RESUMEN
Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy. However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in which deterioration in cellular viability and aging is observed with time.Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity, proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT) expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here we investigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds (AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerative diseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERT levels in a time and dose dependent manner. Prolonged treatments with the compounds increased the average telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations. AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from the toxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated by telomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stem cells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSC as examined by their ability to differentiate into various lineages in the presence of the compounds. These results suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage in regenerative medicine when hMSC therapy is applied.
Asunto(s)
Citoprotección/efectos de los fármacos , Activadores de Enzimas/farmacología , Salud , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/enzimología , Estrés Oxidativo/efectos de los fármacos , Telomerasa/metabolismo , Adulto , Anciano , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Aberraciones Cromosómicas , Daño del ADN , Activadores de Enzimas/química , Femenino , Humanos , Peróxido de Hidrógeno/toxicidad , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Persona de Mediana Edad , Fenoles/química , Fenoles/farmacología , Sustancias Protectoras/farmacología , Telomerasa/genética , Homeostasis del Telómero/efectos de los fármacos , Compuestos de Tritilo/química , Compuestos de Tritilo/farmacologíaRESUMEN
BACKGROUND: Human mesenchymal stem cells (hMSCs) are pluripotent cells that can differentiate to various mesenchymal cell types. Recently, a method to isolate hMSCs from bone marrow and expand them in culture was described. Here we report on the use of hMSCs as a platform for gene therapy aimed at bone lesions. METHODS: Bone marrow derived hMSCs were expanded in culture and infected with recombinant adenoviral vector encoding the osteogenic factor, human BMP-2. The osteogenic potential of genetically engineered hMSCs was assessed in vitro and in vivo. RESULTS: Genetically engineered hMSCs displayed enhanced proliferation and osteogenic differentiation in culture. In vivo, transplanted genetically engineered hMSCs were able to engraft and form bone and cartilage in ectopic sites, and regenerate bone defects (non-union fractures) in mice radius bone. Importantly, the same results were obtained with hMSCs isolated from a patient suffering from osteoporosis. CONCLUSIONS: hMSCs represent a novel platform for skeletal gene therapy and the present results suggest that they can be genetically engineered to express desired therapeutic proteins inducing specific differentiation pathways. Moreover, hMSCs obtained from osteoporotic patients can restore their osteogenic activity following human BMP-2 gene transduction, an important finding in the future planning of gene therapy treatment for osteoporosis.
Asunto(s)
Adenoviridae/genética , Enfermedades Óseas/terapia , Células de la Médula Ósea/citología , Proteínas Morfogenéticas Óseas/genética , Terapia Genética/métodos , Mesodermo/citología , Células Madre , Factor de Crecimiento Transformador beta , Animales , Desarrollo Óseo/genética , Enfermedades Óseas/genética , Proteína Morfogenética Ósea 2 , Regeneración Ósea/genética , Células Cultivadas , Vectores Genéticos , Humanos , Ratones , Proteínas Recombinantes , Transducción GenéticaRESUMEN
Regulated expression of transgene production and function is of great importance for gene therapy. Such regulation can potentially be used to monitor and control complex biological processes. We report here a regulated stem cell-based system for controlling bone regeneration, utilizing genetically engineered mesenchymal stem cells (MSCs) harboring a tetracycline-regulated expression vector encoding the osteogenic growth factor human BMP-2. We show that doxycycline (a tetracycline analogue) is able to control hBMP-2 expression and thus control MSC osteogenic differentiation both in vitro and in vivo. Following in vivo transplantation of genetically engineered MSCs, doxycycline administration controlled both bone formation and bone regeneration. Moreover, our findings showed increased angiogenesis accompanied by bone formation whenever genetically engineered MSCs were induced to express hBMP-2 in vivo. Thus, our results demonstrate that regulated gene expression in mesenchymal stem cells can be used as a means to control bone healing.
