脐带血造血干/祖细胞体外分化为不同阶段红系祖细胞的动力学观察

Dynamics of In Vitro Differentiation of Erythroid Progenitor Cells from Cord Blood Hematopoietic Stem/Progenitor Cells

目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。

Objective： To investigate the dynamics changes of in vitro differentiation of erythroid progenitor cells from cord blood mononuclear cells. Methods： 0.5% methylcellulose sedimentation of cord blood erythrocytes and density gradient centrifugation in human lymphocyte separation medium were used to obtain mononuclear cells. Cells were cultured in serum-free medium containing EPO, SCF, IGF-1 and other factors to differentiate into erythroid progenitor cells. The cell proliferation, survival rate and cell colony formation of cultured erythroid progenitor cells were detected. Results： The different stages erythroid progenitor ceils were identified according to the expression of the cell surface marker CD71 and CD235a. With prolonged incubation time, cell number gradually increased, cells can be amplified 140 times in 14 days. Erythroid progenitor cells can be identified by Wright-Giemsa staining. Colony forming ability increased after induction, and the majority of colonies formed are erythroid colonies. Cultured erythroid progenitor cells were divided into four groups by cell surface markers＇ expression, which corresponding to different stages of the erythroid progenitor cells. In different time point of induction, ratio the early erythroid progenitor cells was decreased, while ratio of late erythroid progenitor cells was increased. Conclusion： Our current research obtained the dynamic data of in vitro differentiation of erythroid progenitor ceils form cord blood mononuclear cells. Our results laid the foundation for further optimization of erythroid differentiation system to inducing homogeneous erythroid progenitor cells. Our research will also be useful for the preparation of uniform erythroid progenitor cells for clinical transfusion.