不同发育阶段拟胚体对小鼠胚胎干细胞造血分化的影响

目的 通过对拟胚体(Embryoid body, EB)在造血发育过程中三个不同发育阶段的初步探讨,以了解它们对胚胎干细胞(Embryonic stem cells, ESCs)造血分化的影响.方法 根据不同阶段拟胚体的发育特点,将拟胚体分为三个阶段:EB 3-5天、EB 6-8天以及EB 9-13天.通过形态学、流式细胞仪分析、Realtime PCR以及造血集落形成试验来分析不同阶段拟胚体在造血分化过程的特点.结果 EB培养至第5天、第7天、第10天时,每6000个ES细胞形成EB的数量分别是97、100、88个;流式细胞仪检测CD34+/Sca-1+细胞,结果分别是8.91%、9.23%、12.73%;Realtime PCR结果分别显示EB培养到第6天左右时中胚层发育最为活跃,第10天时造血发育比前两个阶段要活跃;造血集落形成试验显示EB发育至第5天、第7天以及第10天时,每1×105个EB细胞形成的集落数分别是21、26、32.结论 EB 3-5天处于分化早期,各种检测参数数值都相对比较低,适合于次级拟胚体以及爆发集落的培养;EB 6-8天中胚层发育最为活跃;EB 9-13天造血发育相对比较成熟.根据不同阶段拟胚体发育的特点,可以选择适当的拟胚体作为体外造血分化模型来满足不同研究目的 需要.

The effects of different stages of embryoid bodies derived from embryonic stem cells in hematopoietic differentiation

Objective Embryoid bodies derived from embryonic stem cells may have different characteristics in different developmental stages. We studied the different embryoid bodies to wonder whether they had different characteristics in hematopoietic differentiation. Methods Three types of embryoid bodies were formed during hematopoietic development, including EB-day 3-5, EB day 6-8, EB-day 9-13. EBs were tested by the identification of morphology, and the methods of flow cytometry analysis, Realtime PCR and colony-forming cell (CFC) assay. Results EBs were formed with the numbers of 96, 100 and 88 per 6000 ES cells at day 5, day 7 and day 10 respectively. The cells of CD 34+/Sca-1+ were 8. 91%, 9.23% and 12.73% done by flow cytometry analysis. The results of Realtime PCR also showed the development of mesoderm was most active at the stage of EB-day 6-8 than at other stages, and hematopoiesis was most active at the stage of EB-day 9-13 than at other stages. The numbers of colony were 21, 26 and 32 per 1 × 105 EB cells at EB-day 5, EB-day 7 and EB-day 10 respectively in CFC assay. Conclusion EB-day 3-5 are relative low in many testing parameters. They adapt to make the secondary EBs or blast colonies. EB-day 6-8 are most active than the others in mesoderm development. EB-day 9-13 are most mature relatively in hematopoiesis. Hence, different stages of embryoid bodies can be used to be different models in vitro according to the purpose of study.