Project description:This protocol establishes a tri-culture of hiPSC-derived neurons, astrocytes, and microglia for the study of cellular interactions during homeostasis, injury, and disease. This system allows for mechanistic studies that can identify the roles of individual cell types in disease and injury response in a physiologically relevant, all-human system. This protocol utilizes and modifies prior differentiations. Limitations include the prolonged maturation of human astrocytes and neurons and scalability. For complete details on the use and execution of this protocol, please refer to Ryan et al. (2020).

| S-EPMC7757552 | biostudies-literature

Soluble α-synuclein-antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia.

Project description:Parkinson's disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn-antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

| S-EPMC8054017 | biostudies-literature

Challenges in Physiological Phenotyping of hiPSC-Derived Neurons: From 2D Cultures to 3D Brain Organoids.

Project description:Neurons derived from human induced pluripotent stem cells (hiPSC-derived neurons) offer novel opportunities for the development of preclinical models of human neurodegenerative diseases (NDDs). Recent advances in the past few years have increased substantially the potential of these techniques and have uncovered new challenges that the field is facing. Here, we outline and discuss challenges related to the functional characterization of hiPSC-derived neurons and propose ways to overcome current difficulties. In particular, the enormous variability among studies in the electrical properties of hiPSC-derived neurons and broad differences in cell maturation are factors that impair reproducibility. Furthermore, we discuss how the use of 3D brain organoids are of help in resolving some difficulties posed by 2D cultures. Finally, we elaborate on recent and future advances that may help to overcome the discussed challenges and speed-up progress in the field.

| S-EPMC7479826 | biostudies-literature

Mechanisms of hyperexcitability in Alzheimer's disease hiPSC-derived neurons and cerebral organoids vs isogenic controls.

Project description:Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity, we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids, we found increased excitatory bursting activity, which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.

| S-EPMC6905854 | biostudies-literature

Chromatin accessibility analysis reveals regulatory dynamics of developing human retina and hiPSC-derived retinal organoids.

Project description:Retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs) provide potential opportunities for studying human retinal development and disorders; however, to what extent ROs recapitulate the epigenetic features of human retinal development is unknown. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics over long-term human retinal and RO development. Our results showed that ROs recapitulated the human retinogenesis to a great extent, but divergent chromatin features were also discovered. We further reconstructed the transcriptional regulatory network governing human and RO retinogenesis in vivo. Notably, NFIB and THRA were identified as regulators in human retinal development. The chromatin modifications between developing human and mouse retina were also cross-analyzed. Notably, we revealed an enriched bivalent modification of H3K4me3 and H3K27me3 in human but not in murine retinogenesis, suggesting a more dedicated epigenetic regulation on human genome.

| S-EPMC7007246 | biostudies-literature

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