The role of LncRNA00602 in Ad36-induced differentiation of adipocytes / 中华内分泌代谢杂志

Objective:To investigate the possible role of long non-coding RNA (LncRNA) 00602 in promoting browning in adipocytes induced by adenovirus type 36 (Ad36).Methods:According to Ad36 infection, adipose tissue samples of obese patients were divided into Ad36-negative group and Ad36-infected group. Realtime fluorescent quantitative PCR (qRT-PCR) was used to detect the changes in the expression of LncRNA00602 mRNA in omental adipose tissue of the two groups, and analyze the differences between the two groups. The correlation between waist-to-hip ratio, systolic blood pressure, diastolic blood pressure, fasting blood glucose, triacylglyceride and other indicators of the patients in the group with LncRNA00602 mRNA expression were analyzed. HE staining was used to detect the size of adipocytes in the omental adipose tissue of the Ad36 negative group and the Ad36 infection group. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of uncoupling protein 1 (UCP1) and PR domain containing 16 (PRDM16) in omental adipose tissue of two groups of patients. Human adipose-derived stem cells (hADSC) were isolated and cultured, using Ad36 to induce differentiation, and divided into control group and LncRNA00602 knockdown group. On 0, 2, and 4 days after LncRNA00602 knockdown, fluoroboron dipyrrole (BODIPY) and mitochondrial red fluorescence (Mito-Tracker Red) were used to stain intracellular lipid droplets and mitochondria. At the same time, qRT-PCR and Western blotting were used to detect changes in the expression of UCP1 and PRDM16.Results:The expression of LncRNA00602 gene in the Ad36 infection group was higher than that in the Ad36 negative group (all P<0.05). The expression of LncRNA00602 in the Ad36 negative group was not significantly different from the above clinical indicators, while the expression of LncRNA00602 was negatively correlated with serum fasting blood glucose and triacylglyceride ( r=-0.522, -0.486, P<0.05) in the Ad36 infection group; HE staining showed that the average adipocyte area of the Ad36 infection group was smaller than that of the Ad36 negative group. At the same time, UCP1 and PRDM16 gene expression were higher than the negative group (all P<0.05). At the cellular level, on the 2nd and 4th days after knockdown of LncRNA00602, the lipid droplet area of adipocytes in the LncRNA00602 knockdown group was larger than that of the control group, the number of mitochondria decreased compared with the control group, and difference was statistically significant ( P<0.05 or P<0.01); Compared with the control group, there was significantly lower expression of the browning marker genes UCP1, PRDM16, and protein in the adipocytes in the LncRNA00602 knockdown group (all P<0.05). Conclusion:In Ad36-induced adipocyte differentiation, LncRNA00602 may positively regulate the expression of UCP1, PRDM16 and lipid droplet metabolism, and promote the browning of adipocytes.

Preparation and Characterization of Human Adipose Tissue-Derived Extracellular Matrix, Growth Factors, and Stem Cells: A Concise Review

BACKGROUND: Human adipose tissue is routinely discarded as medical waste. However, this tissue may have valuable clinical applications since methods have been devised to effectively isolate adipose-derived extracellular matrix (ECM), growth factors (GFs), and stem cells. In this review, we analyze the literature that devised these methods and then suggest an optimal method based on their characterization results. METHODS: Methods that we analyze in this article include: extraction of adipose tissue, decellularization, confirmation of decellularization, identification of residual active ingredients (ECM, GFs, and cells), removal of immunogens, and comparing structural/physiological/biochemical characteristics of active ingredients. RESULTS: Human adipose ECMs are composed of collagen type I–VII, laminin, fibronectin, elastin, and glycosaminoglycan (GAG). GFs immobilized in GAG include basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGF-b1), insulin like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), BMP4 (bone morphogenetic protein 4), nerve growth factor (NGF), hepatocyte growth factor (HGF), and epithermal growth factor (EGF). Stem cells in the stromal-vascular fraction display mesenchymal markers, self-renewal gene expression, and multi-differentiation potential. CONCLUSION: Depending on the preparation method, the volume, biological activity, and physical properties of ECM, GFs, and adipose tissue-derived cells can vary. Thus, the optimal preparation method is dependent on the intended application of the adipose tissue-derived products.

Establishment of Efficacy and Safety Assessment of Human Adipose Tissue-Derived Mesenchymal Stem Cells (hATMSCs) in a Nude Rat Femoral Segmental Defect Model

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.

The Effect of Human Adipose Tissue Derived Mesenchymal Stem Cells and Growth Hormone on the Recovery of Neurological Deficits due to Experimental Spinal Cord Injury in Rat

PURPOSE: Human adipose tissue-derived mesenchymal stem cells(hATSCs) can be differentiated into multiple mesenchymal lineages, including bone, cartilage, and muscle. And growth hormone play important roles in the normal growth and development of the CNS. In this study, we explored whether the transplanted hATSCs and growth hormones could improve functional recoveries from rats with contusive spinal cord injury. METHODS: We divided 30 female rats, which were subjected to a weight driven implant spinal cord injury, into 3 groups with 10 rats each; Group A as a control group, group B with hATSCs transplantation on injured region, and group C with hATSCs transplantation and GH administration for 7 days. Then, we researched their neurologic functional recoveries before and 2, 4, and 8 weeks after transplantation using Basso-Beattie-Bresnahan (BBB) locomotor rating scale. And we checked Y- chromosome positive cells by FISH(Fluorescent in situ hybridization) to identify the survival of transplanted mesenchymal stem cells. RESULTS: After 4 weeks of transplantation, the group B and group C showed significant improvement of neurologic function on BBB locomotor rating scale in comparison with the group A(Group A: 13.1+/-0.58, Group B: 14.6+/-0.69, Group C: 14.9+/-0.56). Moreover, the group C displayed meaningful recovery of neurologic function after 8 weeks in comparison with group B (Group B: 15.7+/-0.63, Group C: 16.5+/-1.14). The group A, the control one, improved for 5 weeks after injury, and had no more recovery. On the other hand, Group B and C showed the improvement of neurologic function continuously for 9 weeks after injury. CONCLUSION: In this study, we found out that hATSCs transplantation have an effect on neurologic functional recovery of spinal cord injured rat and GH injection seems to bring the synergistic results on this good tendency.

The Effects of Human Adipose Tissue Derived Mesenchymal Stem Cells on Degenerative Change of Disc in Rabbit Model

OBJECTIVE: To determine whether transplanted human adipose tissue derived stem cells (hATSCs) can survive and increase the amount of proteoglycans in degenerated intervertebral disc. METHOD: Lumbar disc degeneration was induced in thirty New Zealand white rabbits by injection of chondroitinase ABC(R). After 2 weeks, hATSCs were transplanted in degenerated disc in hATSCs group. Control group received phosphate buffered saline. The histologic grading and height of disc were measured at 2, 4, and 8 weeks after transplantation. The viability of donor cells was identified by using beta-Actin gene polymerase chain reaction (PCR). RESULTS: 4 and 8 weeks after hATSCs transplantation, the histologic grading showed significantly high score in hATSCs group (p<0.05), but the amount of proteoglycans was not significantly different between the two groups. The change of disc height was not significantly increased in hATSCs group. In the beta-Actin gene PCR analysis, positive signal in the hATSCs group was observed. CONCLUSION: hATSCs transplantation may be useful in decelerating disc degeneration in experimental models and provide new hopes for treatment of degenerative disc disease in humans

Functional Improvement by Human Adipose Tissue Stromal Cells after Cerebral Ischemia in Rats / 대한해부학회지

Adipose tissue may represent an alternative source of cells capable of neuronal differentiation, potentially enhancing their usefulness in the treatment of neurological disease. We examined that human adipose tissue stromal cells (ATSCs) can be induced to undergo neuronal differentiation. After neuronal induction, the phenotype of hATSCs changed towards neuronal morphology and hATSCs were injected into the lateral ventricle of the rat brain. Implanted cells migrated to brain injury region which was induced by middle cerebral artery occlusion (MCAO). Intracerebral grafting of hATSCs significantly enhances sensory and motor recovery of functional deficits in MCAO rats. These data indicate that transplanted hATSCs survive, migrate and differentiate in the ischemic microenvironment and improve neurological function recovery after stroke in rats. Therefore, we anticipate that transplantation of hATSCs may provide a powerful autoplastic therapy for human neurological injury and degenerative disorders.

Neuronal Phenotypes and Gene Expression Profiles of the Human Adipose Tissue-Derived Stromal Cells in the Neuronal Induction

PURPOSE: Human adipose tissue-derived stromal cells(hADSCs) can be expanded in vitro and induced to differentiate into multiple mesenchymal cell types. In this study we have examined various neuronal phenotypes and gene expression profiles of the hADSCs in the neuronal induction. METHODS: The hADSCs were isolated from human adipose tissue and they were characterized by the flow cytometry analysis using CD13, CD29, CD34, CD45, CD49d, CD90, CD105 and HLA-DR cell surface markers. We differentiated the hADSCs into the neuronal lineage by using chemical induction medium and observed the cells with contrast microscopy. The immunocytochemistry and western blotting were performed using the NSE, NeuN, Trk-A, Vimentin, N-CAM, S-100 and beta-Tubulin III antibodies. RESULTS: The hADSCs were positive for CD13(90.3+/-4%), CD29(98.9+/-0.7%), CD49d(13.6+/-6%), CD90 (99.4+/-0.1%), CD105(96%+/-2.8%) but negative for CD34, CD45 and HLA-DR. The untreated cultures of hADSCs predominately consisted of spindle shaped cells and a few large, flat cells. Three hours after the addition of induction medium, the hADSCs had changed morphology and adopted neuronal-like phenotypes. The result of immunocytochemistry and western blotting showed that NSE, NeuN, Trk-A, Vimentin, N-CAM, S- 100 and beta-Tubulin III were expressed. However, NSE, NeuN, Vimentin were weakly expressed in the control. CONCLUSION: Theses results indicate that hADSCs have the capabillity of differentiating into neuronal lineage in a specialized culture medium. hADSCs may be useful in the treatment of a wide variety of neurological disorders.

The effect of growth factors on osteogenic differentiation of adipose tissue-derived stromal cells

Future cell-based therapies such as tissue engineering will benefit from a source of autogenous pluripotent stem cells. There are embryonic stem cells (ESC) and autologous adult stem cells, two general types of stem cells potentilally useful for these applications. But practical use of ESC is limited due to potential problems of cell regulation and ethical considerations. To get bone marrow stem cells is relatively burden to patients because of pain, anesthesia requirement. The ideal stem cells are required of such as the following advantages: easy to obtain, minimal patient discomfort and a capability of yielding enough cell numbers. Adipose autologus tissue taken from intraoral fatty pad or abdomen may represent such a source. Our study designed to demonstrate the ability of human adipose tissue-derived stromal cells (hATSC) from human abdominal adipose tissue diffentiating into osteocyte and adipocyte under culture in vitro conditions. As a result of experiment, we identified stromal cell derived adipose tissue has the multilineage potentiality under appropriate culture conditions. And the adipose stromal cells expressed several mesenchymal stem cell related antigen (CD29, CD44) reactions. Secondary, we compared the culture results of a group of hATSC stimulated with TGF-beta1, bFGF with a hATSC group without growth factors to confirm whether cytokines have a important role of the proliferation in osteogenic differentiation. The role of cytokines such as TGF-beta1, bFGF increased hATSC's osteogenic differentiation especially when TGF-beta1 and bFGF were used together. These results suggest that adipose stromal cells with growth factors could be efficiently available for cell-based bone regeneration.

The Effect of hATSC on Tactile Threshold in Neuropathic Pain Model in Rats / 대한마취과학회지

BACKGROUND: Mesenchymal stem cells can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types. This study was planned to isolate human adipose tissue stromal cells (hATSCs) from human liposuction tissues and to investigate the changes of tactile threshold after hATSC transplantation in animal neuropathic pain models. METHODS: hATSCs were grown under control conditions in alpha-MEM/10% FBS. To prepare neuropathic pain model rats, thirty male Sprague-Dawley rats that had the average body weight of 208 +/- 5 g, had an experimental nerve injury by cutting or clamping of sural and tibial nerves. The tactile threshold was measured by von Frey hair filament at preinjury and postoperative day (POD) 1, 2, 3, 7, 14. Transplantation of hATSCs was performed after measurement of tactile threshold at POD3. RESULTS: hATSCs grew as a monolayer of large, flat, and spindle-shaped cells. The tactile threshold after spared nerve injury was significantly decreased since one day after cutting or clamping of nerves (P < 0.01). The percent changes of a tactile threshold in clamping and hATSC group were decreased to 59.8 +/- 7.1% (POD7) and 52.6 +/- 5.1% (POD14) (P < 0.05). CONCLUSIONS: This results was suggested that hATSCs could be isolated from human adipose tissue easily. Althogh it needs more long-term investigation hATSCs might be used as a method of therapy for neuropathic pain.