Motor Neuron Progenitors Research Articles

Independent control of neurogenesis and dorsoventral patterning by NKX2-2.

Human neurogenesis is disproportionately protracted, lasting >10 times longer than in mouse, allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord, expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called vpMN (ventral motor neuron progenitor) that uniquely extends and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2, which in vpMNs is co-expressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study, we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that NKX2-2 represses proneural gene NEUROG2 by two distinct, Notch-independent mechanisms that are respectively apparent in rodent and human spinal progenitors: in rodents (and chick), NKX2-2 represses Olig2 and the motor neuron lineage through its tinman domain, leading to loss of Neurog2 expression. In human vpMNs, however, NKX2-2 represses NEUROG2 but not OLIG2, thereby allowing motor neurogenesis to proceed, albeit in a delayed and protracted manner. Interestingly, we found that ectopic expression of tinman-mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs, repressing Neurog2 but not Olig2, and leading to delayed and protracted motor neurogenesis. Our studies identify a Notch- and tinman-independent mode of Nkx2-2-mediated Neurog2 repression that is observed in human spinal progenitors, but is normally masked in rodents and chicks due to Nkx2-2's tinman-dependent repression of Olig2.

A human-specific progenitor sub-domain extends neurogenesis and increases motor neuron production.

Neurogenesis lasts ~10 times longer in developing humans compared to mice, resulting in a >1,000-fold increase in the number of neurons in the CNS. To identify molecular and cellular mechanisms contributing to this difference, we studied human and mouse motor neurogenesis using a stem cell differentiation system that recapitulates species-specific scales of development. Comparison of human and mouse single-cell gene expression data identified human-specific progenitors characterized by coexpression of NKX2-2 and OLIG2 that give rise to spinal motor neurons. Unlike classical OLIG2+ motor neuron progenitors that give rise to two motor neurons each, OLIG2+/NKX2-2+ ventral motor neuron progenitors remain cycling longer, yielding ~5 times more motor neurons that are biased toward later-born, FOXP1-expressing subtypes. Knockout of NKX2-2 converts ventral motor neuron progenitors into classical motor neuron progenitors. Such new progenitors may contribute to the increased production of human motor neurons required for the generation of larger, more complex nervous systems.

Expanded ATXN1 alters transcription and calcium signaling in SCA1 human motor neurons differentiated from induced pluripotent stem cells

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited and lethal neurodegenerative disease caused by the abnormal expansion of CAG repeats in the ATAXIN-1 (ATXN1) gene. Pathological studies identified dysfunction and loss of motor neurons (MNs) in the brain stem and spinal cord, which are thought to contribute to premature lethality by affecting the swallowing and breathing of SCA1 patients. However, the molecular and cellular mechanisms of MN pathogenesis remain unknown.To study SCA1 pathogenesis in human MNs, we differentiated induced pluripotent stem cells (iPSCs) derived from SCA1 patients and their unaffected siblings into MNs. We examined proliferation of progenitor cells, neurite outgrowth, spontaneous and glutamate-induced calcium activity of SCA1 MNs to investigate cellular mechanisms of pathogenesis. RNA sequencing was then used to identify transcriptional alterations in iPSC-derived MN progenitors (pMNs) and MNs which could underlie functional changes in SCA1 MNs. We found significantly decreased spontaneous and evoked calcium activity and identified dysregulation of genes regulating calcium signaling in SCA1 MNs. These results indicate that expanded ATXN1 causes dysfunctional calcium signaling in human MNs.

Isogenic patient-derived organoids reveal early neurodevelopmental defects in spinal muscular atrophy initiation

Whether neurodevelopmental defects underlie postnatal neuronal death in neurodegeneration is an intriguing hypothesis only recently explored. Here, we focus on spinal muscular atrophy (SMA), a neuromuscular disorder caused by reduced survival of motor neuron (SMN) protein levels leading to spinal motor neuron (MN) loss and muscle wasting. Using the first isogenic patient-derived induced pluripotent stem cell (iPSC) model and a spinal cord organoid (SCO) system, we show that SMA SCOs exhibit abnormal morphological development, reduced expression of early neural progenitor markers, and accelerated expression of MN progenitor and MN markers. Longitudinal single-cell RNA sequencing reveals marked defects in neural stem cell specification and fewer MNs, favoring mesodermal progenitors and muscle cells, a bias also seen in early SMA mouse embryos. Surprisingly, SMN2-to-SMN1 conversion does not fully reverse these developmental abnormalities. These suggest that early neurodevelopmental defects may underlie later MN degeneration, indicating that postnatal SMN-increasing interventions might not completely amend SMA pathology in all patients.

Combination of induced pluripotent stem cell-derived motor neuron progenitor cells with irradiated brain-derived neurotrophic factor over-expressing engineered mesenchymal stem cells enhanced restoration of axonal regeneration in a chronic spinal cord injury rat model

BackgroundSpinal cord injury (SCI) is a disease that causes permanent impairment of motor, sensory, and autonomic nervous system functions. Stem cell transplantation for neuron regeneration is a promising strategic treatment for SCI. However, selecting stem cell sources and cell transplantation based on experimental evidence is required. Therefore, this study aimed to investigate the efficacy of combination cell transplantation using the brain-derived neurotrophic factor (BDNF) over-expressing engineered mesenchymal stem cell (BDNF-eMSC) and induced pluripotent stem cell-derived motor neuron progenitor cell (iMNP) in a chronic SCI rat model.MethodA contusive chronic SCI was induced in Sprague-Dawley rats. At 6 weeks post-injury, BDNF-eMSC and iMNP were transplanted into the lesion site via the intralesional route. At 12 weeks post-injury, differentiation and growth factors were evaluated through immunofluorescence staining and western blot analysis. Motor neuron differentiation and neurite outgrowth were evaluated by co-culturing BDNF-eMSC and iMNP in vitro in 2-dimensional and 3-dimensional.ResultsCombination cell transplantation in the chronic SCI model improved behavioral recovery more than single-cell transplantation. Additionally, combination cell transplantation enhanced mature motor neuron differentiation and axonal regeneration at the injured spinal cord. Both BDNF-eMSC and iMNP played a critical role in neurite outgrowth and motor neuron maturation via BDNF expression.ConclusionsOur results suggest that the combined transplantation of BDNF- eMSC and iMNP in chronic SCI results in a significant clinical recovery. The transplanted iMNP cells predominantly differentiated into mature motor neurons. Additionally, BDNF-eMSC exerts a paracrine effect on neuron regeneration through BDNF expression in the injured spinal cord.Graphical

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury

BackgroundSpinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI.MethodsA contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro.ResultsStepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation.ConclusionsOur findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.Graphical

Roxithromycin exposure induces motoneuron malformation and behavioral deficits of zebrafish by interfering with the differentiation of motor neuron progenitor cells

Roxithromycin (ROX), a commonly used macrolide antibiotic, is extensively employed in human medicine and livestock industries. Due to its structural stability and resistance to biological degradation, ROX persists as a resilient environmental contaminant, detectable in aquatic ecosystems and food products. However, our understanding of the potential health risks to humans from continuous ROX exposure remains limited. In this study, we used the zebrafish as a vertebrate model to explore the potential developmental toxicity of early ROX exposure, particularly focusing on its effects on locomotor functionality and CaP motoneuron development. Early exposure to ROX induces marked developmental toxicity in zebrafish embryos, significantly reducing hatching rates (n=100), body lengths (n=100), and increased malformation rates (n=100). The zebrafish embryos treated with a corresponding volume of DMSO (0.1%, v/v) served as vehicle controls (veh). Moreover, ROX exposure adversely affected the locomotive capacity of zebrafish embryos, and observations in transgenic zebrafish Tg(hb9:eGFP) revealed axonal loss in motor neurons, evident through reduced or irregular axonal lengths (n=80). Concurrently, abnormal apoptosis in ROX-exposed zebrafish embryos intensified alongside the upregulation of apoptosis-related genes (bax, bcl2, caspase-3a). Single-cell sequencing further disclosed substantial effects of ROX on genes involved in the differentiation of motor neuron progenitor cells (ngn1, olig2), axon development (cd82a, mbpa, plp1b, sema5a), and neuroimmunity (aplnrb, aplnra) in zebrafish larvae (n=30). Furthermore, the CaP motor neuron defects and behavioral deficits induced by ROX can be rescued by administering ngn1 agonist (n=80). In summary, ROX exposure leads to early-life abnormalities in zebrafish motor neurons and locomotor behavior by hindering the differentiation of motor neuron progenitor cells and inducing abnormal apoptosis.

The multi-lineage transcription factor ISL1 controls cardiomyocyte cell fate through interaction with NKX2.5

Congenital heart disease often arises from perturbations of transcription factors (TFs) that guide cardiac development. ISLET1 (ISL1) is a TF that influences early cardiac cell fate, as well as differentiation of other cell types including motor neuron progenitors (MNPs) and pancreatic islet cells. While lineage specificity of ISL1 function is likely achieved through combinatorial interactions, its essential cardiac interacting partners are unknown. By assaying ISL1 genomic occupancy in human induced pluripotent stem cell-derived cardiac progenitors (CPs) or MNPs and leveraging the deep learning approach BPNet, we identified motifs of other TFs that predicted ISL1 occupancy in each lineage, with NKX2.5 and GATA motifs being most closely associated to ISL1 in CPs. Experimentally, nearly two-thirds of ISL1-bound loci were co-occupied by NKX2.5 and/or GATA4. Removal of NKX2.5 from CPs led to widespread ISL1 redistribution, and overexpression of NKX2.5 in MNPs led to ISL1 occupancy of CP-specific loci. These results reveal how ISL1 guides lineage choices through a combinatorial code that dictates genomic occupancy and transcription.

Arsenic Impairs Differentiation of Human Induced Pluripotent Stem Cells into Cholinergic Motor Neurons.

Arsenic exposure during embryogenesis can lead to improper neurodevelopment and changes in locomotor activity. Additionally, in vitro studies have shown that arsenic inhibits the differentiation of sensory neurons and skeletal muscle. In the current study, human-induced pluripotent stem (iPS) cells were differentiated into motor neurons over 28 days, while being exposed to up to 0.5 μM arsenic. On day 6, neuroepithelial progenitor cells (NEPs) exposed to arsenic had reduced transcript levels of the neural progenitor/stem cell marker nestin (NES) and neuroepithelial progenitor marker SOX1, while levels of these transcripts were increased in motor neuron progenitors (MNPs) at day 12. In day 18 early motor neurons (MNs), choline acetyltransferase (CHAT) expression was reduced two-fold in cells exposed to 0.5 μM arsenic. RNA sequencing demonstrated that the cholinergic synapse pathway was impaired following exposure to 0.5 μM arsenic, and that transcript levels of genes involved in acetylcholine synthesis (CHAT), transport (solute carriers, SLC18A3 and SLC5A7) and degradation (acetylcholinesterase, ACHE) were all downregulated in day 18 early MNs. In day 28 mature motor neurons, arsenic significantly downregulated protein expression of microtubule-associated protein 2 (MAP2) and ChAT by 2.8- and 2.1-fold, respectively, concomitantly with a reduction in neurite length. These results show that exposure to environmentally relevant arsenic concentrations dysregulates the differentiation of human iPS cells into motor neurons and impairs the cholinergic synapse pathway, suggesting that exposure impairs cholinergic function in motor neurons.

Catenin signaling controls phrenic motor neuron development and function during a narrow temporal window.

Phrenic Motor Column (PMC) neurons are a specialized subset of motor neurons (MNs) that provide the only motor innervation to the diaphragm muscle and are therefore essential for survival. Despite their critical role, the mechanisms that control phrenic MN development and function are not well understood. Here, we show that catenin-mediated cadherin adhesive function is required for multiple aspects of phrenic MN development. Deletion of β- and γ-catenin from MN progenitors results in perinatal lethality and a severe reduction in phrenic MN bursting activity. In the absence of catenin signaling, phrenic MN topography is eroded, MN clustering is lost and phrenic axons and dendrites fail to grow appropriately. Despite the essential requirement for catenins in early phrenic MN development, they appear to be dispensable for phrenic MN maintenance, as catenin deletion from postmitotic MNs does not impact phrenic MN topography or function. Our data reveal a fundamental role for catenins in PMC development and suggest that distinct mechanisms are likely to control PMC maintenance.

Enrichment of human embryonic stem cell-derived V3 interneurons using an Nkx2-2 gene-specific reporter

V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.

Fmrp regulates neuronal balance in embryonic motor circuit formation.

Motor behavior requires the balanced production and integration of a variety of neural cell types. Motor neurons are positioned in discrete locations in the spinal cord, targeting specific muscles to drive locomotive contractions. Specialized spinal interneurons modulate and synchronize motor neuron activity to achieve coordinated motor output. Changes in the ratios and connectivity of spinal interneurons could drastically alter motor output by tipping the balance of inhibition and excitation onto target motor neurons. Importantly, individuals with Fragile X syndrome (FXS) and associated autism spectrum disorders often have significant motor challenges, including repetitive behaviors and epilepsy. FXS stems from the transcriptional silencing of the gene Fragile X Messenger Ribonucleoprotein 1 (FMR1), which encodes an RNA binding protein that is implicated in a multitude of crucial neurodevelopmental processes, including cell specification. Our work shows that Fmrp regulates the formation of specific interneurons and motor neurons that comprise early embryonic motor circuits. We find that zebrafish fmr1 mutants generate surplus ventral lateral descending (VeLD) interneurons, an early-born cell derived from the motor neuron progenitor domain (pMN). As VeLD interneurons are hypothesized to act as central pattern generators driving the earliest spontaneous movements, this imbalance could influence the formation and long-term function of motor circuits driving locomotion. fmr1 embryos also show reduced expression of proteins associated with inhibitory synapses, including the presynaptic transporter vGAT and the postsynaptic scaffold Gephyrin. Taken together, we show changes in embryonic motor circuit formation in fmr1 mutants that could underlie persistent hyperexcitability.

Seq’ing the origins of cells in the developing spinal cord

In the developing central nervous system, neurogenesis precedes gliogenesis; however, when and how progenitors are specified for a neuronal versus glial fate and the temporal regulation of this process is unclear. Progenitors within the motor neuron progenitor domain in the developing spinal cord give rise to cholinergic motor neurons and cells of the oligodendroglial lineage sequentially. In a recent study, Xing etal. used single cell RNA-seq to identify previously unknown heterogeneity of these progenitors in zebrafish and to delineate the trajectories that distinct pools of these progenitors take. These data help integrate existing evidence and inform new hypotheses regarding how populations of neural progenitors in the same spatial domain commit to distinct fates.

P.52 Determination of DLC1 isoform 1 (DLC1-i1) as a gene therapy for the treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron (MN) disease caused by loss of the ubiquitously expressed survival motor neuron (SMN) spliceosome protein, resulting in selective degeneration of spinal motor neurons (MNs) but the mechanism underlying the specific loss of MNs remains unknown. A previous report showed that Deleted in Liver Cancer 1 (DLC1) is the most down-regulated gene in MNs derived from a SMA patient but its roles in MN development and SMA pathogenesis remain to be elucidated. Here, we found a specific increase in the expression level of DLC1 isoform 1 (DLC1-i1) in human induced pluripotent stem cells (hiPSCs)-derived MNs compared to other isoforms. Knockdown of DLC1-i1 in MN progenitors resulted in reduced MN formation, axonal outgrowth, synapse number and cell survival, whereas overexpression of DLC1-i1 promoted MN differentiation with extensive neurite projections. Consistent with previous findings, we detected downregulated expression of DLC1-i1 in MNs derived from SMA patients' urine-derived iPSCs compared with healthy controls, likely due to intron retention of DLC1-i1 pre-mRNA. In addition, SMA patients-derived neuromuscular organoids (NMOs) showed reduced MNs and skeletal muscles generation, resulting in defective formation of neuromuscular junctions. Overexpression of DLC1-i1 partly restored both neural and muscle fibre formation in SMA NMOs. Similarly, DLC1-i1 expression was markedly reduced in the lumber spinal cord of SMA mice compared with that of wild-type, implying conservation of dysregulated DLC1-i1 expression in both the human SMA MNs and the spinal neurons of SMA mice. Injection of AAV9-SMN into the brain of SMA mice not only restored their weight and locomotor function but also DLC1-i1 expression level in the lumber spinal cord. Importantly, SMA mice treated with AAV9-DLC1-i1 together with AAV9-SMN resulted in synergistic improvement of their motor activity and lifespan compared with single treatment. Altogether, these findings demonstrate that MN-specific DLC1-i1 functions downstream of SMN to regulate MN maturation, survival, and neuromuscular junctions formation, revealing a new therapeutic target for the treatment of SMA.

Patients' Stem Cells Differentiation in a 3D Environment as a Promising Experimental Tool for the Study of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.

Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells

The pathological consequences leading to primary storage, autophagy impairment, impaired mitochondrial dynamics, and endoplasmic reticulum (ER) stress on neural cell dysfunction and apoptosis in metachromatic leukodystrophy (MLD) have been poorly elucidated. In the present study, we generated 2 cell lines of patient-specific-induced pluripotent stem cells (iPSCs) and modeled the progression of pathological events during the differentiation of iPSCs to motor neuron progenitors (MNPs) and mature motor neurons (MNs). The iPS cells were generated from two late-infantile MLD patient-derived skin fibroblasts using electroporation or the Sendai virus. Olig2+ MNPs were generated from both iPSC lines using a combination of small molecules in a chemically defined neural medium. Furthermore, the MNPs could be differentiated into mature MNs, which was confirmed by RT–PCR and MN markers, including SMI32 and ChAT. The population of MNs was approximately 50% under the culture conditions. Pathological observation of MLD patient-derived iPSCs revealed lysosomal accumulation and impaired autophagy. In addition, both MNPs and MNs derived from MLD-iPSCs showed increased lysosomal accumulation, dysfunctional autophagy, impaired mitophagy, endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation, and premature cellular death.

Motor neuron replacement therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a motor neuron degenerative disease that is also known as Lou Gehrig’s disease in the United States, Charcot’s disease in France, and motor neuron disease in the UK. The loss of motor neurons causes muscle wasting, paralysis, and eventually death, which is commonly related to respiratory failure, within 3–5 years after onset of the disease. Although there are a limited number of drugs approved for amyotrophic lateral sclerosis, they have had little success at treating the associated symptoms, and they cannot reverse the course of motor neuron degeneration. Thus, there is still a lack of effective treatment for this debilitating neurodegenerative disorder. Stem cell therapy for amyotrophic lateral sclerosis is a very attractive strategy for both basic and clinical researchers, particularly as transplanted stem cells and stem cell-derived neural progenitor/precursor cells can protect endogenous motor neurons and directly replace the lost or dying motor neurons. Stem cell therapies may also be able to re-establish the motor control of voluntary muscles. Here, we review the recent progress in the use of neural stem cells and neural progenitor cells for the treatment of amyotrophic lateral sclerosis. We focus on MN progenitor cells derived from fetal central nervous system tissue, embryonic stem cells, and induced pluripotent stem cells. In our recent studies, we found that transplanted human induced pluripotent stem cell-derived motor neuron progenitors survive well, differentiate into motor neurons, and extend axons into the host white matter, not only in the rostrocaudal direction, but also along motor axon tracts towards the ventral roots in the immunodeficient rat spinal cord. Furthermore, the significant motor axonal extension after neural progenitor cell transplantation in amyotrophic lateral sclerosis models demonstrates that motor neuron replacement therapy could be a promising therapeutic strategy for amyotrophic lateral sclerosis, particularly as a variety of stem cell derivatives, including induced pluripotent stem cells, are being considered for clinical trials for various diseases.

Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells.

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.

Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

BackgroundHuman induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities.ResultsEmploying RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation.ConclusionsDetailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.