RESUMEN
Parkinson's disease (PD), characterized by dopaminergic neuron degeneration in the substantia nigra, is caused by various genetic and environmental factors. Current treatment methods are medication and surgery; however, a primary therapy has not yet been proposed. In this study, we aimed to develop a new treatment for PD that induces direct reprogramming of dopaminergic neurons (iDAN). Achaete-scute family bHLH transcription factor 1 (ASCL1) is a primary factor that initiates and regulates central nervous system development and induces neurogenesis. In addition, it interacts with BRN2 and MYT1L, which are crucial transcription factors for the direct conversion of fibroblasts into neurons. Overexpression of ASCL1 along with the transcription factors NURR1 and LMX1A can directly reprogram iDANs. Using a retrovirus, GFP-tagged ASCL1 was overexpressed in astrocytes. One week of culture in iDAN convertsion medium reprogrammed the astrocytes into iDANs. After 7 days of differentiation, TH+/TUJ1+ cells emerged. After 2 weeks, the number of mature TH+/TUJ1+ dopaminergic neurons increased. Only ventral midbrain (VM) astrocytes exhibited these results, not cortical astrocytes. Thus, VM astrocytes can undergo direct iDAN reprogramming with ASCL1 alone, in the absence of transcription factors that stimulate dopaminergic neurons development. [BMB Reports 2024; 57(8): 363-368].
Asunto(s)
Astrocitos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Reprogramación Celular , Neuronas Dopaminérgicas , Mesencéfalo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Enfermedad de Parkinson , Factores de Transcripción , Neuronas Dopaminérgicas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Astrocitos/metabolismo , Animales , Enfermedad de Parkinson/terapia , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Mesencéfalo/metabolismo , Mesencéfalo/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Diferenciación Celular , Neurogénesis , Células Cultivadas , Humanos , Proteínas del Tejido Nervioso , Proteínas con Homeodominio LIMRESUMEN
Neurons derived from human pluripotent stem cells (hPSCs) provide a valuable tool for studying human neural development and neurodegenerative diseases. The investigation of hPSC-based cell therapy, involving the differentiation of hPSCs into target cells and their transplantation into affected regions, is of particular interest. One neurodegenerative disease that is being extensively studied for hPSC-based cell therapy is Parkinson's disease (PD), the second most common among humans. Various research groups are focused on differentiating hPSCs into ventral midbrain dopaminergic (vmDA) progenitors, which have the potential to further differentiate into neurons closely resembling DA neurons found in the substantia nigra pars compacta (SNpc) after transplantation, providing a promising treatment option for PD. In vivo experiments, where hPSC-derived vmDA progenitor cells were transplanted into the striatum or SNpc of animal PD models, the transplanted cells demonstrated stable engraftment and resulted in behavioral recovery in the transplanted animals. Several differentiation protocols have been developed for this specific cell therapy. However, the lack of a reliable live-cell lineage identification method presents a significant obstacle in confirming the precise lineage of the differentiated cells intended for transplantation, as well as identifying potential contamination by non-vmDA progenitors. This deficiency increases the risk of adverse effects such as dyskinesias and tumorigenicity, highlighting the importance of addressing this issue before proceeding with transplantation. Ensuring the differentiation of hPSCs into the target cell lineage is a crucial step to guarantee precise therapeutic effects in cell therapy. To underscore the significance of lineage identification, this review focuses on the differentiation protocols of hPSC-derived vmDA progenitors developed by various research groups for PD treatment. Moreover, in vivo experimental results following transplantation were carefully analyzed. The encouraging outcomes from these experiments demonstrate the potential efficacy and safety of hPSC-derived vmDA progenitors for PD cell therapy. Additionally, the results of clinical trials involving the use of hPSC-derived vmDA progenitors for PD treatment were briefly reviewed, shedding light on the progress and challenges faced in translating this promising therapy into clinical practice.
RESUMEN
Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Neuroblastoma , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Optogenética , Enfermedad de Parkinson/genéticaRESUMEN
Pathological protein inclusion formation and propagation are the main causes of neuronal dysfunction in diverse neurodegenerative diseases; therefore, current disease-modifying therapeutic strategies have targeted this disease protein aggregation process. Recently, we reported that peucedanocoumarin III (PCiii) is a promising therapeutic compound with the ability to disaggregate α-synuclein inclusion and protect dopaminergic neurons in Parkinson's disease (PD). Here, we found that trans-4'-acetyl-3'-tigloylkhellactone (racemic peucedanocoumarin IV [PCiv]), a structural isomer of PCiii with a higher synthetic yield presented a strong anti-aggregate activity to a degree comparable to that of PCiii. PCiv retained effective inhibitory function against ß-sheet aggregate-mimic ß23 cytotoxicities and potently prevented α-synucleinopathy in α-synuclein preformed fibril (PFF)-treated mice cortical neurons. In detailed pharmacokinetic profiling of PCiv, oral administration of PCiv in rats exhibited an approximately 97-min half-life and 10% bioavailability. Moreover, tissue distribution analysis revealed favorable profiles of brain penetration with a 6.4 brain-to-plasma concentration ratio. The therapeutic efficacy of PCiv was further evaluated in a sporadic PD mouse model with a combinatorial co-injection of α-synuclein preformed fibril and recombinant adeno-associated virus expressing α-synuclein. Motor dysfunctions induced in this combinatorial α-synucleinopathy PD mouse model was almost completely rescued by PCiv diet administration, and this therapeutic effect is consistent with the marked prevention of dopaminergic neuron loss and suppression of α-synuclein aggregation. Taken together, our translational study suggests that PCiv is advantageous as a therapeutic agent for neurodegenerative diseases, especially with its good synthetic yield, high brain distribution, and anti-aggregate activity. PCiv may be useful in the management of α-synuclein inclusion formation and propagation at different stages of PD.
Asunto(s)
Cumarinas/farmacología , Enfermedad de Parkinson , Sinucleinopatías , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Ratones , Enfermedad de Parkinson/metabolismo , Ratas , alfa-Sinucleína/metabolismoRESUMEN
Together with neuronal loss, the existence of insoluble inclusions of alpha-synuclein (α-syn) in the brain is widely accepted as a hallmark of synucleinopathies including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy body. Because the α-syn aggregates are deeply involved in the pathogenesis, there have been many attempts to demonstrate the mechanism of the aggregation and its potential causative factors including post-translational modifications (PTMs). Although no concrete conclusions have been made based on the previous study results, growing evidence suggests that modifications such as phosphorylation and ubiquitination can alter α-syn characteristics to have certain effects on the aggregation process in PD; either facilitating or inhibiting fibrillization. In the present work, we reviewed studies showing the significant impacts of PTMs on α-syn aggregation. Furthermore, the PTMs modulating α-syn aggregation-induced cell death have been discussed. [BMB Reports 2022; 55(7): 323-335].
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Cuerpos de Lewy/metabolismo , Enfermedad de Parkinson/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , alfa-Sinucleína/metabolismoRESUMEN
Lipid droplets (LDs) are crucial biological organelles connected with metabolic pathways in biological systems and diseases. To monitor the locations and accumulation of LDs in lipid-related diseases, the development of a visualization tool for LDs has gained importance. In particular, LD visualization using fluorescent probes has gained attention. Herein, a new fluorescent nanoprobe, BMeS-Ali, is developed that can sense LDs based on an amphiphilic single benzene-based fluorophore (SBBF). BMeS-Ali consists of hydrophilic (-NH2) and hydrophobic (-C12H25) moieties and exists as a micelle nanostructure in aqueous media. BMeS-Ali has a weak fluorescence, but its emission was dramatically enhanced upon exposure to the LD components such as oleic acids (OA) by reassembling its nano-formulation. BMeS-Ali showed a selective LD staining ability and great biocompatibility in cells (cancer cells and stem cells). It also showed a practical sensing ability towards biologically derived lipids and can be applied to the visualization of human fingerprints. We found that the nanoprobe BMeS-Ali has significant potential to serve as a practical dye and sensor for lipids, especially for LD imaging in the biomedical research area and broader industrial applications.
Asunto(s)
Colorantes Fluorescentes , Gotas Lipídicas , Benceno , Fluorescencia , Colorantes Fluorescentes/metabolismo , Humanos , Ionóforos , Gotas Lipídicas/química , LípidosRESUMEN
Background/Aims: The objective of this study was to determine the more appropriate wound-closure method by comparing the effectiveness of two methods in a group of patients who underwent ileostomy repair. Methods: The study conducted after obtaining the approval of the Institutional Review Board (IRB) included 58 patients ≥19 years of age who underwent ileostomy at the Department of Surgery at the Presbyterian Medical Center. This was a retrospective, single-center trial. Patients who underwent ileostomy closure between January 2011 and September 2017 were assigned to the primary wound-closure (PC, n=25) group and the purse-string wound-closure (PSC, n=33) group. Post-repair complications, such as wound infection, delayed healing, and patient satisfaction related to wound management, were investigated and compared according to the wound-closure method. Results: The PSC group had a significantly lower surgical site infection rate than the PC group (0% vs. 44%, p<0.001). The wound-healing period was also significantly different between the PC and PSC groups (mean 27.18 days vs. 20.96 days, p=0.023). However, the postoperative wound-healing delay of >30 days was not significantly different (39% vs. 20%, p=0.114). In addition, there were no significant differences in the response to questionnaires on patient satisfaction between the two groups. Conclusions: PSC has a lower surgical site infection rate and the wound-healing delay was not very different from that of PC. Therefore, if patients are at risk of wound infection, such as in severe wound contamination, long operating time, and immunocompromised conditions, we should consider PSC as a wound closure method of choice.
Asunto(s)
Ileostomía , Infección de la Herida Quirúrgica , Humanos , Ileostomía/efectos adversos , Ileostomía/métodos , Satisfacción del Paciente , Estudios Retrospectivos , Técnicas de Sutura/efectos adversosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. Here, we applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1G93A mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Metabolismo de los Lípidos/genética , Ratones , Ratones Transgénicos , Neuronas Motoras/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
A turn-on fluorescent nanoprobe (named AAP-1), based on an aggregation-induced emission luminogen (AIEgen), is disclosed for the detection of adenosine triphosphate (ATP), which is an essential element in the biological system. Organic fluorophore (named TPE-TA) consists of tetraphenylethylene (TPE, sensing and signaling moiety) and mono-triamine (TA, sensing moiety), and it forms an aggregated form in aqueous media as a nanoprobe AAP-1. The nanoprobe AAP-1 has multiple electrostatic interactions as well as hydrophobic interactions with ATP, and it displays superior selectivity toward ATP, reliable sensitivity, with a detection limit around 0.275 ppb, and fast responsive (signal within 10 s). Such a fluorescent probe to monitor ATP has been actively pursued throughout fundamental and translational research areas. In vitro assay and a successful cellular ATP imaging application was demonstrated in cancer cells and embryonic stem cells. We expect that our work warrants further ATP-related studies throughout a variety of fields.
Asunto(s)
Adenosina Trifosfato , Neoplasias , Células Madre Embrionarias , Colorantes Fluorescentes , Interacciones Hidrofóbicas e Hidrofílicas , Electricidad EstáticaRESUMEN
Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.
Asunto(s)
Células Madre Embrionarias , Células Madre Pluripotentes , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Humanos , Transducción de Señal , PorcinosRESUMEN
Articulated structures of naphthalene-based donor (D)-acceptor (A) type dipolar dye and aggregation-induced emission luminogen (AIEgen) based on tetraphenylethylene (TPE) were synthesized, and their photophysical properties were analyzed for the first time. There are many fluorophore backbones, which have dipolar structure and AIEgen. However, there has been neither property analysis nor research that closely articulates DA and AIE through non-conjugation linker. We have therefore prepared two representative fluorophores; DA-AIE series (DA-AIE-M and DA-AIE-D), and characterized their UV/vis absorption and emission properties with quantum chemical calculations. In addition, we utilized the unique photophysical properties of DA-AIE-D for monitoring a trace of dimethyl sulfoxide (DMSO) in aqueous media, including real water samples.
RESUMEN
Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Proteínas Nucleares , Células Madre Pluripotentes/metabolismo , Proteína 1 Relacionada con Twist , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
After the first research declaring the generation of human induced pluripotent stem cells (hiPSCs) in 2007, several attempts have been made to model neurodegenerative disease in vitro during the past decade. Parkinson's disease (PD) is the second most common neurodegenerative disorder, which is mainly characterized by motor dysfunction. The formation of unique and filamentous inclusion bodies called Lewy bodies (LBs) is the hallmark of both PD and dementia with LBs. The key pathology in PD is generally considered to be the alpha-synuclein (α-syn) accumulation, although it is still controversial whether this protein aggregation is a cause or consequence of neurodegeneration. In the present work, the recently published researches which recapitulated the α-syn aggregation phenomena in sporadic and familial PD hiPSC models were reviewed. Furthermore, the advantages and potentials of using patient-derived PD hiPSC with focus on α-syn aggregation have been discussed. [BMB Reports 2019; 52(6): 349-359].
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismo , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/fisiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/metabolismo , Agregación Patológica de Proteínas/patología , alfa-Sinucleína/fisiologíaRESUMEN
Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.
Asunto(s)
Quimiocina CCL2/genética , Quimiocina CXCL1/genética , Enfermedades Genéticas Congénitas , Células Madre Pluripotentes Inducidas/metabolismo , Células de Schwann/metabolismo , Adulto , Animales , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Células Cultivadas , Reprogramación Celular , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocinas , Células Madre Embrionarias/patología , Femenino , Edición Génica , Expresión Génica , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Genética Humana , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones , Ratones Endogámicos NOD , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Ratas , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Células de Schwann/patología , TrasplanteRESUMEN
Zika virus (ZIKV) infection is associated with neurological disorders of both the CNS and peripheral nervous systems (PNS), yet few studies have directly examined PNS infection. Here we show that intraperitoneally or intraventricularly injected ZIKV in the mouse can infect and impact peripheral neurons in vivo. Moreover, ZIKV productively infects stem-cell-derived human neural crest cells and peripheral neurons in vitro, leading to increased cell death, transcriptional dysregulation and cell-type-specific molecular pathology.
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Muerte Celular/fisiología , Células-Madre Neurales/patología , Células-Madre Neurales/virología , Enfermedades del Sistema Nervioso Periférico/patología , Enfermedades del Sistema Nervioso Periférico/virología , Virus Zika/fisiología , Animales , Células Cultivadas , Chlorocebus aethiops , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos ICR , Células Vero , Infección por el Virus Zika/patologíaRESUMEN
Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.
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Citoesqueleto de Actina/metabolismo , Actinas/genética , Movimiento Celular/genética , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Línea Celular , Polaridad Celular , Retroalimentación Fisiológica , Fibroblastos/citología , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Células 3T3 NIH , Neuropéptidos/genética , Neuropéptidos/metabolismo , Optogenética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Polimerizacion , Unión Proteica , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.
Asunto(s)
Diferenciación Celular , Miocitos Cardíacos/citología , Neuronas/citología , Células Madre Pluripotentes/citología , Sistema Nervioso Simpático/citología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Línea Celular , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Ventrículos Cardíacos/citología , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , Optogenética , Fenotipo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Neural crest (NC) generates diverse lineages including peripheral neurons, glia, melanocytes, and mesenchymal derivatives. Isolating multipotent human NC has proven challenging, limiting our ability to understand NC development and model NC-associated disorders. Here, we report direct reprogramming of human fibroblasts into induced neural crest (iNC) cells by overexpression of a single transcription factor, SOX10, in combination with environmental cues including WNT activation. iNC cells possess extensive capacity for migration in vivo, and single iNC clones can differentiate into the four main NC lineages. We further identified a cell surface marker for prospective isolation of iNCs, which was used to generate and purify iNCs from familial dysautonomia (FD) patient fibroblasts. FD-iNC cells displayed defects in cellular migration and alternative mRNA splicing, providing insights into FD pathogenesis. Thus, this study provides an accessible platform for studying NC biology and disease through rapid and efficient reprogramming of human postnatal fibroblasts.