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本研究旨在探讨单纯人脐带间充质干细胞(hUC-MSC)的条件培养基对脐血CD34+细胞体外造血支持作用。分离得到的hUC-MSC以2×106接种于75 cm2培养瓶中,48 h后收集培养上清作为条件培养液。用人脐血CD34阳性分选试剂盒分选CD34+细胞。将CD34+细胞接种在3个培养体系中:hUC-MSC条件培养液+不完全甲基纤维素培养基、完全甲基纤维素阳性培养基和含10%胎牛血清的DMEM/F12+不完全甲基纤维素作为阴性培养基。培养14d观察集落的形态特征,统计细胞数≥50的造血集落单位数。流式细胞术检测组成集落形成单位的细胞免疫表型。结果表明:hUC-MSC条件培养基中能够形成集落形成单位,且以粒系和粒-巨噬集落形成单位为主(CFU-G 47.67±0.58、CFU-GM 48.67±4.73、CFU-M 3.00±2.00),未观察到红细胞系相关的集落形成单位。阳性培养基中各种集落形成单位均可见,阴性培养体系中无集落形成单位。流式细胞术检测条件培养基组CD45+细胞比例为(97.43±2.15)%,显著高于阳性对照中CD45+细胞比例(39.69±0.96)%(P<0.05)。结论:在体外hUC-MSC条件培养基能够促进CD34+细胞的发育分化,单纯hUC-MSC条件培养基具有造血支持功能,并促进CD34+细胞向髓系分化,但不能促进向红细胞分化。
This study aimed to investigate the effect of hUC-MSCs conditioned medium on the hematopoietic function of cord blood CD34 + cells in vitro. The isolated hUC-MSCs were seeded in 75 cm2 flasks at 2 × 106, and the culture supernatants were collected as conditioned medium after 48 h. CD34 + cells were sorted by human cord blood CD34 positive sorting kit. CD34 + cells were seeded in three culture systems: hUC-MSC conditioned medium + incomplete methylcellulose medium, complete methylcellulose positive medium and DMEM / F12 + incomplete methyl group with 10% fetal bovine serum Cellulose serves as negative medium. The morphological characteristics of the colonies were observed after 14 days of culture, and the number of hematopoietic colonies with the number of cells ≥50 was counted. Flow cytometry was used to detect the cellular phenotype of the colony forming units. The results showed that colony-forming units could form in hUC-MSC conditioned medium, and were mainly granulocyte-macrophage colony forming units (CFU-G 47.67 ± 0.58, CFU-GM 48.67 ± 4.73, CFU-M 3.00 ± 2.00), no erythrocyte line related colony-forming units were observed. Positive cells in various colony forming units are visible, negative culture system without colony forming units. The percentage of CD45 + cells in conditioned media group was (97.43 ± 2.15)%, which was significantly higher than that in positive control cells (39.69 ± 0.96)% (P <0.05) by flow cytometry. CONCLUSION: The hUC-MSC conditioned medium can promote the development and differentiation of CD34 + cells in vitro. The hUC-MSC conditioned medium alone has the function of hematopoietic support and promotes the differentiation of CD34 + cells into myeloid but does not promote the differentiation into erythrocytes.