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Obesity has been known to negatively modulate the life-span and immunosuppressive potential of mesenchymal stromal cells (MSC). However, it remains unclear what drives the compromised potency of obese MSC. In this study, we examined the involvement of adiponectin, an adipose tissue-derived hormone, in obesity-induced impaired therapeutic function of MSC. Diet-induced obesity leads to a decrease in serum adiponectin, accompanied by impairment of survival and immunomodulatory effects of adipose-derived MSC (ADSC). Interestingly, priming with globular adiponectin (gAcrp) improved the immunomodulatory potential of obese ADSC. Similar effects were also observed in lean ADSC. In addition, gAcrp potentiated the therapeutic effectiveness of ADSC in a mouse model of DSS-induced colitis. Mechanistically, while obesity inhibited the glycolytic capacity of MSC, gAcrp treatment induced a metabolic shift toward glycolysis through activation of adiponectin receptor type 1/p38 MAPK/hypoxia inducible factor-1α axis. These findings suggest that activation of adiponectin signaling is a promising strategy for enhancing the therapeutic efficacy of MSC against immune-mediated disorders.
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Objetivo Obtener células estromales derivadas del tejido adiposo, medir y comparar las tasas de viabilidad antes e inmediatamente después un ciclo de criopreservación con diferentes combinaciones de criopreservantes de manera de obtener el mejor medio de criopreservación. Material y método Medición de la tasa de viabilidad poscriopreservación de células estromales derivadas del tejido adiposo obtenidas de 5 pacientes utilizando medios definidos (DMEM/Ham F12) libres de suero bovino y suplementados con una de los siguientes combinaciones de compuestos: dimetilsulfóxido (DMSO) 10%; DMSO 10% + trehalosa 7,6%; DMSO 10% + albúmina humana 10% y DMSO 10% + trehalosa 7,6% + albúmina humana 10%, mediante citometría de flujo con ioduro de propidio. Resultados No existen diferencias estadísticamente significativas en las tasas de viabilidad de las células estromales posterior a un ciclo de criopreservación. Sin embargo, se observa una tendencia a mejorar la tasa de recuperación de células vitales al agregar albúmina humana. Conclusiones No se observaron diferencias significativas entre las condiciones estudiadas, sugiriendo que ninguna es superior a las demás en cuanto a rendimiento. Es así como podemos afirmar que la criopreservación de las células estromales derivadas del tejido adiposo en un medio que combine DMEM/F12 con DMSO 10% + trehalosa 7,6% + albúmina humana 10% no logra una tasa de recuperación de células vitales significativamente mayor que las congeladas solo con DMSO 10%.
Aim To obtain stromal cells derived from adipose tissue, to measure and compare viability rates before and immediately after cryopreservation cycle, using different combinations of cryoprotective agents in order to identify the best cryopreservation medium. Material and method Viability rate after cryopreservation of stromal cells derived from adipose tissue were assessed by flow cytometry with propidium iodide. Samples of stromal cells obtained from 5 patients were kept defined, bovine serum-free media (DMEM/Ham-F12), supplemented with one of the following combinations of compounds: 10% dymethylsulfoxide (DMSO); Trehalose 10% DMSO + 7.6%; 10% DMSO + 10% human albumin and 10% DMSO + 7.6% Trehalose + 10% human albumin. Results No statistically significant differences were observed in the viability rates of stromal cells derived from adipose tissue after a cryopreservation cycle. However, we observed a tendency towards improvement of recovery rate when human albumin was added to the medium. Conclusions None of the studied conditions proved superior to others in terms of cell vitality after a cryopreservation cycle. Hence, we conclude that the cryopreservation of stromal cells derived from adipose tissue in an environment that combines DMEM/F12 with 10% DMSO + 7.6% Trehalose + human albumin 10% does not achieve a significantly higher recovery rate than only frozen solely with DMSO 10%.
Subject(s)
Humans , Cryopreservation/methods , Cell Survival/drug effects , Stromal Cells/physiology , Cryoprotective Agents/pharmacology , Trehalose/pharmacology , Dimethyl Sulfoxide/pharmacology , Adipose Tissue/cytology , Serum Albumin, Human/pharmacology , FreezingABSTRACT
Many researchers have focused on the role of adipocytes in increasing efficient bone tissue engineering and osteogenic differentiation of stem cells. Previous reports have not reached a definite consensus on whether adipocytes positively influence in vitro osteogenic differentiation and in vivo bone formation. We investigated the adipocyte influence on osteogenic differentiation from adipose-derived stromal cells (ADSCs) and bone formation through histological analysis in vitro and in vivo. Using the direct co-culture system, we analyzed the influence of adipocytes to promote the differentiation fate of ADSCs. Using co-transplantation of ADSC-derived adipocytes and osteoblasts into the dorsal region of mice, the osteogenesis and bone quality were determined by histological morphology, radiography, and the measurement of the Ca²⁺ concentration. The adipocyte negatively affected the osteoblast differentiation of ADSCs in the in vitro system and induced osteogenesis of osteoblasts in the in vivo system through co-transplantation. Interestingly, in the co-transplanted adipocytes and osteoblasts, the bone formation areas decreased in the osteoblast only group compared with the mixed adipocytes and osteoblast group 6 weeks after transplantation. Conversely, co-transplantation and osteoblast transplantation had similar degrees of calcification as observed from radiography analysis and the measurement of the Ca²⁺ concentrations. Our results revealed that adipocytes inhibited osteoblast differentiation in vitro but enhanced the efficacy of osteogenesis in vivo. In addition, the adipocytes controlled the activity of osteoclasts in the newly formed bone tissue. Our approach can be used to reconstruct bone using stem cell-based tissue engineering and to enhance the understanding of the role adipocytes play.
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Animals , Mice , Adipocytes , Bone and Bones , Coculture Techniques , Consensus , In Vitro Techniques , Osteoblasts , Osteoclasts , Osteogenesis , Radiography , Stem Cells , Stromal Cells , Tissue EngineeringABSTRACT
Objective To evaluate the feasibilty of modified osteogenic culturing of goat adipose derived stromal cells(ADSCs).Methods From March,2013 to September,2014,platelet-rich plasma(PRP) was made from goat autogenous vein blood,and abodimoneal fat was aspirated,following aspetic procedure,primary and series passage of ADSCs was established.Osteoinduced ADSCs was carried out according to following group:combinative osteoinduction group(PRP+rhBMP-2 +ADSCs),growth factor group(rhBMP-2+ADSCs),conventional inductive group(dexamathasone+ascorbic acid + ADSCs) and noninductive group(blank group).Converted microscope was used to observe cellular pattern,cell activity,osteocalcin and collagen type Ⅰ level were detected at 1,3,5,7,9,13,17,21 days.Red Alazarin and Von Kossa staining were also assayed at different interval.Results Under observation of converted microscopy,remarkable cell proliferation with abundant ECM was noticed in combinative osteoinduction group,compared with other groups,level of cell activity,osteocalcin,collagen type Ⅰ [(0.82 ± 2.19)AU,(79.82 ± 1.36)U/L at,(78.51 ± 4.32)μ g/ml at]were significantly higher than other groups (P ＜0.05),and remarkable ALP expression and calcifed nodules were also seen.Conclusion PRP can enhance the inductive effect of rhBMP-2,and combinative osteoinduction procedure acts as a satisfactory culturing method.
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Objective To explore the labeling method of rat adipose-derived stromal cells,and observe the stem cell characteristics and the activities of EGFP-positive adipose-derived stromal cells (EGFP-ADSCs) in vitro and in vivo.Methods ADSCs were transfected for 12 h with enhanced green fluorescent protein gene (EGFP) carried by lentivirus(Lv-EGFP) vector at different value of MOI (0,5,10,25,50,100,respectively).The rate of EGFP expression and fluorescence intensity were evaluated by flow cytometric analysis and fluorescence microscopy,and cell viability was detected by MTT-test after transfection.Secondly,cells were exposed either to adipogenic medium or osteogenic medium,then stained with Oil Red O and Alizarin Red S.Cell growth was investigated on frozen longitudinal sections when EGFP-ADSCs were injected into acellular nerves to build tissue-engineered peripheral nerves repairing sciatic nerve defects in rats for 1 week in vivo.Results EGFP-positive rate and fluorescence intensity peak at 4 days after transfection.The rate of EGFP expression was 0.13％,31.09％,75.33％,92.66％,96.70％,98.38％ for MOI =0,1,5,25,50,100,respectively.The positive rate between the experimental group and control (MOI =0) existed significantly difference (P ＜ 0.05) ; the difference between MOI =1,5 groups and MOI =25,50,100 groups were also observed (P ＜ 0.05).There was no statistical difference in EGFP-positive rate and cell proliferation activity among MOI =25,50,100 groups (P ＞ 0.05).MOI =25 was chosen as best scheme to transfect ADSCs for subsequent experiments.Osteogenic and adipogenic differentiation for 20 days,orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red O staining.At 1 week in vivo,EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections.Conclusion Lv-EGFP transfection does not affect the ADSCs activity and their osteogenic and adipogenic differentiation,so could be as a tracing method for ADSCs-tissue-engineered peripheral nerves repairing nerve defects.
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@#ObjectiveTo evaluate the feasibility of using injective chitosan scaffold and induced- adipose-derived stromal cells(ADSCs) to construct tissue engineered injectable nucleus pulposus (NP).MethodsADSCs were harvested from rabbits to culture 3 passage and induce 2 weeks to NP-like cells. The injective chitosan hydrogel scaffold was made of chitosan and disodium β-glycerophosphate. Its physical properties and gross condition were observed. The tissue engineered NP was constructed by compounding the scaffold and induced-ADSCs. Then, the viability of ADSCs in the scaffold was observed 2 days after compound culture and the growth condition of ADSCs on the scaffold was observed by scanning electron microscope (SEM) 14 days after compound culture. Expression of aggrecan and Col Ⅱ mRNA in ADSCs were analyzed by RT-PCR 14 days after inductive culture and compound culture.ResultsThe injective chitosan hydrogel was liquid at room temperature and solidified into gel at 37 ℃ (10～15 minutes) due to crosslinking reaction. Acridine orange/propidiumiodide staining showed that the viability rate of induced-ADSCs in chitosan scaffoldl was above 90%. Scanning electron microscope observation demonstrated that the ADSCs were distributed in the reticulate scaffold. RT-PCR results showed that the expression of Col Ⅱ and aggrecan mRNA in induced-ADSCs demonstrated differentiation of ADSCs to a phenotype which showed similarities to NP cells, and the co-culture NP-like cells with scaffold didn`t cause dedifferentiation.ConclusionWith good cellular compatibilities, C/Gp scaffold may be a potential NP cells carrier for tissue engineered NP.
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Objective To evaluate the biological potential of surface topography of biomimetic matrix combined PRP gels with RGD modification. Methods Surface topography of nβ-TCP/Cs/PCL matrix was made by Nd: YAG laser, tissue-engineered bone was constructed in the following ways: ADSCs were loaded to nβ-TCP/Cs/PCL matrix with PRP gels plus RGD modification (group A), ADSCs were cultured to nβ-TCP/Cs/PCL matrix with RGD modification (group B), ADSCs implantation with topography-surfaced treatment of matrix (group C), ADSCs cultivation with smooth-surfaced treatment of matrix (group D). SEM and CLM were used to observe cellular pattern, survival rate, cell activity, ALP and collagen type Ⅰ level were detected at 1, 4, 8, 12, 16, 20, 24,28 days. Runx2 and OPG expressions were assayed at different interval. Results Under observation of SEM and CLM, new tissue showed more remarkable cell proliferation with abundant ECM in group A. Compared with other groups, the survival rate in group A was significantly higher (88.16 ± 1.29, P ＜ 0.05),and the level of cell activity, ALP, collagen type Ⅰ were significantly higher (0.92± 0.13, 87.27 ± 3.08, 93.27 ± 3.91, P＜ 0.05), and remarkable expression of Runx2 and OPG was also seen. Conclusion Topography-surfaced treatment of matrix combined PRP gels with RGD modification enhances the cell proliferation and acts as a feasible osteopromotive method.
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PURPOSE: Adipose-derived stromal cells(ADSCs) are multipotent cells that have been found to promote wound healing through the process of angiogenesis and re-epithelialization. Generally, it is well known that the antigenicity of ADSCs doesn't affect stem cell therapy. In this study, we investigated the effect of allogeneic ADSCs in the wound healing process by applying allogeneic ADSCs on the wound healing splint model of mice. METHODS: Adipose tissue was harvested from the epididymal fat pads of BALB/c and C57BL/6 mice. Twenty four mice BALB/c were divided into three groups; control, isogeneic, and allogeneic groups. Two full thickness defects with 6mm diameters were created on the back of BALB/c mice. 1x10(6) ADSCs from BALB/c mice were applied on the isogeneic group. In the allogeneic group, ADSCs from the C57BL/6 mice were applied. No cells were applied to the control group. The sizes of the wounds were evaluated in 3, 5, 7, 10, and 14 days after the wounds were applied, and tissues were harvested in 7 and 14 days for histological analysis. RESULTS: Wound healing rates had showed significant increase in 10, and 14 days when the isogeneic group was compared to the control group, but the allogeneic group showed significantly decrease compared to the isogeneic group(p<0.05). Histological scores in the isogeneic group were significantly high, but significantly lower in the allogeneic group when compared to the isogeneic group in 2 weeks(p<0.05). In the isogeneic group, thick inflammatory cell infiltration with abundant capillaries were observed in 1 week, and thick epithelium with many large capillaries were observed in 2 weeks. CONCLUSION: When isogeneic ADSCs were applied to wounds, they presented a faster wound healing rate compared to controls and the allogeneic group. Unlike general stem cell therapy, these findings suggest that cell therapy targeted at enhancing wound healing may benefit from the use of ADSCs with identical antigenicity, as opposed to allogeneic or xenogenic ADSCs.
Subject(s)
Animals , Mice , Adipose Tissue , Capillaries , Epithelium , Re-Epithelialization , Splints , Stem Cells , Stromal Cells , Cell- and Tissue-Based Therapy , Wound HealingABSTRACT
This study aimed to induce the differentiation of isolated and purified adipose-derived stro-mal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphol-ogy was observed under an inverted microscope. Ultrastructural changes occurring during the differen-tiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression ofdesmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organeUes were observed and the cytoplasm was found to contain many mito-chondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohisto-chemical staining revealed different levels ofdesmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results con-firmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.
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@#Objective To analyze the osteogenic potential of tissue-engineered bone in vitro,and to evaluate an effective way in constructing tissue-engineered bone.Methods Tissue-engineered bone was constructed in the following ways:ADSCs were transfected with BMP-2 gene plus static seeding and static culture (group A),ADSCs were encapsulated with gels containg BMP-2 plus oscillating seeding and static culture (group B),gels encapsulation with BMP-2 gene and oscillating seeding(group C),gels encapsulation with BMP-2 and static seeding(group D).SEM and CLM were used to observe cellular pattern,apoptosis rate,cell activity,ALP level were detected at 1,4,8,12,16,20,24,28 days.New bone formation were observed after transplantation of bone construct in nude mice. Results Under observation of SEM and CLM. new tissue showed more remarkable cell proliferation with abundant ECM in group C,compared with other groups,the apoptosis rate in group C was significantly lower(P＜0.05),and level of cell activity,ALP,DNA and OCN were significantly higher(P＜0.05). Conclusion Gels encapsulation of BMP-2 gene combined with oscillating seeding promotes the cell augmentation and improves the quality of tissue-engineered bone.
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PURPOSE: Skin and soft tissue defect is one of the major challenges faced by plastic surgeons. Adipose derived stromal cells, which can be harvested in large quantities with low morbidity, display multilineage mesodermal potential. Therefore, adipose derived stromal cells have been met with a great deal of excitement by the field of tissue engineering. Recently, Adipose derived stromal cells have been isolated and cultured to use soft tissue restoration. In order to apply cultured cells for clinical purpose, however, FDA approved facilities and techniques are required, which may be difficult for a clinician who cultures cells in a laboratory dedicated to research to utilize this treatment for patients. In addition, long culture period is needed. Fortunately, adipose derived stromal cells are easy to obtain in large quantities without cell culture. The purpose of this study is to present a possibility of using uncultured adipose derived stromal cells for wound coverage. METHODS: Seven patients who needed skin and soft tissue restoration were included. Five patients had diabetic foot ulcers, 1 patient got thumb amputation, and 1 patient had tissue defect caused by resection of squamous cell carcinoma. The patients' abdominal adipose tissues were obtained by liposuction. The samples were digested with type I collagenase and centrifuged to obtain adipose derived stromal cells. The isolated adipose derived stromal cells were applied over the wounds immediately after the wound debridement. Fibrin was used as adipose derived stromal cells carrier. Occlusive dressing was applied with films and foams and the wounds were kept moist until complete healing. RESULTS: One hundred to one hundred sixty thousand adipose derived stromal cells were isolated per ml aspirated adipose tissue. All patients' wounds were successfully covered with the grafted adipose derived stromal cells in a 17 to 27 day period. No adverse events related to this treatment occurred. CONCLUSION: The use of uncultured adipose derived stromal cells was found to be safe and effective treatment for wound coverage without donor site morbidity.
Subject(s)
Humans , Adipose Tissue , Amputation, Surgical , Carcinoma, Squamous Cell , Cell Culture Techniques , Cells, Cultured , Collagenases , Debridement , Diabetic Foot , Fibrin , Lipectomy , Mesoderm , Occlusive Dressings , Quaternary Ammonium Compounds , Skin , Stromal Cells , Thumb , Tissue Donors , Tissue Engineering , Transplants , Ulcer , Wound HealingABSTRACT
Objective To evaluate the effectiveness of using adipose-derived stromal cells (ADSCs)into a tissue-engineered peripheral nerve on bridging sciatic nerve gaps.Methods Forty-eight F344 female rats weighing 200 - 220 g were randomly divided into 6 groups of nerve grafting to repair 15 mm long asiatic nerve lesions,with 8 mrs in each group.Group A:ADSCs-laden acellular nerves;group B:differentiated ADSCs-laden acellular nerves;group C:Schwann cells-laden acellular nerves;group D:acellular nerves without cells;group E:autografts;group F:nonimplanted grafts.The effects were evaluated in terms of electrophysiulogy,Fluorogold retrograde tracing,histology and tracking studies.Results At 12 weeks after surgery,there was no graft bridging nerve gap in nonimplanted grafts.All the examinations of group A and B were better than group D,respectively (P＜0.05 or P＜0.01).But there were no statistically significant differences among group A,B,C,and D (P＞0.05).Conclusion ADSCs and differentiated ADSCs could promote nerve regeneration when used as seed cells to build tissue-engineered peripheral nerves with acellular nerve scaffolds.
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Objective To investigate the applied value of adipose-derived stromal cells(ADSCs) transfected glial cell-derived neurotrophic factor(GDNF)by lentivirus in cerebral transplantation.Methods Primarily isolated and cultured, the rat ADSCs were transfected GDNF by high-titer lentivirus. Then, configuration of cells and expression of neuron-specific enolase (NSE) were observed. By stereotaxic frame, transfected ADSCs were intrastriatally transplanted.1month and 2month after transplantation, survival and migration of marked cells were detected. GDNF expression, verified by Western blot, was compared with control group.Results ADSCs, transfected by lentivirus, can differentiate into neuron-like cells and express NSE in cytolymph. 1 month after transplantation, the majority of cells survived in transplanting area. 2 month later, part of transplanted cells still survived and migrated far from the transplanting area. In addition, the level of GDNF protein in the brain tissue was significantly higher compared with control group (all P