胚胎干细胞来源造血细胞的细胞表型和功能分析

 【目的】 分析经卵黄囊(YS)、胎肝(FL)和骨髓来源(BM)的基质细胞条件培养液(SCCM)诱导产生的胚胎干细胞(ES)来源造血细胞的细胞表型与功能差异。【方法】 制备YS-SCCM、 FL-SCCM及BM-SCCM,将3种基质细胞条件培养液分别加入ESE 14.1细胞分化培养体系培养7 d,通过对分化ESE14.1细胞造血发育表面标志FLK-1、Integrin-α4(整联蛋白-α4、Sca-1(干细胞抗原-1)、CD34的检测、体外高增殖潜能集落形成细胞(HPP-CFC)分析及体内脾集落形成单位(CFU-S)检测,评价3种基质细胞条件培养液对ESE14.1细胞体外造血发育的调控作用。【结果】 经FL-SCCM诱导的EB细胞Flk-1、Integrinα4+ 和Sca-1+ 细胞均高于YS-SCCM和BMSC-CM诱导组,分别为3.03%、2.9%和13.74%;经BMSC-CM诱导产生的CD34+ 细胞比例最高,为1.07% 。经FLSC-CM或BMSC-CM诱导产生的造血细胞其HPP-CFC产率明显高于对照组,分别为7.4个/105细胞(P < 0.01)和5.8个/105细胞(P < 0.05);经FLSC-CM或BMSC-CM诱导产生的造血细胞其CFU-S产率亦明显高于对照组,分别为8.5个/5 × 105细胞和6.75个/5 × 105细胞(P < 0.001)。【结论】 YS-SCCM、 FL-SCCM及BM-SCCM均可诱导ESE14.1向造血细胞分化,FL-SCCM和BM-SCCM造血定向诱导效率较高,所产生的细胞具备造血细胞的正常功能,FL-SCCM诱导产生的造血细胞原始程度高于BM-SCCM诱导产生的造血细胞。

Phenotypic and Functional Analysis of Embryonic Stem Cell Derived Hematopoietic Cells

Objective] To establish an effective and stable method to induce hematopoietic cells from embryonic stem(ES) cells,the phenotype and function of ES-derived hematopoietic cells induced by stromal cell conditioned medium (SCCM) of yolk sac (YS),fetal liver (FL) or bone marrow (BM) were analyzed and compared.Methods] 10% of YS-SCCM,FL-SCCM or BM-SCCM was added to culture system for differentiation of ES cells.Flow cytometric analysis was used to identify expression of Flk1,Integrin α4,Sca-1,and CD34.Colony analysis was used to identify the quantity of high proliferative potential colony-forming cells (HPP-CFC) in differentiated ES cells.The yield of CFU-S (colony-forming unit-spleen) was also analyzed by transplanting ES cell derivatives into lethally irradiated mice.Results] Expression of Flk1,Integrin α4,Sca-1,and CD34 could be tested on induced EB cells.The percentage of Flk-1+,Integrin α4+ and Sca-1+ cells induced by were 3.03%,2.9%,and 13.74%,respectively,which are greater than other groups.The percentage of CD34+ cells induced by BMSC-CM was 1.07% which was greater than other groups.The yields of HPP-CFC from hematopoietic cells induced by FLSC-CM or BMSC-CM were 7.4 /105 cells (P ＜ 0.01) and 5.8 /105 cells (P ＜ 0.05) which were greater than the yields of control group.The yields of CFU-S from hematopoietic cells induced by FLSC-CM or BMSC-CM were 8.5/5 × 105 cells and 6.75/5 × 105 cells which were also greater than the yields of control group (P ＜ 0.001).Conclusion] Both YS-SCCM,FL-SCCM,and BM-SCCM could promote hematopoietic differentiation of ESE14.1 cells.Hematopoietic differentiation induced by FL-SCCM or BM-SCCM is more effective,which generates hematopoietic progenitor cells with normal function.Application of FL-SCCM generates more primitive hematopoietic progenitor cells than that of BM-SCCM.