To isolate and culture adi pose-derived stem cells (ADSCs), and to study the effects of the conditioned medium of ADSCs (ADSC-CM) treated with insul in on HaCaT cells. Methods ADSCs were isolated from adipose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5 × 104 cells/mL. The cells were divided into 2 groups: group A in which the cells were incubated in 1 × 10-7 mol/ Linsul in for 3 days, and group B in which the cells were not treated with insul in. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor (HGF). HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups: group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1 × 10-7 mol/ L insul in; group D1, 1 mL 2%FBS. Prol iferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration abil ity was measured by in vitro wound-heal ing assay 0, 12, 24, 36 and 48 hours after culture. Results The level of VEGF in groups A and B was (643.28 ± 63.57) and (286.52 ± 46.68) pg/mL, respectively, and the level of HGF in groups A and B was (929.95 ± 67.52) and (576.61 ± 84.29) pg/mL, respectively, suggesting differences were significant between two groups (Plt; 0.05). Cell prol iferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881 ± 0.039, 0.804 ± 0.041, 0.663 ± 0.027 and 0.652 ± 0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05). The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23% ± 1.98%, 8.82% ± 2.59%, 31.70% ± 8.85% and 29.60% ± 8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.05), group B1 was significantly higher than group A1 (P lt; 0.05). The migration distance of HaCaT cells in groups A1, B1,C1 and D1 at 36 hours was (0.184 6 ± 0.019 2), (0.159 8 ± 0.029 4), (0.059 2 ± 0.017 6) and (0.058 2 ± 0.012 3) mm, respectively, whereas at 48 hours, it was (0.231 8 ± 0.174 0), (0.205 1 ± 0.012 1), (0.079 2 ± 0.008 1) and (0.078 4 ± 0.011 7) mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05) at 36 and 48 hours, no significant difference was evident at other time points(P gt; 0.05). Conclusion ADSCs treated with insul in can significantly promote the prol iferation and the migration of HaCaT cells and inhibit their apoptosis.
ObjectiveTo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. MethodsThe inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. ResultsAt 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). ConclusionRecombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.
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.
Objective As one of the adult stem cells, adi pose-derived stem cells (ADSCs) have become an important seed cell source for tissue engineering recently. But whether the thawed cryopreserved ADSCs could be used to tissue engineered bone remains unknown. To investigate the effect of cryopreservation on the growth and osteogenesis of ADSCs invitro. Methods The ADSCs were isolated from the adipose aspirates by collagenase digestion method. For the experimental group, the 2nd generation cells were stored with a simple method of cryopreservation by slow cool ing with dimethyl sulphoxide as a cryoprotectant and rapid thawing. After cryopreserved in l iquid nitrogen for 4 weeks, ADSCs were recovered and cultured in osteogenic media, with non-cryopreserved ADSCs as the control group. The osteogenic differentiation was evaluated by alkal ine phosphatase (ALP) staining and Al izarin red O staining at 2 and 3 weeks respectively. The cell growth and osteogenesis of ADSCs were further determined using DNA assay and the ALP activity and calcium content were measured. Results The survival percentage of the cryopreserved cells was 90.44% ± 2.62%. The cell numbers and ALP activity increased with osteogenic induction time, and reach plateaus at 7 days and 11 days, respectively. The ALP staining and Al izarin red O staining results were both positive at 2 weeks and 3 weeks after osteogenic induction, respectively. And no significant difference in the cells number, ALP activity, and calcium content were found between experimental group and control group (P gt; 0.05). Conclusion Cryopreservation does not affect the growth and osteogenesis of ADSCs, and the cryopreserved ADSCs can be used as cell source for tissue engineered bone.
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.
ObjectiveTo study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. MethodsADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. ResultsThe result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P < 0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. ConclusionADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.
Objective To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phe notype. Methods Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, andCD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Li pofectamineTM 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkal ine phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. Results ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P lt; 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P lt; 0.05). Conclusion The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.
ObjectiveTo review the research progress of adipose-derived stem cells (ADSCs) compound with three dimensional (3D) printing scaffold in tissue engineering of fat, bone, cartilage, blood vessel, hepatocyte, and so on. MethodsThe recently published literature about ADSCs compound with 3D printing scaffold in tissue engineering at home and abroad was reviewed, analyzed, and summarized. ResultsA large number of basic researches showed that ADSCs could differentiate into a variety of tissues on 3D printing scaffold and involve in tissue repair and regeneration. But there is still a long way between the basic theory and the clinical practice at the early stages of development. ConclusionIt can effectively improve and restore the structure and function of the damaged tissue and organ to use ADSCs and 3D printing scaffold.
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.