人胚胎干细胞定向诱导分化为红细胞及其生物学特性的研究

Erythrocytes directionally induced by human embryonic stem cells and their biological characteristics

目的探讨人胚胎干细胞(hESC)体外诱导制备成熟红细胞的潜能。方法通过三阶段方案诱导hESC向红细胞分化,并通过流式细胞术检测造血干/祖细胞及红细胞表面标志物的变化,通过细胞染色和形态学分析判断红细胞的成熟程度;用实时定量PCR(qRT-PCR)分析不同时间点关键调控基因和珠蛋白链的表达,并以高效液相色谱法(HPLC)检测诱导所得红细胞(cRBC)不同珠蛋白链的表达量;应用血氧分析仪对诱导所得红细胞的血红蛋白(cRBC-Hb)进行功能分析。结果定向诱导可获得CD235/CD71双阳性>80%的红细胞,细胞具有红系细胞形态和携氧能力,但hESC诱导获得的cRBC中β类珠蛋白的表达以γ-珠蛋白为主,cRBC-Hb氧饱和度为50%时的氧分压(P50)也低于成人Hb标准品检测值。实时定量PCR检测发现,伴随诱导过程胚胎干细胞干性基因表达迅速下调,与造血干细胞自我更新、增殖、分化相关基因SCF、HOXB4、Notch-1、Runx1、WNT5、PU.1和SCL,以及红系定向分化调控基因EPOR、EKLF、GATA1和MYB阶段性开启表达,但调控成人型β-珠蛋白表达的EKLF和BCL11A在诱导结束阶段处于较低的表达水平。结论 hESC在体外能向红系诱导分化,关键调控基因程序化地表达,hESC具有体外制备功能红细胞的潜能,但现有的诱导体系获得的cRBC类似于胎儿红细胞。

Objective To investigate the potential of mature erythrocytes(RBCs)being produced from human embryonic stem cells(hESCs)in vitro. Methods hESCs were induced to differentiate into RBCs by a three-stage protocol,and the changes in hematopoietic stem/progenitor cells and RBC surface markers were detected by flow cytometry. The morphological changes in induced cells were evaluated by cytospin and Wright-Giemsa staining. The expressions of key regulatory genes and globin chains at different time points were analyzed by real-time quantitative PCR,while those of different globin chains in cultured red blood cells(cRBCs)were detected by high-performance liquid chromatography(HPLC). The function of cultured red blood cell hemoglobin(cRBC-Hb)was analyzed by a blood oxygen analyzer. Results More than80% of the CD235/CD71 double positive RBCs could be obtained from hESC induction. These cRBCs had the morphology of typical RBCs,and were capable of oxygen transportation,but they dominantly expressed fetal γ-globin,and the oxygenpressure of c RBC-Hb at 50% oxygen saturation(P50)was lower than that of adults. According to quantitative RT-PCR,the expression of stemness genes of pluripotent stem cells was sharply reduced as soon as the induction was initiated.Genes related to hematopoietic stem cell self-renewal,proliferation and differentiation,such as SCF,HOXB4,Notch-1,Runx1,WNT5,PU.1 and SCL,and genes function in erythroid differentiation,like EPOR,EKLF,GATA1 and MYB,were sequentially activated. However,by the end of the induction,the expression levels of key regulators of adult β-glo-bin,EKLF and BCL11 A,were relatively low. Conclusion h ESCs can be induced to differentiate into RBCs in vitro,and the key regulatory genes are capable of programmed expression. h ESCs have the potential to produce functionalRBCs in vitro,but c RBCs manufactured with the current protocol are more similar to fetal RBCs than to adult RBCs.