ObjectiveTo summarize the isolation procedures, molecular characterization, and differentiation and vascularization capacity of adipose-derived stem cells (ADSCs), in order to discuss the potential value of ADSCs for the repairment and regeneration of adipose tissues. MethodsRelated literatures about ADSCs were retrieved to summarize the potential value of ADSCs for the repairment and regeneration of adipose tissues. ResultsAs mesenchymal stem cells, ADSCs was rich in human adipose tissues. ADSCs possessed the potential to differentiate toward a variety of cell lineages, such as adipogenic, chondrogenic, osteogenic, cardiomyogenic, myogenic, and angiogenic. Besides, its capacity of adipogenic differentiation could maintain several passages. The most importantly, ADSCs could secrete significant amounts of angiogenesis-related cytokines, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), which increased the angiogenesis of adipose tissue. ConclusionsADSCs play a key role in adipose tissue engineering, autologous adipose tissue grafting, and soft tissue wound repairing, which have important application prospect for breast reconstruction.
ObjectiveTo study the feasibility of human adipose-derived stem cells (hADSCs) combined with small intestinal submucosa powder (SISP)/chitosan chloride (CSCl)-β-glycerol phosphate disodium (GP)-hydroxyethyl cellulose (HEC) for adipose tissue engineering. MethodshADSCs were isolated from human breast fat with collagenase type I digestion, and the third passage hADSCs were mixed with SISP/CSCl-GP-HEC at a density of 1×106 cells/mL. Twenty-four healthy female nude mice of 5 weeks old were randomly divided into experimental group (n=12) and control group (n=12), and the mice were subcutaneously injected with 1 mL hADSCs+SISP/CSCl-GP-HEC or SISP/CSCl-GP-HEC respectively at the neck. The degradation rate was evaluated by implant volume measurement at 0, 1, 2, 4, and 8 weeks. Three mice were euthanized at 1, 2, 4, and 8 weeks respectively for general, histological, and immunohistochemical observations. The ability of adipogenesis (Oil O staining), angiopoiesis (CD31), and localized the hADSCs (immunostaining for human Vimentin) were identified. ResultsThe volume of implants of both groups decreased with time, but it was greater in experimental group than the control group, showing significant difference at 8 weeks (t=3.348, P=0.029). The general observation showed that the border of implants was clear with no adhesion at each time point;fat-liked new tissues were observed with capillaries on the surface at 8 weeks in 2 groups. The histological examinations showed that the structure of implants got compact gradually after injection, and SISP gradually degraded with slower degradation speed in experimental group;adipose tissue began to form, and some mature adipose tissue was observed at 8 weeks in the experimental group. The Oil O staining positive area of experimental group was greater than that of the control group at each time point, showing significant difference at 8 weeks (t=3.411, P=0.027). Immunohistochemical staining for Vemintin showed that hADSCs could survive at each time point in the experimental group;angiogenesis was most remarkable at 2 weeks, showing no significant differences in CD31 possitive area between 2 groups (P>0.05), but angiogenesis was more homogeneous in experimental group. ConclusionSISP/CSCl-GP-HEC can use as scaffolds for hADSCs to reconstruct tissue engineered adipose.
Objective To explore the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on the proliferation, migration, and tube-like differentiation of human umbilical vein endothelial cells (HUVECs). Methods Adipose tissue voluntarily donated by liposuction patients was obtained. The ADSCs were harvested by enzyme digestion and identified by flow cytometry and adipogenic induction. The ADSC-Exos were extracted from the supernatant of the 3rd generation ADSCs and the morphology was observed by transmission electron microscopy. The surface proteins (Alix and CD63) were detected by Western blot. The nanoparticle tracking analyzer NanoSight was used to analyze the size distribution of ADSC-Exos. After co-culture of PKH26 fluorescently labeled ADSC-Exos with HUVECs, confocal microscopy had been used to observe whether ADSC-Exos could absorbed by HUVECs. ADSC-Exos and HUVECs were co-cultured for 1, 2, 3, 4, and 5 days. The effect of ADSC-Exos on the proliferation of HUVECs was detected by cell counting kit 8 (CCK-8) assay. The expression of VEGF protein in the supernatant of HUVECs with or without ADSC-Exos had been detected by ELISA after 12 hours. Transwell migration assay was used to detect the effect of ADSC-Exos on the migration ability of HUVECs. The effect of ADSC-Exos on the tubular structure formation of HUVECs was observed by Matrigel experiments in vitro. The formation of subcutaneous tubular structure in vivo was observed in BALB/c male nude mice via the injection of HUVECs and Matrigel with or without ADSC-Exos. After 2 weeks, the neovascularization in Matrigel was measured and mean blood vessel density (MVD) was calculated. The above experiments were all controlled by the same amount of PBS. Results After identification, the cultured cells were consistent with the characteristics of ADSCs. ADSC-Exos were circular or elliptical membranous vesicle with uniform morphology under transmission electron microscopy, and expresses the signature proteins Alix and CD63 with particle size ranging from 30 to 200 nm. Confocal microscopy results showed that ADSC-Exos could be absorbed by HUVECs. The CCK-8 analysis showed that the cell proliferation of the experimental group was better than that of the control group at each time point (P<0.05). The result of Transwell showed that the trans-membrane migration cells in the experimental group were significantly more than that in the control group (t=9.534, P=0.000). In vitro, Matrigel tube-forming experiment showed that the number of tube-like structures in the experimental group was significantly higher than that of the control group (t=15.910, P=0.000). In vivo, the MVD of the experimental group was significantly higher than that of the control group (t=16.710, P=0.000). The ELISA assay showed that the expression of VEGF protein in the supernatant of the experimental group was significantly higher than that of the control group (t=21.470, P=0.000). Conclusion ADSC-Exos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesisin vitro and in vivo.
Objective To review the latest progress in the major biological properties of adipose-derived stem cells (ADSCs) and ADSCs assisted autologous lipotransfer in breast repair and reconstruction. Methods Recent literature about ADSCs assisted autologous lipotransfer in breast repair and reconstruction was reviewed. Results ADSCs have multipotential differentiation capacity, and they could promote angiogenesis and regulate immune reactions. ADSCs assisted autologous lipotransfer can obtain satisfactory effectiveness in breast repair and reconstruction with few complications, but more studies are needed to confirm the long-term safety. Conclusion ADSCs assisted autologous lipotransfer has good effectiveness in breast repaired and reconstruction. But further clinical trials are needed to confirm the long-term safety.
Objective To observe the systemic and local immune response after repair of nerve defect with acellular nerve xenograft laden with allogenic adipose-derived stem cells (ADSCs) in rhesus monkey so as to evaluate the safety of the proposed material for nerve reconstruction. Methods Bilateral tibial nerves were taken from a healthy adult male landrace (weighing 48 kg) to prepare acellular nerve xenograft by chemical extraction. ADSCs were isolated from a healthy adult male rhesus monkey (weighing 4.5 kg), and were seeded into the acellular nerve grafts. The radial nerve defect models with 25 mm in length were established in 10 healthy adult female rhesus monkeys (weighing 3-5 kg), and they were divided into cell-laden group (n=5) and non-cell-laden group (n=5) randomly. Defect was repaired with acellular nerve xenograft laden with allogenic ADSCs in cell-laden group, with acellular nerve xenograft only in non-cell-laden group. The blood samples were taken from peripheral vein preoperatively and at 14, 60, and 90 days after operation for lymphocyte analysis; at 5 months after operation, the grafts were harvested to perform histological examination for local immune response and nerve regeneration. The nerve autograft in rhesus monkey was used as control. Results In cell-laden group and non-cell-laden group, no significant difference was found in the count of lymphocytes and T lymphocytes, the percentage of T lymphocytes, CD8+ T lymphocytes, as well as the ratio of CD4+ T lymphocytes to CD8+ T lymphocytes between pre- and post-operation (P gt; 0.05); in cell-laden group, the percentage of CD4+ T lymphocytes at 14 days was significantly lower than that at 60 and 90 days postoperatively (P lt; 0.05). The percentage of CD4+ T lymphocytes in cell-laden group was significantly lower than that in non-cell-laden group at 14 days (P lt; 0.05), but no significant difference was found in the other indexes at the other time between 2 groups (P gt; 0.05). At 5 months after operation, mild adhesion was found on the surface of nerve xenografts; the epineurium of nerve xenografts was thicker than that of nerve autografts; and neither necrosis nor fibrosis was found. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were scattered within the grafts, in which regenerative axons were revealed. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were comparable in cell-laden group, non-cell-laden group, and autograft group. Conclusion Repair of nerve defect with acellular nerve xenograft elicits neither systemic nor local immune response in rhesus monkeys. Implantation of allogenic ADSCs might result in transient depression of CD4+ T lymphocytes proliferation early after surgery, no immune response can be found.
ObjectiveTo investigate the effect of adipose-derived stem cells (ADSCs) combined with chitosan on the immediate retraction rate of rabbit expanded skin. MethodsADSCs were isolated from rabbit fresh fat under sterile conditions and cultured to the 3rd generation by methods of enzymatic digestion; the specific surface markers and the differentiation into epidermal cells and cartilage cells were identified. Forty New Zealand white rabbits (aged, 2-3 months) were randomly divided into 4 groups (n=10): control group (group A), ADSCs group (group B), chitosan group (group C), and ADSCs+chitosan group (group D). ADSCs cell suspension with the concentration of 5×106 cells/mL was prepared. The skin expansion model was made by embedding 30 mL dilator into the back of rabbit. Chitosan (2%, 5 mL) was coated on the surface of the dilator in groups C and D, and ADSCs cell suspension (1 mL) was injected into the skin in groups B and D. Conventional tissue expansion was performed to expected capacity at 4 weeks, and maintained for 1 week. The expanded skin was harvested to measure the immediate retraction rate, and the thickness of skin, epidermis, and fibrous capsule with HE staining. Masson staining was used to observe the characteristics of collagen in the fibrous capsule, and immunohistochemical staining for CD31 to determine the microvessel density (MVD). ResultsADSCs were successfully isolated, and had multiple differentiation ability. All the animals survived to the end of the experiment. The immediate retraction rate of group D was significantly lower than that of the other groups (P<0.05), groups B and C were significantly lower than group A (P<0.05), and group B was significantly lower than group C (P<0.05). The histological staining revealed that there were more mature fibroblasts and coarse collagen fibers with regular arrangement in groups A and B; there were more naive fibroblasts and tiny and sparse collagen fibers in groups C and D. The thickness of skin and epidermis, and MVD of groups B and D were significantly larger than those of groups A and C (P<0.05); the thickness of fibrous capsule of groups C and D was significantly less than that of groups A and B (P<0.05); but no significant difference was found in the above indexes between other groups (P>0.05). ConclusionADSCs can promote angiogenesis and regeneration of the expanded skin, have no effect on the fibrous capsule. Chitosan can inhibit the proliferation of fibrous capsule, so a combination of ADSCs and chitosan can inhibit the immediate retraction of the expanded skin.
ObjectiveTo study the inducting differentiation effect of the sciatic nerve extracts on rabbit adipose-derived stem cells (ADSCs) in vitro. MethodsThe ADSCs were isolated from 2 healthy 4-month-old New Zealand rabbits (weighing, 2.0-2.5 kg) and cultured to passage 3, which were pretreated with 10 ng/mL basic fibroblast growth factor (bFGF) for 24 hours before induction. Then the induction media containing the extracts of normal sciatic nerve (group B) and injured sciatic nerve at 3, 7, and 14 days (group C, group D, and group E) were used, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed. At 7 days of induction, the gene expressions of neuron-specific enolase (NSE), nestin (NES), and S-100 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunocytochemical staining. ResultsAt 4 days after induction, some ADSCs of groups C, D, and E showed the morphology of Schwann-like cells or neuron-like cells, the change of group D was more obvious; and the ADSCs of group A and B had no obvious change, which were still spindle. The S-100 immunocytochemical staining showed positive expression in groups C, D, and E (more obvious in group D) and negative expression in groups A and B. The gene expression of S-100 displayed time-dependent increases in groups C and D, which was significantly higher than that of groups A, B, and E (P<0.05), but no significant difference was found between groups C and D (P>0.05). The gene expression of NSE showed the same tendency to S-100, which reached the peak in group D; the gene expression of NSE in groups D and E was significantly higher than that of groups A, B, and C (P<0.05), and groups D and E showed significant difference (P<0.05). However, the gene expression of Nestin showed no significant difference among different groups (P>0.05). ConclusionThe ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; and the early stage (3-7 days) after injury is the best time for stem cell transplantation.
ObjectiveTo investigate the differentiation of rat adipose-derived stem cells (ADSCs) into neuronlike cells by indirect co-culture with Schwann cells (SCs) in vitro so as to look for the ideal seed cells for tissue engineering. MethodsSCs were isolated from sciatic nerves of 1-2 days old Sprague-Dawley rats with enzymatic digestion method. Immunofluorescence staining was used to identify SCs with the marker S-100. ADSCs were isolated from the epididymal fat pads of adult male Sprague-Dawley rats by means of differential attachment. And the cell phenotypes (CD29, CD34, CD45, CD73, CD90, and CD105) of ADSCs at passage 3 were determined by flow cytometry analysis. Primary SCs and ADSCs at passage 3 were co-cultured at a ratio of 2:1 in Transwell culture dishes (experimental group), and ADSCs cultured alone served as control group. Immunofluorescence and flow cytometry were adopted to investigate the neural differentiation of ADSCs at 14 days. The expression differences for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), neuronal nuclei protein (NeuN), and glial fibrillary acidic protein (GFAP) were detected, and the percentage of positive cells was calculated. ResultsADSCs were successfully extracted and can passage in a considerable large amount. Flow cytometry analysis showed that ADSCs at passage 3 were positive for CD29, CD90, CD73, and CD105 expression, but negative for CD34 and CD45 expression. The ADSCs of the experimental group showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body. The cell shape had no obvious change in the control group. Both immunofluorescence and flow cytometry analysis results showed the expressions of MAP2, NSE, NeuN, and GFAP at 14 days after co-cultured with SCs, and the positive cell ratios were significantly higher than those in the control group (P<0.01). ConclusionCo-culture with SCs not only can promote the survival regeneration of ADSCs, but also can induce the differentiation of ADSCs into neuron-like cells.
Objective To introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared with BMSCs which are commonly used as adult stem cells, ADSCs and DFAT cells have extensive sources and can be easily acquired. While compared with ADSCs, DFAT cells have good homogeneity and b proliferation capacity. Conclusion As a potential source of stem cells, adipose tissue will provide a new promising for regenerative medicine.
Objective To evaluate the synergistic effect of bone morphogenetic protein 14 (BMP-14) and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells (ADSCs) so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits (weighing, 1.5 kg and 2.0 kg). The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction (alizarin red staining), chondrogenic induction (alcian blue staining), and adipogenic induction (oil red O staining). The optimum multiplicity of infection (MOI) of transfection of adenovirus-cytomegalovirus (CMV)-BMP-14-internal ribosome entry site (IRES)-human renilla reniformis green fluorescent protein 1 (hrGFP-1) was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups: group A, co-culture of ADSCs transfected by BMP-14 and chondrocytes (1 ∶ 1 in Transwell chambers); group B, co-culture of ADSCs and chondrocytes (1 ∶ 1 in Transwell chambers); group C, culture of ADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan (GAG) content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C (P lt; 0.05), and groups B and D were significantly higher than group E (P lt; 0.05), but no significant difference was found between groups B and D (P gt; 0.05). Conclusion It can promote differentiation of ADSCs into chondrocytes by BMP-14 co-culture with chondrocytes, and they have a synergistic effect.