Microglia-containing cerebral organoids derived from induced pluripotent stem cells for the study of neurological diseases.

Microglia play an important role in neuroinflammation and neurodegeneration. Here, we report an approach for generating microglia-containing cerebral organoids derived from human pluripotent stem cells involving the supplementation of growth factors (FGF, EGF, heparin) and 10% CO2 culture conditions. Using this platform, Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS-PDC) cerebral organoids were generated from patient-derived induced pluripotent stem cells (iPSCs). These ALS-PDC-affected organoids had more reactive astrocytes and M1 microglia, and had fewer M2 microglia than their unaffected counterparts, leading to impaired microglia-mediated phagocytosis. RNA-seq analysis of ALS-PDC and control organoids indicated that the most significant changes were microglia- and astrocyte-related genes (IFITM1/2, TGF-ß, and GFAP). The most significantly downregulated pathway was type I interferon signaling. Interferon-gamma supplementation increased IFITM expression, enhanced microglia-mediated phagocytosis, and reduced beta-amyloid accumulation in ALS-PDC-affected network. The results demonstrated the feasibility of using microglia-containing organoids for the study of neurodegenerative diseases.

Bisphenol-A exposure induced neurotoxicity in glutamatergic neurons derived from human embryonic stem cells.

Bisphenol-A (BPA) is a lipophilic, organic, synthetic compound that has been used as an additive in polycarbonate plastics manufacturing since 1957. Studies have shown that BPA interferes with the development and functions of the brain, but little is known about the effects of BPA on human glutamatergic neurons (hGNs) at the molecular and cellular levels. We investigated the impact of chronic exposure to BPA to hGNs derived from human embryonic stem cells (hESCs). The results showed that chronic exposure of different concentrations of BPA (0, 0.1, 1.0 and 10 µM) to hGNs for 14 days reduced neurite outgrowth in a concentration-dependent manner. Using presynaptic protein synaptophysin and postsynaptic protein PSD-95 antibodies, immunofluorescence staining and western blotting results indicated that BPA exposure altered the morphology of dendritic spines and increased synaptophysin and PSD-95 expression. Furthermore, BPA exposure at concentrations higher than 1.0 µM resulted in the increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression and deterioration of dendritic spines. In addition, our results suggested that these BPA mediated neurotoxicity effects were due to an increased production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via increased nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine expression and Ca2+ influx. These results imply that hESC-based neuronal differentiation is an excellent cellular model to examine BPA-induced neurotoxicity on human neurons at the cellular and molecular level.

Silver nanoparticles induce neurotoxicity in a human embryonic stem cell-derived neuron and astrocyte network.

Silver nanoparticles (AgNPs) are among the most extensively used nanoparticles and are found in a variety of products. This ubiquity leads to inevitable exposure to these particles in everyday life. However, the effects of AgNPs on neuron and astrocyte networks are still largely unknown. In this study, we used neurons and astrocytes derived from human embryonic stem cells as a cellular model to study the neurotoxicity that is induced by citrate-coated AgNPs (AgSCs). Immunostaining with the astrocyte and neuron markers, glial fibrillary acidic protein and microtubule-associated protein-2 (MAP2), respectively, showed that exposure to AgSCs at the concentration of 0.1 µg/mL increased the astrocyte/neuron ratio. In contrast, a higher concentration of AgSCs (5.0 µg/ml) significantly changed the morphology of astrocytes. These results suggest that astrocytes are sensitive to AgSC exposure and that low concentrations of AgSCs promote astrogenesis. Furthermore, our results showed that AgSCs reduced neurite outgrowth, decreased the expression of postsynaptic density protein 95 and synaptophysin, and induced neurodegeneration in a concentration-dependent manner. Our findings additionally suggest that the expression and phosphorylation status of MAP2 isoforms, as modulated by the activation of the Akt/glycogen synthase kinase-3/caspase-3 signaling pathway, may play an important role in AgSC-mediated neurotoxicity. We also found that AgNO3 exposure only slightly reduced neurite outgrowth and had little effect on MAP2 expression, suggesting that AgSCs and AgNO3 have different neuronal toxicity mechanisms. In addition, most of these effects were reduced when the cell culture was co-treated with AgSCs and the antioxidant ascorbic acid, which implies that oxidative stress is the major cause of AgSC-mediated astrocytic/neuronal toxicity and that antioxidants may have a neuroprotective effect.

Aqueous cigarette tar extracts disrupt corticogenesis from human embryonic stem cells in vitro.

BACKGROUND: Cigarette butts are the most common form of litter in the world, and approximately 4.5 trillion smoked cigarettes are discarded every year worldwide. Cigarette butts contain over 4000 chemicals, many of which are known to have neurotoxic effects. Stem cell neuronal differentiation provides an excellent cellular model with which to examine the impact of aqueous cigarette tar extracts (ACTEs) on neurodevelopment. METHODS: We have developed a neurosphere-based stem cell neuronal differentiation protocol that can recapitulate corticogenesis and produce cell types that are similar to upper and lower layer cortical projection neurons found in the germinal zone of the developing human cortex. In this study, ACTEs were generated from smoked cigarette butts and then applied at different concentrations to neuronal progenitors and cortical neurons derived from human embryonic stem cells. RESULTS: ACTEs reduced the expression of the cortical neuronal progenitor markers pax6, tbr2, and neuroD and decreased the number of cortical layer neurons (tbr1, satb2, foxp2, and brn2) after exposure to as low as 1.87% of the extract from one smoked cigarette butt. Furthermore, our results showed that ACTEs increased reactive oxygen species (ROS) production in cortical neurons, which caused a substantial loss of the synaptic proteins PSD95, synaptophysin, vesicular glutamate transporter1 (vGlut1), and the extracellular matrix molecule reelin; all of those molecules are important for the maintenance of cortical neuron identity and activity. CONCLUSION: ACTEs from smoked cigarettes have significant effects on cortical neuron development and neurodegeneration. The stem cell neuronal differentiation model holds great promise as a potentially powerful tool for the assessment of ACTEs on neurotoxicity.

Triptonide Effectively Inhibits Wnt/ß-Catenin Signaling via C-terminal Transactivation Domain of ß-catenin.

Abnormal activation of canonical Wnt/ß-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/ß-catenin signaling by targeting the downstream C-terminal transcription domain of ß-catenin or a nuclear component associated with ß-catenin. In addition, triptonide treatment robustly rescued the zebrafish "eyeless" phenotype induced by GSK-3ß antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide.

Silver nanoparticles exhibit coating and dose-dependent neurotoxicity in glutamatergic neurons derived from human embryonic stem cells.

Silver nanoparticles (AgNPs) are used extensively as anti-microbial agents in various products, but little is known about their potential neurotoxic effects. In this study, we used glutamatergic neurons derived from human embryonic stem cells as a cellular model to study 20nm citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated AgNPs (AgSPs) induced neurotoxicity. AgSCs significantly damaged neurite outgrowths; increased the production of reactive oxygen species and Ca2+ influxes; reduced the expression of MAP2, PSD95, vGlut1 and NMDA receptor proteins at concentrations as low as 0.1µg/ml. In contrast, AgSPs exhibited neurotoxicity only at higher concentration. Furthermore, our results showed that AgSCs induced glutamate excitotoxicity by the activation of calmodulin and the induction of nitric oxide synthase; increased the phosphorylation of glycogen synthase kinase-3 α/ß at Tyr216 and Tau at Ser396 and reduced the expression of Tau46, which are typically observed in Alzheimer's disease. This study indicated that stem cells can provide an excellent platform for studying nanoparticle induced neurotoxicity.

Directed cardiomyogenesis of human pluripotent stem cells by modulating Wnt/ß-catenin and BMP signalling with small molecules.

Cardiomyocytes derived from human pluripotent stem cells (PSCs) are a potential cell source for regenerative medicine, disease modelling and drug development. However, current approaches for in vitro cardiac differentiation of human PSCs are often time-consuming, heavily depend on expensive growth factors and involve the tedious formation of embryonic bodies whose signalling pathways are difficult to precisely modulate due to their complex microenvironments. In the present study, we report a new small molecule-based differentiation approach, which significantly promoted contracting cardiomyocytes in human PSCs in a monolayer format in as little as 7 days, in contrast with most traditional differentiation methods that usually take up to 3 weeks for cardiomyogenesis. This approach consists in activation of the Wnt/ß-catenin signalling at day 0-1 with small molecule CHIR99021 (CH) followed by inhibition of bone morphogenetic protein (BMP) signalling at day 1-4 with DMH1 [termed as CH(0-1)/DMH1(1-4) treatment], a selective small molecule BMP inhibitor reported by us previously. Our study further demonstrated that the CH(0-1)/DMH1(1-4) treatment significantly promotes cardiac formation via mesoderm and mesoderm-derived cardiac progenitor cells without impacts on either endoderm or ectoderm differentiation of human PSCs. This rapid, efficient and inexpensive small molecule-based cardiomyogenic method may potentially harness the use of human PSCs in regenerative medicine as well as other applications.

Nanoimaging of focal adhesion dynamics in 3D.

Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.

Deep tissue fluorescence imaging and in vivo biological applications.

We describe a novel technical approach with enhanced fluorescence detection capabilities in twophoton microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

Effecto of the methylxantine derivative pentoxifylline on benzodiazepine and muscarinic binding sites in rats cerebral cortex

La inyección subcutánea del derivado metilxantínico pentoxifilina (3, 7, dimetil-1-(5-oxohexil)-xantina) redujo 3 h más tarde la concentración de sitios receptores para benzodiazepinas en la corteza cerebral de la rata. In vitro la pentoxifilina compitió efectivamente por la unión 3H-flunitrazepam a membranas cerebrales, demostrando poseer una potencia 10 veces mayor que la cafeína. Inyectada 2 veces por día durante 5 días, la pentoxifilina indujo un aumento de la concentración cerebral de receptores para benzodiazepinas. Estos resultados indican que la pentoxifilina modifica en forma distinta la concentración de receptores cerebrales para benzodiazepinas dependiendo del tiempo de tratamiento

Effecto of the methylxantine derivative pentoxifylline on benzodiazepine and muscarinic binding sites in rats cerebral cortex

La inyección subcutánea del derivado metilxantínico pentoxifilina (3, 7, dimetil-1-(5-oxohexil)-xantina) redujo 3 h más tarde la concentración de sitios receptores para benzodiazepinas en la corteza cerebral de la rata. In vitro la pentoxifilina compitió efectivamente por la unión 3H-flunitrazepam a membranas cerebrales, demostrando poseer una potencia 10 veces mayor que la cafeína. Inyectada 2 veces por día durante 5 días, la pentoxifilina indujo un aumento de la concentración cerebral de receptores para benzodiazepinas. Estos resultados indican que la pentoxifilina modifica en forma distinta la concentración de receptores cerebrales para benzodiazepinas dependiendo del tiempo de tratamiento (AU)