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.

Opsonization-independent antigen-specific recognition by myeloid phagocytes expressing monoclonal antibodies.

This report demonstrates a novel class of innate immune cells designated "variable immunoreceptor-expressing myeloids" (VIREMs). Using single-cell transcriptomics and genome-wide epigenetic profiling, we establish that VIREMs are myeloid cells unrelated to lymphocytes. We visualize the phenotype of B-VIREMs that are capable of genetically recombining and expressing antibody genes, the exclusive hallmark function of B lymphocytes. These cells, designated B-VIREMs, display monoclonal antibody cell surface signatures and regularly circulate in the blood of healthy individuals. Single-cell data reveal clonal expansion of circulating B-VIREMs as a dynamic response to disease stimuli. Live-cell imaging models suggest that B-VIREMs load their own Fc receptors with endogenous antibodies during vesicle transport to the cell surface. A first cloned B-VIREM-derived antibody (Vab1) specifically binds stomatin, a ubiquitous scaffold protein that is strictly expressed intracellularly, allowing Vab1-bearing macrophages to phagocytose cell debris without requiring prior opsonization. Our results suggest important antigen-specific tissue maintenance functionalities in these innate immune cells.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Analysis of calcium signaling in live human Tongue cell 3D-Cultures upon tastant perfusion.

Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include (i) the stabilization of these structures under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, (iii) and the data analysis that ranges in complexity from whole specimens to single cells. Here, we addressed these issues for the time-lapse analysis of Ca2+ signaling in spheroids composed of human tongue-derived HTC-8 cells upon perfusion of gustatory substances. Live cell imaging setups for confocal and light sheet microscopy were developed that allow simple and robust spheroid stabilization and high-resolution microscopy with perfusion. Visualization of spheroids made of HTC-8 cells expressing the G-GECO fluorescent Ca2+ sensor revealed Ca2+ transients that showed similar kinetics but different amplitudes upon perfusion of bitter compounds Salicine and Saccharin. Dose-dependent responses to Saccharin required extracellular Ca2+. From the border towards the center of spheroids, compound-induced Ca2+ signals were progressively delayed and decreased in amplitude. Stimulation with ATP led to strong Ca2+ transients that were faster than those evoked by the bitter compounds and blockade of purinergic receptors with Suramin abutted the response to Saccharin, suggesting that ATP mediates a positive autocrine and paracrine feedback. Imaging of ATP-induced Ca2+ transients with light sheet microscopy allowed acquisition over a z-depth of 100 µm without losing spatial and temporal resolution. In summary, the presented approaches permit the study of fast cellular signaling in three-dimensional cultures upon compound perfusion.

Basal glucocorticoid receptor activation induces proliferation and inhibits neuronal differentiation of human induced pluripotent stem cell-derived neuronal precursor cells.

Glucocorticoids (GC) have first been shown to originate from the adrenal glands where synthesis and release is controlled by the hypothalamic-pituitary-adrenal (HPA) axis. Recently, it was shown that GC and other steroid hormones are also synthesized in the central nervous system, so-called neurosteroids. GC bind to specific GC receptors (GR) which function as ligand-activated transcription factors. GR are expressed in nearly all cell types in the brain, and therefore GC have a strong impact on neuronal development. Most knowledge of the influence of GC on neurodevelopment has been obtained from animal research. Recent advances in stem cell technology made it possible to generate neuronal precursor cells (NPCs) and neurons from human induced pluripotent stem cells (hiPSCs). To explore the cellular mechanism of GC affecting human neuronal development, we quantified the proliferation and differentiation of hiPSCs-derived NPCs in the absence and presence of the selective high-affinity GR agonist dexamethasone and the selective GR antagonist mifepristone, respectively. Our results show that inhibition of GR significantly reduced proliferation of NPCs and promoted differentiation whereas GR activation suppressed neuronal differentiation. This implies that neuronal GC must be present in NPCs for proliferation. Consequently we identified the presence of 11-ß-hydroxylase CYP11B1, which hydroxylates the respective steroid precursors to bioactive GC, in NPCs. We propose that hiPSC technology offers an ideal system to get more insight into the synthesising and regulatory pathways in steroidogenesis in human neurons and to differentiate between the mechanism by which adrenal GC and neuronal GC impact on neurodevelopment.