Abercrombie and,Human adipose stem cells to induce

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Human adipose stem cells to induce endothelial differentiation of Establishment of the best

Of: Yang Xufang He Xu Rui Zhang Lihong any prop Zhang Mu Li Yulin
Abstract the purpose of human adipose stem cells (human Adipose  derived stem cells, hADSCs) to differentiate into endothelial cells in vitro induction system for the best. Method using collagenase digestion and adherence screening from human adipose tissue isolation, culture and amplification hADSCs, divided into 3 groups induced endothelial respectively: that of fibronectin (fibronectin, FN) coated group, gelatin coated group and not shop group; also induce liquid divided into two groups: pure endothelial cell growth medium (EGM2  MV) group and the endothelial cell growth medium (EGM2  MV) +50 ng / ml vascular endothelial growth factor (VEGF165) group. 15 d after induction by flow cytometry to detect the endothelial cell-specific expression of surface antigen CD34; observed by phase contrast microscopy before and after induction of general morphological characteristics of cells. Results Isolation, culture, a high degree of homology hADSCs, CD29, CD44, CD90, CD105 and CD166-positive expression, while CD34, CD45 and negative expression of HLA  DR; induced cells showed that flow induced by a simple EGM  2MV 3 cells negative for endothelial cell specific markers CD34 expression, and EGM2  MV +50 ng / ml VEGF165-induced cells, which express CD34 FN coated group was 8.4%, gelatin coated group was 5.4%, did not shop group 4.2%; the difference can be seen under the microscope after the cells were induced by triangular or polygonal, integration at the cobblestone-like growth. Conclusion FN as the extracellular matrix and EGM2  MV +50 ng / ml VEGF165 induction medium composed of synergies, constitute hADSCs vitro differentiation into endothelial cells induced the best system for ischemic disease in clinical autologous cell transplantation therapy source of a large number of seed cells.
Key words of human adipose stem cells; flow cytometry; endothelial differentiation; induction system
】【Abstract Objective To discuss the best method for endothelial differentiation of human adipose  derived stem cells (hADSCs). Methods hADSCs were isolated from human adipose tissue by collagenase digestion and adherence to flasks, divided into three groups and induced into endothelia respectively,[link widoczny dla zalogowanych], including Fibronectin (FN)  coated, gelatin  coated group and non  coated group. At the same time, inducing medium was divided into pure endothelial cell growth medium (EGM2  MV) and endothelial cell growth medium (EGM2  MV) +50 ng / ml vascular endothelial growth factor (VEGF165) groups. After induced 15 d, CD34, which was endothelial cell  specific surface antigen, was detected by flow cytometry. General morphological characteristics of hADSCs and induced hADSCs were observed by phase  contrast microscope. Results A high degree of homology and the CD34 negative hADSCs were succeed to isolate and cultivate. At the end of differentiation, flow results of the three groups induced by simple EGM2  MV all showed CD34 negative expression, but the other three groups induced by EGM2  MV plus 50 ng / ml VEGF165 showed that CD34 expression rate of FN  coated group was 8.4%, gelatin  coated group was 5.6%, not  coated group was 4.2%; Induced hADSCs were triangular or polygonal, paving stone  like growth was observed at the place of local integration. Conclusions Combination of FN  coated and EGM2  MV +50 ng / ml VEGF165 is the best method for endothelial differentiation of hADSCs. The results will offer abundant of seeding cells for autologous cell transplantation therapy of ischemic disease.
】【Key words Human adipose  derived stem cells (hADSCs); Flow cytometry; Endothelial differentiation; Induction system
Ischemic disease is one of the world's common diseases, particularly atherosclerotic vascular disease as the main representative. Currently used drug therapy can not completely solve the narrow blood vessels has already occurred anatomy, and arterial bypass surgery is faced with the long-term patency rate is not ideal and so on,[link widoczny dla zalogowanych], so look for new methods of treatment and late graft ischemic diseases has become an important research subject. Recent years, with the progress of stem cell research, stem cells as seed cells to ischemic tissue angiogenesis (vasculogenesis) provided new opportunities 〔1,2〕, but how convenient and effective access to the seed cells, clinical application of tissue engineered blood vessel an important issue. This study was to investigate the human adipose stem cells (human Adipose  derived stem cells, hADSCs) to induce endothelial differentiation of the best system for a variety of clinical patients with advanced ischemic disease easily obtained, suitable for cultivation in vitro the proliferation of large,[link widoczny dla zalogowanych], non-immune epitope for the seed cells for autologous transplantation.
1 Materials and methods
1.1 Reagents LG  DMEM, 10% high-quality fetal bovine serum (Hyclone, USA), 0.25% trypsin, collagenase Ⅰ (Invitrogen), mouse anti-human monoclonal antibody CD44, CD90 and CD166 (BD, USA ), mouse anti-human monoclonal antibody CD29, CD105,[link widoczny dla zalogowanych], CD34, CD45, HLA  DR (BioLegend, USA), EGM2  MV (Lonza, USA), VEGF165 (Peprotech, USA).
1.2 hADSCs Isolation and culture and identification
1.2.1 hADSCs Isolation and culture of adipose tissue obtained by plastic surgery, repeated washing with sterile PBS solution, carefully remove the connective tissue and blood vessels separated, and finally cut into pieces the size of 1 mm3. Collagenase with 0.1% Ⅰ digested 60 min 37 ℃ 〔3,4〕 cells with conditioned medium obtained (LG  DMEM, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) resuspended 〔5 〕, 37 ℃, 5% CO2, saturated humidity inside the culture incubation, 48 h after the first exchange of medium, every 3 d after the replacement of 1 medium until the cells grew in the culture dish bottom area of 80%, with 0.25% trypsin digestion and passage, the following experiments were taken from the P2 generation of hADSCs.
1.2.2 hADSCs digested collection of phenotypic identification hADSCs, each taking 2 × 105 cells, and mouse anti-human monoclonal antibody CD29, CD44, CD90, CD105,[link widoczny dla zalogowanych], CD166, CD31, C45 and HLA  DR ( 1:100 dilution) were incubated at room temperature 30 min, and FITC-conjugated secondary antibody (1:50 dilution), 4 ℃ dark incubated for 20 min, resuspended with 300 μl PBS, flow cytometry.
1.3 hADSCs system, explore the use of endothelial cells induced by cytokines and extracellular matrix and other factors differentiate into endothelial cells induced by hADSCs. To investigate the cytokines and extracellular matrix on hADSCs the differentiation of endothelial cells, divided into 6 experimental groups were fibronectin (FN) coated with pure endothelial cell growth medium (EGM2  MV) induction group; FN package been with the EGM2  MV +50 ng / ml vascular endothelial growth factor (VEGF165) induced group; gelatin-coated with pure EGM2  MV induction group; gelatin-coated with EGM2  MV +50 ng / ml VEGF165 induced group; no package was induced with simple EGM2  MV group; not coated with EGM2  MV +50 ng / ml VEGF165 induced group. hADSCs by 2 × 103 个 / cm2 cell density of inoculation, medium was changed every 3 d, induced 15 d.
1.4 hADSCs endothelial induced endothelial phenotype after induction, digested cells were collected, respectively, with mouse anti-human monoclonal antibody CD34 (1:100 dilution) were incubated at room temperature 30 min, and FITC-conjugated secondary antibody (1: 50 dilution) 4 ℃ dark incubated 20 min, resuspended with 300 μl PBS, flow cytometry.
1.5 hADSCs endothelial morphology after the induction of endothelial and inducible induction of 4,15 d resurfaced after the end of digestion, respectively, were observed under phase contrast microscope, camera.
2 results
2.1 hADSCs Isolation and culture and identification of visible laser confocal microscope: Typical hADSCs was fibroblast-like cell bodies narrow, elongated spindle-shaped, arranged in order, a certain direction, was fish-like growth. Flow cytometry results showed in Figure 1 after the induction of endothelial CD34 by flow cytometry in Figure 2 at different times during the induction of endothelial cell morphology (× 100)

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