Towards adipose tissue-derived stromal cells-based therapy for diabetic retinopathy

In this thesis, we investigated the impact of hyperglycemia on adipose tissue-derived stromal cells (ASC) as a prelude to their use in the future therapeutic treatment of diabetic retinopathy (DR). Ultrastructure analyses of co-cultured ASC-endothelial cells featured the pericytic role of ASC in the maintenance of the vascular architecture under normal and high glucose conditions. The ROS-induced mitochondrial dysfunction and hyperglycemia-induced apoptosis partially influenced the pericytic functions of ASC. Injected ASC into an angiogenic mouse models, were detected at pericytic positions on newly formed vessels. Our recommendation is to preculturing of ASC under ‘chronic’ hyperglycemia, before injecting into a hyperglycemic environment to reduce their expression of pro-inflammatory and pro-angiogenic genes. ASC-conditioned medium (ASC-Cme) delivered from ASC cultured in chronically HG, protected bovine retinal endothelial cells (BREC) from hyperglycemia-induced apoptosis and inflammatory activation. This effect on BREC was evoked by the ROS-neutralizing capacity of ASC-Cme in culture that was associated with a reduced NF-κB activation which showed as a downmodulation of HG-upregulated pro-inflammatory genes in BREC. Towards using ASC for treatment of DR, we present a new hypothermic storing technique of cells in their own culture medium to well-maintain and transport the prepared cells to their clinical destination. The new pharmacologic compound, (6-hydroxyl-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl) methanone (SUL-109) shields ASC during cell preservation from hypothermic cell death without influencing their multi-potency capacity and proliferation through maintenance of the mitochondrial membrane potential and promoting the activation of mitochondrial complexes I and IV, consequently sustaining ATP production and preventing the overproduction of ROS under hypothermic conditions

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells

The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-kappa B dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy

Assessment of Energy Metabolic Changes in Adipose Tissue-Derived Stem Cells

Adipose tissue-derived stem cells (ADSC) are promising candidates for therapeutic applications in cardiovascular regenerative medicine. By definition, the phenotype ADSCs, e.g., the ubiquitous secretion of growth factors, cytokines, and extracellular matrix components is not met in vivo, which renders ADSC a culture "artefact." The medium constituents therefore impact the efficacy of ADSC. Little attention has been paid to the energy source in medium, i.e., glucose, which feeds the cell's power plants: mitochondria. The role of mitochondria in stem cell biology goes beyond their function in ATP synthesis, because it includes cell signaling, reactive oxygen species (ROS) production, regulation of apoptosis, and aging. Appropriate application of ADSC for stem cells therapy of cardiovascular disease warrants knowledge of their mitochondrial phenotype and function. We discuss several methodologies for assessing ADSC mitochondrial function and structural changes under environmental cues, in particular, increased ROS caused by hyperglycemia.</p