改良三维条件下人胚胎干细胞向内皮细胞的分化及功能

Differentiation of Human Embryonic Stem Cells to Endothelial Cells via Improved Three-dimension Approach

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-dimension（3D） and serum-free approach to differentiate human embryonic stem cells（hESCs） into endothelial cells,and detect the endothelial functions of the obtained cells.Methods We cultured undifferentiated H9 human embryonic stem cell line in low-adhesion dishes to form embryonic bodies（EBs）.After 12 days,EBs were harvested,re-suspended into rat tail collagen type I,and put into the incubator（37℃）.After 30 minutes,EGM-2 culture medium was added to the solidified collagen,and the EBs were cultured for another 3 days to form embryonic body-sproutings（EB-sproutings）.EB-sproutings were digested with 0.25% collagenase I and 0.56 U/ml Liberase Blendzyme for 20 minutes respectively,and the CD31＋ cells were sorted by FACS.The endothelial functions were tested by Dil-ac-LDL uptake assay and tube formation assay.Results This approach raised the efficiency of endothelial differentiation to 18%,and also avoided the contamination with animal materials.The obtained hESC-derived endothelial cells（hESC-ECs） had the similar pattern of surface biomarkers as human umbilical vein endothelial cells（HUVECs）,and their endothelial functions were confirmed by the uptake of Dil-ac-LDL and the tube formation on Matrigel.Conclusions The improved 3D approach can enhance the efficiency of differentiation from hESCs into endothelial cells.Furthermore,serum free differentiation system may be applied in future hESC-based therapies for various ischemic diseases.