Puesta a punto de un protocolo de diferenciación condrogénica en células madre pluripotentes inducidas (iPSCs) generadas a partir de pacientes con artrosis de manos y un donante sano.

[Resumen] El objetivo del presente trabajo fue poner a punto un protocolo de diferenciación condrogénica en líneas de células madre pluripotentes inducidas (iPSCs) obtenidas de pacientes con artrosis de manos (OAm) y un donante sano. Las iPSCs, cultivadas en un medio en feeder, se adaptaron a un sistema libre de feeder compuesto por Laminina y Stemflex. A continuación, se evaluaron 2 protocolos: un protocolo de diferenciación condrogénica dirigida y otro con cuerpos embrionarios (EBs) espontáneos. Además, también se testó la formación de EBs mediante la técnica de hanging drop. Una vez transcurridas las 5 semanas de cultivo, se evaluaron las diferenciaciones de las líneas mediante tinciones histológicas de los esferoides y análisis moleculares de ARN. La línea TC28a2 fue utilizada como control positivo de la diferenciación condrogénica. Las líneas de iPSCs fueron adaptadas al cultivo libre de feeder tras varios intentos. Tras la adaptación, se realizaron los protocolos de diferenciación obteniéndose esferoides de ambas líneas. El análisis histológico y molecular mostró diferenciación condrogénica, aunque los resultados no son concluyentes.[Resumo] O obxectivo deste traballo foi desenrolar un protocolo de diferenciación condroxénica nas liñas de células nai pluripotentes inducidas (iPSCs) obtidas a partir de pacientes con artrose de mans (OAm) e dun doante san. As iPSCs, cultivadas sobre feeder, adaptáronse a un sistema libre de feeder composto por Laminina e Stemflex. Posteriormente, avaliáronse dous protocolos: un protocolo de diferenciación condroxénica dirixida e outro con corpos embrionarios (EBs) espontáneos. Ademáis, tamén se probou a formación de EBs usando a técnica de hanging drop. Despois das 5 semanas de cultivo, a diferenciación das liñas foi avaliada por tinción histolóxica dos esferoides e análise molecular do ARN. A liña TC28a2 utilizouse como control positivo da diferenciación condroxénica. As liñas de iPSCs adaptáronse ao sistema libre de feeder tras varios intentos. Despois da adaptación, realizáronse os protocolos de diferenciación obtendo esferoides de ambas liñas. As análises histolóxicas e moleculares mostraron diferenciación condroxénica, aínda que os resultados non son concluíntes.[Abstract] The aim of the present work was to develop a chondrogenic differentiation protocol for induced pluripotent stem cell lines (iPSCs) obtained from patients with hand osteoarthritis (OAm) and a healthy donor. The iPSCs, cultured onto feeder cells, were adapted to a feeder-free system composed of Laminin and Stemflex. Then, 2 protocols were evaluated: a protocol of directed chondrogenic differentiation and another with spontaneous embryonic bodies (EBs). In addition, the formation of EBs was also tested using the hanging drop technique. After the 5 weeks in culture, the differentiation of the lines was evaluated by histological staining of the spheroids and RNA molecular analysis. The TC28a2 line was used as a positive control for chondrogenic differentiation. The iPSCs lines were adapted to free feeder culture after several attempts. After the adaptation, the differentiation protocols were tested obtaining spheroids of both lines. Histological and molecular analysis showed chondrogenic differentiation, although the results are not conclusive.Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en Fuentes de investigación biomédica.Curso 2018/201

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes

[Abstract] This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.This research was funded by the Wellcome Trust (grant no. 206194/Z/17/Z) and by Plan Estatal I+D+i, Instituto Carlos III (ISCIII, Spain) (grants no. PI14/01031 and PI18/01417) cofunded by the Fondo Europeo de Desarrollo Regional-FEDER (The European Regional Development Fund-ERDF) “A way of Making Europe”, and by Xunta de Galicia (Consolidación Grupos Referencia Competitiva grant no. ED431C 2020-08)Reino Unido. Wellcome Trust; 206194/Z/17/ZXunta de Galicia; ED431C 2020-0

The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa