[en] Human articular cartilage is affected by several rheumatic diseases such as osteoarthritis (OA) and has a very limited regeneration capacity. Cell therapies with mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represent a promising field in cartilage repair medicine. The following thesis is a compendium of researches designed to better understand the processes that lead to the degradation of this tissue and, especially, to develop strategies to get a better repair in the future. In the first study, we examined the effect of the over-expression of LMNA, or its mutant form progerin (PG), on the mesoderm differentiation potential of mesenchymal stem cells (MSCs) from human umbilical cord (UC) stroma using a recently described differentiation model employing spheroid formation. Accumulation of lamin A (LMNA) was previously associated with the osteoarthritis (OA) chondrocyte phenotype. Mutations of this protein are linked to laminopathies and specifically to Hutchinson–Gilford Progeria Syndrome (HGPS), an accelerated aging disease. Some authors have proposed that a deregulation of LMNA affects the differentiation potential of stem cells. The chondrogenic potential is defective in PG-MSCs, although both PG and LMNA transduced MSCs, have an increase in hypertrophy markers during chondrogenic differentiation. Furthermore, both PG and LMNA-MSCs showed a decrease in manganese superoxide dismutase (MnSODM), an increase of mitochondrial MnSODM-dependent reactive oxygen species (ROS) and alterations in their migration capacity. Finally, defects in chondrogenesis are partially reversed by periodic incubation with ROS-scavenger agent that mimics MnSODM effect. Our results indicate that over-expression of LMNA or PG by lentiviral gene delivery leads to defects in chondrogenic differentiation potential partially due to an imbalance in oxidative stress. The second study, aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 30 , 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qPCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3b gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. In the third study, genetically modified cells capable of resisting the inflammation process were generated by deletion of the interleukin 1 receptor. Pro-inflammatory cytokines such as IL-1β are found in elevated levels in diseased or injured tissues and promote rapid Tissue degradation while preventing the differentiation of stem cells. Because of this cells immune to their signals could represent a useful strategy in cell therapy. Human articular chondrocytes (CAs) and immortalized CMM (3A6 line) were modified by the CRISPR-Cas system in combination with a vector designed to silence the IL-1β (IL1R1) receptor. Cells were stimulated with rIL-1β and rTNFα in monolayer to evaluate the inflammatory response in IL1R1-KO cells. Chondrogenesis was performed on alginate disks and with chondrogenic differentiation medium with or without rIL-1β. qPCR and western blot analyzes were performed to investigate the expression of the mRNAs and proteins of IL1R1-resistant cells, in addition to a biological characterization and by flow cytometry in the case of CMM. Gene expression of IL1R1 was significantly reduced in the modified cells. The addition of rIL-1β did not increase the expression of IL-1B, IL-6 and IL-8 in IL1R1-KO cells whereas it did significantly in the unedited control cells. Chondrogenic differentiation studies indicated that IL1R1-KO cells in the presence of rIL-1β reach a level of redifferentiation / differentiation equivalent similar to controls in the absence of rIL-1β, and exhibited a significant increase in expression of SOX9, ACAN and COL2A1 genes. In the presence of rIL-1β it was observed that in the control cells the expression of the IL-1β, IL6 and IL8 genes increased, whereas the transcription and synthesis of the SOX9, ACAN and COL2A1 genes was blocked. In contrast, IL-1B, IL-6 and IL8 did not increase in IL1R1-KO cells, and the inhibitory effect of rIL-1β on the SOX9, ACAN and COL2A1 genes was completely suppressed. In the last study , we have injected via intravenous and directly into the monkey joint a CD105+ enriched population of mesenchymal stem cells (CD105+-MSCs) from human synovial membrane, previously characterized by flow cytometry. CD105+-MSCs were labelled with oxacarbocyanine (DiO) when they were injected via intravenous (IV) and with octadecyl (C18) indocarbocyanine (DiI) when they were intra-articular (IA) injected directly into the knee, to follow their evolutions and location through the animals. The animal models used were adult Macaca Fascicularis which had been injured into the left knee to create an Osteoarthritis (OA) animal model and the right knee was used as control. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were sacrificed one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. Some labelled cells were found in the spleen, lung, liver and ganglion and teratomes were not found in any organs from any animals. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. This study was validated injecting gene modified MSCs expressing GFP in OA monkeys and the results were similar. We concluded that a characterized MSCs subset could be a safer alternative for cell therapy in clearly localized pathologies as osteoarthritis in the knee.

[gl] A cartilaxe articular humana vese afectada por varias enfermidades reumáticas como a osteoartrosis (OA) e ten unha capacidade de rexeneración moi limitada. As terapias celulares con células estaminais mesenquimais (CSMs) e condrócitos articulares (CAs) representan un campo esperanzador na medicina reparadora da cartilaxe. A seguinte tese é un compendio de investigacións deseñadas para entender mellor os procesos que levan á degradación deste tecido e, sobre todo, para desenvolver diversas estratexias para unha mellor e máis eficaz reparación do mesmo no futuro. No primeiro estudo, foi examinado o efecto sobre-expresión de lamina A (LMNA) ou a súa variante mutante proxerina (PG) sobre o potencial de diferenciación das CSMs procedentes de cordón umbilical humano (CU). A acumulación de lámina A (LMNA) foi previamente asociada ó fenotipo de condrócitos artrósicos (OA). As mutacións nesta proteína están ligadas a laminopatias e, especialmente, ó síndrome de Hutchinson-Gilford (HGPS), unha enfermidade de envellecemento acelerado. Algúns autores suxeriron que a desregulación da LMNA afecta o potencial de diferenciación das células estaminais. O noso conxunto de resultados mostra que o potencial condroxénico é defectuoso en células que sobre-expresan PG (PG-CSMs), e que as transduzidas con lámina A (LMNA-CSMs) presentan un aumento nos marcadores de hipertrofia durante condroxénese. Ambas liñas mostraron unha diminución de manganeso superóxido-dismutasa (MnSODM), e cambios na súa capacidade migratoria. Por último, os defectos na condroxénese foron parcialmente revertidos coa incubación periódica cun axente de eliminación de ROS. Os nosos resultados indican que a sobre-expresión de LMNA e PG produce defectos no potencial de diferenciación condrogénica, en parte, debido a un desequilibrio no estrés oxidativo. No segundo estudo, investigouse unha posible mellora nos medios condroxénicas actuales para CSMs-CU. O obxectivo era determinar se as hormonas prolactina (PRL) e 3,3'-5-triiodo-L-tironina (T3), a hormona activa da tiroide, modula a condroxénese no noso modelo in vitro, e se a vía de sinalización Wnt toma parte na referida modulación. As CSMs-CU sometéronse a condroxénese usando un sistema de esferóides. A adición de T3 no medio condroxénico aumentou a expresión de xenes ligados á condroxénese como Col2, integrinas alfa-10 e beta-1 (ITGα10, ITG β 1) e SOX9, segundo a análise por qPCR. Os niveis de COL2, e ACAN analizados por inmuno-histoquímica e tintura con safranina O aumentaron tras 14 días de condroxénese en esferoides con T3, en comparación cos esferóides sen T3 ou con PRL. A expresión de β -catenina, a GSK-3β Frizzled e foron significativamente maiores nos esferóides cultivados con T3. Tales melloras inhibíase cando as células foron tratadas con T3 máis ML151, un inhibidor do receptor esteroideo de T3. Este traballo demostra, por primeira vez que T3 promove a diferenciación condrogénica no noso modelo in vitro, e esta diferenciación é mediada polo receptor de esteroides coactivator (SRC2). No terceiro estudo, xeramos células modificadas xeneticamente, capaces de resistir o proceso de inflamación por supresión do receptor da interleucina 1. As citocinas pro-inflamatorias, como IL-1 β son elevados en tecidos enfermos ou feridos e promoven a rápida degradación do tecido, evitando ademáis a diferenciación de células estaminais. Debido a esto as células inmunes aos seus sinais poden representar unha estratexia útil en terapia celular. Condrócitos articulares humanos (CAs) e células estaminais mesenquimais foron xenéticamente editadas mediante o sistema CRISPR-Cas en combinación con un vector deseñado para silenciar o receptor de IL-1 β (IL-1R1). As células foron estimuladas con IL-1β e TNFα en monocapa para avaliar a resposta inflamatoria nas células IL-1R1-KO. A condroxénese realizouse en discos de alxinato con medio condroxénico sen e con IL-1β. Leváronse a cabo análises de qPCRs e western- blot para investigar a expresión dos ARNm e proteínas nas células resistentes a IL1R1. A expresión do xen IL1R1 foi significativamente reducida nas células modificadas. A adición de IL-1β non aumentou a expresión de IL-1B, IL-6 e IL-8 nas células IL-1R1-KO mentres que so o fixo nas células control non editadas. Os estudos de diferenciación condroénica indican que as células de IL1R1-KO en presenza de IL-1β atinxen un nivel de diferenciación/rediferenciación equivalente ou similar ós cos controis en ausencia de IL-1β, e mostraron un aumento significativo na expresión de xenes SOX9 , ACAN e COL2A1. En presenza de IL-1β, as células control aumentaron á expresión dos xenes IL-6 e IL-8, mentres a transcrición e síntese de SOX9, ACAN e COL2A1 foi bloqueada. En contraste, a IL-1B, IL-6 e IL-8, non aumentou nas células IL-1R1-KO, e o efecto inhibidor de IL-1β en SOX9, ACAN e COL2A1 foi suprimido por completo. O cuarto estudo é un modelo animal en primates. Estudouse o proceso de migración espécifica de CSMs de membrana sinovial dende o torrente sanguíneo ás articulacións danadas. Inxectamos intravenosamente e directamente na articulación unha poboación de CSMs positivas para o marcador de superfície CD105 (CD105+-CSM). As células marcáronse con oxacarbocyanine (DIO) cando eran inxectadas por vía intravenosa (IV) ou octadecilo (C18) indocarbocianina (DII), cando a inxección era intra-articular (IA) directamente ao xeonllo, para seguir os seus movementos e situación través dos animais. Os animais utilizados foron Macaca fascicularis adultos ós que provocóuselles unha lesión no xeonllo esquerdo para crear un modelo animal osteoartrósico (OA). O xeonllo dereito foi usado como control. As células CD105 + marcadas inxectaronse dúas veces cun intervalo dunha semana. Os animais foron sacrificados un mes despois do tratamento. O análise inmunohistoqumico de diferentes órganos: bazo, corazón, graxa, fígado, intestino, páncreas, pulmón, músculo esquelético e ril revelaron que as CD105 +-CMM migrado cara á articulación danada. Algunhas células marcadas foron atopados nos nódulos do bazo, pulmón, fígado e nódulos linfáticos. Non se atoparon teratomas. Apareceu un aumento significativo de CSM nativas no xeonllo lesionado frente ó sano. As células CD105+-CMM foron negativas para CD68 e a zona onde se atoparon mostrou un aumento de SDF-1 en comparación co xeonllo saudable. Este estudo foi validado por inxección de células macadas con GFP e os resultados foron semellantes. Concluímos que a poboación CD105+-CSM foi reclutada polo xeonllo danado e que pode ser unha alternativa segura para a terapia celular en patoloxías claramente situadas coma a OA de xeonllo.