Human induced pluripotent stem cell (hiPSC)-derived neurons offer a promising, physiologically relevant alternative to animal-based neurotoxicity models. However, the influence of the surface coating, a basic but important variable, has often been neglected. Here, the impact of commonly used surface coatings including polymers (polyethylenimine, poly-L-ornithine, poly-D-lysine, poly-L-lysine) as well as extracellular matrix proteins (laminin, fibronectin, Matrigel) on NGN2 neurons, primary human astrocytes, and neuron/astrocyte co-cultures was systematically investigated. Neurons cultured on polymer-only coatings exhibited a less mature neuronal network. Interestingly, neurons on polymers with laminin, laminin only, fibronectin and Matrigel exhibited no differences in protein and gene expression but displayed different electrophysiological profiles in co-culture with human primary astrocytes. The peak in number of network bursts was reached at different time points, with fibronectin and Matrigel as early as week 2. Co-cultures on fibronectin exhibited stable cell-electrode coupling and robust activity patterns up to week 6, despite being rarely used in MEA. In a proof-of-principle experiment, laminin, fibronectin and Matrigel were further evaluated in a donor-matched hiPSC-derived co-culture system, supporting the observed effects. This study highlights the importance of the coating selection for establishing neural cultures, ultimately improving the maturation and robustness of hiPSC-derived neuronal models for drug development.

Colorectal cancer (CRC) is one of the most commonly diagnosed and globally spread malignant diseases. Cancer-associated fibroblasts (CAFs) are key architects of the tumor microenvironment, yet their origin, stability, and interconvertibility remain poorly understood. Using transcriptomic profiling of fibroblasts from colorectal cancer (CRC) patients, we identify highly expressed (HEX) markers that define fibroblast subpopulations and uncover mechanisms governing their plasticity. We find that ADH1B marks normal colon-associated fibroblasts (NAFs), which consist of PI16-NAFs and ADAMDEC1-NAFs. ITGA3 delineates the total CAF population, which comprises myofibroblastic CAFs (myCAFs), whose characterizing markers were associated with poor prognosis and proteolytic inflammatory CAFs (piCAFs), characterized by markers not associated with prognosis. An AGT/TGM2-expressing fibroblast subset is present in both healthy and tumor tissues, suggesting alternative trajectories to the classical NAF-to-CAF transition model. While PI16-NAFs, AGT/TGM2-fibroblasts, and myCAFs maintain stable identities in long-term culture, the ADAMDEC1-NAF and piCAF phenotypes are lost in vitro. ITGA3-CAFs demonstrate dynamic plasticity, with TGF-β stably inducing myCAF formation and TNF-α or inhibition of DNA methylation promoting transient piCAF emergence. These findings redefine fibroblast heterogeneity in CRC and reveal a coexisting stable and plastic fibroblast network that may be amenable to modulation and provides a framework for future functional and translational studies. We identified highly expressed markers (HEX markers) to distinguish CAFs, NAFs and corresponding subpopulations in CRC. ADH1B characterized NAFs, which consisted of stable (solid outline) PI16-NAFs and unstable (dashed outline) ADAMDEC1-NAFs. ITGA3 identified CAFs consisting of stable myCAFs associated with poor prognosis and unstable piCAFs not associated with prognosis. AGT/TGM2 fibroblasts did not express ADH1B or ITGA3, were stable in culture and could be detected in both healthy colon and CRC. Treatment of PI16-NAFs with LPS or IFN-γ induced ADAMDEC1-NAFs, TGF-β the formation of myCAFs, while treatment with TNF-α led to the formation of piCAFs. Reduced DNA methylation converted myCAFs and PI16-NAFs into piCAFs.

BACKGROUND & AIMS:The gut-vascular barrier plays a pivotal role in inflammatory bowel disease pathogenesis. We introduce a novel 3-dimensional multiphoton endomicroscopy approach for real-time and sensitive detection of vascular permeability in the colon to identify colitis-associated vascular changes in the early stages. METHODS:Using fluorescence-based multiphoton endomicroscopy, we visualized dynamic changes in vascular permeability in vivo during longitudinal observations in different experimental colitis models (dextran sodium sulfate- and T cell transfer-induced colitis). Vascular permeability changes were systematically compared with conventional inflammatory markers, including weight loss, endoscopic scoring, colon length, histopathology, and immune cell infiltration. To assess molecular regulation of barrier functions, the expression of key molecules of vascular (plasmalemma vesicle-associated protein, vascular endothelial cadherin) and epithelial (epithelial cadherin) barriers was investigated during development of colitis at the single-cell level. RESULTS:Multiphoton endomicroscopy provided unprecedented 3-dimensional visualization of vascular permeability dynamics and showed that vascular dysfunction occurs prior to epithelial barrier breakdown and the detection of traditional inflammatory markers across all colitis models. Distinct spatial vascular permeability patterns strongly correlated with mucosal damage severity, further supporting that early gut-vascular barrier disruption precedes mucosal barrier breakdown. Additionally, this sequence was confirmed at the molecular level, with the vascular upregulation of the transendothelial permeability channel plasmalemma vesicle-associated protein occurring before downregulation of the epithelial barrier molecule E-cadherin. CONCLUSIONS:Multiphoton endomicroscopy with 3-dimensional imaging demonstrated gut-vascular barrier dysfunction in the very early stages of experimental colitis. Further development of multiphoton endomicroscopy-based vascular permeability analysis for use in routine clinical monitoring of patients may provide new perspectives to improve diagnosis and clinical decision-making in inflammatory bowel disease.

Abstract Background Progression and metastasis of solid cancers are orchestrated by activation of epithelial-mesenchymal transition (EMT) in the primary tumor. This process is typically restricted to a limited number of cells that acquire partial or hybrid EMT states to unleash cellular plasticity. Capturing such dynamic and often reversible events in vivo on the single cell level is hampered by the lack of proper labeling tools that yet often induce permanent staining persisting beyond transient EMT activation. Methods To enable live-tracking of EMT in vivo, we utilized CRISPaint and homologous recombination to endogenously tag ZEB1, one key transcription factor to activate EMT during tumorigenesis. Using the bright fluorescent protein mNeonGreen, we generated ZEB1-Neon fusion knock-in alleles in MDA-MB-231 and MCF10A cells, as well as in mice. Results We demonstrate that mNeonGreen fluorescence is suitable to faithfully report on ZEB1 expression in vitro over time, becomes properly upregulated by TGFβ, and allows separation of ZEB1hi and ZEB1lo cells to capture different cellular properties, e.g., handling of DNA damage. The fusion does not affect ZEB1 function as evident by proper EMT induction, embryogenesis, and tissue homeostasis when present homozygously. Moreover, introducing Zeb1-Neon into the KPC mouse model of pancreatic cancer permits tracking of ZEB1+ cells in precision-cut slices and time-lapse imaging of isolated tumor cells. Conclusions In summary, we provide a versatile tool that allows precise detection and live cell imaging of EMT, which will help to more accurately decipher the role of EMT in tumor progression and to identify therapeutic agents that can specifically manipulate EMT for novel combination therapies. Graphical Abstract

Neurite growth is regulated by NADPH Oxidase (NOX1 and 2) and in this study, we investigate whether neuritic abnormalities observed in stem cell models of Huntington's disease relates to altered NOX function during NGF-driven differentiation of PC12 neuronal cells. NOX1 and 2 were contained in separate vesicular compartments, and by overexpression inhibited or promoted neurite extension, respectively. Expression of mutant Htt (mHtt; exon 1 fragment) accelerated neuronal induction causing longer neurites in the first phase of differentiation, but fewer and shorter mature neurites. Htt/mHtt increased NOX2 protein levels but did not change global oxidant production; However, Htt/mHtt prominently redistributed NOX activity to neurites. Oxidant production was concentrated in intraluminal vesicles in multivesicular bodies, and mHtt specifically increased secretion of NOX1 in exosomes, which demonstrated oxidant production capacity, while rerouting NOX2 to lysosomal degradation. Knockdown of TSG101, required for intraluminal vesicle formation, increased cellular levels of NOX2/p22phox and neurite growth.Our study provides new insights on the disposition of NOX enzymes in nerve cells, indicating that deficient neurites in HD may be a correlate of altered trafficking, distribution, and activity of NOX.

Integrin β6 is associated with poor prognosis in colorectal cancer (CRC) patients, with metastasis being a crucial determinant. Capillary endothelial cells (EC) in the liver and lung are the primary sites of contact for circulating tumour cells during metastasis. Here, we analysed the role of integrin β6 in tumour cells for their interaction with EC. Integrin β6 functions as a heterodimer with integrin αv. Interestingly, we found that liver and lung EC strongly express fibronectin, a high-affinity ligand of αvβ6. Expression of ITGB6 in CRC tumour cells closely correlated with their adhesion to EC. This interaction was greatly reduced by silencing ITGB6 in the tumour cells and was integrin β6 dependent under both static and flow conditions. Binding assays with fibronectin-coated surfaces, competing RGD peptides, and integrin β6-neutralizing antibodies confirmed the crucial role of β6-fibronectin binding in the interaction between tumour cells and EC. Since metastatic tumours exhibit increased proteolytic activity, we examined integrin β6 stability under these conditions. Remarkably, β6 remained resistant to trypsin and the matrix metalloprotease 12, underscoring its role in maintaining tumour cell adhesion in proteolytic microenvironments. Furthermore, ITGB6 expression was significantly elevated in liver metastases compared to corresponding primary tumours from the same patients, suggesting an enrichment of β6-expressing cells in metastatic sites. These results suggest that tumour cell integrin β6 binding to EC-derived fibronectin may serve as a critical first step in metastasis formation. Targeting this interaction could provide a promising therapeutic strategy to repress CRC metastasis.

Dysregulation at the intestinal epithelial barrier is a driver of inflammatory bowel disease (IBD). However, the molecular mechanisms of barrier failure are not well understood. Here, we demonstrate dysregulated mitochondrial fusion in intestinal epithelial cells (IECs) of patients with IBD and show that impaired fusion is sufficient to drive chronic intestinal inflammation. We found reduced expression of mitochondrial fusion–related genes, such as the dynamin-related guanosine triphosphatase (GTPase) optic atrophy 1 ( OPA1 ), and fragmented mitochondrial networks in crypt IECs of patients with IBD. Mice with Opa1 deficiency in the gut epithelium ( Opa1 i∆IEC ) spontaneously developed chronic intestinal inflammation with mucosal ulcerations and immune cell infiltration. Intestinal inflammation in Opa1 i∆IEC mice was driven by microbial translocation and associated with epithelial progenitor cell death and gut barrier dysfunction. Opa1 -deficient epithelial cells and human organoids exposed to a pharmacological OPA1 inhibitor showed disruption of the mitochondrial network with mitochondrial fragmentation and changes in mitochondrial size, ultrastructure, and function, resembling changes observed in patient samples. Pharmacological inhibition of the GTPase dynamin-1–like protein in organoids derived from Opa1 i∆IEC mice partially reverted this phenotype. Together, our data demonstrate a role for epithelial OPA1 in regulating intestinal immune homeostasis and epithelial barrier function. Our data provide a mechanistic explanation for the observed mitochondrial dysfunction in IBD and identify mitochondrial fusion as a potential therapeutic target in this disease.

B cells can recognize soluble and membrane bound antigens, enabling them to initiate and execute versatile immune responses. This study examines Swiprosin-1/EFhd2 (EFhd2) in regulating mitochondrial function and organization in B cells during B cell receptor (BCR) activation and immune synapse formation. Using EFhd2 knockout (KO) and wild-type (WT) murine B cells, we assessed mitochondrial abundance, membrane potential, and respiratory capacity using soluble anti-IgM and anti-CD40/IL-4 stimulation. EFhd2KO B cells exhibit more functional mitochondria and mitochondrial spare capacity selectively in activated but not in resting cells. This phenotype changed absolutely upon BCR synapse formation: While activated WT B cells enhance basal mitochondrial respiration, ATP production and maximal respiration, with large increments of spare capacity, EFhd2KO B cells fail completely to do so. Actin depolymerization and microtubule destabilization impair functional mitochondrial upregulation in WT but not in EFhd2KO B cells, while microtubule stabilization restores full spare capacity in EFhd2KO B cells at the BCR synapse. Live-cell imaging reveals that EFhd2KO B cells fail to organize mitochondria, microtubules, and BCRs symmetrically. Super-resolution 3D imaging shows that WT B cells condense mitochondria at the synapse, whereas EFhd2KO B cells display dispersed, unorganized mitochondria and BCR clusters. EFhd2 re-expression restores mitochondrial polarization in EFhd2KO B cells, confirming its role in coordinating mitochondrial positioning. These findings highlight EFhd2 as a key integrator of cytoskeletal and mitochondrial functions for optimal B cell responses to membrane bound antigens. ### Competing Interest Statement The authors have declared no competing interest.

INTRODUCTION:The putative proton/organic cation (H+/OC) antiporter has been shown to mediate transport of CNS drug compounds like oxycodone and pyrilamine across the blood-brain barrier (BBB). This transporter has a broad substrate profile and is able to transport substrates against their concentration gradient, making it an interesting target for brain drug delivery. However, the molecular identity of this transporter remains unknown. Recent studies have indicated that the two proteins TM7SF3 and LHFPL6 might be components of this transporter. The present study aimed to investigate the roles of TM7SF3 and LHFPL6 in the H+/OC antiporter function to advance understanding of its molecular identity and potential in CNS drug delivery. METHODS:CRISPR-Cas9 gene-editing was used to generate three hCMEC/D3 knockout (KO) cell lines: TM7SF3 KO (TM-KO), LHFPL6 KO (LH-KO), and a double KO of TM7SF3 and LHFPL6 (TMLH-KO). The uptake of pyrilamine analogue (EDMPG) and [3H]-pyrilamine was assessed in wild type (WT) and KO lines. Quantitative Realtime Polymerase Chain Reaction (qRT-PCR) confirmed successful gene knockouts. Passive diffusion properties and the expression and functionality of known BBB transporters, including LAT1 (SLC7A5), GLUT1 (SLC2A1), and MCT1 (SLC16A1), were also examined. RESULTS:The EDMPG uptake was significantly reduced in TM-, LH-, and TMLH-KO cells, suggesting that TM7SF3 and LHFPL6 contribute to the H+/OC antiporter function. However, [3H]-pyrilamine uptake remained unchanged across all KOs, indicating a TM7SF3- and LHFPL6-independent transport mechanism. This was further supported by the persistent inhibition of [3H]-pyrilamine uptake in the presence of known H+/OC antiporter substrates. While passive diffusion and GLUT1- and MCT1-mediated transport were unaffected, LAT1-mediated uptake of [3H]L-leucine and gabapentin (Neurontin) was significantly reduced in LH- and TMLH-KO cells, correlating with decreased LAT1 mRNA expression in these cells. CONCLUSIONS:This study suggests that the H+/OC antiporter operates via two distinct mechanisms: a high-capacity, TM7SF3- and LHFPL6-independent pathway and a low-capacity, TM7SF3- and LHFPL6-dependent pathway. These findings underscore the complexity of the H+/OC antiporter molecular composition and highlight the need for further research to fully elucidate its identity.

Microglia are the primary immune cells of the brain and represent the main line of defense against brain environmental insults. In recent years, microglia have been implicated in Alzheimer’s disease (AD) pathogenesis by having interconnected yet opposing roles: beneficial as they clear amyloid beta (Aβ) and amyloid plaques, and detrimental as being responsible for synaptic and neuronal loss. These activities are tightly regulated by microglia receptors CD33 and TREM2. Microglial expression of both CD33 and TREM2 is upregulated and correlates with Aβ plaque load in the brain of AD patients, and genome-wide association studies have associated genetic variants of CD33 and TREM2 with AD progression. Interestingly, humans carrying the polymorphic allele rs12459419(T) of CD33, which results in the loss of exon 2 in the CD33 transcript giving rise to a shorter isoform of CD33 (CD33-D2), show higher CD33 expression in microglia and decreased Aβ deposition, lowering AD risk. In contrast, the heterozygous variant R47H TREM2 increases AD risk by four-fold. We characterized the phagocytic and surveillance activity of human iPSC-derived microglia carrying R47H TREM2 or Exon 2-deleted CD33 (CD33 ΔE2 ) in monoculture. Moreover, we analyzed how these microglia genotypes impact Aβ accumulation and neuronal integrity in a robust AD in-vitro model represented by iPSC-derived neurogenin-2 (NGN2) neurons carrying the PSEN1 E280A mutation. iPSC-derived microglia carrying R47H TREM2 or CD33 ΔE2 show altered phagocytic and migratory activities. When co-cultured with NGN2 neurons carrying PSEN1 E280A, R47H TREM2 microglia and CD33 ΔE2 microglia affect Aβ40 and Aβ42 levels and neuronal integrity. Insights into how CD33 ΔE2 and R47H TREM2 mutations regulate microglial activity in an AD context can help to characterize them as potential therapeutic targets to arrest AD progression.

The human MDR1 gene encodes the efflux transporter P-glycoprotein, which plays an important part of the blood–brain barrier function of brain microvascular endothelial cells (BMECs). Here, we report the generation of an iPSC line, where a construct of the human MDR1 gene was inserted into the safe-site locus AAVS1. This iPSC line (BIONi010-C-48) shows functional expression of P-gp and can further be differentiated and cultured into electrically tight BMEC-like monolayers exhibiting polarized expression of P-gp in the apical membrane.

Late-onset Alzheimer’s disease (AD) has become the paradigm of a non-mendelian complex neurodegenerative disease, for which a major genetic determinant is known, the APOE locus. A rare APOE variant named Christchurch (APOEch) yielding a missense mutation from Arginine to Serine at amino acid 136, has been suggested to exert a protective effect in an individual carrying the most penetrant form of Familial AD (Paisa mutation in PSEN1 gene, E280A). We describe here a new set of induced pluripotent stem cell (iPSC) lines, where the Christchurch mutation (Ch) has been introduced by gene editing into the APOE locus of three isogenic iPSC lines carrying the more common APOE variants (APOE 2/2, APOE 3/3, and an APOE 4/4) in homozygosity. Brain cells derived from these iPSC lines will enable a better understanding of APOE biology in general and facilitate the study of how the Christchurch variant affects the function of each APOE genotype. This set of iPSC lines are globally available via the European Bank of iPSCs, EBiSC.org.

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

In this chapter, the authors present some of the commercial or open-source systems available. Now, it is important to remember that most part of the systems described are relatively recent and they are evolving rapidly as the technology and the integration of multimodal systems improve at a very fast pace. The history of ZEISS and light sheet microscopy reaches back over a hundred years and was always characterized by a close collaboration between engineers and scientist. A classical light sheet setup implicates new features compared with conventional microscopy, and ZEISS was aware that a bundle of questions had to be answered to derive a consistent concept for a commercial system. The illumination units are designed to work with a fibered laser source and allow direct imaging of an optical section with a single frame at full camera resolution. Special attention has been given to the chamber design and its sample mounting accessories.

Background The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. Methods Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. Results Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. Conclusions We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches.

Functional and structural alterations of peritubular capillaries (PTCs) are a major determinant of chronic kidney disease (CKD). Using a software-based algorithm for semiautomatic segmentation and morphometric quantification, this study analyzes alterations of PTC shape associated with chronic tubulointerstitial injury in three mouse models and in human biopsies. In normal kidney tissue PTC shape was predominantly elongated, whereas the majority of PTCs associated with chronic tubulointerstitial injury had a rounder shape. This was reflected by significantly reduced PTC luminal area, perimeter and diameters as well as by significantly increased circularity and roundness. These morphological alterations were consistent in all mouse models and human kidney biopsies. The mean circularity of PTCs correlated significantly with categorized glomerular filtration rates and the degree of interstitial fibrosis and tubular atrophy (IFTA) and classified the presence of CKD or IFTA. 3D reconstruction of renal capillaries revealed not only a significant reduction, but more importantly a substantial simplification and reconfiguration of the renal microvasculature in mice with chronic tubulointerstitial injury. Computational modelling predicted that round PTCs can deliver oxygen more homogeneously to the surrounding tissue. Our findings indicate that alterations of PTC shape represent a common and uniform reaction to chronic tubulointerstitial injury independent of the underlying kidney disease.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor. In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.