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目的建立一种改良的三维分化无血清方法,对人胚胎干细胞(hESCs)向内皮细胞进行分化,并对这种方法得到的人胚胎干细胞来源内皮细胞(hESC-ECs)进行功能检测。方法在低附着培养皿中培养未分化的H9 hESCs12 d,使其形成类胚体(EBs),12 d后将已形成的EBs收集起来,用浓度为1.5 mg/ml的Ⅰ型鼠尾胶原重悬,加入六孔板中,在37℃待胶原凝固后加入EGM-2培养基培养3 d,获得出芽类胚体后用0.25%胶原酶Ⅰ和0.56 U/ml LiberaseBlendzyme消化芽生类胚体各20 min,采用流式技术分选出其中CD31阳性的细胞,并用乙酰化低密度脂蛋白(ac-LDL)摄取实验和成管实验验证这部分细胞的内皮细胞功能。结果建立了一种基于胶原培养环境的三维分化方法,采用这种方法能够将hESCs向内皮细胞分化的效率提高到18%,最终获得的hESC-ECs具有和人脐带静脉内皮细胞(HU-VECs)相似的细胞表面标志物,并在乙酰化LDL吞噬实验和血管新生实验中表现出良好的内皮细胞功能。结论建立的改良三维分化方法能够明显提高hESCs向内皮细胞的分化效率,是一种简单高效的分化方法,同时无血清培养方法为将来hESC-ECs的临床应用提供了可能。
Objective To establish an improved three-dimensional differentiation-free serum-based method to differentiate human embryonic stem cells (hESCs) into endothelial cells and to detect the function of hESC-ECs derived from this method. Methods Undifferentiated H9 hESCs were cultured on low attachment petri dishes for 12 days to form embryoid bodies (EBs). After 12 days, EBs that had formed were collected and cultured at a concentration of 1.5 mg / ml Suspended, added to a six-well plate, cultured in EGM-2 medium at 37 ℃ for 3 days to obtain the budding embryoid body, digested with 0.25% collagenase I and 0.56 U / ml LiberaseBlendzyme to digest 20 min, CD31-positive cells were sorted by flow cytometry. The endothelial cell function of these cells was verified by ac-LDL uptake and tube formation tests. Results A three-dimensional differentiation method based on collagen culture environment was established. By using this method, the efficiency of hESCs differentiating into endothelial cells was increased to 18%. The resulting hESC-ECs had the same characteristics as human umbilical vein endothelial cells (HU-VECs) Similar cell surface markers and showed good endothelial cell function in acetylated LDL phagocytosis experiments and angiogenesis experiments. Conclusion The improved three-dimensional differentiation method can significantly improve the differentiation efficiency of hESCs into endothelial cells, which is a simple and efficient method for differentiation. Meanwhile, the serum-free culture method may provide the possibility for future clinical application of hESCs.