Combinación de terapias celulares para la reparación de lesiones de cartílago

Abstract

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.