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

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

The 6-chromanol derivate SUL-109 enables prolonged hypothermic storage of adipose tissue-derived stem cells

Encouraging advances in cell therapy research with adipose derived stem cells (ASC) require an effective short-term preservation method that provides time for quality control and transport of cells from their manufacturing facility to their clinical destination. Hypothermic storage of cells in their specific growth media offers an alternative and simple preservation method to liquid nitrogen cryopreservation or commercial preservation fluids for short-term storage and transport. However, accumulation of cell damage during hypothermia may result in cell injury and death upon rewarming through the production of excess reactive oxygen species (ROS). Here, the ability of the cell culture medium additive SUL-109, a modified 6-chromanol, to protect ASC from hypothermia and rewarming damage is examined. SUL-109 conveys protective effects against cold-induced damage in ASC as is observed by preservation of cell viability, adhesion properties and growth potential. SUL-109 does not reduce the multilineage differentiation capacity of ASC. SUL-109 conveys its protection against hypothermic damage by the preservation of the mitochondrial membrane potential through the activation of mitochondrial membrane complexes I and IV, and increases maximal oxygen consumption in FCCP uncoupled mitochondria. Consequently, SUL-109 alleviates mitochondrial ROS production and preserves ATP production. In summary, here we describe the generation of a single molecule cell preservation agent that protects ASC from hypothermic damage associated with short-term cell preservation that does not affect the differentiation capacity of ASC. (C) 2016 Elsevier Ltd. All rights reserved

Hyperglycemia Induces Bioenergetic Changes in Adipose-Derived Stromal Cells While Their Pericytic Function Is Retained

Diabetic retinopathy (DR) is a hyperglycemia (HG)-mediated microvascular complication. In DR, the loss of pericytes and subsequently endothelial cells leads to pathologic angiogenesis in retina. Adipose-derived stromal cells (ASC) are a promising source of therapeutic cells to replace lost pericytes in DR. To date, knowledge of the influence of HG on the bioenergetics and pericytic function of ASC is negligible. Human ASC were cultured in normoglycemia medium (5mM d-glucose) or under HG (30mM D-glucose) and assessed. Our data showed that HG increased the level of apoptosis and reactive oxygen species production in ASC, yet their proliferation rate was not affected. HG induced alterations in mitochondrial function and morphology in ASC. HG also strongly affected the bioenergetic status of ASC in which both the maximum oxygen consumption rate and extracellular acidification rate were decreased. This was corroborated by a reduced uptake of glucose under HG. In spite of these observations, in vitro, ASC promoted the formation of vascular-like networks of human umbilical vein endothelial cells on monolayers of ASC under HG with minimally affected

Pontocerebellar hypoplasia with spinal muscular atrophy (PCH1): identification of SLC25A46 mutations in the original Dutch PCH1 family

Molecular Epidemiolog