Biological Characteristics Of Human Skeletal Muscle-derived Pericytes/Perivascular Cells And Their Supporting Effects On Hematopoietic Stem/Progenitor Cell In Vitro

Objective To identify the biological characteristics of pericytes/perivascular cells,which isolated and cultured from human skeletal muscle,and to investigate their supporting effect on umbilical cord blood CD34~+cells in vitro.Methods 1.Human skeletal muscle-derived pericytes/perivascular cells(hMD-PCs)with phenotype CD146~+CD56~-CD34~-CD144~-CD45~-were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting(MP-FACS),and their biological characteristic were conducted by detecting the surface antigen of mesenchymal stem cells(MSCs)and inducing multilineage differentiation.2.Take advantage of density gradient centrifugation to isolate mononuclear cells of human umbilical cord blood.Sorting high purity human UCB CD34~+cells by immunomagnetic beads;3.Established the in vitro culture system of UCB CD34+cells co-culture with human CD146~+hMD-PCs(experimental group),human bone marrow MSCs for feeder layer(positive control)and UCB CD34+cells alone culture system(blank control);After 1 week,2 weeks and 4 weeks of co-culture,the number of cells,the colony formation ability,immunophenotype of each system and the hematopoietic factor expression level in culture supernatant were measured and statistical analysis was performed.Result(1)CD146~+hMD-PCs were sorted by MP-FACS and the purity was(91.5±1.85)%(n=5);CD146~+hMD-PCs expressed mesenchymal surface markers CD105,CD90,CD73,CD44,and did not express endothelial cell and hematopoietic cell marker CD31,CD34,CD45;After induced differentiation culture,CD146~+hMD-PCs can differentiate into chondrogenesis,osteoblasts,adipocytes and retain their myogenicity.(2)The positive rate of cord blood CD34~+cells obtained by immunomagnetic bead sorting was(85.35±3.25)%;(3)UCB CD34~+cells co-cultured with CD146~+hMD-PCs and human BM-MSCs at a density of 5×10~4/well:(1)After 1 week of co-culture,the number of cells in the CD146~+hMD-PCs group and the BM-MSCs group were(5.36±2.63)×10~4 and(4.08±1.88)×10~4,respectively,there was no significant increase in the two groups.The cells in the blank control group was(0.78±0.47)×10~4,and the number of cells decreased significantly;After 2 weeks of co-cultivation,the number of cells in the CD146~+hMD-PCs group and the BM-MSCs group were(5.78±1.41)×10~4 and(5.2±1.59)×10~4,respectively,the number of cells increased,and the blank control group had almost no cells survive;After 4 weeks of co-culture,the number of cells in the CD146~+hMD-PCs group and the BM-MSCs group showed a downward trend,which were(3.55±2.65)×10~4,(2.86±1.27)×10~4;Almost no cell survival at 5 weeks of co-culture;After statistical analysis,there was no significant difference in the number of cells between the experimental group and the positive control group at 1 week,2weeks,and 4 weeks(P>0.05,n=6);Compared with the experimental group and the positive control group,the number of cells in the blank control group at 1 week and 2 weeks was significantly different(1 week P<0.01;2 week P<0.001,respectively,n=6).(2)The results of colony culture showed that there was no statistically significant difference in colony forming ability of CD146~+hMD-PCs group and BM-MSCs group after co-culture for 1 week,2 weeks,and 4 weeks(P>0.05,n=5),the blank control group failed to perform colony culture due to a significant decrease in the number of cells.(3)Detection by flow cytometry,the proportion of CD45~+cells,CD34~+CD33~-cells,CD14~+cells,CD10~+/CD19~+cells in the CD146~+hMD-PCs group after 1 week,2 weeks,and 4 weeks co-culture was not statistically different from the BM-MSCs group(P>0.05,n=6).(4)Detection of cytokine expression in culture supernatant by ELISA,there was no significant difference in the expression levels of IL-3,IL-6,and VEGF between the CD146~+hMD-PCs group and the BM-MSCs group.The expression levels of HGF,MCSF,TNF-?and IFN-?in CD146~+hMD-PCs group were higher than those in BM-MSCs group(P<0.05);and the expression levels of SCF,TPO in CD146~+hMD-PCs group were lesser than BM-MSCs group those in(P<0.05).Conclusion CD146~+hMD-PCs has similar biological characteristics to BM-MSCs,CD146~+hMD-PCs as the feeder cells have the same hematopoietic support effects as BM-MSCs in vitro.