reprogramming-induced-pluripotent-stem-cell-lines-from-frozen-buffy

Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

Human pluripotent stem cells (hPSCs) are a valuable tool for disease modeling. Further stem cells can be reprogrammed from adult somatic cells, called ...

University of Georgia

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.

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

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..

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

Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the  () gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the  T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the  transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs . Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain  splicing defects in a humanized FD mouse model carrying the  T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.

Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia.

Accessing untransformed human primary parasympathetic neurons is extremely challenging due to their inaccessibility in patients. Therefore, a human pluripotent stem cell-based approach to generate pure parasympathetic neurons is valuable for studying their development, maturation, and pathology. Here, we present a protocol to derive parasympathetic neurons from human pluripotent stem cells. We describe steps for human pluripotent stem cell replating, neural crest replating, cryobanking, and parasympathetic neuron differentiation. We then detail procedures for functional assays via multielectrode array (MEA) analysis. For complete details on the use and execution of this protocol, please refer to Wu et al..

Protocol to derive postganglionic parasympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessment.

Familial dysautonomia (FD) is a rare genetic, neurodevelopmental and neurodegenerative disorder, where a homozygous mutation in the  gene is responsible for defects and symptoms found in 99% of patients (1). FD symptoms mainly affect the peripheral nervous system (PNS) (2), including the autonomic and sensory nervous systems (ANS, SNS) (3). The ANS regulates unconscious physiological responses and maintains body homeostasis, such as heart rate, blood pressure, gland secretion, and breathing, which are vital for bodily function. The SNS is the key mediator that processes and relays sensory information from the internal and external environment to the brain, including limb position, temperature, and pain.

Article	Year
O-GlcNAcylation is crucial for sympathetic neuron development, maintenance, functionality and contributes to peripheral neuropathy. Frontiers in neuroscience\| PubMed ID: 37229433	2023
Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessments. STAR protocols\| PubMed ID: 38517897	2024
Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia. bioRxiv : the preprint server for biology\| PubMed ID: 39651221	2024
Protocol to derive postganglionic parasympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessment. STAR protocols\| PubMed ID: 40748761	2025
Peripheral neuron phenotypes of familial dysautonomia are rescued by AAV-mediated gene therapy. Research square\| PubMed ID: 41041527	2025
Peripheral neuron phenotypes of familial dysautonomia are rescued by AAV-mediated gene therapy. Clinical autonomic research : official journal of the Clinical Autonomic Research Society\| PubMed ID: 41957238	2026

Jennifer Art

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