Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

Low glucose induced Alzheimer's disease-like biochemical changes in human induced pluripotent stem cell-derived neurons is due to dysregulated O-GlcNAcylation.

INTRODUCTION: Sporadic Alzheimer's disease (sAD) is the leading type of dementia. Brain glucose hypometabolism, along with decreased O-GlcNAcylation levels, occurs before the onset of symptoms and correlates with pathogenesis. Heretofore, the mechanisms involved and the roles of O-GlcNAcylation in sAD pathology largely remain unknown due to a lack of human models of sAD. METHODS: Human cortical neurons were generated from pluripotent stem cells (PSCs) and treated with glucose reduction media. RESULTS: We found a narrow window of glucose concentration that induces sAD-like phenotypes in PSC-derived neurons. With our model, we reveal that dysregulated O-GlcNAc, in part through mitochondrial dysfunction, causes the onset of sAD-like changes. We demonstrate the therapeutic potential of inhibiting O-GlcNAcase in alleviating AD-like biochemical changes. DISCUSSION: Our results suggest that dysregulated O-GlcNAc might be a direct molecular link between hypometabolism and sAD-like alternations. Moreover, this model can be exploited to explore molecular processes and for drug development. HIGHLIGHTS: Lowering glucose to a critical level causes AD-like changes in cortical neurons. Defective neuronal structure and function were also recapitulated in current model. Dysregulated O-GlcNAcylation links impaired glucose metabolism to AD-like changes. Mitochondrial abnormalities correlate with O-GlcNAcylation and precede AD-like phenotype. Our model provides a platform to study sAD as a metabolic disease in human neurons.

Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

Induced pluripotent stem cells for disease modeling, cell therapy and drug discovery in genetic autonomic disorders: a review.

The autonomic nervous system (ANS) regulates all organs in the body independent of consciousness, and is thus essential for maintaining homeostasis of the entire organism. Diseases of the ANS can arise due to environmental insults such as injury, toxins/drugs and infections or due to genetic lesions. Human studies and animal models have been instrumental to understanding connectivity and regulation of the ANS and its disorders. However, research into cellular pathologies and molecular mechanisms of ANS disorders has been hampered by the difficulties in accessing human patient-derived ANS cells in large numbers to conduct meaningful research, mainly because patient neurons cannot be easily biopsied and primary human neuronal cultures cannot be expanded.Human-induced pluripotent stem cell (hiPSC) technology can elegantly bridge these issues, allowing unlimited access of patient-derived ANS cell types for cellular, molecular and biochemical analysis, facilitating the discovery of novel therapeutic targets, and eventually leading to drug discovery. Additionally, such cells may provide a source for cell replacement therapy to replenish lost or injured ANS tissue in patients.Here, we first review the anatomy and embryonic development of the ANS, as this knowledge is crucial for understanding disease modeling approaches. We then review the current advances in human stem cell technology for modeling diseases of the ANS, recent strides toward cell replacement therapy and drug discovery initiatives.

Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessments.

Assessing the development and function of the sympathetic nervous system in diseases on a large scale is challenging. Here, we present a protocol to generate human pluripotent stem cell (hPSC)-derived postganglionic sympathetic neurons (symNs) differentiated via neural crest cells (NCCs), which can be cryopreserved. We describe steps for hPSC replating, NCC replating and cryobanking, and symN differentiation. We then demonstrate the functionality of the hPSC-derived symNs, focusing on electrophysiological activity, calcium flux, and norepinephrine dynamics. For complete details on the use and execution of this protocol, please refer to Wu et al.1,2.

A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development.

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren's syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.

Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning.

Sensory neurons (SNs) detect a wide range of information from the body and the environment that is critical for homeostasis. There are three main subtypes of SNs: nociceptors, mechanoreceptors, and proprioceptors, which express different membrane proteins, such as TRKA, TRKB, or TRKC, respectively. Human pluripotent stem cell technology provides an ideal platform to study development and diseases of SNs, however there is not a viable method to isolate individual SN subtype for downstream analysis available. Here, we employ the method immunopanning to isolate each SN subtype. This method is very gentle and allows proper survival after the isolation. We use antibodies against TRKA, TRKB, and TRKC to isolate nociceptors, mechanoreceptors, and proprioceptors, respectively. We show that our cultures are enriched for each subtype and express their respective subtype markers. Furthermore, we show that the immunopanned SNs are electrically active and respond to specific stimuli. Thus, our method can be used to purify viable neuronal subtypes using respective membrane proteins for downstream studies.

O-GlcNAcylation is crucial for sympathetic neuron development, maintenance, functionality and contributes to peripheral neuropathy.

O-GlcNAcylation is a post-translational modification (PTM) that regulates a wide range of cellular functions and has been associated with multiple metabolic diseases in various organs. The sympathetic nervous system (SNS) is the efferent portion of the autonomic nervous system that regulates metabolism of almost all organs in the body. How much the development and functionality of the SNS are influenced by O-GlcNAcylation, as well as how such regulation could contribute to sympathetic neuron (symN)-related neuropathy in diseased states, remains unknown. Here, we assessed the level of protein O-GlcNAcylation at various stages of symN development, using a human pluripotent stem cell (hPSC)-based symN differentiation paradigm. We found that pharmacological disruption of O-GlcNAcylation impaired both the growth and survival of hPSC-derived symNs. In the high glucose condition that mimics hyperglycemia, hPSC-derived symNs were hyperactive, and their regenerative capacity was impaired, which resembled typical neuronal defects in patients and animal models of diabetes mellitus. Using this model of sympathetic neuropathy, we discovered that O-GlcNAcylation increased in symNs under high glucose, which lead to hyperactivity. Pharmacological inhibition of O-GlcNAcylation rescued high glucose-induced symN hyperactivity and cell stress. This framework provides the first insight into the roles of O-GlcNAcylation in both healthy and diseased human symNs and may be used as a platform for therapeutic studies.

Genipin Crosslinks the Extracellular Matrix to Rescue Developmental and Degenerative Defects, and Accelerates Regeneration of Peripheral Neurons.

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration.

Site-specific integration of adeno-associated virus involves partial duplication of the target locus.

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.

A protocol to differentiate nociceptors, mechanoreceptors, and proprioceptors from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) show promise for studying diseases affecting cell populations that are not easily available, including sensory neurons (SNs). Here, we present a differentiation protocol in chemically defined conditions to generate peripheral SNs from hPSCs. We describe four main steps: expansion of hPSCs, neural crest cell (NCC) differentiation, NCC dissociation and replating, and sensory neuron (SN) differentiation. This protocol enables generation of a mechanoreceptor-enriched culture or a population containing all three SN subtypes (nociceptors, mechanoreceptors, and proprioceptors). For complete details on the use and execution of this protocol, please refer to Saito-Diaz et al. (2021).

Norepinephrine transporter defects lead to sympathetic hyperactivity in Familial Dysautonomia models.

Familial dysautonomia (FD), a rare neurodevelopmental and neurodegenerative disorder affects the sympathetic and sensory nervous system. Although almost all patients harbor a mutation in ELP1, it remains unresolved exactly how function of sympathetic neurons (symNs) is affected; knowledge critical for understanding debilitating disease hallmarks, including cardiovascular instability or dysautonomic crises, that result from dysregulated sympathetic activity. Here, we employ the human pluripotent stem cell (hPSC) system to understand symN disease mechanisms and test candidate drugs. FD symNs are intrinsically hyperactive in vitro, in cardiomyocyte co-cultures, and in animal models. We report reduced norepinephrine transporter expression, decreased intracellular norepinephrine (NE), decreased NE re-uptake, and excessive extracellular NE in FD symNs. SymN hyperactivity is not a direct ELP1 mutation result, but may connect to NET via RAB proteins. We found that candidate drugs lowered hyperactivity independent of ELP1 modulation. Our findings may have implications for other symN disorders and may allow future drug testing and discovery.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells.

The three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development.

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

A Multiplex Human Pluripotent Stem Cell Platform Defines Molecular and Functional Subclasses of Autism-Related Genes.

Autism is a clinically heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restricted interests, and repetitive behaviors. Despite significant advances in the genetics of autism, understanding how genetic changes perturb brain development and affect clinical symptoms remains elusive. Here, we present a multiplex human pluripotent stem cell (hPSC) platform, in which 30 isogenic disease lines are pooled in a single dish and differentiated into prefrontal cortex (PFC) lineages to efficiently test early-developmental hypotheses of autism. We define subgroups of autism mutations that perturb PFC neurogenesis and are correlated to abnormal WNT/ßcatenin responses. Class 1 mutations (8 of 27) inhibit while class 2 mutations (5 of 27) enhance PFC neurogenesis. Remarkably, autism patient data reveal that individuals carrying subclass-specific mutations differ clinically in their corresponding language acquisition profiles. Our study provides a framework to disentangle genetic heterogeneity associated with autism and points toward converging molecular and developmental pathways of diverse autism-associated mutations.