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The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.
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Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.
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Diabetes Mellitus , Retinopatía Diabética , Animales , Humanos , Barrera Hematorretinal/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Endoteliales/metabolismo , Fenotipo , Dispositivos Laboratorio en un Chip , Vasos Retinianos/metabolismo , Retina/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
The degeneration of axon terminals before the soma, referred to as "dying back", is a feature of Parkinson's disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to "dying back" pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics.
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Data integration methods are used to obtain a unified summary of multiple datasets. For multi-modal data, we propose a computational workflow to jointly analyze datasets from cell lines. The workflow comprises a novel probabilistic data integration method, named POPLS-DA, for multi-omics data. The workflow is motivated by a study on synucleinopathies where transcriptomics, proteomics, and drug screening data are measured in affected LUHMES cell lines and controls. The aim is to highlight potentially druggable pathways and genes involved in synucleinopathies. First, POPLS-DA is used to prioritize genes and proteins that best distinguish cases and controls. For these genes, an integrated interaction network is constructed where the drug screen data is incorporated to highlight druggable genes and pathways in the network. Finally, functional enrichment analyses are performed to identify clusters of synaptic and lysosome-related genes and proteins targeted by the protective drugs. POPLS-DA is compared to other single- and multi-omics approaches. We found that HSPA5, a member of the heat shock protein 70 family, was one of the most targeted genes by the validated drugs, in particular by AT1-blockers. HSPA5 and AT1-blockers have been previously linked to α-synuclein pathology and Parkinson's disease, showing the relevance of our findings. Our computational workflow identified new directions for therapeutic targets for synucleinopathies. POPLS-DA provided a larger interpretable gene set than other single- and multi-omic approaches. An implementation based on R and markdown is freely available online.
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Biología Computacional , Sinucleinopatías , Humanos , Biología Computacional/métodos , Multiómica , Evaluación Preclínica de Medicamentos , Proteómica/métodosRESUMEN
Hummingbirds possess distinct metabolic adaptations to fuel their energy-demanding hovering flight, but the underlying genomic changes are largely unknown. Here, we generated a chromosome-level genome assembly of the long-tailed hermit and screened for genes that have been specifically inactivated in the ancestral hummingbird lineage. We discovered that FBP2 (fructose-bisphosphatase 2), which encodes a gluconeogenic muscle enzyme, was lost during a time period when hovering flight evolved. We show that FBP2 knockdown in an avian muscle cell line up-regulates glycolysis and enhances mitochondrial respiration, coincident with an increased mitochondria number. Furthermore, genes involved in mitochondrial respiration and organization have up-regulated expression in hummingbird flight muscle. Together, these results suggest that FBP2 loss was likely a key step in the evolution of metabolic muscle adaptations required for true hovering flight.
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Adaptación Fisiológica , Aves , Vuelo Animal , Fructosa-Bifosfatasa , Gluconeogénesis , Músculo Esquelético , Animales , Aves/genética , Aves/metabolismo , Metabolismo Energético/genética , Vuelo Animal/fisiología , Gluconeogénesis/genética , Adaptación Fisiológica/genética , Fructosa-Bifosfatasa/genética , Músculo Esquelético/enzimologíaRESUMEN
The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.
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Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.
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Técnica del Anticuerpo Fluorescente/métodos , Hibridación Fluorescente in Situ/métodos , ARN/metabolismo , Células Cultivadas , Fijadores/química , Colorantes Fluorescentes/química , Células HeLa , Humanos , Nanopartículas/química , ARN/química , Procesamiento Postranscripcional del ARN , Transporte de ARNRESUMEN
Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.
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Endocitosis , Endosomas/metabolismo , Liposomas/metabolismo , Nanopartículas/metabolismo , ARN Mensajero/metabolismo , Células HeLa , Humanos , ARN Mensajero/genética , Transferrina/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
ß cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of ß cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo-labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.
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Animales Modificados Genéticamente/metabolismo , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Exocitosis , Glucosa/metabolismo , Secreción de Insulina , Masculino , PorcinosRESUMEN
Phagocytosis is a key function in various cells throughout the body. A deficiency in photoreceptor outer segment (POS) phagocytosis by the retinal pigment epithelium (RPE) causes vision loss in inherited retinal diseases and possibly age-related macular degeneration. To date, there are no effective therapies available aiming at recovering the lost phagocytosis function. Here, we developed a high-throughput screening assay based on RPE derived from human embryonic stem cells (hRPE) to reveal enhancers of POS phagocytosis. One of the hits, ramoplanin (RM), reproducibly enhanced POS phagocytosis and ensheathment in hRPE, and enhanced the expression of proteins known to regulate membrane dynamics and ensheathment in other cell systems. Additionally, RM rescued POS internalization defect in Mer receptor tyrosine kinase (MERTK) mutant hRPE, derived from retinitis pigmentosa patient induced pluripotent stem cells. Our platform, including a primary phenotypic screening phagocytosis assay together with orthogonal assays, establishes a basis for RPE-based therapy discovery aiming at a broad patient spectrum.
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Células Madre Embrionarias Humanas/metabolismo , Fagocitosis , Células Fotorreceptoras de Vertebrados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Línea Celular , Células Madre Embrionarias Humanas/citología , Humanos , Células Fotorreceptoras de Vertebrados/citología , Epitelio Pigmentado de la Retina/citologíaRESUMEN
In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.
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Axones/metabolismo , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN/genética , Técnicas de Inactivación de Genes , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Compuestos de Bencidrilo/farmacología , Proteína C9orf72/metabolismo , Diferenciación Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Mutación con Ganancia de Función/genética , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Pirrolidinonas/farmacología , Transcriptoma/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) arises from an interplay of genetic mutations and environmental factors. ssRNA viruses are possible ALS risk factors, but testing their interaction with mutations such as in FUS, which encodes an RNA-binding protein, has been difficult due to the lack of a human disease model. Here, we use isogenic induced pluripotent stem cell (iPSC)-derived spinal neurons (SNs) to investigate the interaction between ssRNA viruses and mutant FUS. We find that rabies virus (RABV) spreads ALS phenotypes, including the formation of stress granules (SGs) with aberrant composition due to increased levels of FUS protein, as well as neurodegeneration and reduced restriction activity by FUS mutations. Consistent with this, iPSC-derived SNs harboring mutant FUS are more sensitive to human immunodeficiency virus (HIV-1) and Zika viruses (ZIKV). We demonstrate that RABV and HIV-1 exacerbate cytoplasmic mislocalization of FUS. Our results demonstrate that viral infections worsen ALS pathology in SNs with genetic risk factors, suggesting a novel role for viruses in modulating patient phenotypes.
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Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.
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Glioblastoma/metabolismo , Proteínas Represoras/metabolismo , Línea Celular Tumoral , Descubrimiento de Drogas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Glioblastoma/genética , Humanos , Interferencia de ARN , Proteínas Represoras/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
High-content screening (HCS) has established itself in the world of the pharmaceutical industry as an essential tool for drug discovery and drug development. HCS is currently starting to enter the academic world and might become a widely used technology. Given the diversity of problems tackled in academic research, HCS could experience some profound changes in the future, mainly with more imaging modalities and smart microscopes being developed. One of the limitations in the establishment of HCS in academia is flexibility and cost. Flexibility is important to be able to adapt the HCS setup to accommodate the multiple different assays typical of academia. Many cost factors cannot be avoided, but the costs of the software packages necessary to analyze large datasets can be reduced by using open-source software. We present and discuss the open-source software CellProfiler for image analysis and KNIME for data analysis and data mining that provide software solutions, which increase flexibility and keep costs low.
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Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Animales , Descubrimiento de Drogas/métodos , Humanos , Flujo de TrabajoRESUMEN
Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3ß is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3ß. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3ß in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.
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Quinasa 5 Dependiente de la Ciclina/metabolismo , Citoesqueleto/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Inflamación/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Citoesqueleto/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Degeneración Nerviosa/tratamiento farmacológico , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismoRESUMEN
PURPOSE OF REVIEW: We provide an overview about unbiased screens to identify modifiers of alpha-synuclein (αSyn)-induced toxicity, present the models and the libraries that have been used for screening, and describe how hits from primary screens were selected and validated. RECENT FINDINGS: Screens can be classified as either genetic or chemical compound modifier screens, but a few screens do not fit this classification. Most screens addressing αSyn-induced toxicity, including genome-wide overexpressing and deletion, were performed in yeast. More recently, newer methods such as CRISPR-Cas9 became available and were used for screening purposes. Paradoxically, given that αSyn-induced toxicity plays a role in neurological diseases, there is a shortage of human cell-based models for screening. Moreover, most screens used mutant or fluorescently tagged forms of αSyn and only very few screens investigated wild-type αSyn. Particularly, no genome-wide αSyn toxicity screen in human dopaminergic neurons has been published so far. Most unbiased screens for modifiers of αSyn toxicity were performed in yeast, and there is a lack of screens performed in human and particularly dopaminergic cells.
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alfa-Sinucleína/genética , alfa-Sinucleína/toxicidad , Animales , Neuronas Dopaminérgicas , HumanosRESUMEN
Phenotypic screens using automated microscopy allow comprehensive measurement of the effects of compounds on cells due to the number of markers that can be scored and the richness of the parameters that can be extracted. The high dimensionality of the data is both a rich source of information and a source of noise that might hide information. Many methods have been proposed to deal with this complex data in order to reduce the complexity and identify interesting phenotypes. Nevertheless, the majority of laboratories still only use one or two parameters in their analysis, likely due to the computational challenges of carrying out a more sophisticated analysis. Here, we present a novel method that allows discovering new, previously unknown phenotypes based on negative controls only. The method is compared with L1-norm regularization, a standard method to obtain a sparse matrix. The analytical pipeline is implemented in the open-source software KNIME, allowing the implementation of the method in many laboratories, even ones without advanced computing knowledge.
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Farmacología , Fenotipo , Automatización , Microscopía/métodos , Programas InformáticosRESUMEN
Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self-renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into ß-cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient ß-cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI-5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640-651.
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Familia de Aldehído Deshidrogenasa 1/genética , Aldehído Deshidrogenasa Mitocondrial/genética , Diferenciación Celular/genética , Diabetes Mellitus/terapia , Células Madre Pluripotentes/trasplante , Proteína Metiltransferasas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Benzoatos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/metabolismo , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/enzimología , Proteínas del Tejido Nervioso/genética , Páncreas/efectos de los fármacos , Páncreas/crecimiento & desarrollo , Proteína Metiltransferasas/antagonistas & inhibidores , Xantenos/farmacologíaRESUMEN
Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cis-regulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits. We first show that limb loss in snakes is associated with widespread divergence of limb regulatory elements. We next show that eye degeneration in subterranean mammals is associated with widespread divergence of eye regulatory elements. In both cases, sequence divergence results in an extensive loss of transcription factor binding sites. Importantly, diverged regulatory elements are associated with genes required for normal limb patterning or normal eye development and function, suggesting that regulatory divergence contributed to the loss of these phenotypes. Together, our results show that genome-wide decay of the phenotype-specific cis-regulatory landscape is a hallmark of lost morphological traits.