人骨骼肌源性血管外膜细胞对人脐血CD34^+细胞的体外造血支持作用

In vitro hematopoietic support of human umbilical cord blood CD34^+ cells by human skeletal muscle-derived pericytes/perivascular cells

背景:研究显示血管外膜细胞是间充质干细胞的前体细胞,其通过细胞接触或旁分泌效应调节造血干细胞的行为并支持造血,人骨骼肌源性血管外膜细胞对造血的支持作用有待于研究。目的:从人骨骼肌中分离培养血管外膜细胞并进行生物学特性鉴定,研究其对脐血CD34^+细胞的体外支持作用。方法:①利用多参数流式细胞术从人骨骼肌中分选表型为CD146^+CD56^-CD34^-CD144^-CD45^-的血管外膜细胞,并对其进行生物学鉴定;②建立以CD146^+人骨骼肌源性血管外膜细胞为滋养层的脐血CD34^+细胞体外培养体系(实验组),以人骨髓间充质干细胞为滋养层的脐血CD34^+细胞体外培养体系为阳性对照组,共培养1,2,4周检测培养体系的细胞数量、集落形成能力及免疫表型并进行统计分析。结果与结论:①通过多参数流式细胞术分选出CD146+人骨骼肌源性血管外膜细胞,回测纯度为(91.5±1.85)%(n=5);其表达间充质干细胞表面抗原CD73、CD90、CD105、CD44,不表达造血细胞及内皮细胞表面抗原CD45、CD34、CD31;经诱导培养可向骨细胞、软骨细胞、脂肪细胞及肌细胞分化;②实验组与阳性对照组相比,在细胞数、集落形成能力及免疫表型方面(CD45^+、CD34^+CD33^-、CD14^+、CD10^+/CD19)差异均无显著性意义(P> 0.05,n=6);无滋养层的空白对照组培养1周时细胞数量明显减少,2周时几乎无细胞存活;③结果表明,CD146^+人骨骼肌源性血管外膜细胞与人骨髓间充质干细胞一样对脐血CD34^+细胞具有体外支持作用。

BACKGROUND: Perivascular cells have been shown to be the precursor cells of mesenchymal stem cells, which regulate the behavior of hematopoietic stem cells and support hematopoiesis through cell-to-cell contact or paracrine effects. Hematopoietic support of human skeletal muscle-derived pericytes/perivascular cells(hMD-PCs) remains to be studied. OBJECTIVE: To identify the biological characteristics of hMD-PCs isolated from human skeletal muscle and to study their supporting effect on umbilical cord blood CD34^+ cells in vitro. METHODS:(1) hMD-PCs with phenotype CD146^+CD56^-CD34^-CD144^-CD45^-were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting, and their biological characteristics were identified.(2) The in vitro culture system of umbilical cord blood CD34^+ cells co-cultured with human CD146^+ hMD-PCs(experimental group) or with human bone marrow mesenchymal stem cells(positive control group) was established. After 1, 2 and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were measured and statistically analyzed. RESULTS AND CONCLUSION:(1) CD146^+ hMD-PCs were sorted by multiparameter fluorescence-activated cell sorting and the purity was(91.5±1.85)%(n=5). CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell markers CD45, CD34, and CD31. After induced culture, CD146+ hMD-PCs could differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts.(2) There were no significant differences in the cell number, colony formation ability or immunophenotype(CD45^+, CD34^+CD33^-, CD14^+, CD10^+/CD19^+) between experimental and positive control groups(P > 0.05, n=6). The number of cells in the blank control group without feeder was significantly decreased at 1 week of culture, and there was almost no cell survival at 2 weeks of culture.(3) In summary, CD146^+ hMD-PCs, like human bone marrow mesenchymal stem cells, have hematopoietic support capacity in vitro.