Biology subjects

Wingens, L. J. A.

Publications and source records attributed to Wingens, L. J. A..

3 recordsLinked to original sources

bioRxiv2025

Long-term co-maturation of stem cell-derived microglia and neuronal networks: an optimized platform to assess human microglial contribution to neuronal function

Microglia-neuron interactions play a central role in a variety of central nervous system disorders. Technologies using human induced pluripotent stem cells (hiPSCs) have been developed to model human brain cells with the goal to understand their function. To effectively study neuro-immune crosstalk and investigate microglial contributions to neuronal network development and function, both microglia and neurons should co-mature allowing for long-term interactions throughout their differentiation. Here, we present a co-maturation protocol that robustly generates glutamatergic neuronal networks containing human iPSC-derived microglia. We validated the long-term co-cultures using single-cell transcriptomics, imaging and neuronal activity readouts. We show that astrocytes were required for long-term survival of microglia and for their integration into neuronal networks. Our co-maturation approach induced the typical ramified microglia morphology and characteristic microglia-neuron interactions. Homeostatic markers like P2RY12 and TMEM119 and neuronal remodeling associated genes were upregulated compared to microglia monocultures, highlighting the necessity of the environment to generate and maintain the context-dependent microglia signature in vitro. In this manuscript, we include the full optimization process of our co-maturation approach, a comprehensive description of the protocol, practical guidelines and troubleshooting tips. Our co-maturation model provides a powerful tool to assess the role of human microglia in modulating neuronal function and development in health and disease. HighlightsO_LIptimized human iPSC differentiation protocol that allows for co-maturation of microglia and Neurogenin2-induced neuronal networks. C_LIO_LIare required for long-term survival and integration of microglia into neuronal networks. C_LIO_LICo-maturation approach enables characteristic neuron-microglia interactions and induces signature morphology, transcriptome and proteins of human microglia. C_LI

neuroscience↗

bioRxiv2023

Signalling-state dependent drug-tolerance in head and neck squamous cell carcinoma.

Intratumor heterogeneity negatively impacts therapeutic response and patient prognosis. Besides the established role of genetic heterogeneity, non-genetic mechanisms of persistence to drug treatment are emerging. Here, we characterise cells selected for their persistence to control, epidermal growth factor inhibition (EGFRi), radiation and combined treatment from low passage head and neck squamous cell carcinoma (HNSCC) cultures. Using a panel of 70 (phospho-)specific DNA-conjugated antibodies we measured activities of 8 signalling pathways, self-renewal, differentiation, DNA damage and cell-cycle, in conjunction with the transcriptional output in single cells, using our RNA and Immuno-Detection (RAID) technology. Six recurrent transcriptional programs reflecting processes including proliferation, differentiation and metabolic activity, as well as protein-based signalling-states, were associated with drug persistence, while copy number variation inference indicated involvement of non-genetic tolerance mechanisms. Projecting RNA velocity onto the antibody-derived signalling-states suggested a key role for integrin-mediated focal-adhesion signalling in drug-persistence in our cell system. Using machine-learning we derived a core transcriptional signature connected to adhesion-based drug-persistence, which was predictive of poor prognosis in a TGCA HNSCC cohort (hazard-ratio 1.87, p<10-5). Furthermore, functional analyses confirmed that cells expressing high levels of integrin alpha-6 (ITGA6) were tolerant to EGFRi treatment, and that forcing cells out of this cell-state through transient targeted inhibition of Focal Adhesion Kinase activity re-instated EGFRi sensitivity in drug persistent cells. Taken together, our single-cell multi-omics analysis identified an actionable adhesion-signalling mediated cell-state driving drug tolerance in HNSCC.

systems biology↗

bioRxiv2022

Integrated single-cell (phospho-)protein and RNA detection uncovers phenotypic characteristics of human antibody secreting cells.

Antibody-secreting cells (ASCs) secrete IgM, IgA, or IgG antibodies and are key components of humoral immunity; however, little is known about unique characteristics of the Ig-classes due to limited availability of material and challenges to quantify many intracellular molecular modalities at a single-cell resolution. We combined a method to in vitro differentiate peripheral B-cells into ASCs with integrated multi-omic single-cell sequencing technologies to quantify subclass-specific hallmark surface markers, transcriptional profiles and signaling transduction pathway components. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors and IL-2, IL-6, JAK/STAT and mTOR signaling activity across Ig-subclasses. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a complex sample of in vitro differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets. TeaserIntegrated single-cell analysis allows tri-modal phenotypic analysis of in-vitro generated human antibody-secreting cells.

immunology↗