 human donors ages 22-97 years, including healthy individuals and persons with schizophrenia. Latent-factor analysis of these data revealed that in persons whose cortical neurons more strongly expressed genes for synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the Synaptic Neuron- and-Astrocyte Program (SNAP). In schizophrenia and aging - two conditions that involve declines in cognitive flexibility and plasticity 1,2 - cells had divested from SNAP: astrocytes, glutamatergic (excitatory) neurons, and GABAergic (inhibitory) neurons all reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy persons of similar age, may underlie many aspects of normal human interindividual differences and be an important point of convergence for multiple kinds of pathophysiology.

neuroscience↗

bioRxiv2023

Maximizing the potential of genomic and transcriptomic studies by nanopore sequencing

Nucleic acid sequencing is the process of identifying the sequence of DNA or RNA, with DNA used for genomes and RNA for transcriptomes. Deciphering this information has the potential to greatly advance our understanding of genomic features and cellular functions. In comparison to other available sequencing methods, nanopore sequencing stands out due to its unique advantages of processing long nucleic acid strands in real time, within a small portable device, enabling the rapid analysis of samples in diverse settings. Evolving over the past decade, nanopore sequencing remains in a state of ongoing development and refinement, resulting in persistent challenges in protocols and technology. This article employs an interdisciplinary approach, evaluating experimental and computational methods to address critical gaps in our understanding in order to maximize the information gain from this advancing technology. Here we present both overview and analysis of all aspects of nanopore sequencing by providing statistically supported insights. Thus, we aim to provide fresh perspectives on nanopore sequencing and give comprehensive guidelines for the diverse challenges that frequently impede optimal experimental outcomes.

bioinformatics↗

bioRxiv2022

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Astrocytes play essential roles in normal brain function, with dysfunction implicated in diverse developmental and degenerative disease processes. Emerging evidence of profound species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy, incompletely characterized, or require intermediate purification steps, limiting their utility for multi-cell line, adequately powered functional studies. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs) based upon transient Neurogenin 2 (NGN2) induction of neural progenitor-like cells followed by maturation in astrocyte media, which demonstrate remarkable homogeneity within the population and across 11 independent cell lines in the absence of additional purification steps. These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal maturation in vitro. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, most closely resembling human fetal astrocytes, and highly similar to hPSC-derived astrocytes generated using more complex approaches. Finally, the hiAs capture key molecular hallmarks in a trisomy 21 disease model. Thus, hiAs provide a valuable and practical resource well-suited for study of basic human astrocyte function and dysfunction in disease.

neuroscience↗

bioRxiv2022

Neurodevelopmental Patterns of Early Postnatal White Matter Maturation Represent Distinct Underlying Microstructure and Histology

During the early postnatal period, cerebral white matter undergoes rapid maturation through a complex series of interrelated cellular and histogenetic processes. Accurately quantifying these processes is important for improving understanding of early brain development, developmental abnormalities related to prematurity, and neurodevelopmental diseases. Past efforts have used magnetic resonance imaging (MRI) to track these developmental processes in vivo. However, most previous studies have relied on single imaging modality data and have often been limited by small samples and analytics that do not evaluate complex multivariate imaging patterns. Here, we applied an advanced unsupervised multivariate pattern analysis technique, non-negative matrix factorization (NMF), to T2w/T1w signal ratio maps from a large cohort of newborns (Developing Human Connectome Project [dHCP], n=342), revealing patterns of synchronous white matter maturation. These patterns showed divergent age-related maturational trajectories and differential susceptibility to premature birth, which were replicated in an independent large sample of newborns (Early Life Adversity, Biological Embedding, and Risk for Developmental Precursors of Mental Disorders [eLABE], n=239). Furthermore, we showed that T2w/T1w signal variations in white matter maturational patterns are explained by differential contributions of white matter microstructure indices (i.e., free water content and neurite density index) derived from neurite orientation dispersion and density imaging (NODDI) modeling of diffusion-weighted MRI. Finally, we demonstrated how white matter maturation patterns relate to distinct histological features by comparing our findings with postmortem late fetal/early postnatal brain tissue staining. Together, these results delineate a novel MRI representation of white matter microstructural and histological reorganization during the early postnatal development.

neuroscience↗

bioRxiv2022

Cytotoxic T lymphocytes targeting a conserved SARS-CoV-2 spike epitope are efficient serial killers

Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening can provide insights into the skewing of a T-cell response elicited upon vaccination or infection, they unfortunately provide no assessment into the functional capacity of T cells or their ability to eliminate virally infected targets. We have used time-lapse imaging microscopy in nanowell grids (TIMING) to integrate the migration of individual T cells with analysis of effector functions including cytokine secretion and cytotoxicity. Machine learning is then applied to study thousands of videos of dynamic interactions as T cells with specificity for SARS-CoV-2 eliminate targets bearing spike protein as a surrogate for viral infection. Our data provide the first direct evidence that cytotoxic T lymphocytes from a convalescent patient targeting an epitope conserved across all known variants of concern (VoC) are serial killers capable of eliminating multiple infected targets. These data have implications for development of vaccines to provide broad and sustained cellular immunity and for the recovery and monitoring of individuals who have been exposed to SARS-CoV-2. Multidisciplinary abstractWe present an imaging platform that uses artificial intelligence (AI) to track thousands of individual cell-cell interactions within nanowell arrays. We apply this platform to quantify how the T cell component of adaptive immunity responds to infections. Our results show that T cells specific for a conserved epitope within the SARS-CoV-2 spike protein are serial killers that can rapidly eliminate virally infected targets. The ability to map the functional capacity of T cells and their ability to kill infected cells provides fundamental insights into the immunology of vaccines and recovery from infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/477535v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3e3eaborg.highwire.dtl.DTLVardef@844cacorg.highwire.dtl.DTLVardef@1c5ecfdorg.highwire.dtl.DTLVardef@14a0b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

neuroscience↗