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Background: CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such "disease in a dish" models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious., Methods: Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones., Results: We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches., Conclusions: This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening., (© 2023. The Author(s).)

Aims: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1., Methods: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells., Results: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1., Conclusions: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays., (© 2022 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data., Methods: We investigated the potential of hiPSC-derived motor neurons (hMNs) optical stimulation combined with calcium imaging in cocultured muscle cells activity to investigate BoNT-sensitivity of an in vitro model of human muscle-nerve system., Results: Functional muscle-nerve coculture system was developed using hMNs and human immortalized skeletal muscle cells. Our results demonstrated that hMNs can innervate myotubes and induce contractions and calcium transient in muscle cells, generating an in vitro human motor endplate showing dose-dependent sensitivity to BoNTs intoxication. The implementation of optogenetics combined with live calcium imaging allows to monitor the impact of BoNTs intoxication on synaptic transmission in human motor endplate model., Conclusions: Altogether, our findings demonstrate the promise of optogenetically hiPSC-derived controlled muscle-nerve system for pharmaceutical BoNTs testing and development., (© 2021. The Author(s).)

Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response-which is activated in late Wolfram degenerative processes-was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address., Methods: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts., Results: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model., Conclusions: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.

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.

With the development of stem cell (SC) biology, cell-based therapy has become a highly challenging field for experimental and clinical research. Among neurological disorders, stroke has pioneered the clinical application of SC. Safety concerns have prevailed for pilot clinical studies and important preclinical work is ongoing to help SC therapy reach the level of generalization. Stroke is classically divided into an acute, a subacute and a chronic phase. Each phase is defined by a complex array of events with overlapping and distinct kinetics that lead to both rapid tissue degeneration and long-lasting remodeling. Each SC type possesses intrinsic properties - transposed via cell-autonomous and non-cell-autonomous signaling - that would more specifically address some of these events. A better definition of what is expected from SC therapy in stroke might help assign SC sources to the acute or chronic phases and possibly optimize their use in the clinic., (Copyright © 2011 S. Karger AG, Basel.)

Background and Purpose: Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors., Methods: Four differentiation stages were identified on the basis of quantitative polymerase chain reaction expression of pluripotency, proliferation, and differentiation markers. Neural progenitors were transplanted at these 4 stages into rats with no, small, or large middle cerebral artery occlusion lesions. The fate of each transplant was compared with their pretransplantation status 1 to 4 months posttransplantation., Results: The influence of the postischemic environment was limited to graft survival and occurrence of nonneuroectodermal structures after transplantation of very immature neural progenitors. Both effects were lost with differentiation. We identified a particular stage of differentiation characterized in vitro by a rebound of proliferative activity that produced highly proliferative grafts susceptible to threaten surrounding host tissues., Conclusions: The effects of the ischemic environment on the formation of teratoma by transplanted human embryonic stem cell-derived neural progenitors are limited to early differentiation stages that will likely not be used for stem cell therapy. In contrast, hyperproliferation observed at later stages of differentiation corresponds to an intrinsic activity that should be monitored to avoid tumorigenesis.

Research on spinal cord trauma requires models reflecting the contusion mechanisms encountered in clinical situation. The aim of this study was to develop in the adult rat a reproducible model of upper cervical spinal cord contusion inducing persistent unilateral diaphragm deficit. After dura and pia matter removal, weight drop and compression were targeted at the ventro-lateral funiculi which contain the bulbospinal descending respiratory pathways that command the phrenic motoneurons innervating the diaphragm. At 7 days post-injury, the left diaphragm activity recorded in contused rats (27.4 +/- 5.1% of the contralateral activity) was significantly lower than in the sham group (97.6 +/- 1.2%). This respiratory deficit still persisted 1 month later. Histology showed a reproducible left C2-lateralized lesion that involved both white and gray matter including the ventro-lateral funiculi. This C2 contusion model provides a basis for testing both regenerative and neuroprotective strategies aimed at improving functional respiratory recovery after spinal cord trauma.

Over recent years, a certain number of experimental investigations have studied the effect of the transplantation of olfactory ensheathing glial cells (OEC) after spinal traumatism in animal, the rat in particular. Some of these studies have reported improvements in motor (mainly locomotor, postural and respiratory) and sensory function. While these new data provide additional support for the interest of the strategy of EOC transplantation to minimise the incapacitating effects of spinal pathologies in clinical therapy, it nonetheless remains necessary to continue experiments on animal models in order to better understand and master certain important points: beneficial effects according to the nature and composition of the transplants; therapeutic impact according to the type of pathology and the nature of the traumatism; influence of the dose effect; migration of the transplanted OECs (distance, pathways); active principles of the transplants; beneficial effect on various functions, in particular at the level of the vesico-sphincteric area; long-term innocuousness; long-term posttraumatic efficacy. Although therapeutic trials are in progress in certain countries (Australia, China, Portugal), it would nonetheless appear essential that these somewhat obscure points should be better understood before any clinical application might be seriously envisaged, in order to respect the principles of precaution, maximum efficacy and observance of the prevailing ethical rules.

Ensheathing olfactory glial cells (OEC) can be considered, with stem cells, as the other most important cell type for developing therapeutic cellular transplantation strategies following lesion of the central nervous system (CNS) and particularly in the case of spinal cord injury. OECs are macroglial cells whose precursors are located in the olfactory mucosa. OEC ensheath the axons of the sensory olfactory neurons, from the peripheral mucosa to the central olfactory bulbs. These glial cells constitute one of the rare macroglial cells which, after removal in the adult mammal, can survive in culture and multiply. After post-traumatic transplantation in the CNS, these cells have induced several instances of functional recovery after injury of different neural systems. The "OEC transplantation effect" consists in modifying the central inhibitory environment to make it more propitious for axonal regrowth and cell survival (reduction of the glial scar; releasing of numerous survival and neurotrophic factors, and of surface, extracellular matrix and adhesion molecules). In addition to the fact that OEC can ensheath and/or myelinate central axons, migrate in the CNS and accompany the growing axons over a relatively long distance, they also can be obtained from olfactory mucosa. OEC thus constitute a preferential candidate for autologous transplantation for the purposes of repair.

Functional respiratory recovery was evaluated by recording diaphragm and phrenic nerve activity several months after cervical cord hemisection followed by olfactory ensheathing cell (OEC) transplantation. The intact side was taken as a control in each rat. Sham-transplanted rats did not recover respiratory activity from the ipsilateral lesioned side. By contrast, ipsilateral phrenic and diaphragmatic activities recovered in transplanted rats amounted to 80.7% and 73% of their controls, respectively. After contralateral acute C1 section eliminating any contralateral influence from crossed compensatory pathways, the ipsilateral phrenic activity remained at 57.5% of the control, indicating that the phrenic recovery originated from the ipsilateral side. Supralesional stimulation in these rats elicited sublesional ipsilateral postsynaptic phrenic responses showing that transplantation helped ipsilateral fibers to again transmit nervous messages to the phrenic target, leading to substantial functional recovery. The origin of mechanisms involved in respiratory recovery (regeneration, resurrection, sprouting, sparing, demasking of latent pathways) is discussed.

The present work investigated, in adult rats, the long-term functional properties and terminal reconnections of central respiratory neurons regenerating axons within a peripheral nerve autograft bridging two separated central structures. A nerve graft was first inserted into the left medulla oblongata, in which the respiratory centers are located. Three months later, a C3 left hemisection was performed, and the distal tip of the graft was implanted into the C4 left spinal cord at the level of the phrenic nucleus, a natural central inspiratory target. Six to eight months after medullary implantation, the animals (n = 12) were electrophysiologically investigated to test 1) the phrenic target reinnervation by analyzing the phrenic responses elicited by bridge electrical stimulation and 2) the bridge innervation by unitary recordings of the spontaneous activity of regenerated axons within the nerve bridge. In the control group (n = 6), the medullary site of implantation corresponded to the dorsolateral medulla, a region known to be an unsuitable site for inducing respiratory axonal regrowth after nerve grafting. Stimulation of the nerve bridge never elicited phrenic nerve response, and no respiratory units were found within the nerve bridge. In the experimental group (n = 6), the proximal tip of the nerve bridge was implanted within the ventrolateral medulla at the level of the respiratory centers. Electrical stimulation of the nerve bridge induced phrenic nerve responses that reflected a postsynaptic activation of the phrenic target. Subsequent unitary recordings from teased fibers within the bridge revealed the presence of regenerated inspiratory fibers exhibiting discharge patterns typical of medullary inspiratory neurons, which normally make synaptic contacts with the inspiratory phrenic target. These results indicate that, when provided with an appropriate denervated target, central respiratory neurons with regenerated axons along a nerve bridge can remain functional for a long period and can make precise and specific functional reconnections with central homotypic target neurons., (Copyright 2002 Wiley-Liss, Inc.)