RESUMEN
Tissue damage by ischemia/reperfusion (I/R) results from a temporary cessation of blood flow followed by the restoration of circulation. The injury depresses mitochondrial respiration, increases the production of reactive oxygen species (ROS), decreases the mitochondrial transmembrane potential, and stimulates invasion by inflammatory cells. The primary objective of this work was to address the potential use of bone marrow stem cells (BMSCs) to preserve and restore mitochondrial function in the kidney after I/R. Mitochondria from renal proximal tubule cells were isolated by differential centrifugation from rat kidneys subjected to I/R (clamping of renal arteries followed by release of circulation after 30 min), without or with subcapsular administration of BMSCs. Respiration starting from mitochondrial complex II was strongly affected following I/R. However, when BMSCs were injected before ischemia or together with reperfusion, normal electron fluxes, electrochemical gradient for protons, and ATP synthesis were almost completely preserved, and mitochondrial ROS formation occurred at a low rate. In homogenates from cultured renal cells transiently treated with antimycin A, the coculture with BMSCs induced a remarkable increase in protein S-nitrosylation that was similar to that found in mitochondria isolated from I/R rats, evidence that BMSCs protected against both superoxide anion and peroxynitrite. Labeled BMSCs migrated to damaged tubules, suggesting that the injury functions as a signal to attract and host the injected BMSCs. Structural correlates of BMSC injection in kidney tissue included stimulus of tubule cell proliferation, inhibition of apoptosis, and decreased inflammatory response. Histopathological analysis demonstrated a score of complete preservation of tubular structures by BMSCs, associated with normal plasma creatinine and urinary osmolality. These key findings shed light on the mechanisms that explain, at the mitochondrial level, how stem cells prevent damage by I/R. The action of BMSCs on mitochondrial functions raises the possibility that autologous BMSCs may help prevent I/R injuries associated with transplantation and acute renal diseases.
Asunto(s)
Adenosina Trifosfato/metabolismo , Riñón/metabolismo , Mitocondrias/metabolismo , Animales , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Estrés Oxidativo/fisiología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers.