Study On The Biological Characteristics Of Human Adipose-derived Pericytes/Perivascular Cells And Their Support For Hematopoietic Stem/Progenitor Cells In Vitro

Objective To investigate the feasibility of obtaining and purifying vascular adventitia cells from human adipose tissue,and perform general biological characteristics and analysis of the isolated human adipose-derived pericyte/perivascular cells(CD146~+hAD-PCs)To identify the characteristics of mesenchymal stem cells,establish an in vitro co-culture system to explore whether they have the ability to maintain cord blood(UCB)CD34~+hematopoietic stem/progenitor cells(HSPCs)in vitro expansion.Methods 1.Human adipose-derived adventitia cells CD146~+hAD-PCs were isolated and purified from human omentum adipose tissue by a combination of enzymatic hydrolysis and multi-parameter flow cytometry(MFC)and expanded in vitro.2.Biological identification of CD146~+hAD-PCs:(1)Continuously observe the cell morphology and proliferation activity of CD146~+hAD-PCs at different passages by CCK-8 method.(2)The expression of CD146~+hAD-PCs cell surface markers was detected by MFC.(3)Induction of CD146~+hAD-PCs into adipose,osteogenic and chondrogenic differentiation to identify their multi-directional differentiation potential in vitro.3.CD34~+HSPCs were sorted from human cord blood tissue(UCB)by density gradient centrifugation and immunomagnetic bead sorting.The blank group only cultured UCB CD34~+HSPCs,the positive control group used BM-MSCs as stromal cells co-cultured with UCB CD34~+HSPCs,and the experimental group used CD146~+hAD-PCs as stromal cells co-cultured with UCB CD34~+HSPCs.The number of expanded UCB CD34~+HSPCs,the colony-forming ability of expanded human cord blood UCB CD34~+HSPCs and the expression of hematopoietic markers were analyzed and counted,and the supernatant of different culture systems was subjected to ELISA to detect the secretion of hematopoietic factors when different stromal cells were co-cultured,During 1week,2 weeks and 4 weeks of co-cultivation.Results 1.CD146~+hAD-PCs can be obtained from human adipose tissue by enzymatic hydrolysis and multi-parameter flow cytometry.In vitro culture can be stably passaged and the purity is(92.08±0.86)%,(n=5).2.The results of biological characteristics show:(1)The CCK-8 test results showed that the CD146~+hAD-PCs of the P6 and P9 generations were in the logarithmic growth phase on the 4rd day,and the growth curve was similar to the"S"type.The proliferation activity of P12 generation cells was poor,which was significantly different from that of P6 generation cells(P<0.01,n=5).(2)MFC detected that CD146~+hAD-PCs highly expressed MSCs surface markers CD44,CD73,CD90,CD105.(3)CD146~+hAD-PCs can differentiate into adipocytes,osteoblasts and chondrocytes in vitro.3.The results of in vitro co-culture of UCB CD34~+HSPCs showed:(1)Analysis of UCB CD34~+HSPCs proliferation:The number of viable cells in the experimental group(CD146~+hAD-PCs group)and the positive control group(BM-MSCs group)increased slightly in the first week,while the number of cells in the blank group(without stromal cell group)decreased,and there was no change in the number of cells Statistical difference(P>0.05,n=5);In the second week,the number of cells in the CD146~+hAD-PCs group and BM-MSCs group continued to increase and reached a peak,while the number of cells in the stromal cell-free group continued to decrease.The difference was statistically significant(P<0.05,n=5);In the 4th week,the number of cells in the CD146~+hAD-PCs group and the BM-MSCs group decreased slightly,and all cells in the stromal cell-free group died,with a significant difference(P<0.01,n=5),CD146~+hAD-PCs group and BM-MSCs group had no significant difference in cell count at each time point(P>0.05,n=5).(2)UCB CD34~+HSPCs colony formation results show:The number of colonies formed in the CD146~+hAD-PCs group and the BM-MSCs group was not statistically different at each time point(P>0.05,n=5).(3)Analysis of the expression of blood cell markers:There was no significant difference between CD146~+hAD-PCs group and BM-MSCs group by MFC in the expression ratios of CD45~+,CD34~+CD33~-,CD10~+/CD19~+and CD14~+for 1,2,and 4 weeks of co-culture(P>0.05,n=5).(4)Analysis of cytokine secretion:The CD146~+hAD-PCs group had higher G-CSF than BM-MSCs in the first and second weeks of SCF,and the difference was statistically significant(P<0.01,n=4);IL-2 in the CD146~+hAD-PCs group was expressed at 2 weeks,and the expression levels of G-CSF,VEGF and SCF at 4 weeks were lower than those in the BM-MSCs group,the difference was statistically significant(P<0.05 or 0.01,n=4);between the CD146~+hAD-PCs group and the BM-MSCs group There was no significant difference in the expression of IL-3,IL-6,TPO,IFN-?and TNF-?(P>0.05,n=4).Because the blank group cells did die rapidly without stromal cell trophoblast cells,statistical analysis of colony formation,blood cell marker expression and cytokine expression was not performed.Conclusion Human adipose tissue can be used to separate high-purity CD146~+hAD-PCs through collagenase digestion and multi-parameter flow cytometry.It can be stably cultured and passaged in vitro,and has the characteristics of mesenchymal stem cells.It is used as a stromal cell in vitro to support UCB CD34~+HSPCs,which has the same support ability as BM-MSCs.