Objective To review research progress of adipose tissuederived stromal cells (ADSCs).Methods The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated.Results A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes,endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate intomultiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells.Conclusion ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.
Objective
To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury.
Methods
ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells.
Results
The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05).
Conclusion
The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene.
MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12.
ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05).
ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
Objective
To introduce the related issues in the clinical translational application of adipose-derived stem cells (ASCs).
Methods
The latest papers were extensively reviewed, concerning the issues of ASCs production, management, transportation, use, and safety during clinical application.
Results
ASCs, as a new member of adult stem cells family, bring to wide application prospect in the field of regenerative medicine. Over 40 clinical trials using ASCs conducted in 15 countries have been registered on the website (http://www.clinicaltrials.gov) of the National Institutes of Health (NIH), suggesting that ASCs represents a promising approach to future cell-based therapies. In the clinical translational application, the related issues included the quality control standard that management and production should follow, the prevention measures of pathogenic microorganism pollution, the requirements of enzymes and related reagent in separation process, possible effect of donor site, age, and sex in sampling, low temperature storage, product transportation, and safety.
Conclusion
ASCs have the advantage of clinical translational application, much attention should be paid to these issues in clinical application to accelerate the clinical translation process.
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 study biological rule of recombinant human bone morphogenetic protein 2 (rhBMP-2) in regulating the expression of vascular endothelial growth factor (VEGF) of adipose-derived stem cells (ADSCs) at different induced concentrations and time points at gene level and protein level.
Methods
ADSCs were separated from adult human adipose tissues and cultured until passage 3. After ADSCs were induced by rhBMP-2 in concentrations of 0, 50, 100, and 200 ng/ mL respectively for 24 hours, and by 100 ng/mL rhBMP-2 for 3, 6, 12, 18, 24, 36, and 48 hours (ADSCs were not induced at corresponding time point as controls) respectively, the VEGF mRNA and protein expressions were detected by RT-PCR and ELISA.
Results
The VEGF mRNA and protein expressions induced by rhBMP-2 were concentration-dependent; the expressions were highest in a concentration of 100 ng/mL. The VEGF mRNA expression in concentrations of 50, 100, and 200 ng/mL were significantly higher than that in a concentration of 0 ng/mL (P lt; 0.05); and the expression in concentration of 100 ng/ mL was significantly higher than that in concentrations of 50 and 200 ng/mL (P lt; 0.05). The VEGF protein expression in a concentration of 100 ng/mL was significantly higher than that in the other concentrations (P lt; 0.05). The VEGF mRNA and protein expressions induced by rhBMP-2 were time-dependent. The VEGF mRNA and protein expressions at 3 and 6 hours after induction were significantly lower than those of non-induced ADSCs (P lt; 0.05); the expressions were lower at 12 hours after induction, showing no significant difference when compared with those of non-induced ADSCs (P gt; 0.05); the expressions reached peak at 18 and 24 hours after induction, and were significantly higher than those of non-induced ADSCs (P lt; 0.05); the expressions decreased in induced and non-induced ADSCs at 36 and 48 hours, showing no significant difference between induced and non-induced ADSCs (P gt; 0.05).
Conclusion
rhBMP-2 adjusts VEGF expression of ADSCs in a concentration- and time-dependent manner. The optimum inductive concentration of rhBMP-2 is 100 ng/mL, induced to 18-24 hours is a key period when rhBMP-2 is used to promote tissue engineering bone vascularization.
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.
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 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.
Objective To find a kind of simple and effective method for purifying and label ing stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for cl inical application of SVFs. Methods The subcutaneous adi pose tissue were harvested form volunteers. The adi pose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase,respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, 1’-dioctadecyl-3, 3, 3’, 3’-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent label ing and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation. Results The number of SVFs was (138.68 ± 11.64) × 104, (183.80 ± 10.16) × 104, and (293.07 ± 8.31) × 104 in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P lt; 0.01). The rates of viable cells were 91% ± 2%, 90% ± 2%, and 81% ± 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P lt; 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs. Conclusion The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.
