RESUMEN
BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. OBJECTIVE: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. METHODS: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. RESULTS: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. CONCLUSIONS: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.
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Hipersensibilidad a los Alimentos , Mutación con Ganancia de Función , Niño , Humanos , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Citocinas/metabolismo , ADNRESUMEN
Janus kinase 2 (JAK2) gene mutations are the main drivers for polycythemia vera (PV) and essential thrombocythemia (ET). The mechanisms of single altered gene causing two different diseases are unclear. Additionally, novel treatments specifically targeting mutated JAK2 proteins are needed. In this study, the induced pluripotent stem cells (iPSCs) were virally transduced to express wild-type JAK2 (JAK2WT), JAK2p.V617F (JAK2V617F) or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs which were differentiated into megakaryocytes in the presence vs. absence of doxycycline were compared to ensure that the differences were solely from mutated JAK2 expressions. The JAK2V617-expressing iPSCs yielded significantly higher numbers of megakaryocytes consistent with the ET phenotype, while there was no enhancement by JAK2exon12 expression compatible with the pure erythrocytosis in humans. Capillary Western analyses revealed significantly greater JAK2 phosphorylation in iPSCs carrying JAK2V617F but not in JAK2WT and JAK2exon12 iPSCs. Activation of STAT3, STAT5 and AKT was increased by JAK2V617F, while they were decreased in JAK2exon12 iPSCs. Notably, interferon alpha and/or arsenic trioxide inhibited megakaryocytes proliferation and reduced JAK2, STAT3, STAT5 and AKT phosphorylation in mutant JAK2-expressing iPSCs compared with those without induction. In conclusion, JAK2V617F expression in iPSCs preferentially promoted megakaryocytes with a signaling profile distinctive from JAK2exon12 expression. Treatments with interferon alpha or arsenic trioxide preferentially suppressed the mutated over wild-type JAK2 signaling. This iPSC model is helpful in mechanistic studies and novel therapy screen for myeloproliferative neoplasm.
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Células Madre Pluripotentes Inducidas , Janus Quinasa 2 , Transducción de Señal , Trombocitemia Esencial , Trióxido de Arsénico/farmacología , Doxiciclina , Humanos , Interferón-alfa/farmacología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Trombocitemia Esencial/genéticaRESUMEN
Either the glycoprotein (GP) Ib deficiency or hyper-function in humans can cause macrothrombocytopenia, the molecular mechanisms of which remain unclear. Herein, the investigations for disease pathogenesis were performed in the human induced pluripotent stem cell (hiPSC) model. The hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD) were generated using CRISPR/Cas9. The GP1BA-null hiPSCs were previously derived from a Bernard-Soulier syndrome (BSS) patient. After full megakaryocyte differentiation in culture, both hiPSC mutations showed large proplatelet tips under fluorescence microscopy and yielded fewer but larger platelets compared with those of wild-type cells. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to wild-type hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and wild-type hiPSCs with a MAPK inhibitor. In conclusion, the genetically modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.
Asunto(s)
Megacariocitos/metabolismo , Recuento de Plaquetas/métodos , Humanos , Transducción de SeñalRESUMEN
Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-ß peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lisina/análogos & derivados , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Código Genético , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Methanosarcina/enzimología , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-PostraduccionalRESUMEN
OBJECTIVE: To evaluate the effects of pre-treatment with low-dose simvastatin on angiogenesis and wound healing in a diabetic mouse model. MATERIAL AND METHODS: Balb/c nude mice were divided into three groups, including control (CON), diabetic (DM, and diabetic pre-treated with low-dose simvastatin (DM+ SIM). Seven days prior to wounding, the DM + SIM group was started on oral simvastatin (0.25 mg/kg/day). Eleven weeks after diabetes was induced, all mice were subjected to a bilateral full-thickness excisional skin wound on the back (0.6 x 0.6 cm²). On day 14 after wounding, percentage of wound closure (%WC), percentage of capillary vascularity (%CV), and neutrophil infiltration were determined using Image Pro-Plus, confocal fluorescence microscopy, and hematoxylin and eosin (H&E) staining, respectively. Tissue vascular endothelial growth factor (VEGF) was detected by ELISA at days 7 and 14, post-wounding. RESULTS: On day 14, %WC and %CV in CON and DM + SIM groups were significantly increased, with no significant change observed in the DM group. Neutrophil infiltration in the CON and DM + SIM groups was signficantly lower than that of the DM group. VEGF levels in the CON and DM + SIM groups were significantly higher than levels in the DM group on day 7, but not different among groups on day 14. CONCLUSION: The present study demonstrated that pre-treatment with low-dose simvastatin could increase angiogenesis, reduce inflammation, and improve wound healing in diabetic mice.
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Diabetes Mellitus Experimental/etiología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Neovascularización Fisiológica/efectos de los fármacos , Simvastatina/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Masculino , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
A series of novel styryl dye derivatives incorporating indolium and quinolinium core structures were successfully synthesized to explore their interacting and binding capabilities with tau aggregates inâ vitro and in cells. The synthesized dyes exhibited enhanced fluorescence emission in viscous environments due to the rotatable bond confinement in the core structure. Dye 4, containing a quinolinium moeity and featuring two cationic sites, demonstrated a 28-fold increase in fluorescence emission upon binding to tau aggregates. This dye could also stain tau aggregates in living cells, confirmed by cell imaging using confocal fluorescence microscopy. A molecular docking study was conducted to provide additional visualization and support for binding interactions. This work offers novel and non-cytotoxic fluorescent probes with desirable photophysical properties, which could potentially be used for studying tau aggregates in living cells, prompting further development of new fluorescent probes for early Alzheimer's disease detection.
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Colorantes Fluorescentes , Colorantes Fluorescentes/química , Simulación del Acoplamiento Molecular , Microscopía FluorescenteRESUMEN
Since its establishment in 2015, the transcriptomics-based consensus molecular subtype (CMS) classification has unified our understanding of colorectal cancer. Each of the four CMS exhibited distinctive high-level molecular signatures that correlated well with prognosis and treatment response. Nonetheless, many key aspects of colorectal cancer progression and intra-subtype heterogeneity remain unresolved. This is partly because the bulk transcriptomic data used to define CMS contain substantial interference from non-tumor cells. Here, we propose a concise panel of 62 genes that not only accurately recapitulates all key characteristics of the four original CMS but also identifies three additional subpopulations with unique molecular signatures. Validation on independent cohorts confirms that the new CMS4 intra-subtypes coincide with single-cell-derived intrinsic subtypes and that the panel consists of many immune cell-type markers that can capture the status of tumor microenvironment. Furthermore, a 2D embedding of CMS structure based on the proposed gene panel provides a high-resolution view of the functional pathways and cell-type markers that underlie each CMS intra-subtype and the continuous progression from CMS2 to CMS4 subtypes. Our gene panel and 2D visualization refined the delineation of colorectal cancer subtypes and could aid further discovery of molecular mechanisms in colorectal cancer. IMPLICATIONS: : Well-selected gene panel and representation can capture both the continuum of cancer cell states and tumor microenvironment status.
Asunto(s)
Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/patología , Perfilación de la Expresión Génica , Transcriptoma , Biomarcadores de Tumor/genética , Microambiente Tumoral/genéticaRESUMEN
Many studies have shown that cellular morphology can be used to distinguish spiked-in tumor cells in blood sample background. However, most validation experiments included only homogeneous cell lines and inadequately captured the broad morphological heterogeneity of cancer cells. Furthermore, normal, non-blood cells could be erroneously classified as cancer because their morphology differ from blood cells. Here, we constructed a dataset of microscopic images of organoid-derived cancer and normal cell with diverse morphology and developed a proof-of-concept deep learning model that can distinguish cancer cells from normal cells within an unlabeled microscopy image. In total, more than 75,000 organoid-drived cells from 3 cholangiocarcinoma patients were collected. The model achieved an area under the receiver operating characteristics curve (AUROC) of 0.78 and can generalize to cell images from an unseen patient. These resources serve as a foundation for an automated, robust platform for circulating tumor cell detection.
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Línea Celular Tumoral , Neoplasias , Humanos , Área Bajo la Curva , Aprendizaje Profundo , Microscopía , Línea Celular Tumoral/clasificación , Línea Celular Tumoral/patología , Neoplasias/diagnóstico por imagen , Neoplasias/patologíaRESUMEN
Corneal grafts are the imperative clinical treatment for canine corneal blindness. To serve the growing demand, this study aimed to generate tissue-engineered canine cornea in part of the corneal epithelium and underlying stroma based on canine limbal epithelial stem cells (cLESCs) seeded silk fibroin/gelatin (SF/G) film and canine corneal stromal stem cells (cCSSCs) seeded SF/G scaffold, respectively. Both cell types were successfully isolated by collagenase I. SF/G corneal films and stromal scaffolds served as the prospective substrates for cLESCs and cCSSCs by promoting cell adhesion, cell viability, and cell proliferation. The results revealed the upregulation of tumor protein P63 (P63) and ATP-binding cassette super-family G member 2 (Abcg2) of cLESCs as well as Keratocan (Kera), Lumican (Lum), aldehyde dehydrogenase 3 family member A1 (Aldh3a1) and Aquaporin 1 (Aqp1) of differentiated keratocytes. Moreover, immunohistochemistry illustrated the positive staining of tumor protein P63 (P63), aldehyde dehydrogenase 3 family member A1 (Aldh3a1), lumican (Lum) and collagen I (Col-I), which are considerable for native cornea. This study manifested a feasible platform to construct tissue-engineered canine cornea for functional grafts and positively contributed to the body of knowledge related to canine corneal stem cells.
Asunto(s)
Materiales Biocompatibles/química , Epitelio Corneal/patología , Regeneración , Células Madre/citología , Células del Estroma/citología , Células 3T3 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Acuaporina 1/metabolismo , Proliferación Celular , Colágeno Tipo I/metabolismo , Trasplante de Córnea , Perros , Proteínas del Ojo/metabolismo , Fibroblastos/citología , Fibroínas/química , Gelatina/química , Genes Supresores de Tumor , Inmunohistoquímica , Técnicas In Vitro , Lumican/metabolismo , Ratones , Resistencia a la Tracción , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Cholangiocarcinoma (CCA), a lethal malignancy of the biliary epithelium, is the second most common primary liver cancer. The poor prognosis of CCA is due to the high rate of tumour invasion and distant metastasis. We found that the RNA-binding protein LIN28B, a known regulator of microRNA biogenesis, stem cell maintenance, and oncogenesis, is expressed in a subpopulation of CCA patients. To further investigate the potential role of LIN28B in CCA pathogenesis, we studied the effect of LIN28B overexpression in the cholangiocyte cell line MMNK-1 and cholangiocarcinoma cell lines HuCCT-1 and KKU-214. Here, we show that enhanced LIN28B expression promoted cancer stem cell-like properties in CCA, including enhanced cell migration, epithelial-to-mesenchymal transition (EMT), increased cell proliferation and spheroid formation. Proteomic analysis revealed TGF-ß-induced protein (TGFBI) as a novel LIN28B target gene, and further analysis showed upregulation of other components of the TGF-ß signalling pathway, including TGF-ß receptor type I (TGFBRI) expression and cytokine TGFB-I, II and III secretion. Importantly, the small molecule TGF-ß inhibitor SB431542 negated the effects of LIN28B on both cell migration and clonogenic potential. Overexpression of TGFBI alone promoted cholangiocarcinoma cell migration and EMT changes, but not spheroid formation, suggesting that TGFBI partially contributes to LIN28B-mediated aggressive cell behaviour. These observations are consistent with a model in which TGF-ß and LIN28B work together to form a positive feedback loop during cholangiocarcinoma metastasis and provide a therapeutic intervention opportunity.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Proteínas de Unión al ARN , Factor de Crecimiento Transformador beta , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Transición Epitelial-Mesenquimal/genética , Retroalimentación , Humanos , Proteómica , Proteínas de Unión al ARN/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Circulating tumor cells (CTCs) have been shown as a surrogate for cancer progression and prognostication. We aimed to determine an association between CTCs and survival of hepatocellular carcinoma (HCC) patients. Peripheral blood was obtained from 73 HCC patients to enumerate for epithelial CTCs/8 mL blood. CTCs were detected by immunoaffinity-based method using epithelial cell adhesion molecule (EpCAM) and mucin1 (MUC1). The CTCs detection rates of BCLC stages A, B, and C patients were 65.4% (17/26), 77.3% (17/22), and 96% (24/25), respectively, p = 0.018. Patients with CTCs < 5 cells/8 mL had significantly longer survival than those with CTCs ≥ 5 cells/8 mL (>36 vs. 4.6 months, p < 0.001). In multivariate analysis, CTP B, BCLC B, BCLC C, AFP ≥ 400 ng/mL, and CTC ≥ 5 cells/8 mL were independently associated with survival, with adjusted HRs (95%CI) of 4.1 (2.0-8.4), 3.5 (1.1-11.4), 4.7 (1.4-15.4), 2.4 (1.1-5.0), and 2.6 (1.2-8.4); p < 0.001, 0.036, 0.011, 0.025 and 0.012, respectively. The combination of CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL provided additively increased HR to 5.3 (2.5-11.1), compared to HRs of 4.0 (2.0-8.0) and 3.5 (1.8-6.7) for CTCs ≥ 5 cells/8 mL and AFP ≥ 400 ng/mL, p < 0.001, respectively. The larger number of peripheral CTCs is correlated with higher tumor aggressive features and poorer survival of HCC patients. CTCs can potentially become novel prognostic biomarker in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Células Neoplásicas Circulantes , Humanos , Carcinoma Hepatocelular/metabolismo , Células Neoplásicas Circulantes/patología , Neoplasias Hepáticas/patología , Pronóstico , alfa-Fetoproteínas , Molécula de Adhesión Celular Epitelial , Biomarcadores de Tumor/metabolismoRESUMEN
Members of the Hes and Hey families of basic helix-loop-helix transcription factors are regarded as Notch target genes that generally inhibit neuronal differentiation of neural progenitor cells. We found that HeyL, contrary to the classic function of Hes and Hey factors, promotes neuronal differentiation of neural progenitor cells both in culture and in the embryonic brain in vivo. Furthermore, null mutation of HeyL decreased the rate of neuronal differentiation of cultured neural progenitor cells. HeyL binds to and activates the promoter of the proneural gene neurogenin2, which is inhibited by other Hes and Hey family members, and HeyL is a weak inhibitor of the Hes1 promoter. HeyL is able to bind other Hes and Hey family members, but it cannot bind the Groucho/Tle1 transcriptional corepressor, which mediates the inhibitory effects of the Hes family of factors. Furthermore, although HeyL expression is only weakly augmented by Notch signaling, we found that bone morphogenic protein signaling increases HeyL expression by neural progenitor cells. These observations suggest that HeyL promotes neuronal differentiation of neural progenitor cells by activating proneural genes and by inhibiting the actions of other Hes and Hey family members.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Antígenos/genética , Astrocitos/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Proteína Morfogenética Ósea 4/metabolismo , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Inmunoprecipitación/métodos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Proteoglicanos/genética , Transfección/métodos , Tubulina (Proteína)/metabolismoRESUMEN
Activating mutations affecting the JAK-STAT signal transduction is the genetic driver of myeloproliferative neoplasms (MPNs) which comprise polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. The JAK2p.V617F mutation can produce both erythrocytosis in PV and thrombocytosis in ET, while JAK2 exon 12 mutations cause only erythrocytosis. We hypothesized that these two mutations activated different intracellular signals. In this study, the induced pluripotent stem cells (iPSCs) were used to model JAK2-mutated MPNs. Normal iPSCs underwent lentiviral transduction to overexpress JAK2p.V617F or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs were differentiated into erythroid cells. Compared with JAK2V617F-iPSCs, JAK2exon12-iPSCs yielded more total CD71+GlycophorinA+ erythroid cells, displayed more mature morphology and expressed more adult hemoglobin after doxycycline induction. Capillary Western immunoassay revealed significantly higher phospho-STAT1 but lower phospho-STAT3 and lower Phospho-AKT in JAK2exon12-iPSCs compared with those of JAK2V617F-iPSCs in response to erythropoietin. Furthermore, interferon alpha and arsenic trioxide were tested on these modified iPSCs to explore their potentials for MPN therapy. Both agents preferentially inhibited proliferation and promoted apoptosis of the iPSCs expressing mutant JAK2 compared with those without doxycycline induction. In conclusion, the modified iPSC model can be used to investigate the mechanisms and search for new therapy of MPNs.
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Janus Quinasa 2/genética , Policitemia Vera/genética , Policitemia/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Eritropoyesis/genética , Exones , Regulación de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Policitemia/patología , Policitemia Vera/patología , Mielofibrosis Primaria , Factores de Transcripción STAT/genética , Trombocitemia Esencial/genética , Trombocitemia Esencial/patología , Trombocitosis/genética , Trombocitosis/patologíaRESUMEN
Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-ß peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.
RESUMEN
Platelet demand has increased around the world. However, the inadequacy of donors, the risk of transfusion-transmitted infections and associated reactions, and the refractory nature of platelet transfusions are among the limitations of allogeneic platelet transfusions. To alleviate these problems, we propose generating platelets in a laboratory that do not induce alloimmunity to human leukocyte antigen (HLA) class I, which is a major cause of immune reaction in platelet transfusion refractoriness. Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) of a healthy Thai woman. We then knocked out the ß2-microglobulin (ß2m) gene in the cells using paired CRISPR/Cas9 nickases and sequentially differentiated the cells into haematopoietic stem cells (HSCs), megakaryocytes (MKs) and platelets. Silencing of HLA class I expression was observed on the cell surface of ß2m-knockout iPSCs, iPSC-derived HSCs, MKs and platelets. The HLA-universal iPSC-derived platelets were shown to be activated, and they aggregated after stimulation. In addition, our in vivo platelet survival experiments demonstrated that human platelets were detectable at 2 and 24 hours after injecting the ß2m-KO MKs. In summary, we successfully generated functional iPSC-derived platelets in vitro without HLA class I expression by knocking out the ß2m gene using paired CRISPR/Cas9 nickases.
Asunto(s)
Plaquetas/citología , Células Madre Hematopoyéticas/citología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Madre Pluripotentes Inducidas/citología , Leucocitos Mononucleares/citología , Megacariocitos/citología , Animales , Plaquetas/metabolismo , Diferenciación Celular , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1-AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.
RESUMEN
Bernard-Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.
Asunto(s)
Síndrome de Bernard-Soulier/patología , Plaquetas/fisiología , Membrana Celular/ultraestructura , Células Madre Pluripotentes Inducidas/fisiología , Megacariocitos/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Síndrome de Bernard-Soulier/genética , Síndrome de Bernard-Soulier/terapia , Coagulación Sanguínea/genética , Plaquetas/ultraestructura , Diferenciación Celular , Línea Celular , Forma de la Célula/genética , Tratamiento Basado en Trasplante de Células y Tejidos , Técnicas de Reprogramación Celular , Femenino , Terapia Genética , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Lentivirus/genética , Megacariocitos/ultraestructura , Microscopía Electrónica , Complejo GPIb-IX de Glicoproteína Plaquetaria/genéticaRESUMEN
Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Miocitos Cardíacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Conejos , Fosfatasa Alcalina/fisiología , Animales , Línea Celular , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/fisiología , Proteína Homeótica Nanog/fisiología , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Factores de Transcripción SOXB1/fisiología , Antígenos Embrionarios Específico de Estadio/fisiologíaRESUMEN
Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.
Asunto(s)
Citocinas/biosíntesis , Parálisis/virología , Rabia/patología , Rabia/fisiopatología , Animales , Citocinas/sangre , Citocinas/genética , Diagnóstico por Imagen , Perros , Imagen por Resonancia Magnética , Rabia/diagnóstico , Rabia/inmunología , Virus de la Rabia/inmunología , Médula Espinal/patologíaRESUMEN
Uses of micro-/nano-sized particles to deliver biologically active entities into cells are common for medical therapeutics and prophylactics and also for cellular experiments. Enhancing cellular uptake and avoiding destruction by lysosomes are desirable for general particulate drug delivery systems. Here, we show that the relatively nontoxic, negatively charged oxidized carbon black particles (OCBs) can enhance cellular penetration of micro- and nano-particles. Experiments with retinal-grafted chitosan particles (PRPs) with hydrodynamic sizes of 1200 ± 51.5, 540 ± 29.0, and 430 ± 11.0 nm (three-sized model particles) indicate that only the sub-micron-sized particles can penetrate the first layer of multilayered liposomes. However, in the presence of OCBs, the micron-sized PRPs and the two submicron-sized PRPs can rapidly enter the interiors of all layers of the multilayered liposomes. Very low cellular uptakes of micro- and submicron-sized PRPs into keratinocytes cells are usually observed. However, in the presence of OCBs, faster and higher cellular uptakes of all of the three-sized PRPs are clearly noticed. Intracellular traffic monitoring of PRP uptake into HepG2 cells in the presence of OCBs revealed that the PRPs did not co-localize with endosomes, suggesting a nonendocytic uptake process. This demonstration of OCB's ability to enhance cellular uptake of micro- and submicron-particles should open up an easy strategy to effectively send various carriers into cells.