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To improve human hepatotoxicity prediction, in vitro liver cell models replicating hepatocyte function, drug metabolism, and toxicity are required. Here, we present a protocol for creating 3D primary human hepatocyte (PHH) cell models using the RASTRUM Platform. We describe the process for PHH model generation; procedures for characterizing the PHH model, including viability, albumin production, and CYP450 inducibility; and drug treatment using acetaminophen and troglitazone. This protocol has applications in upscaling phenotypic hepatotoxicity applications.
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Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans.
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Longitudinal studies that continuously generate data enable the capture of temporal variations in experimentally observed parameters, facilitating the interpretation of results in a time-aware manner. We propose IL-VIS (incrementally learned visualizer), a new machine learning pipeline that incrementally learns and visualizes a progression trajectory representing the longitudinal changes in longitudinal studies. At each sampling time point in an experiment, IL-VIS generates a snapshot of the longitudinal process on the data observed thus far, a new feature that is beyond the reach of classical static models. We first verify the utility and correctness of IL-VIS using simulated data, for which the true progression trajectories are known. We find that it accurately captures and visualizes the trends and (dis)similarities between high-dimensional progression trajectories. We then apply IL-VIS to longitudinal multi-electrode array data from brain cortical organoids when exposed to different levels of quinolinic acid, a metabolite contributing to many neuroinflammatory diseases including Alzheimer's disease, and its blocking antibody. We uncover valuable insights into the organoids' electrophysiological maturation and response patterns over time under these conditions.
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Aprendizaje Automático , Estudios Longitudinales , Humanos , Organoides , Enfermedad de Alzheimer/metabolismo , Encéfalo/fisiologíaRESUMEN
In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high-throughput production of reproducible cell-laden structures with high fidelity. The current stiffness range of printable matrices surrounding the cells that mimic the extracellular matrix environment remains limited. The work presented herein aims to expand the range of stiffnesses by utilizing a four-armed polyethylene glycol with maleimide-functionalized arms. The complementary cross-linkers comprised a matrix metalloprotease-degradable peptide and a four-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated using MCF-7 cells, which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviors.
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Bioimpresión , Hidrogeles , Humanos , Matriz Extracelular , Vidrio , Células MCF-7RESUMEN
Current research tools for preclinical drug development such as rodent models and two-dimensional immortalized monocultures have failed to serve as effective translational models for human central nervous system (CNS) disorders. Recent advancements in the development of induced pluripotent stem cells (iPSCs) and three-dimensional (3D) culturing can improve the in vivo-relevance of preclinical models, while generating 3D cultures though novel bioprinting technologies can offer increased scalability and replicability. As such, there is a need to develop platforms that combine iPSC-derived cells with 3D bioprinting to produce scalable, tunable, and biomimetic cultures for preclinical drug discovery applications. We report a biocompatible poly(ethylene glycol)-based matrix which incorporates Arg-Gly-Asp and Tyr-Ile-Gly-Ser-Arg peptide motifs and full-length collagen IV at a stiffness similar to the human brain (1.5 kPa). Using a high-throughput commercial bioprinter we report the viable culture and morphological development of monocultured iPSC-derived astrocytes, brain microvascular endothelial-like cells, neural progenitors, and neurons in our novel matrix. We also show that this system supports endothelial-like vasculogenesis and enhances neural differentiation and spontaneous activity. This platform forms a foundation for more complex, multicellular models to facilitate high-throughput translational drug discovery for CNS disorders.
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Bioimpresión , Células Madre Pluripotentes Inducidas , Humanos , Astrocitos , Bioimpresión/métodos , Diferenciación Celular , Sistema Nervioso Central , Células Madre , Impresión TridimensionalRESUMEN
AIM: To examine to what extent the high frame rate contrast-enhanced ultrasound (HiFR) diagnostic enables the conclusive diagnosis of liver changes with suspected malignancy. MATERIAL/METHODS: Ultrasound examinations were performed by an experienced examiner using a multifrequency probe (SC6-1) on a high-end ultrasound system (Resona 7, Mindray) to clarify liver changes that were unclear on the B-scan. A bolus of 1-2.4âml of the Sulphur hexafluoride ultrasound microbubbles contrast agent SonoVue™ (Bracco SpA, Italy) was administered with DICOM storage of CEUS examinations from the early arterial phase (5-15âs) to the late phase (5-6 min). Based on the image files stored in the PACS, an independent reading was performed regarding image quality and finding-related diagnostic significance (0 not informative/non-diagnostic to 5 excellent image quality/confident diagnosis possible). References were clinical follow-up, if possible, comparison to promptly performed computed tomography or magnetic resonance imaging, in some cases also to histopathology. RESULTS: We examined 100 patients (42 women, 58 men, from 18 years to 90 years, mean 63±13 years) with different entities of focal and diffuse liver parenchymal changes, which could be detected in all cases with sufficient image quality with CEUS and with high image quality with HiFR-CEUS. Proportionally septate cysts were found in nâ=â19 cases, scars after hemihepatectomy with local reduced fat in nâ=â5 cases, scars after microwave ablation in nâ=â19 cases, hemangiomas in nâ=â9 cases, focal nodular hyperplasia in nâ=â8 cases, colorectal metastases in nâ=â15 cases, hepatocellular carcinoma (HCC) in nâ=â11 cases, Osler disease in nâ=â8 cases. The size of lesions ranged from 5âmm to 200âmm with a mean value of 33.1±27.8âmm. Conclusive diagnoses could be made by the experienced investigator in 97/100 cases with CEUS, confirmed by reference imaging, in parts by histopathology or follow-up. The image quality for HiFR CEUS was rated with a score of 3 to 5; 62 cases were assessed with an average of good (4 points), 27 cases with very good (5 points), and in 11 cases (3 points) still satisfactory despite aggravated acoustic conditions. The specificity of HIFR-CEUS was 97%, the sensitivity 97%, the positive predictive value 94%, the negative predictive value 99% and the accuracy 97%. CONCLUSION: HIFR-CEUS has demonstrated has demonstrated an improved image quality resulting in a high diagnostic accuracy. In the hands of an experienced investigator, HiFR-CEUS allows the assessment of focal and diffuse unclear liver parenchymal changes on B-scan and dynamic assessment of microcirculation in solid and vascular changes.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Masculino , Humanos , Femenino , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Cicatriz/patología , Medios de Contraste , Ultrasonografía/métodos , Imagen por Resonancia MagnéticaRESUMEN
Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.
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Bioimpresión , Neoplasias , Movimiento Celular , Humanos , Hidrogeles , Impresión Tridimensional , Reproducibilidad de los ResultadosRESUMEN
Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter RASTRUM with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3ß phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.
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Bioimpresión , Neoplasias de la Mama , Neoplasias de la Mama/tratamiento farmacológico , Técnicas de Cultivo Tridimensional de Células , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Descubrimiento de Drogas/métodos , Femenino , Humanos , Esferoides Celulares , Flujo de TrabajoRESUMEN
Introducción: Los tumores luminales presentan diferencias moleculares y distinto comportamiento. El antígeno ki67 (ki67) es uno de los factores que sirve para diferenciar entre luminal A y B. Las plataformas genómicas pueden identificar qué pacientes se benefician con quimioterapia. Objetivo: Establecer si existe asociación entre ki67 y Score de Oncotype Dx o score de recurrencia (SR). Evaluar la influencia del ki67 y el SR en la decisión terapéutica, evaluar la asociación entre riesgo clínico y SR, entre invasión linfovascular (ILV) y SR y entre axila positiva (hasta 1 ganglio) y SR. Material y método: Estudio retrospectivo, observacional, descriptivo. Se incluyeron 68 pacientes con tumores luminales Her2Neu negativos, T1-T2, axila negativa o positiva hasta 1 ganglio las cuales realizaron Oncotype DX entre 2009 y 2020 en el Hospital Alemán. Se clasificaron en SR menor o igual a 25 y mayor a 25 en base al estudio TAILORX donde se demostró que globalmente no hay beneficio con quimioterapia entre 0-25. Resultados: Se observó asociación entre ki67 y SR en 44 (64,7%) pacientes y fue mayor entre ki67 bajo y SR menor o igual a 25 (77,3%). El tratamiento se basó en el SR. Se observó asociación entre riesgo clínico y SR en 43 (63,2%) pacientes y fue mayor entre bajo riesgo clínico y SR menor o igual a 25 (87,5%). En un 88,8% no existió asociación entre ILV y SR, como así tampoco, entre axila positiva hasta 1 ganglio y SR en un 85,7%. Conclusiones: Es menester ofrecer a toda paciente con un tumor luminal una plataforma genómica ya que tanto el ki67 como otros factores clínicopatológicos por sí solos no demostraron ser superiores ni suficientes.
Introduction: Luminal tumors show molecular differences and different behavior. The antigen ki67 (ki67) is one of the factors that differentiate between luminal A and B. Genomic platforms can identify which patients will benefit from chemotherapy. Objective: To establish if there is an association between ki67 and Oncotype Dx Score or recurrence score (RS). To assess the influence of ki67 and RS on the therapeutic decision, to evaluate the association between clinical risk and RS, between lymphovascular invasion (LVI) and RS, and between positive armpit (up to 1 node) and RS. Material and method: Retrospective, observational, descriptive study. We included 68 patients with negative Her2Neu luminal tumors, T1-T2, negative or positive axillary up to 1 node, who performed Oncotype DX between 2009 and 2020 at Hospital Alemán. They were classified into RS less than or equal to 25 and greater than 25 based on the TAILORX study, where it was shown that overall there is no benefit from chemothe- rapy between 0-25. Results: An association was observed between ki67 and RS in 44 (64.7%) patients and it was greater between low ki67 and RS less than or equal to 25 (77.3%). The treatment was based on RS. An association between clinical risk and RS was observed in 43 (63.2%) patients, and it was greater between low clinical risk and RS less than or equal to 25 (87.5%). In 88.8% there was no association between LVI and RS, as well as between positive axillary up to 1 node and RS in 85.7%. Conclusions: It is necessary to offer every patient with a luminal tumor a genomic platform since both ki67 and other clinicopathological factors alone did not prove to be superior or sufficient.
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Femenino , Neoplasias de la Mama , Terapéutica , Antígeno Ki-67 , Quimioterapia , AntígenosRESUMEN
Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.
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Técnicas de Cultivo de Célula , Descubrimiento de Drogas , Evaluación Preclínica de MedicamentosRESUMEN
The life expectancy of patients with high-grade glioma (HGG) has not improved in decades. One of the crucial tools to enable future improvement is advanced models that faithfully recapitulate the tumour microenvironment; they can be used for high-throughput screening that in future may enable accurate personalised drug screens. Currently, advanced models are crucial for identifying and understanding potential new targets, assessing new chemotherapeutic compounds or other treatment modalities. Recently, various methodologies have come into use that have allowed the validation of complex models-namely, spheroids, tumouroids, hydrogel-embedded cultures (matrix-supported) and advanced bioengineered cultures assembled with bioprinting and microfluidics. This review is designed to present the state of advanced models of HGG, whilst focusing as much as is possible on the paediatric form of the disease. The reality remains, however, that paediatric HGG (pHGG) models are years behind those of adult HGG. Our goal is to bring this to light in the hope that pGBM models can be improved upon.
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Antineoplásicos/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Glioblastoma/tratamiento farmacológico , Esferoides Celulares/efectos de los fármacos , Adulto , Antineoplásicos/química , Bioimpresión/métodos , Niño , Glioblastoma/patología , Humanos , Hidrogeles/química , Hidrogeles/uso terapéutico , Microfluídica/métodos , Microambiente Tumoral/efectos de los fármacosRESUMEN
AIM: The aim of this study was to assess the success of irreversible electroporation (IRE) in prostate cancer and to differentiate between reactive changes and tumor. MATERIAL AND METHODS: This is a retrospective pilot study of 50 patients after irreversible electroporation (IRE) in prostate cancer between 50-79 years (mean age 65 years). Each patient received a transabdominal sonography using a 1-6 MHz convex matrix probe. Contrast-enhanced ultrasound (CEUS) was performed after i.v. bolus injection of 2.0âml sulphur hexafluoride microbubbles. DICOM loops were continuously stored up to one minute. Parametric images were calculated by integrated perfusion analysis software. A comparison was drawn to a follow-up MRI six months after ablation. RESULTS: While 13 patients showed local recurrence, 37 patients were successfully treated, meaning no local recurrence within six months after ablation. 18 patients showed signs of prostatitis after IRE. Tumorous changes were visually characterized by dynamic early nodular hypervascularization with fast and high wash-in. Correspondingly, nodular red and yellow shades were seen in parametric imaging. All patients with remaining tumor were correctly identified with CEUS and parametric imaging. After IRE there is a relevant decrease in tumor microcirculation in all patients, as seen in more purple shades of the prostate. The sensitivity for detecting residual tumor with CEUS compared to MRI was 76%, the specificity was 81%. The corresponding positive predictive value (PPV) was 73% and the negative predictive value (NPV) was 83%. CONCLUSION: CEUS and parametric imaging enable a critical analysis of post-ablation defects after IRE for prostate cancer even with a transabdominal approach. Remaining tumor can be detected with the help of pseudo-colors.
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Medios de Contraste/uso terapéutico , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/terapia , Ultrasonografía/métodos , Anciano , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios RetrospectivosRESUMEN
The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
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Diferenciación Celular/efectos de los fármacos , Neuronas Colinérgicas/efectos de los fármacos , Medios de Cultivo/farmacología , Cuerpos Embrioides/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Cultivo Primario de Células/métodos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/patología , Benzamidas/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Línea Celular , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Medios de Cultivo/química , Dioxoles/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Factor 2 de Diferenciación de Crecimiento/farmacología , Proteínas Hedgehog/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Modelos Biológicos , Factor de Crecimiento Nervioso/farmacología , Técnicas de Placa-Clamp , Pirazoles/farmacología , Pirimidinas/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the "mini-brain," the "brain in a dish". At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell-generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term "dementia." We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model "dementia in a dish."
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Enfermedad de Alzheimer/patología , Encéfalo/patología , Demencia/patología , Células Madre Pluripotentes Inducidas/citología , Organoides/citología , Animales , Diferenciación Celular/fisiología , Demencia/fisiopatología , HumanosRESUMEN
Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1E101G mutation.
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Esclerosis Amiotrófica Lateral/genética , Fibroblastos/metabolismo , Superóxido Dismutasa-1/genética , Línea Celular , Humanos , Factor 4 Similar a Kruppel , ARN Mensajero/metabolismoRESUMEN
Dermal fibroblasts from a 59â¯year old male patient with amyotrophic lateral sclerosis (symptomatic at the time of collection), attributed to a mutation in the cyclin F gene (CCNFS621G), were reprogrammed using mRNA and microRNA-delivered OSKM factors to induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed pluripotent, expressing typical pluripotency markers and were capable of three germ layer differentiation. This is the first reported reprogramming of cells with a mutation in the cyclin F gene, and represents a novel resource for the study of amyotrophic lateral sclerosis.
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Esclerosis Amiotrófica Lateral/patología , Ciclinas/genética , Dermis/citología , Células Madre Pluripotentes Inducidas/citología , Esclerosis Amiotrófica Lateral/genética , Diferenciación Celular , Línea Celular , Reprogramación Celular , Fibroblastos/citología , Estratos Germinativos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation.
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Diferenciación Celular , Neuronas Motoras/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Supervivencia Celular , Femenino , Fibroblastos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Proteoma/metabolismoRESUMEN
Targeting epigenetic mechanisms has shown promise against several cancers but has so far been unsuccessful against glioblastoma (GBM). Altered histone 3 lysine 4 methylation and increased lysine-specific histone demethylase 1A (LSD1) expression in GBM tumours nonetheless suggest that epigenetic mechanisms are involved in GBM. We engineered a dual-action prodrug, which is activated by the high hydrogen peroxide levels associated with GBM cells. This quinone methide phenylaminecyclopropane prodrug releases the LSD1 inhibitor 2-phenylcyclopropylamine with the glutathione scavenger para-quinone methide to trigger apoptosis in GBM cells. Quinone methide phenylaminocyclopropane impaired GBM cell behaviours in two-dimensional and three-dimensional assays, and triggered cell apoptosis in several primary and immortal GBM cell cultures. These results support our double-hit hypothesis of potentially targeting LSD1 and quenching glutathione, in order to impair and kill GBM cells but not healthy astrocytes. Our data suggest this strategy is effective at selectively targeting GBM and potentially other types of cancers. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/patología , Glioblastoma/patología , Profármacos/farmacología , Compuestos de Anilina/farmacología , Células Cultivadas , Ciclopropanos/farmacología , Diseño de Fármacos , Glutatión/antagonistas & inhibidores , Histona Demetilasas/antagonistas & inhibidores , Humanos , Indolquinonas/farmacologíaRESUMEN
It has been suggested that electromagnetic fields could be used to differentiate neurons in culture but how to do this is not clear. We investigated the effect of external magnetic fields (DC and AC MF) on neuronal viability, differentiation, and neurite outgrowth of human SH-SY5Y neuroblastoma cells in vitro. A strong low frequency DC MF or a weak AC MF improved retinoic acid-mediated neuronal differentiation and increased neurite length, without any adverse effects on neuronal viability. Even in the absence of the conventional differentiation factor, retinoic acid, DC and AC MF promoted neurite outgrowth. No significant negative effect on cell viability was observed after MF exposure and the DC MF had greater effects on neurite length and branch number than AC MF. Thus, we have identified a novel, simple and cost-effective method that is easy to set up in any cell culture laboratory that can be used to efficiently differentiate neuronal-like cells, using a DC MF without the need for expensive reagents. This research provides a fresh approach to promote neurite outgrowth in a commonly used neuronal-like cell line model and may be applicable to neural stem cells or primary neurons.
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Peripheral dermal fibroblasts (DF) from a healthy 56â¯year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage. Resource table.