Biology subjects

Hagemann, C.

Publications and source records attributed to Hagemann, C..

11 recordsLinked to original sources

bioRxiv2026

Dynamic modelling of human neural crest development using a bioengineered stem cell organoid system

The neural crest (NC) is a transient stem cell population which migrates throughout the developing embryo to contribute to diverse tissues dependent on axial origin. For example, cranial NC can give rise to bone and cartilage, while more posterior NC populations give rise to peripheral nervous system and neuroendocrine tissues. Perturbations in neural crest development can lead to severe congenital anomalies and cancers, with over 700 neurocristopathies reported. In humans, early NC development remains poorly understood due to the inaccessibility of tissue samples, thus necessitating the development of in vitro models. Currently, a limited number of NC organoid protocols are available, but these mainly focus on cranial NC and lack relevant tissue architecture. Here, we describe a novel bioengineered pipeline to derive human pluripotent stem cell (hPSC)-derived neuroepithelial organoids, "neurocrestoids" featuring physiologically-relevant tissue architecture. We show that neurocrestoids recapitulate the dynamics of induction, delamination, and migration of human neural crest cells (NCCs), and can be directly compared to murine NC explants for cross-species validation. Organoids express an array of HOX genes indicating the successful generation of cranial, vagal and trunk NCCs. Moreover, we have integrated our neurocrestoids with a customised micropatterned substrate suitable for live visualisation and guided separation of SOX10-positive migratory human NCCs. Our "NCC migration on-chip" are reproducible across multiple hPSC lines and should be scalable for future diagnostic and therapeutic applications, significantly improving our ability to study human NC pathologies.

developmental biology↗

bioRxiv2025

VISIBLE: An imaging-driven system for sampling, biofabrication, and manipulation of complex biological models

Complex in vitro models (CIVMs), including organoids, spheroids, and bioprinted constructs, have emerged as powerful platforms for recapitulating human tissue architecture and function. However, their inherent heterogeneity and dynamic nature pose significant challenges for standardization, reproducibility, and real-time manipulation. Here, we present VISIBLE (Versatile Imaging-Based Interactive Sampling and Bioprinting System), a modular, imaging-guided platform that integrates real-time monitoring with automated manipulation and 3D bioprinting that addresses these challenges. VISIBLE employs a dual-axis system comprising independently controlled tool-heads and microscope stages, enabling precise spatiotemporal interventions within live cultures. We demonstrate the system capabilities across a range of applications, including morphology- and function-based sampling of organoids and neurospheres, interactive 3D bioprinting, serial scratch assays, and real-time cell-cell interaction studies. Furthermore, we illustrate the system utility in translational contexts through the selective sampling and implantation of patient-derived xenograft organoids. VISIBLE supports long-term culture within an integrated incubation environment and accommodates interchangeable tool-heads for scalable, high-throughput workflows. By enabling closed-loop, feedback-controlled experimentation, VISIBLE addresses some of the critical limitations in existing CIVM platforms and offers a new versatile solution for a wide range of biomedical research applications. This work represents a significant step toward the development of autonomous experimental systems for complex tissue modelling and preclinical investigation.

bioengineering↗

bioRxiv2025

Engineering Cortical Networks: An Open Platform for Controlled Human Circuit Formation and Synaptic Analysis In vitro

Neuronal circuits are complex networks formed by specific neuron connections across brain regions. Understanding their development is key to studying circuit-related dysfunctions in brain diseases. Human-induced pluripotent stem cell (iPSC) models aid in this research but lack precise architecture, limiting insights into neuronal interactions and activity-dependent processes. Microfluidic technologies offer structural control but are restricted by closed systems that hinder 3D integration, scalability, and cell retrieval. To address these limitations, we developed an open cortical network platform integrating iPSC-derived cortical neurons with bioengineering techniques. Using a polydimethylsiloxane (PDMS)-based microgroove topography and a cell plating guide, we created neuronal nodes for controlled circuit assembly. This design enables large-scale functional cortical circuits without physical barriers, allowing optogenetic control of neural activity and flexible network modifications, including cellular composition, neurite directionality, and synapse formation. The open design facilitates neuronal material accessibility, supporting multi-level analyses such as proteomics. This platform serves as a powerful tool for investigating neuronal network development and function, offering new opportunities to study both normal and pathological states, including molecular changes linked to connectivity loss in brain diseases.

neuroscience↗

bioRxiv2025

A novel CAR T cell blend targeting PDPN and GD2 to overcome glioblastoma heterogeneity

BackgroundWhile chimeric antigen receptor (CAR) T cells have achieved encouraging remission rates in hematological malignancies, they demonstrated limited success in treating Glioblastoma (GBM), particularly due to high intra- and intertumoral heterogeneity. In this study, we identified a relevant and preserved target antigen, Podoplanin (PDPN), and evaluated the potential of a PDPN- and GD2-CAR T cell blend to overcome GBM heterogeneity. MethodsTarget antigen screening included clinical samples, healthy tissues and cell lines, as well as publicly available RNA sequencing datasets. The anti-tumor function of CAR T cells were examined in co-culture experiments with GBM cell lines and patient-derived organoids (PDOs), and in vivo after locoregional delivery in orthotopic xenograft models. ResultsThe generated CAR T cells demonstrated strong anti-tumor activity against several cell lines and PDOs from multiple patients. PDPN and GD2 expression was detectable in all PDOs at varying densities and regardless of the antigenic profile, the CAR T cell blend induced significantly higher levels of apoptosis in organoids than single antigen targeting counterparts. In vivo, we observed efficient tumor regression after locoregional administration of monospecific CAR T cells. While heterogeneous orthotopic tumors eventually relapsed in these groups, blended therapy resulted in a significantly increased overall survival and even achieved cure in the majority of mice. ConclusionThis novel PDPN-/GD2-CAR T cell blend demonstrated strong efficacy in advanced preclinical models of glioblastoma. The results suggest that this approach can overcome GBM heterogeneity in clinical application and address previous limitations of single antigen CAR T cell therapies. KEYPOINTSO_LIPDPN is a relevant and consistent CAR T cell target antigen in primary and recurrent GBM C_LIO_LIPDPN- and GD2-CAR T cells display synergistic anti-tumor activity in patient-derived organoids C_LIO_LILocal delivery of our CAR T cell blend confers long-term survival and cure in GBM-bearing mice C_LI IMPORTANCE OF THE STUDYThe highly variable landscape of tumor antigens in GBM represents a serious obstacle to single antigen CAR T cell therapies. In this study, we identified Podoplanin (PDPN) and GD2 as the most preserved target antigens in a screening campaign, with an even increased density in recurrent GBM compared to primary GBM. We combined PDPN- and GD2-CAR T cells in a blended treatment approach to counter antigen heterogeneity, and achieved a substantial gain in efficacy against patient-derived organoids (PDOs). In an orthotopic xenograft model, locoregional administration of the PDPN- and GD2-CAR T cell blend conferred long-term complete remission and increased survival compared to single antigen targeting. This study provides a hierarchy of CAR target antigens for treating GBM and illustrates the therapeutic potential of combinatorial antigen targeting using a PDPN/GD2-CAR T cell blend. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/636223v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@109a9aaorg.highwire.dtl.DTLVardef@16215faorg.highwire.dtl.DTLVardef@910623org.highwire.dtl.DTLVardef@129f79c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACT:C_FLOATNO C_FIG

immunology↗

bioRxiv2023

BDNF controls phosphorylation and transcriptional networks governing cytoskeleton organization and axonal regeneration

The cell-intrinsic capacity of neurons to regenerate axons requires widespread coordination of the transcriptome, activation of multiple kinases, and reorganization of the cytoskeleton. Axonal repair is also influenced by extrinsic activating factors, such as neurotrophins. Here, we reveal that brain-derived neurotrophic factor (BDNF) amplifies multiple neuron-intrinsic programs to foster axonal regeneration in human motor neurons. Through metabolic RNA sequencing and phosphoproteomic profiling, we elucidate BDNF signalling and its role in axonal regeneration. We discover that BDNF controls RNA stability and transcriptional programs that converge with regeneration-associated gene (RAG) sets. We further unveil that BDNF governs the phosphorylation of multiple proteins essential for cytoskeletal dynamics, a major determinant of effective nerve regeneration. Using compartmentalized neuronal cultures, we demonstrate that the regeneration driven by BDNF depends on the axon-specific activation of ERK/RSK/S6K kinase pathway. We propose a model in which BDNF augments neuron-intrinsic pathways to drive axonal regeneration in human motor neurons. TeaserBDNF aids nerve repair by fine-tuning the metabolism of RNA and by changing the building blocks of the nerve cell cytoskeleton.

molecular biology↗

bioRxiv2023

Nondestructive Spatial Lipidomics for Glioma Classification

Mapping the molecular composition of tissues using spatial biology provides high-content information for molecular diagnostics. However, spatial biology approaches require invasive procedures to collect samples and destroy the investigated tissue, limiting the extent of analysis, particularly for highly functional tissues such as those of the brain. To address these limitations, we developed a workflow to harvest biomolecules from brain tissues using nanoneedles and characterise the distribution of lipids using desorption electrospray ionization mass spectrometry imaging. The nanoneedles preserved the original tissue while harvesting a reliable molecular profile and retaining the original lipid distribution for mouse and human brain samples, accurately outlining the morphology of key regions within the brain and tumour lesions. The deep neural network analysis of a cohort containing 23 human glioma biopsies showed that nanoneedle samples maintain the molecular signatures required to accurately classify disease state. Thus, nanoneedles provide a route for tissue-preserving spatial lipidomic and molecular diagnostics.

bioengineering↗

bioRxiv2022

Combining SLA 3D printing and soft lithography for fast, versatile, and accessible high-resolution fabrication of customised multiscale cell culture devices with complex designs

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models whilst retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices and microfluidic systems to larger constructs for specific applications such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques and tools required for their manufacture, and the physical science background sometimes needed, the design and fabrication of such devices directly by biological labs remains a relatively high investment in terms of costs, use of facilities, needed collaborations and time. Whilst commercially available systems are available, these are also often costly, and importantly lack the potential for customisation by each single lab. This combination of factors still limits widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent important advancements in the fields of bioengineering and microfabrication, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised low-cost and highly reproducible microfabrication pipeline, capable of generating a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with little to none prior exposure to these fields technique and enables the manufacture of complex devices across the {micro}m to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D printed constructs for PDMS curing, using a combination of curing steps, washes and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilization of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multi-part hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

bioengineering↗

bioRxiv2021

Axonal length determines distinct homeostatic phenotypes in human iPSC derived motor neurons on a bioengineered platform

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a "threshold" size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.

neuroscience↗

bioRxiv2021

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with click chemistry. UAAs are site-specifically incorporated into a protein of interest by genetic code expansion. If the UAA carries a strained alkene or alkyne moiety it can be conjugated to a tetrazine-bearing fluorophore via a strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC), a variant of bioorthogonal click chemistry. The minimal size of the incorporated tag and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision make SPIEDAC live-cell labeling unique. However, until now, this type of labeling has not been used in complex, non-dividing cells, such as neurons. Using neurofilament light chain as a target protein, we established SPIEDAC labeling in living primary neurons and applied it for fixed-cell, live-cell, dual-color pulse--chase and super-resolution microscopy. We also show that SPIEDAC labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced microscopy techniques, we anticipate that SPIEDAC labeling will contribute to novel discoveries in neurobiology.

neuroscience↗

bioRxiv2020

Automated and unbiased classification of motor neuron phenotypes with single cell resolution in ALS tissue.

Histopathological analysis of tissue sections is an invaluable resource in neurodegeneration research. Importantly, cell-to-cell variation in both the presence and severity of a given phenotype is however a key limitation of this approach, reducing the signal to noise ratio and leaving unresolved the potential of single-cell scoring for a given disease attribute. Here, we developed an image processing pipeline for automated identification and profiling of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS) pathological tissue sections. This approach enabled unbiased analysis of hundreds of cells, from which hundreds of features were readily extracted. Next by testing different machine learning methods, we automated the identification of phenotypically distinct MN subpopulations in VCP- and SOD1-mutant transgenic mice, revealing common aberrant phenotypes in cellular shape. Additionally we established scoring metrics to rank cells and tissue samples for both disease probability and severity. Finally, by adapting this methodology to human post-mortem tissue analysis, we validated our core finding that morphological descriptors strongly discriminate ALS from control healthy tissue at the single cell level. In summary, we show that combining automated image processing with machine learning methods substantially improves the speed and reliability of identifying phenotypically diverse MN populations. Determining disease presence, severity and unbiased phenotypes at single cell resolution might prove transformational in our understanding of ALS and neurodegenerative diseases more broadly.

neuroscience↗

bioRxiv2018

Formation of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Reelin signaling regulates tangential migration of SN-mDA neurons, but whether Reelin acts directly on SN-mDA neurons and how it affects their cellular morphology and migratory behavior has not been explored. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons with 3D-time-lapse imaging and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. By speci1cally inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. We show that Reelin signaling promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.

developmental biology↗