Identification of signaling pathways modifying human dopaminergic neuron development using a pluripotent stem cell-based high-throughput screening automated system: purinergic pathways as a proof-of-principle

Introduction: Alteration in the development, maturation, and projection of dopaminergic neurons has been proposed to be associated with several neurological and psychiatric disorders. Therefore, understanding the signals modulating the genesis of human dopaminergic neurons is crucial to elucidate disease etiology and develop effective countermeasures.Methods: In this study, we developed a screening model using human pluripotent stem cells to identify the modulators of dopaminergic neuron genesis. We set up a differentiation protocol to obtained floorplate midbrain progenitors competent to produce dopaminergic neurons and seeded them in a 384-well screening plate in a fully automated manner.Results and Discussion: These progenitors were treated with a collection of small molecules to identify the compounds increasing dopaminergic neuron production. As a proof-of-principle, we screened a library of compounds targeting purine- and adenosine-dependent pathways and identified an adenosine receptor 3 agonist as a candidate molecule to increase dopaminergic neuron production under physiological conditions and in cells invalidated for the HPRT1 gene. This screening model can provide important insights into the etiology of various diseases affecting the dopaminergic circuit development and plasticity and be used to identify therapeutic molecules for these diseases

Human cytomegalovirus infection is associated with increased expression of the lissencephaly gene PAFAH1B1 encoding LIS1 in neural stem cells and congenitally infected brains

peer reviewedCongenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these ndings, western blotting and immunouorescence analyses conrmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our ndings. Of note, PAFAH1B1 mutations (resulting in either haploinsufciency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufciency cause classic lissencephaly. Taken together, our ndings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infectio

Notch Promotes Neural Lineage Entry by Pluripotent Embryonic Stem Cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice

Contribution of Human Pluripotent Stem Cell-Based Models to Drug Discovery for Neurological Disorders

One of the major obstacles to the identification of therapeutic interventions for central nervous system disorders has been the difficulty in studying the step-by-step progression of diseases in neuronal networks that are amenable to drug screening. Recent advances in the field of human pluripotent stem cell (PSC) biology offers the capability to create patient-specific human neurons with defined clinical profiles using reprogramming technology, which provides unprecedented opportunities for both the investigation of pathogenic mechanisms of brain disorders and the discovery of novel therapeutic strategies via drug screening. Many examples not only of the creation of human pluripotent stem cells as models of monogenic neurological disorders, but also of more challenging cases of complex multifactorial disorders now exist. Here, we review the state-of-the art brain cell types obtainable from PSCs and amenable to compound-screening formats. We then provide examples illustrating how these models contribute to the definition of new molecular or functional targets for drug discovery and to the design of novel pharmacological approaches for rare genetic disorders, as well as frequent neurodegenerative diseases and psychiatric disorders

Participation des caspases à la mort neuronale induite par une ischémie focale chez la souris

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

CRISPR/Cas9-mediated generation of human embryonic stem cell sub-lines with HPRT1 gene knockout to model Lesch Nyhan disease

Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder

R26NotchIC Cells Undergo Accelerated Neural Specification

<div><p>R26NotchIC ES cells or parental control 46C cells cultured under monolayer differentiation conditions.</p> <p>(A) Typical FACS profile of <i>Sox1</i>GFP expression after 48 h. <i>Sox1</i>GFP+ cells were scored using gate M1. </p> <p>(B) Proportion of <i>Sox1</i>GFP+ cells at various time points (average of triplicates). </p> <p>(C–H) Intact cultures at 72 h of monolayer differentiation, shown in phase contrast or stained for markers as indicated.</p> <p>(I) Typical FACS profile of <i>Sox1</i>GFP expression after 5 d. M1 is the gate used throughout to quantify the proportion <i>Sox1</i>GFP+ cells: note that this FACS profile indicates a more striking difference between control and NotchIC populations than is reflected by our conservative quantification. </p> <p>(J and K) Quantitative RT-PCR for Oct4 and for FGF5 during monolayer differentiation of R26NotchIC cells (NotchIC) or control parental 46C cells. Levels were normalised to GAPDH and are displayed relative to expression in 46C ES cells.</p> <p>(L and M) FACS analysis of the proportion of <i>Sox1</i>GFP+ cells for R26-NotchIC cells or parental 46C cells at various time points from triplicate cultures at normal density (10⁴ cells/cm²) or higher density (3 × 10⁴ cells/cm²) (average of triplicates). </p></div

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells.

International audienceDendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis (1). The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases

NotchIC Inhibits Nonneural Differentiation

<div><p>(A–D) R26NotchIC cells (NotchIC) or parental 46C cells (control) cultured under monolayer differentiation conditions and stained for Oct4 (red) to indicate ES cells together with a combination of BLBP and GFP (green) to indicate both types of neural progenitor together.</p> <p>(E) qRT-PCR for various markers in R26NotchIC cells relative to parental 46C cells after 6 d of monolayer differentiation. Data are averaged from at least three independent experiments and normalised to GAPDH. Notch1 primers amplify within the NotchIC and detect both endogenous and exogenous transcripts.</p> <p>(F) R26NotchIC cells (NotchIC) or parental 46C cells (control) cultured under monolayer differentiation conditions and stained for cytokeratin 8, a marker of nonneural differentiation (red) and counterstained with DAPI (blue)</p></div