Schwann cells in neuromuscular in vitro models.

Neuromuscular cell culture models are used to investigate synapse formation and function, as well as mechanisms of de-and regeneration in neuromuscular diseases. Recent developments including 3D culture technique and hiPSC technology have propelled their ability to complement insights from in vivo models. However, most cultures have not considered Schwann cells, the glial part of NMJs. In the following, a brief overview of different types of neuromuscular cocultures is provided alongside examples for studies that included Schwann cells. From these, findings concerning the effects of Schwann cells on those cultures are summarized and future lines of research are proposed.

Quantitative Analysis of Whole-Mount Fluorescence-Stained Tumor Spheroids in Phenotypic Drug Screens.

Three-dimensional cell cultures, such as spheroids or organoids, serve as important models for drug screening purposes. Optical tissue clearing (OTC) enhances the visualization of fluorescence stainings and enables in toto microscopy of 3D cell culture models. Furthermore, subsequent automated image analysis tools convert qualitative confocal image sets into quantitative data. In this chapter, we describe a detailed protocol for preparation of HT29 cancer spheroids, 3D in toto immunostaining, glycerol-based OTC, whole-mount imaging, and semi-automated downstream image processing and segmentation for nuclear image analysis using open-source software.

Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model.

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including SOD1. So far, clinical records, rodent studies, and in vitro models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC, the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings, we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model, myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers, lack of sarcomeres, morphologically different nAChR clusters, and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably, trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary, a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS.

A multiparametric analysis including single-cell and subcellular feature assessment reveals differential behavior of spheroid cultures on distinct ultra-low attachment plate types.

Spheroids have become principal three-dimensional models to study cancer, developmental processes, and drug efficacy. Single-cell analysis techniques have emerged as ideal tools to gauge the complexity of cellular responses in these models. However, the single-cell quantitative assessment based on 3D-microscopic data of the subcellular distribution of fluorescence markers, such as the nuclear/cytoplasm ratio of transcription factors, has largely remained elusive. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. However, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study developed a novel AI-based pipeline for the analysis of 3D-confocal data of optically cleared large spheroids at the wholemount, single-cell, and sub-cellular levels. To identify relevant samples for the pipeline, automated brightfield microscopy was employed to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines. This showed that all plate types exhibited similar spheroid-forming capabilities and the gross patterns of growth or shrinkage during 4 days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. Based on this prescreen, spheroids of HaCaT keratinocytes and HT-29 cancer cells were further assessed. In HaCaT spheroids, the in-depth analysis revealed a correlation between spheroid size, cell proliferation, and the nuclear/cytoplasm ratio of the transcriptional coactivator, YAP1, as well as an inverse correlation with respect to cell differentiation. These findings, yielded with a spheroid model and at a single-cell level, corroborate earlier concepts of the role of YAP1 in cell proliferation and differentiation of keratinocytes in human skin. Further, the results show that the plate type may influence the outcome of experimental campaigns and that it is advisable to scan different plate types for the optimal configuration during a specific investigation.