Diabetic Adipose-derived Stem Cells Research Articles

Photobiomodulation preconditioned diabetic adipose derived stem cells with additional photobiomodulation: an additive approach for enhanced wound healing in diabetic rats with a delayed healing wound.

In this preclinical investigation, we examined the effects of combining preconditioned diabetic adipose-derived mesenchymal stem cells (AD-MSCs) and photobiomodulation (PBM) on a model of infected ischemic delayed healing wound (injury), (IIDHWM) in rats with type I diabetes (TIDM). During the stages of wound healing, we examined multiple elements such as stereology, macrophage polarization, and the mRNA expression levels of stromal cell-derived factor (SDF)-1α, vascular endothelial growth factor (VEGF), hypoxia-induced factor 1α (HIF-1α), and basic fibroblast growth factor (bFGF) to evaluate proliferation and inflammation. The rats were grouped into: (1) control group; (2) diabetic-stem cells were transversed into the injury site; (3) diabetic-stem cells were transversed into the injury site then the injury site exposed to PBM; (4) diabetic stem cells were preconditioned with PBM and implanted into the wound; (5) diabetic stem cells were preconditioned with PBM and transferred into the injury site, then the injury site exposed additional PBM. While on both days 4, and 8, there were advanced histological consequences in groups 2-5 than in group 1, we found better results in groups 3-5 than in group 2 (p < 0.05). M1 macrophages in groups 2-5 were lower than in group 1, while groups 3-5 were reduced than in group 2 (p < 0.01). M2 macrophages in groups 2-5 were greater than in group 1, and groups 3-5 were greater than in group 2. (p ≤ 0.001). Groups 2-5 revealed greater expression levels of bFGF, VEGF, SDF- 1α, and HIF- 1α genes than in group 1 (p < 0.001). Overall group 5 had the best results for histology (p < 0.05), and macrophage polarization (p < 0.001). AD-MSC, PBM, and AD-MSC + PBM treatments all enhanced the proliferative stage of injury repairing in the IIDHWM in TIDM rats. While AD-MSC + PBM was well than the single use of AD-MSC or PBM, the best results were achieved with PBM preconditioned AD-MSC, plus additional PBM of the injury.

Dual Glyoxalase-1 and β-Klotho Gene-Activated Scaffold Reduces Methylglyoxal and Reprograms Diabetic Adipose-Derived Stem Cells: Prospects in Improved Wound Healing.

Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic β-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (-84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (-65%) and fibronectin (-76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (-62%), collagen IV (-70%) and collagen VII (-86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (-59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and β-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.

Low-glucose culture environment can enhance the wound healing capability of diabetic adipose-derived stem cells

BackgroundApplication of autologous adipose-derived stem cells (ASC) for diabetic chronic wounds has become an emerging treatment option. However, ASCs from diabetic individuals showed impaired cell function and suboptimal wound healing effects. We proposed that adopting a low-glucose level in the culture medium for diabetic ASCs may restore their pro-healing capabilities.MethodsASCs from diabetic humans and mice were retrieved and cultured in high-glucose (HG, 4.5 g/L) or low-glucose (LG, 1.0 g/L) conditions. Cell characteristics and functions were investigated in vitro. Moreover, we applied diabetic murine ASCs cultured in HG or LG condition to a wound healing model in diabetic mice to compare their healing capabilities in vivo.ResultsHuman ASCs exhibited decreased cell proliferation and migration with enhanced senescence when cultured in HG condition in vitro. Similar findings were noted in ASCs derived from diabetic mice. The inferior cellular functions could be partially recovered when they were cultured in LG condition. In the animal study, wounds healed faster when treated with HG- or LG-cultured diabetic ASCs relative to the control group. Moreover, higher collagen density, more angiogenesis and cellular retention of applied ASCs were found in wound tissues treated with diabetic ASCs cultured in LG condition.ConclusionsIn line with the literature, our study showed that a diabetic milieu exerts an adverse effect on ASCs. Adopting LG culture condition is a simple and effective approach to enhance the wound healing capabilities of diabetic ASCs, which is valuable for the clinical application of autologous ASCs from diabetic patients.

Comparable Effects on Healing between Autologous Diabetic Adipose-Derived Stem Cells and Allogeneic Normal Counterparts.

Adipose-derived stem cell (ASC) therapy is considered a promising strategy for improving impaired wound healing, especially in diabetics. Although the therapeutic potential of allogeneic ASCs from healthy donors is naturally limited, that of autologous ASCs from diabetic patients is questionable. The aim of this study was to investigate the impact of diabetic ASCs in the treatment of diabetic wounds. Diabetic ASCs (DMA) and nondiabetic ASCs were isolated from db/db and C57BL/6J mice, and characterized by immunocytochemistry, proliferation, differentiation, and gene expression assays. The effects of both ASCs on healing were investigated using 36 male 10- to 12-week-old db/db mice. Wound size was measured semiweekly until day 28, and histologic and molecular analyses were performed at day 14. Both ASCs had fibroblast-like morphology and were CD44 + /CD90 + /CD34 - /CD45 - at passage 4. Compared with nondiabetic ASCs in vitro, DMA proliferative capability was restored by passage 4 ( P > 0.05). Although DMA osteogenesis was attenuated ( P < 0.01), both ASCs had similar adipogenesis and expressions of PPARγ/LPL/OCN/RUNX2 ( P > 0.05). In vivo experiments showed that, compared with phosphate-buffered saline control, both ASCs are comparable in improving wound healing ( P < 0.0001), angiogenesis ( P < 0.05), epithelial cell proliferation ( P < 0.05), and granulation tissue formation ( P < 0.0001). In both in vitro and in vivo murine models, DMAs have shown a comparable therapeutic capacity to normal ASCs in promoting diabetic wound healing by improving angiogenesis, reepithelialization, and granulation tissue formation. These results support clinical applications of autologous ASCs in diabetic wound treatments. This work has particular surgical relevance as it highlights a theoretical and clinical pathway to use diabetic patients' own ASCs to treat their wounds, bypassing any concerns of cross-host sourcing issues in regenerative medicine.

Knockdown of Adra2a Increases Secretion of Growth Factors and Wound Healing Ability in Diabetic Adipose-Derived Stem Cells.

Studies showed that compared to normal adipose-derived stem cells (ASCs), ASCs from type 2 diabetic (T2D) mice were less effective in treating diabetic cutaneous wounds. However, the mechanisms remain unclear. Our transcriptomic profiling comparison showed that the expression of α2A-adrenergic receptor (Adra2a) was significantly increased in ASCs from T2D mice (T2D ASCs). Therefore, the purpose of this study was to investigate whether the elevated Adra2a is involved in the diminished wound-healing capabilities of T2D ASCs. RNA-seq was used to compare the transcriptomic profiles of T2D and normal ASCs. The differential genes were verified by real-time RT-qPCR. Clonidine was used to active Adra2a, and lentivirus-mediated RNAi was used to knockdown Adra2a. The secretion and expression of growth factors were detected by enzyme-linked immunosorbent assay (ELISA) and real-time RT-qPCR, respectively. The cAMP and PKA activity were also detected. Wound healing abilities of various ASCs were assessed in T2D mouse excisional wound models. The results showed Adra2a agonist clonidine decreased the expression and secretion of growth factors, cAMP content, and activity of PKA in ASCs, while Adra2a knockdown T2D ASCs showed the opposite effects. Adra2a knockdown T2D ASCs also showed increased wound-healing capabilities compared to untreated T2D ASCs. Altogether, T2D increased Adra2a expression, which may subsequently decrease the expression and secretion of growth factors and eventually diminish the wound-healing capabilities of T2D ASCs. Adra2a knockdown can restore the secretion of growth factors in T2D ASCs and then accelerate the wound healing, which may provide a new possibility in the treatment of diabetic wounds.

Effects of photobiomodulation on mitochondrial function in diabetic adipose-derived stem cells in vitro

Herein are reported the effects of photobiomodulation (PBM) on adenosine triphosphate (ATP) and reactive oxygen species (ROS) quantification and mitochondria membrane potential (MMP) of the mitochondria of diabetic adipose-derived stem cells (ADSCs) in vitro. Additionally, the expression of PTEN-induced kinase 1 (PINK1) and RBR E3 ubiquitin-protein ligase (PARKIN) genes, which are involved in mitochondrial quality, were quantified.First, type one diabetes was induced in 10 rats. The rats were then kept for 1 month, after which fat tissue was excised from subcutaneous regions, and stem cells were selected from the fat, characterized as ADSC, and cultivated and increased in elevated sugar conditions in vitro; these samples were considered diabetic-ADSC. Two groups were formed, namely, diabetic-control-ADSC and PBM–diabetic-ADSC. ATP, ROS quantification, and MMP of mitochondria of diabetic ADSCs in vitro were measured, and the expression of PINK1 and Parkin genes was quantified in vitro.The results revealed that PBM significantly increased ATP quantification (p = 0.05) and MMP activity (p = 0.000) in diabetic-ADSCs in vitro compared to the control diabetic-ADSCs; however, it significantly decreased ROS quantification (p = 0.002) and PINK1(p = 0.003) and PARKIN gene expression (p = 0.046) in diabetic-ADSCs.The current findings indicate for the first time that PBM has the potential to maintain the function and quality of mitochondrial diabetic-ADSCs by significantly increasing ATP quantification and MMP activity in diabetic-ADSCs in vitro while significantly decreasing ROS quantification and PINK1 and PARKIN gene expression, making PBM an attractive candidate for use in improving the efficacy of autologous stem cell remedies for diabetic patients with infected diabetic foot ulcers.

SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells.

Non-healing diabetic foot ulcers (DFUs) can lead to leg amputation in diabetic patients. Autologous stem cell therapy holds some potential to solve this problem; however, diabetic stem cells are relatively dysfunctional and restrictive in their wound healing abilities. This study sought to explore if a novel collagen–chondroitin sulfate (coll–CS) scaffold, functionalized with polyplex nanoparticles carrying the gene encoding for stromal-derived factor-1 alpha (SDF-1α gene-activated scaffold), can enhance the regenerative functionality of human diabetic adipose-derived stem cells (ADSCs). We assessed the impact of the gene-activated scaffold on diabetic ADSCs by comparing their response against healthy ADSCs cultured on a gene-free scaffold over two weeks. Overall, we found that the gene-activated scaffold could restore the pro-angiogenic regenerative response in the human diabetic ADSCs similar to the healthy ADSCs on the gene-free scaffold. Gene and protein expression analysis revealed that the gene-activated scaffold induced the overexpression of SDF-1α in diabetic ADSCs and engaged the receptor CXCR7, causing downstream β-arrestin signaling, as effectively as the transfected healthy ADSCs. The transfected diabetic ADSCs also exhibited pro-wound healing features characterized by active matrix remodeling of the provisional fibronectin matrix and basement membrane protein collagen IV. The gene-activated scaffold also induced a controlled pro-healing response in the healthy ADSCs by disabling early developmental factors signaling while promoting the expression of tissue remodeling components. Conclusively, we show that the SDF-1α gene-activated scaffold can overcome the deficiencies associated with diabetic ADSCs, paving the way for autologous stem cell therapies combined with novel biomaterials to treat DFUs.

Anti-Ageing Protein β-Klotho Rejuvenates Diabetic Stem Cells for Improved Gene-Activated Scaffold Based Wound Healing.

Skin wounds can lead to serious morbidity complications in diabetic patients due to the reduced healing potential of autologous stem cells. One reason for the low functional potency of stem cells from diabetic patients (diabetic stem cells) is attributed to their senescent-like nature. Here, we investigated if an anti-ageing protein, β-klotho, could be used to rejuvenate diabetic stem cells and to promote pro-angiogenic gene-activated scaffold (GAS)-induced functional response for wound healing applications. Human stem cells derived from the adipose tissue (adipose-derived stem cells (ADSCs)) of normal and diabetic (type 2) donors were used for the study. We report that the β-klotho priming facilitated inflammatory signal pruning by reducing interleukin-8 release by more than half while concurrently doubling the release of monocyte chemoattractant protein-1. Additionally, β-klotho priming enhanced the pro-angiogenic response of diabetic ADSCs on GAS by dampening the release of anti-angiogenic factors (i.e., pigment epithelium-derived factor, tissue inhibitor of metalloproteinase-1 and thrombospondin-1) while simultaneously supporting the expression of pro-angiogenic factors (i.e., Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 and angiogenin). Finally, we show that β-klotho pre-treatment expedites the cellular expression of matrix proteins such as collagen IV and collagen VI, which are implicated in tissue maturation. Taken together, our study provides evidence that the synergistic effect of the pro-angiogenic GAS and β-klotho activation effectively accelerates the functional development of diabetic ADSCs for wound healing applications.

New Amphiphilic Squalene Derivative Improves Metabolism of Adipocytes Differentiated From Diabetic Adipose-Derived Stem Cells and Prevents Excessive Lipogenesis.

Squalene (Sq) is a natural compound, found in various plant oils, algae, and larger quantity in deep-sea shark liver. It is also known as an intermediate of cholesterol synthesis in plants and animals including humans. Although evidences demonstrated its antioxidant, anticancer, hypolipidemic, and hepatoprotective and cardioprotective effects, its biological effects in cellular function might have been underestimated because of the water-insoluble property. To overcome this hydrophobicity, we synthesized new amphiphilic Sq derivative (HH-Sq). On the other hand, adipose-derived stem cells (ASCs) are a valuable source in regenerative medicine for its ease of accessibility and multilineage differentiation potential. Nevertheless, impaired cellular functions of ASCs derived from diabetic donor have still been debated controversially. In this study, we explored the effect of the HH-Sq in comparison to Sq on the adipocyte differentiation of ASCs obtained from subjects with type 2 diabetes. Gene expression profile by microarray analysis at 14 days of adipogenic differentiation revealed that HH-Sq induced more genes involved in intracellular signaling processes, whereas Sq activated more transmembrane receptor pathway-related genes. In addition, more important number of down-regulated and up-regulated genes by Sq and HH-Sq were not overlapped, suggesting the compounds might not only have difference in their chemical property but also potentially exert different biological effects. Both Sq and HH-Sq improved metabolism of adipocytes by enhancing genes associated with energy homeostasis and insulin sensitivity, SIRT1, PRKAA2, and IRS1. Interestingly, Sq increased significantly early adipogenic markers and lipogenic gene expression such as PPARG, SREBF1, and CEBPA, but not HH-Sq. As a consequence, smaller and fewer lipid droplet formation was observed in HH-Sq-treated adipocytes. Based on our findings, we report that both Sq and HH-Sq improved adipocyte metabolism, but only HH-Sq prevented excessive lipogenesis without abrogating adipocyte differentiation. The beneficial effect of HH-Sq provides an importance of synthesized derivatives from a natural compound with therapeutic potentials in the application of cell therapies.

Deferoxamine enhances the regenerative potential of diabetic Adipose Derived Stem Cells

Diabetes mellitus remains a significant public health problem, consuming over $400 billion every year. While Diabetes itself can be controlled effectively, impaired wound healing still occurs frequently in diabetic patients. Adipose-derived mesenchymal stem cells (ASCs) provide an especially appealing source for diabetic wound cell therapy. With autologous approaches, the functionality of ASCs largely underlie patient-dependent factors. Diabetes is a significant diminishing factor of MSC functionality. Here, we explore a novel strategy to enhance diabetic ASC functionality through deferoxamine (DFO) preconditioning. Human diabetic ASCs have been preconditioned with 150 µM and 300 µM DFO in vitro and analyzed for regenerative cytokine expression. Murine diabetic ASCs have been preconditioned with 150 µM DFO examined for their in vitro and in vivo vasculogenic capacity in Matrigel assays. Additionally, a diabetic murine wound healing model has been performed to assess the regenerative capacity of preconditioned cells. DFO preconditioning enhances the VEGF expression of human diabetic ASCs through hypoxia-inducible factor upregulation. The use of 150 µM of DFO was an optimal concentration to induce regenerative effects. The vasculogenic potential of preconditioned diabetic ASCs is significantly greater in vitro and in vivo. The enhanced regenerative functionality of DFO preconditioned ASCs was further confirmed in a model of diabetic murine wound healing. These results demonstrate that DFO significantly induced the upregulation of hypoxia-inducible factor-1 alpha and VEGF in diabetic ASCs and showed efficacy in the treatment of diabetes-associated deficits of wound healing. The favorable status of DFO as a small molecule drug approved since decades for multiple indications makes this approach highly translatable.

Investigating The Alterations of Oxidative Stress Status, Antioxidant Defense Mechanisms, MAP Kinase and Mitochondrial Apoptotic Pathway in Adipose-Derived Mesenchymal Stem Cells from STZ Diabetic Rats.

Objective This study aimed to investigate the reliability of diabetic adipose-derived stem cells (ADSCs) for autologous cell-based therapies by exploring the functionality of signalling pathways involved in regulating oxidative stress and apoptosis. Materials and Methods In this experimental study, ADSCs were isolated from streptozotocin (STZ)-induced diabetic rats (dADSCs) and normal rats (nADSCs). The colonies derived from dADSCs and nADSCs were compared by colony-forming unit (CFU) assay. Reactive oxygen species (ROS) formation and total antioxidant power (TAP) were also measured. Furthermore, the expression of antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod)-1 and -3, glutathione peroxidase (Gpx)-1, -3 and -4 was measured at mRNA level by semi-quantitative reverse transcriptase polymerase chain reaction assay. The expression of Bax, Bcl2, caspase-3, total and phosphorylated c-Jun N-terminal kinase (JNK) and P38 Mitogen-Activated Protein Kinase (MAPK) at protein level was analyzed by western blotting. Results The results of this study indicated that viability and plating efficiency of dADSCs were significantly lower than those of nADSCs. ROS generation and TAP level were respectively higher and lower in dADSCs. The gene expression of antioxidant enzymes, including Cat, Sod-1, Gpx-3 and Gpx-4 in dADSCs was significantly greater than that in nADSCs. However, Sod-3 and Gpx-1 mRNA levels were decreased in dADSCs. Moreover, Bax/Bcl-2 protein ratio, caspase-3 protein expression and phosphorylation of JNK and P38 proteins were increased in dADSCs compared to nADSCs. ConclusionTaken together, diabetes might impair the cellular functions of dADSCs as candidates for autologous cell- based therapies. This impairment seems to be mediated by JNK, P38 MAPKs, and mitochondria pathway of apoptosis and partly by disruption of antioxidant capacity.

Pretreatment of Diabetic Adipose-derived Stem Cells with mitoTEMPO Reverses their Defective Proangiogenic Function in Diabetic Mice with Critical Limb Ischemia

Adipose-derived stem cells (ADSCs) have the ability to migrate to injury sites and facilitate tissue repair by promoting angiogenesis. However, the therapeutic effect of ADSCs from patients with diabetes is impaired due to oxidative stress. Given that diabetes is a group of metabolic disorders and mitochondria are a major source of reactive oxygen species (ROS), it is possible that mitochondrial ROS plays an important role in the induction of diabetic ADSC (dADSC) dysfunction. ADSCs isolated from diabetic mice were treated with mitoTEMPO, a mitochondrial ROS scavenger, or TEMPO, a universal ROS scavenger, for three passages. The results showed that pretreatment with mitoTEMPO increased the proliferation, multidifferentiation potential, and the migration and proangiogenic capacities of dADSCs to levels similar to those of ADSCs from control mice, whereas pretreatment with TEMPO showed only minor effects. Mechanistically, mitoTEMPO pretreatment enhanced the mitochondrial antioxidant capacity of dADSCs, and knockdown of superoxide dismutase reduced the restored mitochondrial antioxidant capacity and attenuated the proangiogenic effects induced by mitoTEMPO pretreatment. In addition, mitoTEMPO pretreatment improved the survival of dADSCs in diabetic mice with critical limb ischemia, showing protective effects similar to those of control ADSCs. Pretreatment of dADSCs with mitoTEMPO decreased limb injury and improved angiogenesis in diabetic mice with critical limb ischemia. These findings suggested that short-term pretreatment of dADSCs with a mitochondrial ROS scavenger restored their normal functions, which may be an effective strategy for improving the therapeutic effects of ADSC-based therapies in patients with diabetes.

Diabetes mellitus type 2 reduces the viability, proliferation, and angiogenic marker of adipose-derived stem cells cultured in low-glucose anti-oxidant-serum supplemented medium

Introduction: Hyperglycemia in diabetic patients induces elevated pro-inflammatory cytokine production, resulting in cellular damage, which may affect the regenerative function of mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs). Identifying the effect of diabetes on ADSCs and optimization of culture conditions is therefore an important starting point for the application of autologous stem cells to improve clinicial outcomes. The aim of this study was to investigate the effect of diabetes on ADSCs that cultured in low-glucose anti-oxidant-serum supplemented medium.&#x0D; Methods: In this study, freshly isolated stromal vascular fraction (SVF) and expanded ADSCs were compared between diabetic and non-diabetic donors. SVF were isolated from the abdominal fat, and total viable cells and viability were estimated. Fresh SVF were cultured in low-glucose (100 mg/dL) culture medium supplemented with an anti-oxidant and fetal bovine serum (complete culture medium) at a low density for 14 days for the colony formation unit-fibroblast (CFU-F) assay. The remaining SVF were expanded to obtain ADSCs in the complete culture medium, which were evaluated based on MSCs surface marker expression and three lineage differentiation potential. Diabetic and non-diabetic ADSCs were compared with respect to population doubling time and viability after serial passage.&#x0D; Results: Total viable counts (0.97 +/- 0.39 x 109 cells/10 mL of adipose tissue, 0.56 +/- 0.39 x 109 cells/10 mL of adipose tissue, p=0.02, independent t-test), but not viability (98.63 +/- 1.12%, 98.20 +/- 1.21%, p= 0.38, independent t-test), were significantly higher for SVF cells from adipose tissues of non-diabetic donors than diabetic donors. Fewer CFU-F were obtained from cultured diabetic SVF than from non-diabetic SVF. Diabetic and non-diabetic ADSCs had similar differentiation potency and CD73 (99.44 +/- 0.34%, 97.15 +/- 5.37%, p= 0.21, Mann-Whitney U test) and CD90 (97.30 +/- 2.86%, 95.06 +/- 6.32%, p= 0.90, Mann- Whitney U test) expression, but significantly fewer diabetic ADSCs expressed CD105 or endoglin, a marker for angiogenesis (89.91 +/- 7.14%, 57.90 +/- 21.36% for non-diabetic and diabetic groups, p&lt; 0.001, Mann-Whitney U test). Diabetic ADSCs tended to exhibit slower proliferation (4.43 +/- 2.70 days, 3.04 +/- 0.55 days, p= 0.27 in passage 2 (P2); 3.95 +/- 1.55 days, 2.96 +/- 0.91 days, p= 0.21 in P3, independent t-test) and lower viability than those of non-diabetic ADSCs (77.65 +/- 10.61%, 87.13 +/- 10.06%, p= 0.25 in P2; 82.70 +/- 8.07%, 91.15 +/- 3.77%, p= 0.04 in P3, independent t-test). Culture in low-glucose anti-oxidant-serum supplemented medium did not improve CD105 expression (65.14 +/- 5.86%, 71.06 +/- 10.27%, 64.05 +/- 10.04%, p= 0.70, for P1, P2, and P3, respectively, repeated measure ANOVA) and cell proliferation (p= 0.50 for P2 vs. P3, paired t-test) of diabetic ADSCs.&#x0D; Conclusions: Overall, diabetes reduced CD105 expression and ADSCs proliferation, suggesting that the angiogenic potency of diabetic ADSCs is reduced. The diabetic ADSCs in this study were also more prone to cell death caused by handling technique compared to non-diabetic ADSCs. Therefore, more advanced culture techniques should be applied to expand ADSCs from diabetic patients to achieve expected clinical outcomes.&#x0D;

Advances in the research of influence of diabetes in biological function of adipose-derived stem cells

Adipose-derived stem cells (ADSCs) are adult mesenchymal stem cells in adipose tissue with self-renewal and multi-directional differentiation potential. The application of ADSCs in the treatment of wounds has achieved good results. Because of its extensive sources, high content in vivo, low immunogenicity, slight injury to body when obtained, the clinical application prospect of ADSCs is promising. The reasons why diabetic wound is difficult to heal may be closely related to the increase of advanced glycation end products, long-term chronic inflammatory response, and peripheral neurologic dysfunction. The abnormal internal environment of diabetic patients can affect the biological function of ADSCs, which further affects wound healing. This article reviews the general feature, differentiation, proliferation, migration, secretion, and pro-angiogenic function of diabetic ADSCs.

Glyoxalase-1 Overexpression Reverses Defective Proangiogenic Function of Diabetic Adipose-Derived Stem Cells in Streptozotocin-Induced Diabetic Mice Model of Critical Limb Ischemia.

Adipose‐derived stem cell (ADSC)‐based therapy is promising for critical limb ischemia (CLI) treatment, especially in patients with diabetes. However, the therapeutic effects of diabetic ADSCs (D‐ADSCs) are impaired by the diabetes, possibly through intracellular reactive oxygen species (ROS) accumulation. The objective of the present study was to detect whether overexpression of methylglyoxal‐metabolizing enzyme glyoxalase‐1 (GLO1), which reduces ROS in D‐ADSCs, can restore their proangiogenic function in a streptozotocin‐induced diabetic mice model of CLI. GLO1 overexpression in D‐ADSCs (G‐D‐ADSCs) was achieved using the lentivirus method. G‐D‐ADSCs showed a significant decrease in intracellular ROS accumulation, increase in cell viability, and resistance to apoptosis under high‐glucose conditions compared with D‐ADSCs. G‐D‐ADSCs also performed better in terms of migration, differentiation, and proangiogenic capacity than D‐ADSCs in a high‐glucose environment. Notably, these properties were restored to the same level as that of nondiabetic ADSCs under high‐glucose conditions. G‐D‐ADSC transplantation induced improved reperfusion and an increased limb salvage rate compared D‐ADSCs in a diabetic mice model of CLI. Histological analysis revealed higher microvessel densities and more G‐D‐ADSC‐incorporated microvessels in the G‐D‐ADSC group than in the D‐ADSC group, which was comparable to the nondiabetic ADSC group. Higher expression of vascular endothelial growth factor A and stromal cell‐derived factor‐1α and lower expression of hypoxia‐induced factor‐1α were also detected in the ischemic muscles from the G‐D‐ADSC group than that of the D‐ADSC group. The results of the present study have demonstrated that protection from ROS accumulation by GLO1 overexpression is effective in reversing the impaired biological function of D‐ADSCs in promoting neovascularization of diabetic CLI mice model and warrants the future clinical application of D‐ADSC‐based therapy in diabetic patients. Stem Cells Translational Medicine 2017;6:261–271

Deferoxamine preconditioning to restore impaired HIF‐1α‐mediated angiogenic mechanisms in adipose‐derived stem cells from STZ‐induced type 1 diabetic rats

Both excessive and insufficient angiogenesis are associated with progression of diabetic complications, of which poor angiogenesis is an important feature. Currently, adipose-derived stem cells (ADSCs) are considered to be a promising source to aid therapeutic neovascularization. However, functionality of these cells is impaired by diabetes which can result from a defect in hypoxia-inducible factor-1 (HIF-1), a key mediator involved in neovascularization. In the current study, we sought to explore effectiveness of pharmacological priming with deferoxamine (DFO) as a hypoxia mimetic agent, to restore the compromised angiogenic pathway, with the aid of ADSCs derived from streptozotocin (STZ)-induced type 1 diabetic rats ('diabetic ADSCs'). Diabetic ADSCs were treated with DFO and compared to normal and non-treated diabetic ADSCs for expression of HIF-1α, VEGF, FGF-2 and SDF-1, at mRNA and protein levels, using qRT-PCR, western blotting and ELISA assay. Activity of matrix metalloproteinases -2 and -9 were measured using a gelatin zymography assay. Angiogenic potential of conditioned media derived from normal, DFO-treated and non-treated diabetic ADSCs were determined by invitro (in HUVECs) and invivo experiments including scratch assay, three-dimensional tube formation testing and surgical wound healing models. DFO remarkably enhanced expression of noted genes by mRNA and protein levels and restored activity of matrix metalloproteinases -2 and -9. Compromised angiogenic potential of conditioned medium derived from diabetic ADSCs was restored by DFO both invitro and invivo experiments. DFO preconditioning restored neovascularization potential of ADSCs derived from diabetic rats by affecting the HIF-1α pathway.

Adipose-derived stem cells from diabetic mice show impaired vascular stabilization in a murine model of diabetic retinopathy.

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose-derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose-derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high-throughput enzyme-linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.

Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications

To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber–based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing.

Endothelial differentiation of diabetic adipose-derived stem cells

BackgroundDiabetic (DM) patients frequently lack autologous vascular tissue required for revascularization procedures and dialysis access creation. We have developed a tissue-engineered graft that uses adipose-derived stem cells (ASC) as endothelial cell substitutes. Here, we compare DM versus nondiabetic (NDM) ASC in terms of isolation efficiency, proliferation, commitment toward endothelial lineage, and seeding onto the luminal surface of a graft. MethodsASC were isolated from liposuction specimens of vascular surgery patients. Proliferation was assessed by constructing growth curves over 14 d. ASC were differentiated in endothelial growth medium (EGM2). Endothelial commitment was assessed by measuring endothelial cell-specific gene expression (CD31, von Willebrand factor) and by cord formation on Matrigel. Finally, ASC were seeded onto a vascular scaffold, flow conditioned, and imaged with confocal microscopy. ResultsDiabetes did not alter ASC isolation efficiency (224,028 ± 20,231 cells/g adipose for DM (n = 53) versus 259,345 ± 15,441 cells/g adipose for NDM (n = 145; P = 0.21). Growth curves for DM (n = 6) and NDM (n = 6) also appeared similar. After culture in EGM2, upregulation of CD31 and von Willebrand factor message was observed in NDM; these markers were found within the primary cultures of DM but no upregulation was observed after culture in EGM2. Both groups exhibited similar cord formation on Matrigel and retention to vascular scaffolds. ConclusionsIsolation and proliferation studies suggest that adipose is a promising source of stem cells for tissue engineering in the DM population. The angiogenic potential of DM ASC appears intact; however, differences in acquisition of endothelial cell markers suggest that differentiation may be inhibited or delayed by diabetes.

Diabetic Adipose Derived Stem Cells Display Functional Impairments in the Setting of a Regenerative Biomimetic Hydrogel Scaffold

Diabetic Adipose Derived Stem Cells Display Functional Impairments in the Setting of a Regenerative Biomimetic Hydrogel Scaffold