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1.
Pathology ; 55(1): 94-103, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36175183

RESUMEN

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma worldwide, accounting for about 40% of cases. The role of cytokines in the pathogenesis of lymphomas has been rarely addressed, although cytokines have a close immunological relationship with lymphocytes. We observed overexpression of interleukin (IL)-20 in reactive germinal centres (GCs) leading us to hypothesise that IL-20 may play a role in lymphomagenesis. In this study, we surveyed for IL-20 expression in various types of lymphoma and found that IL-20 was expressed most frequently in follicular lymphoma (94%), but also in Burkitt lymphoma (81%), mantle cell lymphoma (57%), nodal marginal zone lymphoma (56%), Hodgkin lymphomas (50%), small lymphocytic lymphoma (50%) and diffuse large B-cell lymphoma (DLBCL, 48%). IL-20 was not expressed in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), lymphoplasmacytic lymphoma, and plasmacytoma. T-cell lymphomas were largely negative for IL-20 expression, except for anaplastic large cell lymphoma (ALCL, 61%), which frequently expressed IL-20, especially in cutaneous ALCL, and showed an inverse association with ALK expression (p=0.024). We further tested IL-20 expression in another large cohort of DLBCL and found IL-20 expression more frequently in germinal centre B-cell (GCB) than in non-GCB subtype [16/26 (62%) versus 24/64 (38%), p=0.038]. In this cohort, IL-20 was associated with a lower rate of extranodal involvement (p=0.009), bone marrow involvement (p=0.040), and better overall survival (p=0.020). Mechanistically, IL-20 overexpression promoted G1 cell cycle arrest and subsequent apoptosis of DLBCL cells and vice versa in vitro. We conclude that IL-20 may be involved in lymphomagenesis and may be useful as a prognostic marker in patients with DLBCL. In addition, IL-20 plays an inhibitory role in DLBCL growth, probably through cell cycle regulation.


Asunto(s)
Linfoma de Células B de la Zona Marginal , Linfoma de Células B Grandes Difuso , Adulto , Humanos , Citocinas , Interleucinas , Linfoma de Células B de la Zona Marginal/patología , Linfoma de Células B Grandes Difuso/metabolismo , Pronóstico
2.
Cancer Sci ; 112(4): 1589-1602, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33525055

RESUMEN

Hodgkin lymphoma (HL) is composed of neoplastic Hodgkin and Reed-Sternberg cells in an inflammatory background. The neoplastic cells are derived from germinal center B cells that, in most cases, are infected by Epstein-Barr virus (EBV), which may play a role in tumorigenesis. Given that EBV-latent membrane protein 1 (LMP1) regulates autophagy in B cells, we explored the role of autophagy mediated by EBV or LMP1 in HL. We found that EBV-LMP1 transfection in HL cells induced a modest increase in autophagy signals, attenuated starvation-induced autophagic stress, and alleviated autophagy inhibition- or doxorubicin-induced cell death. LMP1 knockdown leads to decreased autophagy LC3 signals. A xenograft mouse model further showed that EBV infection significantly increased expression of the autophagy marker LC3 in HL cells. Clinically, LC3 was expressed in 15% (19/127) of HL samples, but was absent in all cases of nodular lymphocyte-predominant and lymphocyte-rich classic HL cases. Although expression of LC3 was not correlated with EBV status or clinical outcome, autophagic blockade effectively eradicated LMP1-positive HL xenografts with better efficacy than LMP1-negative HL xenografts. Collectively, these results suggest that EBV-LMP1 enhances autophagy and promotes the viability of HL cells. Autophagic inhibition may be a potential therapeutic strategy for treating patients with HL, especially EBV-positive cases.


Asunto(s)
Autofagia/genética , Supervivencia Celular/genética , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/patología , Regulación hacia Arriba/genética , Proteínas de la Matriz Viral/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Muerte Celular/genética , Línea Celular Tumoral , Niño , Preescolar , Doxorrubicina/uso terapéutico , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Femenino , Centro Germinal/efectos de los fármacos , Xenoinjertos , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/virología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Adulto Joven
3.
Cell Mol Life Sci ; 78(5): 2199-2212, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32910224

RESUMEN

It is critical to specify a signal that directly drives the transition that occurs between cell states. However, such inferences are often confounded by indirect intercellular communications or secondary transcriptomic changes due to primary transcription factors. Although FGF is known for its importance during mesoderm-to-endothelium differentiation, its specific role and signaling mechanisms are still unclear due to the confounding factors referenced above. Here, we attempted to minimize the secondary artifacts by manipulating FGF and its downstream mediators with a short incubation time before sampling and protein-synthesis blockage in a low-density angioblastic/endothelial differentiation system. In less than 8 h, FGF started the conversion of KDRlow/PDGFRAlow nascent mesoderm into KDRhigh/PDGFRAlow angioblasts, and the priming by FGF was necessary to endow endothelial formation 72 h later. Further, the angioblastic conversion was mediated by the FGFR1/BRAF/MEK/ERK pathway in mesodermal cells. Finally, two transcription factors, ETV2 and LMO2, were the early direct functional responders downstream of the FGF pathway, and ETV2 alone was enough to complement the absence of FGF. FGF's selective role in mediating the first-step, angioblastic conversion from mesoderm-to-endothelium thus allows for refined control over acquiring and manipulating angioblasts. The noise-minimized differentiation/analysis platform presented here is well-suited for studies on the signaling switches of other mesodermal-lineage fates as well.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Vasos Sanguíneos/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Proteínas con Dominio LIM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología
4.
Sci Rep ; 10(1): 21342, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33288848

RESUMEN

Diffuse large B-cell lymphoma (DLBCL) may present initially in bone marrow, liver and spleen without any lymphadenopathy (referred to as BLS-type DLBCL), which is aggressive and frequently associated with hemophagocytic syndrome. Its tumorigenesis and molecular mechanisms warrant clarification. By gene microarray profiling with bioinformatics analysis, we found higher expression of the stem cell markers HOXA9 and NANOG, as well as BMP8B, CCR6 and S100A8 in BLS-type than conventional DLBCL. We further validated expression of these markers in a large cohort of DLBCL including BLS-type cases and found that expression of HOXA9 and NANOG correlated with inferior outcome and poor prognostic parameters. Functional studies with gene-overexpressed and gene-silenced DLBCL cell lines showed that expression of NANOG and HOXA9 promoted cell viability and inhibited apoptosis through suppression of G2 arrest in vitro and enhanced tumor formation and hepatosplenic infiltration in a tail-vein-injected mouse model. Additionally, HOXA9-transfected tumor cells showed significantly increased soft-agar clonogenic ability and tumor sphere formation. Interestingly, B cells with higher CCR6 expression revealed a higher chemotactic migration for CCL20. Taken together, our findings support the concept that tumor or precursor cells of BLS-type DLBCL are attracted by chemotaxis and home to the bone marrow, where the microenvironment promotes the expression of stem cell characteristics and aggressiveness of tumor cells.


Asunto(s)
Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Células Madre/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Muerte Celular/genética , Muerte Celular/fisiología , Línea Celular Tumoral , Biología Computacional , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones SCID , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , ARN Mensajero/metabolismo , Células Madre/fisiología
5.
J Biomed Sci ; 27(1): 92, 2020 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-32887585

RESUMEN

BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.


Asunto(s)
Diferenciación Celular , Variaciones en el Número de Copia de ADN , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Reprogramación Celular , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Taiwán , Adulto Joven
6.
Mol Neurobiol ; 56(4): 2440-2449, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30032422

RESUMEN

Previous studies report detection of high concentrations of circulating nucleic acids (CNAs), which are likely related to cell apoptosis, in the plasma of patients with cancers, stroke, trauma, and relapsing-remitting multiple sclerosis. However, the relationship between Alzheimer's disease (AD) and CNAs is unclear. A total of 36 adult participants (9 non-demented controls and 27 patients with AD) and patients with mild AD, who met the criteria for probable AD, were enrolled in the present study, which was conducted at the Department of Neurology of National Cheng Kung University Hospital. The CNA levels were increased in the plasma of patients with AD, culture medium of amyloid-ß-treated SH-SY5Y cells, and plasma from a mouse model of AD. The CNA concentrations in the plasma were positively correlated with the cognitive scores. Further, CNAs in patients with AD contained neuronal tissue-specific methylated LHX2, at CpG sites 1 and 5. These results showed that the increased levels of plasma CNAs could be related to neuronal cell death that was induced by ß-amyloid toxicity. Thus, the results suggested that the levels of plasma CNAs and LHX2 methylation might serve as potential biomarkers for the diagnosis of AD, particularly during the early stages of the disease.


Asunto(s)
Enfermedad de Alzheimer/sangre , Biomarcadores/sangre , Ácidos Nucleicos Libres de Células/sangre , Metilación de ADN/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Cognición , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Especificidad de Órganos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
World J Gastroenterol ; 24(10): 1152-1166, 2018 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-29563759

RESUMEN

AIM: To investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC). METHODS: We performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion. RESULTS: We found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects. CONCLUSION: PGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Receptores de Progesterona/metabolismo , Carcinogénesis/patología , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/cirugía , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Hígado/patología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/cirugía , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Pronóstico , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/genética
8.
BMC Biol ; 15(1): 22, 2017 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-28327113

RESUMEN

BACKGROUND: In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable. METHODS: Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing. RESULTS: Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power. CONCLUSIONS: Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.


Asunto(s)
Endotelio/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcriptoma/genética , Tampones (Química) , Recuento de Células , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Límite de Detección , Magnesio/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nucleótidos/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Poliadenilación/efectos de los fármacos , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Reversa/efectos de los fármacos , Análisis de la Célula Individual , Transcriptoma/efectos de los fármacos
9.
Mol Neurobiol ; 54(8): 6198-6212, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27709493

RESUMEN

Recent studies reported granulocyte colony-stimulating factor (G-CSF) treatment can improve the cognitive function of Alzheimer's disease (AD) mice, and the mobilized hematopoietic stem cells (HSCs) or bone marrow mesenchymal stem cells (BM-MSCs) are proposed to be involved in this recovery effect. However, the exact role of mobilized HSC/BM-MSC in G-CSF-based therapeutic effects is still unknown. Here, we report that C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor 1 (SDF-1) chemotaxis was a key mediator in G-CSF-based therapeutic effects, which was involved in the recruitment of repair-competent cells. Furthermore, we found both mobilized HSCs and BM-MSCs were able to infiltrate into the brain, but only BM-MSCs replenished the neural lineage cells and contributed to neurogenesis in the brains of AD mice. Together, our data show that mobilized BM-MSCs are involved in the replenishment of neural lineages following G-CSF treatment via CXCR4/SDF-1 chemotaxis and further support the potential use of BM-MSCs for further autogenically therapeutic applications.


Asunto(s)
Linaje de la Célula/fisiología , Quimiocina CXCL12/metabolismo , Quimiotaxis/fisiología , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Mesenquimatosas/metabolismo , Receptores CXCR4/metabolismo , Enfermedad de Alzheimer , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Linaje de la Célula/efectos de los fármacos , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo
10.
Sci Rep ; 6: 27358, 2016 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-27264956

RESUMEN

Stem cell-based therapy is a potential treatment for neurodegenerative diseases, but its application to Alzheimer's disease (AD) remains limited. Brain-derived neurotrophic factor (BDNF) is critical in the pathogenesis and treatment of AD. Here, we present a novel therapeutic approach for AD treatment using BDNF-overexpressing neural stem cells (BDNF-NSCs). In vitro, BDNF overexpression was neuroprotective to beta-amyloid-treated NSCs. In vivo, engrafted BDNF-NSCs-derived neurons not only displayed the Ca(2+)-response fluctuations, exhibited electrophysiological properties of mature neurons and integrated into local brain circuits, but recovered the cognitive deficits. Furthermore, BDNF overexpression improved the engrafted cells' viability, neuronal fate, neurite complexity, maturation of electrical property and the synaptic density. In contrast, knockdown of the BDNF in BDNF-NSCs diminished stem cell-based therapeutic efficacy. Together, our findings indicate BDNF overexpression improves the therapeutic potential of engrafted NSCs for AD via neurogenic effects and neuronal replacement, and further support the feasibility of NSC-based ex vivo gene therapy for AD.


Asunto(s)
Enfermedad de Alzheimer/terapia , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trasplante de Células/métodos , Expresión Génica , Células-Madre Neurales/metabolismo , Animales , Diferenciación Celular , Fenómenos Fisiológicos Celulares , Células Cultivadas , Modelos Animales de Enfermedad , Ratones , Resultado del Tratamiento
11.
Cell Rep ; 12(12): 1960-7, 2015 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-26365191

RESUMEN

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.


Asunto(s)
MicroARNs/genética , Miocitos Cardíacos/metabolismo , Receptor ErbB-4/genética , Animales , Biomarcadores/metabolismo , Calcio/metabolismo , Comunicación Celular , Diferenciación Celular , Técnicas de Cocultivo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Potenciales de la Membrana , Ratones , MicroARNs/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Miocitos Cardíacos/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor ErbB-4/antagonistas & inhibidores , Receptor ErbB-4/metabolismo , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Transducción de Señal
12.
Sci Rep ; 5: 9718, 2015 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-25864432

RESUMEN

It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFß and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Madre Embrionarias/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glucógeno Sintasa Quinasas/antagonistas & inhibidores , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Factor de Crecimiento Transformador beta/agonistas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Wnt/agonistas
13.
Int J Mol Sci ; 14(10): 20079-111, 2013 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-24113586

RESUMEN

Frontotemperal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are two common neurodegenerative diseases. TDP-43 is considered to be a major disease protein in FTLD/ALS, but it's exact role in the pathogenesis and the effective treatments remains unknown. To address this question and to determine a potential treatment for FTLD/ALS, the disease animal models of TDP-43 proteinopathy have been established. TDP-43 proteinopathy is the histologic feature of FTLD/ALS and is associated with disease progression. Studies on the disease animal models with TDP-43 proteinopathy and their pre-clinical applications are reviewed and summarized. Through these disease animal models, parts of TDP-43 functions in physiological and pathological conditions will be better understood and possible treatments for FTLD/ALS with TDP-43 proteinopathy may be identified for possible clinical applications in the future.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteinopatías TDP-43/metabolismo , Proteinopatías TDP-43/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Humanos
14.
Mediators Inflamm ; 2013: 512978, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23533308

RESUMEN

Several studies have provided evidence with regard to the neuroprotection benefits of hyperbaric oxygen (HBO) therapy in cases of stroke, and HBO also promotes bone marrow stem cells (BMSCs) proliferation and mobilization. This study investigates the influence of HBO therapy on the migration of BMSCs, neurogenesis, gliosis, and inflammation after stroke. Rats that sustained transient middle cerebral artery occlusion (MCAO) were treated with HBO three weeks or two days. The results were examined using a behavior test (modified neurological severity score, mNSS) and immunostaining to evaluate the effects of HBO therapy on migration of BMSCs, neurogenesis, and gliosis, and expression of neurotrophic factors was also evaluated. There was a lower mNSS score in the three-week HBO group when compared with the two-day HBO group. Mobilization of BMSCs to an ischemic area was more improved in long course HBO treatments, suggesting the duration of therapy is crucial for promoting the homing of BMSCs to ischemic brain by HBO therapies. HBO also can stimulate expression of trophic factors and improve neurogenesis and gliosis. These effects may help in neuronal repair after ischemic stroke, and increasing the course of HBO therapy might enhance therapeutic effects on ischemic stroke.


Asunto(s)
Isquemia Encefálica/terapia , Oxigenoterapia Hiperbárica , Inflamación/metabolismo , Accidente Cerebrovascular/terapia , Animales , Inmunohistoquímica , Masculino , Células Madre Mesenquimatosas/citología , Microscopía Confocal , Neurogénesis/fisiología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
15.
Brain Res ; 1462: 26-39, 2012 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-22608070

RESUMEN

Recently, missense mutations in the gene TARDBP encoding TDP-43 have been linked to familial ALS. The discovery of genes encoding these RNA binding proteins, such as TDP-43 and FUS/TLS, raised the notion that altered RNA metabolism is a major factor underlying the pathogenesis of ALS. To begin to unravel how mutations in TDP-43 cause dysfunction and death of motor neurons, investigators have employed both gain- and loss-of-function studies in rodent model systems. Here, we will summarize major findings from the initial sets of TDP-43 transgenic and knockout rodent models, identify their limitations, and point to future directions toward clarification of disease mechanism(s) and testing of therapeutic strategies that ultimately may lead to novel therapy for this devastating disease. This article is part of a Special Issue entitled RNA-Binding Proteins.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/patología , Adiposidad/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , ADN/genética , Modelos Animales de Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/patología , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Neuronas Motoras/patología
16.
Neurobiol Aging ; 33(2): 277-85, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20382452

RESUMEN

γ-Secretase, a multisubunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective one, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on nine mutations distributed in six different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1aS prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/química , Péptido Hidrolasas/química , Presenilinas/metabolismo , Transducción de Señal/fisiología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Sitios de Unión , Línea Celular , Membrana Celular/química , Endopeptidasas , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Péptido Hidrolasas/metabolismo , Presenilinas/química , Presenilinas/genética , Unión Proteica , Estructura Terciaria de Proteína
17.
Development ; 138(13): 2833-43, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21613326

RESUMEN

Current approaches to differentiate embryonic stem (ES) cells to hematopoietic precursors in vitro use either feeder cell, serum, conditioned culture medium or embryoid body, methods that cannot avoid undefined culture conditions, precluding analysis of the fate of individual cells. Here, we have developed a defined, serum-free and low cell-density differentiation program to generate endothelial and hematopoietic cells within 6 days from murine ES cells. Our novel approach identifies a set of factors that are necessary and sufficient to differentiate ES cells into definitive hematopoietic precursors, as documented by the time-lapse video microscopy of the stepwise differentiation processes from single progenitors. Moreover, this defined milieu revealed the essential role of bone morphogenetic protein 4 (BMP4) in determining the hematopoietic/endothelial fate and demonstrated that the hemogenic fate in mesoderm is determined as early as day 4 of our differentiation protocol. Our ability to directly convert ES cells to endothelial and hematopoietic precursors should have important utilities for studies of hematopoietic development and personalized medicine in the future.


Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Hemangioblastos/citología , Activinas/farmacología , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Medio de Cultivo Libre de Suero , Factores de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/farmacología
18.
Proc Natl Acad Sci U S A ; 107(37): 16325-30, 2010 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-20736350

RESUMEN

TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein involved in RNA transcription and splicing, has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here we use both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions composed of TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals, and immature neuromuscular junctions. Whereas an elevated level of TDP-43 disrupts the normal nuclear distribution of survival motor neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that a decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm.


Asunto(s)
Cuerpos Enrollados/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Animales , Supervivencia Celular , Proteínas de Unión al ADN/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Neuronas Motoras/citología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo
19.
Proc Natl Acad Sci U S A ; 107(37): 16320-4, 2010 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-20660762

RESUMEN

Tat activating regulatory DNA-binding protein (Tardbp or TDP-43), a highly conserved metazoan DNA/RNA binding protein thought to be involved in RNA transcription and splicing, has been linked to the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration and is essential for early embryonic development. However, neither the physiological role of TDP-43 in the adult nor its downstream targets are well defined. To address these questions, we developed conditional Tardbp-KO mice and embryonic stem (ES) cell models. Here, we show that postnatal deletion of Tardbp in mice caused dramatic loss of body fat followed by rapid death. Moreover, conditional Tardbp-KO ES cells failed to proliferate. Importantly, high-throughput DNA sequencing analysis on the transcriptome of ES cells lacking Tardbp revealed a set of downstream targets of TDP-43. We show that Tbc1d1, a gene known to mediate leanness and linked to obesity, is down-regulated in the absence of TDP-43. Collectively, our results establish that TDP-43 is critical for fat metabolism and ES cell survival.


Asunto(s)
Tejido Adiposo/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Eliminación de Gen , Proteínas Nucleares/metabolismo , Obesidad/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas Activadoras de GTPasa , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Obesidad/genética , Células Madre/citología , Células Madre/metabolismo
20.
J Formos Med Assoc ; 103(12): 939-42, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15624045

RESUMEN

Disseminated tuberculosis usually occurs in immunocompromised hosts. Involvement of bone marrow, liver, and spleen is infrequent. A previously healthy 15-year-old boy presented with body weight loss, prolonged fever, neck lymphadenopathy, pancytopenia, and hepatosplenic microabscesses within the recent month and was transferred to our hospital. Bone marrow studies showed hypocellular marrow. Based on his clinical manifestations, hemophagocytic syndrome was initially suspected. Pancytopenia resolved after administration of intravenous immunoglobulin but caseous necrosis and/or positive acid-fast stain were subsequently demonstrated in the lymph node biopsy and sputum. Cultures from these 2 specimens grew Mycobacterium tuberculosis. Fever continued in a low-grade pattern even under antituberculous therapy with rifampin, isoniazid, pyrazinamide and ethambutol. Five months after admission, fever subsided after splenectomy and liver wedge resection. Microscopic examinations of both the liver and spleen showed mycobacteria-related granulomatous inflammation and caseating necrosis. This report suggests that tuberculosis infection should be considered in the differential diagnosis in patients with prolonged fever, pancytopenia and hepatosplenic abscesses.


Asunto(s)
Absceso/microbiología , Absceso Hepático/microbiología , Pancitopenia/microbiología , Enfermedades del Bazo/microbiología , Tuberculosis/diagnóstico , Adolescente , Humanos , Masculino
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