Terapia celular de la diabetes mellitus : optimización de los procedimientos de diferenciación e inducción de tolerancia

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Fundamentos y Aplicaciones de las Células TroncalesClave Programa: DBICódigo Línea: 17La Diabetes Mellitus es una enfermedad metabólica crónica caracterizada por la presencia de elevados niveles de glucosa en sangre, como consecuencia de un fallo en la producción de insulina por el páncreas y/o por la ineficacia de su uso por el organismo. El trasplante de páncreas, al igual que sucede en cualquier trasplante de órganos, está sometido a la escasez de donantes, y debido a la alta prevalencia de la diabetes a nivel mundial y la estimación de su incremento en los próximos años, este problema se ve agravado. Por ello, científicos de todo el mundo tratan de encontrar fuentes alternativas para la terapia de la diabetes, que puede ser enfocada desde dos perspectivas distintas. Por un lado, la sustitución de las células beta pancreáticas productora de insulina, ámbito en el cual las células madre (CM) han ganado terreno en las últimas décadas, gracias a su capacidad de proliferación y diferenciación hacia los distintos linajes. Y, por otro lado, la detección de la destrucción de las células beta mediante el control del sistema inmune. En este sentido, el papel de las células T reguladoras (Treg) parecen ser fundamental debido a su capacidad de mediar el balance entre la tolerancia y la autoinmunidad. Este trabajo se ha centrado en el estudio de la regulación del factor de transcripción Pdx1 en células madre embrionarias de ratón (CMEr), debido a su implicación en la embriogénesis y función pancreática. El estudio epigenético del promotor de Pdx1, así como el estudio de la regulación por distintos factores de transcripción, nos ha permitido desarrollar un protocolo de diferenciación de CMEr hacia células productoras de insulina. Este protocolo esta basado en la adición de pequeñas moléculas, y consigue la obtencion de células productoras de insulina maduras in vitro. Asimismo, este protocolo parece ser reproducible en células madre humanas, por lo que puede ser una alternativa o proporcionar complementariedad a los protocolos de diferenciación existentes, reduciendo de forma significativa su costo y complejidad. En este trabajo nos hemos centrado también en el estudio y caracterización de células Treg procedentes de sangre de cordón umbilical y sangre periférica de adulto, además de comparar dos técnicas de selección distintas. Este estudio ha revelado que existen diferencias significativas en función de la procedencia y técnica de aislamiento empleada, por lo que este análisis proporciona datos interesantes a tener en cuenta para el uso de las células Treg en terapia celular.Universidad Pablo de Olavide. Escuela de Doctorad

The Role of Nitric Oxide in Stem Cell Biology

Nitric oxide (NO) is a gaseous biomolecule endogenously synthesized with an essential role in embryonic development and several physiological functions, such as regulating mitochondrial respiration and modulation of the immune response. The dual role of NO in embryonic stem cells (ESCs) has been previously reported, preserving pluripotency and cell survival or inducing differentiation with a dose-dependent pattern. In this line, high doses of NO have been used in vitro cultures to induce focused differentiation toward different cell lineages being a key molecule in the regenerative medicine field. Moreover, optimal conditions to promote pluripotency in vitro are essential for their use in advanced therapies. In this sense, the molecular mechanisms underlying stemness regulation by NO have been studied intensively over the current years. Recently, we have reported the role of low NO as a hypoxia-like inducer in pluripotent stem cells (PSCs), which supports using this molecule to maintain pluripotency under normoxic conditions. In this review, we stress the role of NO levels on stem cells (SCs) fate as a new approach for potential cell therapy strategies. Furthermore, we highlight the recent uses of NO in regenerative medicine due to their properties regulating SCs biology

Evaluation of NAB2-STAT6 Fusion Variants and Other Molecular Alterations as Prognostic Biomarkers in a Case Series of 83 Solitary Fibrous Tumors

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location

Breakthrough Technologies Reshape the Ewing Sarcoma Molecular Landscape

Ewing sarcoma is a highly aggressive round cell mesenchymal neoplasm, most often occurring in children and young adults. At the molecular level, it is characterized by the presence of recurrent chromosomal translocations. In the last years, next-generation technologies have contributed to a more accurate diagnosis and a refined classification. Moreover, the application of these novel technologies has highlighted the relevance of intertumoral and intratumoral molecular heterogeneity and secondary genetic alterations. Furthermore, they have shown evidence that genomic features can change as the tumor disseminates and are influenced by treatment as well. Similarly, next-generation technologies applied to liquid biopsies will significantly impact patient management by allowing the early detection of relapse and monitoring response to treatment. Finally, the use of these novel technologies has provided data of great value in order to discover new druggable pathways. Thus, this review provides concise updates on the latest progress of these breakthrough technologies, underscoring their importance in the generation of key knowledge, prognosis, and potential treatment of Ewing SarcomaJunta de Andalucía, Consejería de Salud PI-0036-2017 y PI-0040-2017Fundación María García EstradaUniversidad de SevillaJuan de la Cierva Incorporación IJC-2018-036767-ILaboratorio de EdA compatible por el proyecto AECC GCB13-1578, ISCIIIFEDER PI14 / 01466, PI17 / 00464, CIBERONC CB16 / 12/00361Fundación CRIS Contra el Cánce

A Novel NFIX-STAT6 Gene Fusion in Solitary Fibrous Tumor: A Case Report

Solitary fibrous tumor is a rare subtype of soft-tissue sarcoma with a wide spectrum of histopathological features and clinical behaviors, ranging from mildly to highly aggressive tumors. The defining genetic driver alteration is the gene fusion NAB2–STAT6, resulting from a paracentric inversion within chromosome 12q, and involving several different exons in each gene. STAT6 (signal transducer and activator of transcription 6) nuclear immunostaining and/or the identification of NAB2–STAT6 gene fusion is required for the diagnostic confirmation of solitary fibrous tumor. In the present study, a new gene fusion consisting of Nuclear Factor I X (NFIX), mapping to 19p13.2 and STAT6, mapping to 12q13.3 was identified by targeted RNA-Seq in a 74-year-old female patient diagnosed with a deep-seated solitary fibrous tumor in the pelvis. Histopathologically, the neoplasm did not display nuclear pleomorphism or tumor necrosis and had a low proliferative index. A total of 378 unique reads spanning the NFIXexon8–STAT6exon2 breakpoint with 55 different start sites were detected in the bioinformatic analysis, which represented 59.5% of the reads intersecting the genomic location on either side of the breakpoint. Targeted RNA-Seq results were validated by RT-PCR/ Sanger sequencing. The identification of a new gene fusion partner for STAT6 in solitary fibrous tumor opens intriguing new hypotheses to refine the role of STAT6 in the sarcomatogenesis of this entity

A Novel NFIX-STAT6 Gene Fusion in Solitary Fibrous Tumor: A Case Report

Solitary fibrous tumor is a rare subtype of soft-tissue sarcoma with a wide spectrum of histopathological features and clinical behaviors, ranging from mildly to highly aggressive tumors. The defining genetic driver alteration is the gene fusion NAB2–STAT6, resulting from a paracentric inversion within chromosome 12q, and involving several different exons in each gene. STAT6 (signal transducer and activator of transcription 6) nuclear immunostaining and/or the identification of NAB2–STAT6 gene fusion is required for the diagnostic confirmation of solitary fibrous tumor. In the present study, a new gene fusion consisting of Nuclear Factor I X (NFIX), mapping to 19p13.2 and STAT6, mapping to 12q13.3 was identified by targeted RNA-Seq in a 74-year-old female patient diagnosed with a deep-seated solitary fibrous tumor in the pelvis. Histopathologically, the neoplasm did not display nuclear pleomorphism or tumor necrosis and had a low proliferative index. A total of 378 unique reads spanning the NFIXexon8–STAT6exon2 breakpoint with 55 different start sites were detected in the bioinformatic analysis, which represented 59.5% of the reads intersecting the genomic location on either side of the breakpoint. Targeted RNA-Seq results were validated by RT-PCR/ Sanger sequencing. The identification of a new gene fusion partner for STAT6 in solitary fibrous tumor opens intriguing new hypotheses to refine the role of STAT6 in the sarcomatogenesis of this entity

Nitric oxide prevents mouse embryonic stem cell differentiation through regulation of gene expression, cell signaling, and control of cell proliferation

Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation.Ministerio de Economía y Competitividad, Secretaria de Estado de Investigacion Desarrollo e Innovacion co-funded by Fondos FEDER; Grant number: SAF2005-08014, SAF2006-06673, SAF2007/60105, CYT-836, IPT-2011-1615-900000; Grant sponsor: Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-funded by Fondos FEDER; Grant number: RED-TERCEL RD06/0010/0025, FIS-052106 and CIBERDEM initiative; Grant sponsor: Junta de Andalucía, Consejería de Economía, Innovacion, Ciencia y Empleo-CEICE; Grant number: PAI/CTS576, PI-0022/2008, Proyecto Motriz/CTS-7127/2011; Grant sponsor: Junta de Andalucía, Consejería de Salud; Grant number: PI-0105/2010, PI-0095/2007; Grant sponsor: Junta de Andalucía, Consejería de Salud, Servicio Andaluz de Salud; Grant number: SAS 11245.Peer Reviewe

Regulación de la Expresión del Factor de Transcripción Pdx1 por Altas Concentraciones de Óxido Nítrico en Células Madre Embrionarias de Ratón

Estudios previos en nuestro laboratorio muestran que el óxido nítrico a altas concentraciones promueve la diferenciación de células madre embrionarias de ratón (mESC), e induce la expresión de genes de endodermo, entre ellos el factor de transcripción Pdx1, “Pancreas/duodenum homeobox protein 1”. Pdx1, tiene un rol importante en el desarrollo embrionario del páncreas y en la células beta madura. Con el fin de conocer los mecanismos por los que el NO incrementa la expresión de Pdx1, hemos estudiado la región promotora de este gen, encontrando que la expresión de Pdx1 se asocia con la liberación del factor de transcripción Egr1. Además, análisis epigenéticos del promotor muestran que la expresión de Pdx1 va asociada a un cambio en el patrón de metilación de su promotor y a la ocupación de marcas de histonas activadoras (H3Ac y H3K4me3) y a la desocupación de modificadores represores de histonas (HDAC) en el promotor de Pdx1

Endoglin and MMP14 contribute to Ewing sarcoma spreading by modulation of cell-matrix interactions

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell-matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.E.A.’s laboratory is supported by ISCIII-FEDER (PI20/00003), CIBERONC (CB16/12/00361), PAIDI-Junta de Andalucía (P18-RT-735), Fundación CRIS Contra el Cáncer, Asociación Candela Riera and Asociación Pablo Ugarte. A.T.A. is supported by Juan de la Cierva Incorporación fellowship (IJC-2018-036767-I); P.P.-C. is sponsored by the Fundación María García Estrada. J.O.-P is supported by Ph.D. Grant Plan Propio from the University of Seville. J.D.-M is supported by CIBERONC (CB16/12/00361). C.S.-A. is supported by the European Social Fund and the Junta de Andalucía (Talento Doctores 2020, DOC_01473). This work was supported by grants from the Consejería de Salud (Junta de Andalucía, grants No PI-0036-2017, PI-0040-2017, and PI-0061-2020) awarded to J.D.-M, A.T.A. and C. S.-A., respectively. This work was also supported by the GEIS-Fundación Mari Paz Jiménez Casado (IV beca trienal) granted to J.D.-M, the 13ª GEIS-Beca Buesa granted to A.T.A. and CRIS (Cancer Research Innovation Spain) granted to J.D.-M and E.A. The laboratory of T.G.P.G. is supported by the Barbara and Wilfried Mohr Foundation. The lab of J.A. is supported by the Instituto de Salud Carlos III (ISCIII), grant number PI20CIII/00020; Asociación Pablo Ugarte, grant numbers TRPV205/18, TPI-M 1149/13; Asociación Candela Riera; Asociación Todos Somos Iván & Fundación Sonrisa de Alex, grant reference: TVP333-19.S

Characterizing the Invasive Tumor Front of Aggressive Uterine Adenocarcinoma and Leiomyosarcoma

The invasive tumor front (the tumor-host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors