Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions

Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.Peer reviewe

Microglial amyloid beta clearance is driven by PIEZO1 channels

Background Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer's disease (AD), toxic amyloid beta (A beta) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of A beta. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia had been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. Methods Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and A beta pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. Results We show that PIEZO1 orchestrates A beta clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. A beta inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in A beta clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single-cell datasets. Conclusion These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated A beta burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.Peer reviewe

EFFETS BIOLOGIQUES DE L'ACIDE LYSOPHOSPHATIDIQUE ET DE LA SPHINGOSINE-1-PHOSPHATE DANS LES CELLULES GLIALES

PARIS-BIUSJ-Physique recherche (751052113) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid

We previously reported that lysophosphatidic acid (LPA) inhibits the neuronal differentiation of human embryonic stem cells (hESC). We extended these studies by analyzing LPA\u27s effects on the expansion of neural stem/ progenitor cells (NS/PC) derived from hESCs and human induced pluripotent stem cells (iPSC), and we assessed whether data obtained on the neural differentiation of hESCs were relevant to iPSCs. We showed that hESCs and iPSCs exhibited comparable mRNA expression profiles of LPA receptors and producing enzymes upon neural differentiation. We demonstrated that LPA inhibited the expansion of NS/PCs of both origins, mainly by increased apoptosis in a Rho/Rho-associated kinase (ROCK)-dependent mechanism. Furthermore, LPA inhibited the neuronal differentiation of iPSCs. Lastly, LPA induced neurite retraction of NS/ PC-derived early neurons through Rho/ROCK, which was accompanied by myosin light chain (MLC) phosphorylation. Our data demonstrate the consistency of LPA effects across various sources of human NS/PCs, rendering hESCs and iPSCs valuable models for studying lysophospholipid signaling in human neural cells. Our data also highlight the importance of the Rho/ROCK pathway in human NS/ PCs. As LPA levels are increased in the central nervous system (CNS) following injury, LPA-mediated effects on NS/ PCs and early neurons could contribute to the poor neurogenesis observed in the CNS following injury

G-protein coupled receptors in stem cell self-renewal and differentiation

Stem cells have great potential for understanding early development, treating human disease, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidence now exists demonstrating the potent effects of various G-protein coupled receptor (GPCR) ligands on the biology of stem cells. This review aims to give an overview of the current knowledge of the regulation of embryonic and somatic stem cell maintenance and differentiation by GPCR ligands

Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish

Lysophosphatidic acid (LPA) is a unique bioactive lysophospholipid that induces pleiotropic effects in various cell types and organisms by acting on its specific receptors. LPA is mainly synthetised extracellularly by the ectonucleotide pyrophosphatase/phosphodiesterase 2/autotaxin (enpp2). Altered LPA signalling is associated with embryonic abnormalities, suggesting critical roles for LPA during development. However, the role of LPA signalling during early embryogenesis is not well established. We demonstrate that enpp2/LPA signalling in the early zebrafish embryo results in altered axis and midline formation, defects in left right (L-R) patterning, ciliogenesis of the Kupffer’s vesicle (KV), through the modulation of cell migration during gastrulation in a lpar(1–3) Rho/ROCK-dependant manner. Overall, this study demonstrates an essential role of enpp2/LPA signalling during early embryogenesis

Optimized Bombyx mori silk sericin biomaterial for retinal pigment epithelium regeneration.

Purpose: A transplantable cell carrier is sought to restore the retinal pigment epithelium (RPE) in age-related macular degeneration. Preparations of Bombyx mori silk sericin (BMSS) have shown in vivo and in vitro protective, wound healing, and antioxidative properties; however, current isolation techniques lead to inconsistent structure and bioactivity. We present an optimized method for BMSS isolation and manufacture into a biomaterial capable ofsupporting RPE.Methods: Gravimetric yield analysis, SDS-PAGE, solubility, transparency, and the ability to support viable human adult RPE cells (ARPE-19) were determined for the BMSS films created from four increasing durations of high temperature high pressure (HTHP) extraction, without dialysis. The new preparations were compared to both the original HTHPmethod (30-min HTHP with 4-h dialysis) and to uncoated tissue culture plastic (TCP), as a positive control. The effect of water annealing the films was also investigated. The optimized method was tested as a biomaterial for humanembryonic stem cell derived-RPE (hESC-RPE) cells against Geltrex coated and uncoated transwell inserts (Fig. 1).Results: The yield was not adversely affected by increased duration of HTHP (n=5) and surpassed previous methods. The polypeptide component of BMSS degraded in a time-dependent manner, increasing solubility of the lyophilised product. Films were suitable for biomaterial applications if cast from freshly prepared solutions but not if reconstituted from lyophilised BMSS. Films prepared from both the new and original 30-min HTHP methods (and not annealed) performed comparably to TCP in terms of ARPE19 cell viability at 4 h and 7 days (2-way ANOVA, n=3, p>0.05). As dialysis proved unnecessary, the new 30 min method was selected for improved speed, yield, and theoretical retention of potentially bioactive components. Long term hESC-RPE established for 8 weeks upon optimized BMSS filmsdisplayed a monolayer culture (1-2 cells deep) with correct cobblestone morphology (Fig. 2) and pigmentation.Conclusions: Films prepared by the new optimized method of 30-min HTHP without dialysis or water annealing supported ARPE19 cells with viability equivalent to TCP controls, and outperformed Geltrex and uncoated transwells in developing correct hESC-RPE morphology and pigmentation

Generation of a gene-corrected human isogenic iPSC line from an Alzheimer\u27s disease iPSC line carrying the London mutation in APP (V717I)

We report the genome-editing of an existing iPSC line carrying the London mutation in APP (V717I) into an iPSC line in which the pathogenic mutation was corrected. The resulting isogenic iPSC line maintained pluripotent stem cell morphology, a normal karyotype, expression of pluripotency markers and the ability to differentiate into the three germ-layers in vitro

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

© The Author(s) 2020. Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.