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.

Dual Blockade of Misfolded Alpha-Sarcoglycan Degradation by Bortezomib and Givinostat Combination.

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.

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.

Rescuing compounds for Lesch-Nyhan disease identified using stem cell-based phenotypic screening.

Lesch-Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). LND is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected with LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic-based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency-associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S-adenosylmethionine, a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to patients with LND via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.

Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain.

Human induced pluripotent stem cells (hiPSCs) are a most appealing source for cell replacement therapy in acute brain lesions. We evaluated the potential of hiPSC therapy in stroke by transplanting hiPSC-derived neural progenitor cells (NPCs) into the postischemic striatum. Grafts received host tyrosine hydroxylase-positive afferents and contained developing interneurons and homotopic GABAergic medium spiny neurons that, with time, sent axons to the host substantia nigra. Grafting reversed stroke-induced somatosensory and motor deficits. Grafting also protected the host substantia nigra from the atrophy that follows disruption of reciprocal striatonigral connections. Graft innervation by tyrosine hydoxylase fibers, substantia nigra protection, and somatosensory functional recovery were early events, temporally dissociated from the slow maturation of GABAergic neurons in the grafts and innervation of substantia nigra. This suggests that grafted hiPSC-NPCs initially exert trophic effects on host brain structures, which precede integration and potential pathway reconstruction. We believe that transplantation of NPCs derived from hiPSCs can provide useful interventions to limit the functional consequences of stroke through both neuroprotective effects and reconstruction of impaired pathways.

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis.

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother's uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.

Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system.

In parallel to the active search for therapeutic and industrial applications of human embryonic stem cells (hESCs), designing automated means of producing those cells is a timely goal. Slow-turning lateral vessels (STLVs) with low shear stress have shown promise for expanding the cells at the embryoid body stage. We have improved this technology by developing two complementary systems, allowing continuous optimization of the culture conditions. First, perfused STLV bioreactors were set up, to provide continuous delivery of culture medium to the cells growing in the rotating chamber. This allowed the external control of the culture medium, and consequently optimized oxygenation, pH, nutrient supply, and waste elimination. Second, a dialysis chamber was adapted. This led to a further enhanced controlled environment and a decrease in the quantity of adjunct products (e.g., growth factors) necessary to the cells inside the bioreactor chamber. hESC aggregation and initial differentiation-taking neural induction as an example-were compared between the perfused and dialyzed STLV system and static cultures. Perfused and dialyzed STLV bioreactors promoted formation of embryoid bodies that were differentiated more rapidly and were homogeneously synchronized in a statistically significant manner.