One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive ...impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage.
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► Lamin A is specifically repressed in iPSC neurons derivatives ► miR-9 knocks down lamin A and progerin expression ► miR-9 overexpression improves HGPS nuclear abnormalities
Hutchinson-Gilford progeria syndrome is a rare genetic disorder principally due to a heterozygous silent mutation in the lamin A/C gene. Its clinical features are associated with global and dramatic premature aging with the notable exception of the central nervous system. By generating patient-specific induced pluripotent stem cells (iPSCs), Nissan and colleagues were able to explore the molecular machinery of this disease, and they identified miR-9 as a key regulator of lamin A expression in human iPSC neural derivatives.
Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells ...carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the
SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of
SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to
SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.
► Neural differentiation of hESC lines from myotonic dystrophy type 1 (DM1) embryos ► Coculture assays reveal defects in neuritis and neuromuscular connections ► Gene expression analysis highlights reduction in
SLITRK family proteins ► SLITRK knockdown mimics the phenotype, and SLITRK overexpression rescues it
MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function ...screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS--Suppressor Target Screen--approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.
There is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing ...expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases.
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•Myotonic dystrophy type 1 hPSCs were adapted for high content screening•FDA-approved cardiac glycosides normalize in vitro and in vivo DM1 biological markers•Cardiac glycosides synergize with the ERK pathway to normalize DM1 biomarkers•This study emphasizes the value of human pluripotent stem cells for drug discovery
Physiology; Molecular Biology; Cell Biology
Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of ...patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.
Considerable hope surrounds the use of disease‐specific pluripotent stem cells to generate models of human disease allowing exploration of pathological mechanisms and search for new treatments. ...Disease‐specific human embryonic stem cells were the first to provide a useful source for studying certain disease states. The recent demonstration that human somatic cells, derived from readily accessible tissue such as skin or blood, can be converted to embryonic‐like induced pluripotent stem cells (hiPSCs) has opened new perspectives for modelling and understanding a larger number of human pathologies. In this review, we examine the opportunities and challenges for the use of disease‐specific pluripotent stem cells in disease modelling and drug screening. Progress in these areas will substantially accelerate effective application of disease‐specific human pluripotent stem cells for drug screening.
Wnt-ligands are among key morphogens that mediate patterning of the anterior territories of the developing brain in mammals. We qualified the role of Wnt-signals in regional specification and ...subregional organization of the human telencephalon using human pluripotent stem cells (hPSCs). One step neural conversion of hPSCs using SMAD inhibitors leads to progenitors with a default rostral identity. It provides an ideal biological substrate for investigating the role of Wnt signaling in both anteroposterior and dorso-ventral processes. Challenging hPSC-neural derivatives with Wnt-antagonists, alone or combined with sonic hedgehog (Shh), we found that Wnt-inhibition promote both telencephalic specification and ventral patterning of telencephalic neural precursors in a dose-dependent manner. Using optimal Wnt-antagonist and Shh-agonist signals we produced human ventral-telencephalic precursors, committed to differentiation into striatal projection neurons both in vitro and in vivo after homotypic transplantation in quinolinate-lesioned rats. This study indicates that sequentially organized Wnt-signals play a key role in the development of human ventral telencephalic territories from which the striatum arise. In addition, the optimized production of hPSC-derived striatal cells described here offers a relevant biological resource for exploring and curing Huntington disease.
Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain ...tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.
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•Specific derivation of MGE-type GABAergic pallial interneurons (pINs) from human ESCs•Post-mitotic human pINs migrate, persist long term, and functionally integrate in vivo•Dose-dependent suppression of seizures and hippocampal pathology in MTLE mouse model•POC data supporting phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE
Bershteyn and colleagues developed a human GABAergic pallial-type interneuron cell therapy from pluripotent stem cells for the treatment of drug-resistant mesial temporal lobe epilepsy (MTLE). One-time intrahippocampal delivery of human interneurons into a chronic mouse model of MTLE resulted in robust and stable seizure suppression.
Patients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive dysfunctions, the origin of which has remained elusive, partly ...because of the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-gene carriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whose function is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.
Les cellules souches pluripotentes (CSP) humaines sont devenues en quelques années des modèles de choix pour étudier les mécanismes cellulaires et moléculaires qui gouvernent l'apparition de maladies ...monogéniques, mais également pour le développement de criblages à haut débits afin d'identifier parmi plusieurs milliers de molécules chimiques celles qui ont un potentiel thérapeutique. C'est dans ce contexte de criblage que mes travaux de thèse s'inscrivent, alliant automatisation et miniaturisation de la biologie des CSP dans le cadre de deux maladies monogéniques, l'amyotrophie spinale infantile (SMA) et la dystrophie myotonique de type I (DM1). De manière générale, la mise en place d'une telle stratégie repose sur trois étapes essentielles qui sont l'obtention de CSP porteuses d'une mutation donnée, l'identification d'un modèle d'étude pertinent et la réalisation du criblage à proprement parlé. L'obtention de CSP humaines repose sur deux approches principales. La première consiste en la dérivation de cellules embryonnaires humaine (hES) issues de diagnostiques préimplantatoires et la seconde repose sur la reprogrammation de cellules somatiques par l'induction de pluripotence (iPS). Une partie de mon travail a consisté en la création de cellules iPS modèles de la SMA et leur caractérisation par une approche à haut débit. Par la suite un travail d'optimisation du protocole de génération de motoneurones à partir de CSP humaines a permis d'accélérer et augmenter les rendements de production de ces cellules qui sont principalement affectées dans la SMA. Enfin, l'utilisation de cellules hES porteuses de la mutation causale de la DM1 a permis le criblage de 12000 molécules et a conduit à l'identification d'une famille chimique capable de restaurer plusieurs défauts typiques de cette maladie tels que des défauts d'épissage et de fusion moléculaire.
For only few years, Human pluripotent stem cells (PSC) have become wide spread models in order to study and decipher cellular or molecular mechanims involved in monogenic diseases, but also for the development of large scale screening strategies allowing the identification of new therapeutics among thousands of chemicals. Mythesis research aimed at the development of such strategies, miniaturizing and automating PSC biology within the framework of two monogenic diseases, namely spinal muscular atrophy (SMA) and myotonic dystrophy type 1 (DM1).Basically, PSC based screening programs are generally built around three main steps which are the access to a stem cell model, the identification of a relevant cell type and lastly the screening campaign. There is actually two main ways to generate human PSC. Firstly, human embryonic stem cells (hES) can be derived from the inner cell mass of blastocyte through a pre-implantation diagnosis and secondly, induced pluripotent stem cells (iPS) can be generated after somatic cell reprogramming in vitro. A part of my work has consisted in the generation of hiPS cellular models for SMA by reprogramming fibroplasts that carried SMN1 gene deletion, followed bay the characterization of several dozen of independant clones with high throughput. Then an optimization process of the protocol for the generation of Motoneuron from PSC has been done multiplying experimental conditions. This finally allowed the description of a fast and efficient protocol to generate the most affected cell type in SMA. Finally, DM1 mutated hES were uded for the screening of 12.000 compounds among which a chemical family has been identified to rescue DM1 typical splicing and myogenesis defects.
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